11.3G: Inflammation - Biology

11.3G: Inflammation - Biology

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Learning Objectives

  1. Describe the 4 processes that make up the inflammatory mechanism.
  2. Briefly describe the various beneficial effects of inflammation that are associated with plasma leakage and with diapedesis.
  3. Briefly describe the process of diapedesis, indicating the role of P-selectins, integrins, and adhesion molecules.
  4. Briefly describe the healing stage of inflammation.
  5. Briefly describe the problems that arise from chronic inflammation.

The inflammatory response is an attempt by the body to restore and maintain homeostasis after injury and is an integral part of body defense. Most of the body defense elements are located in the blood and inflammation is the means by which body defense cells and defense chemicals leave the blood and enter the tissue around the injured or infected site. Inflammation is essentially beneficial, however, excess or prolonged inflammation can cause harm.

The Mechanism of Inflammation

Essentially, four processes make up the inflammatory mechanism:

a. Smooth muscles around larger blood vessels contract to slow the flow of blood through the capillary beds at the infected or injured site. This gives more opportunity for leukocytes to adhere to the walls of the capillary and squeeze out into the surrounding tissue.

b. The endothelial cells that make up the wall of the smaller blood vessels contract. This increases the space between the endothelial cells resulting in increased capillary permeability. Since these blood vessels get larger in diameter as a result of this, the process is called vasodilation (see Figure (PageIndex{1})).

Scanning electron micrographs of a cross section of a capillary showing an endothelial cell and a capillary with a red blood cell; courtesy of Dennis Kunkel's Microscopy).

c. Molecules called selectins are produced on the membrane of the leukocyte and are able to reversibly bind to corresponding selectin glycoprotein receptors on the inner wall of the venule. This reversible binding enables the leukocyte to roll along the inner wall of the venule. Adhesion molecules are activated on the surface of the endothelial cells on the inner wall of the capillaries. Corresponding molecules on the surface of leukocytes called integrins attach to these adhesion molecules allowing the leukocytes to flatten and squeeze through the space between the endothelial cells. This process is called diapedesis or extravasation.

d. Activation of the coagulation pathway causes fibrin clots to physically trap the infectious microbes and prevent their entry into the bloodstream. This also triggers blood clotting within the surrounding small blood vessels to both stop bleeding and further prevent the microorganisms from entering the bloodstream.

You Tube movie and animation of leukocyte extravasation (diapedesis)
from ImmuneDocumentary

3D animation illustrating illustrating white blood cells leaving capillaries and entering tissue (diapedesis) as well as the endomembrane system in the leukocyte.
From Harvard University, The Inner Life of the Cell. This animation takes some time to load.

These four events are triggered and enhanced by a variety of chemical inflammatory mediators. We will now divide the inflammatory response into two stages: early inflammation and late inflammation.

Early Inflammation and Diapedesis

Most leukocyte diapedesis (extravasation) occurs in post-capillary venules because hemodynamic shear forces are lower in these venules. This makes it easier for leukocytes to attach to the inner wall of the vessel and squeeze out between the endothelial cells. We will look at this process in more detail below.

  1. During the very early stages of inflammation, stimuli such as injury or infection trigger the release of a variety of mediators of inflammation such as leukotrienes, prostaglandins, and histamine. The binding of these mediators to their receptors on endothelial cells leads to vasodilation, contraction of endothelial cells, and increased blood vessel permeability. In addition, the basement membrane surrounding the capillaries becoming rearranged so as to promote the migration of leukocytes and the movement of plasma macromolecules from the capillaries into the surrounding tissue. Mast cells in the connective tissue as well as basophils, neutrophils and platelets leaving the blood from injured capillaries, release or stimulate the synthesis of vasodilators such as histamine, leukotrienes, kinins, and prostaglandins. Certain products of the complement pathways (C5a and C3a) can bind to mast cells and trigger their release their vasoactive agents. In addition, tissue damage activates the coagulation cascade and production of inflammatory mediators like bradykinins.
  2. The binding of histamine to histamine receptors on endothelial cells triggers an upregulation of P-selectin molecules and platelet-activating factor or PAF on the endothelial cells that line the venules.
  3. The P-selectins then are able to reversibly bind to corresponding P-selectin glycoprotein ligands (PSGL-1) on leukocytes. This reversible binding enables the leukocyte to now roll along the inner wall of the venule.
  4. The binding of PAF to its corresponding receptor PAF-R on the leukocyte upregulates the surface expression of an integrin called leukocyte function-associated molecule-1 (LFA-1) on the surface of the leukocyte.
  5. The LFA-1 molecules on the rolling leukocytes can now bind firmly to an an adhesion molecule called intercellular adhesion molecule-1 (ICAM-1) found on the surface of the endothelial cells forming the inner wall of the blood vessel (see Figure (PageIndex{4})).
  6. The leukocytes flatten out, squeeze between the constricted endothelial cells, and use enzymes to breakdown the matrix that forms the basement membrane surrounding the blood vessel. The leukocytes then migrate towards chemotactic agents such as the complement protein C5a and leukotriene B4 generated by cells at the site of infection or injury (see Figure (PageIndex{5})).

Late Inflammation and Diapedesis

1) Usually within two to four hours of the early stages of inflammation, activated macrophages and vascular endothelial cells release inflammatory cytokines such as TNF and IL-1 when their toll-like receptors bind pathogen-associated molecular patterns - molecular components associated with microorganisms but not found as a part of eukaryotic cells. This enables vascular endothelial cells of nearby venules to increase their expression of adhesion molecules such as P-selectins, E-selectins, intercellular adhesion molecules (ICAMs), and chemokines.

2) The binding of TNF and IL-1 to receptors on endothelial cells triggers an maintains the inflammatory response by upregulation the production of the adhesion molecule E-selectin and maintaining P-selectin expression on the endothelial cells that line the venules.

3). The E-selectins on the inner surface of the endothelial cells can now bind firmly to its corresponding integrin E-selectin ligand-1 (ESL-1) on leukocytes (see Figure (PageIndex{4})).

4) The leukocytes flatten out, squeeze between the constricted endothelial cells, and move across the basement membrane as they are are attracted towards chemokines such as interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) generated by cells at the site of infection or injury (see Figure (PageIndex{5})). Leakage of fibrinogen and plasma fibronectin then forms a molecular scaffold that enhances the migration and retention of leukocytes at the infected site.

Benefits of Inflammation

As a result of this increased permeability:

a. Plasma flows out of the blood into the tissue.

Beneficial molecules in the plasma (see Figure (PageIndex{2})) include:

1. Clotting factors. Tissue damage activates the coagulation cascade causing fibrin clots to form to localize the infection, stop the bleeding, and chemotactically attract phagocytes.

2. Antibodies. These help remove or block the action of microbes through a variety of methods that will be explained in Unit 6.

3. Proteins of the complement pathways. These, in turn: 1) stimulate more inflammation (C5a, C3a, and C4a), 2) stick microorganisms to phagocytes (C3b and C4b), 3) chemotactically attract phagocytes ( C5a), and 4) lyse membrane-bound cells displaying foreign antigens (membrane attack complex or MAC).

4. Nutrients. These feed the cells of the inflamed tissue.

5. Lysozyme, cathelicidins, phospholipase A2,and human defensins. Lysozyme degrades peptidoglycan. Cathelicidins are cleaved into two peptides that are directly toxic to microbes and can neutralize LPS from the gram-negative bacterial cell wall. Phospholipase A2 hydrolyzes the phospholipids in the bacterial cytoplasmic membrane. Human defensins put pores in the cytoplasmic membranes of many bacteria. Defensins also activate cells involved in the inflammatory response.

6. Transferrin.Transferrin deprives microbes of needed iron.

b. Leukocytes enter the tissue through a process called diapedesis or extravasation, discussed above under early inflammation and late inflammation.

Benefits of diapedesis include (see Figure (PageIndex{2})):

1. Increased phagocytosis. Neutrophils, monocytes that differentiate into macrophages when they enter the tissue, and eosinophils are phagocytic leukocytes.

2. More vasodilation. Basophils, eosinophils, neutrophils, and platelets enter the tissue and release or stimulate the production of vasoactive agents that promote inflammation.

3. Cytotoxic T-lymphocytes (CTLs), effector T4-cells, and NK cells enter the tissue to kill cells such as infected cells and cancer cells that are displaying foreign antigens on their surface (discussed in Unit 6).

Cytokines called chemokines are especially important in this part of the inflammatory response. They play key roles in diapedesis -enabling white blood cells to adhere to the inner surface of blood vessels, migrate out of the blood vessels into the tissue, and be chemotactically attracted to the injured or infected site. They also trigger extracellular killing by neutrophils.

Finally, within 1 to 3 days, macrophages release the cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-a). These cytokines stimulate NK cells and T-lymphocytes to produce the cytokine interferon-gamma. (IF-?). The IF-? then binds to receptors on macrophages causing them to produce fibroblast growth factor and angiogenic factors for tissue remodeling. With the proliferation of endothelial cells and fibroblasts, endothelial cells form a fine network of new capillaries into the injured area to supply blood, oxygen, and nutrients to the inflamed tissue. The fibroblasts deposit the protein collagen in the injured area and form a bridge of connective scar tissue to close the open, exposed area. This is called fibrosis or scarring, and represents the final healing stage.

Inflammation is normally carefully regulated by cytokines. Inflammatory cytokines such as interferon-gamma and interleukin-12 enhance the inflammatory response whereas the cytokine interleukin-10 inhibits inflammation by decreasing the expression of inflammatory cytokines.

So as can be seen, acute inflammation is essential to body defense. Chronic inflammation, however, can result in considerable tissue damage and scarring. With prolonged increased capillary permeability, neutrophils continually leave the blood and accumulate in the tissue at the infected or injured site. As they discharge their lysosomal contents and reactive oxygen species or ROS, surrounding tissue is destroyed and eventually replaced with scar tissue. Anti-inflammatory agents such as antihistamines or corticosteroids may have to be given to relieve symptoms or reduce tissue damage.

For example, as learned in Unit 3, during severe systemic infections with large numbers of microorganisms present, high levels of pathogen-associated molecular patterns (PAMPs) are released resulting in excessive cytokine production by macrophages and this can harm the body. In addition, neutrophils start releasing their proteases and reactive oxygen species that kill not only the bacteria, but the surrounding tissue as well. Harmful effects include high fever, hypotension, tissue destruction, wasting, acute respiratory distress syndrome or ARDS, disseminated intravascular coagulation or DIC, damage to the vascular endothelium, hypovolemia, and reduced perfusion of blood through tissues and organs resulting to shock, multiple system organ failure (MOSF), and often death. This excessive inflammatory response is referred to as Systemic Inflammatory Response Syndrome or SIRS or the Shock Cascade.

Exercise: Think-Pair-Share Questions

  1. Briefly describe the mechanisms that enable to slow the flow of blood at an infection site and get phagocytes, complement proteins and antibodies to the infection site.
  2. Why is it important to deliver plasma to an infection site?
  3. Why is it important for diapedesis to occur during inflammation?

Chronic inflammation also contributes to heart disease, Alzheimer's disease, diabetes, and cancer.

  • In the case of cancer,it is proposed that when macrophages produce inflammatory cytokines, such as TNF-alpha, these cytokines activate a gene switch in the cancer cell that turns on the synthesis of proteins that promote cell replication and inflammation while blocking apoptosis of the cancer cell.
  • In heart disease, it is thought that macrophages digest low density lipoprotein or LDL, the bad cholesterol, and are then encased in a fibrous cap that forms arterial plaque.
  • With diabetes, it is thought that the metabolic stress of obesity triggers innate immune cells and fat cells to produce cytokines such as TNF-alpha that can interfere with the normal function of insulin.
  • In the case of Alzheimer's disease, microglial cells, macrophage-like cells in the brain, interact with the beta-amyloid proteins that build up in neurons of those with Alzheimer's and subsequently produce inflammatory cytokines and free radicals that destroy the neurons.


  1. Most of the body defense elements are located in the blood and inflammation is the means by which body defense cells and defense chemicals leave the blood and enter the tissue around the injured or infected site.
  2. As part of the mechanism for inflammation, smooth muscles around larger blood vessels contract to slow the flow of blood through the capillary beds at the infected or injured site. This gives more opportunity for leukocytes to adhere to the walls of the capillary and squeeze out into the surrounding tissue.
  3. As part of the mechanism for inflammation, the endothelial cells that make up the wall of the smaller blood vessels contract. This increases the space between the endothelial cells resulting in increased capillary permeability.
  4. As part of the mechanism for inflammation, adhesion molecules are activated on the surface of the endothelial cells on the inner wall of the capillaries and corresponding molecules on the surface of leukocytes called integrins attach to these adhesion molecules allowing the leukocytes to flatten and squeeze through the space between the endothelial cells. This process is called diapedesis or extravasation.
  5. As part of the mechanism for inflammation, activation of the coagulation pathway causes fibrin clots to physically trap the infectious microbes and prevent their entry into the bloodstream.
  6. Acute inflammation is essential to body defense.
  7. As a result of this increased permeability, plasma flows out of the blood into the tissue delivering clotting factors, antibody molecules, complement pathway proteins, nutrients, antibacterial enzymes and peptides, and transferrin for innate body defense.
  8. As a result of this increased permeability, leukocytes enter the tissue delivering phagocytic cells, inflammation-inducing cells, cytotoxic T-lymphocytes, effector T4-lymphocytes, and NK cells.
  9. Inflammatory cytokines also, enable endothelial cells form a fine network of new capillaries into the injured area to supply blood, oxygen, and nutrients to the inflamed tissue, and enable fibroblasts to deposit the protein collagen in the injured area and form a bridge of connective scar tissue to close the open, exposed area.
  10. Chronic inflammation can result in considerable tissue damage and scarring, primarily to extracellular killing by phagocytes and hypoperfusion.
  11. Chronic inflammation is thought to also contribute to heart disease, Alzheimer's disease, diabetes, and cancer.

Inflammation can be either short-lived (acute) or long-lasting (chronic). Acute inflammation goes away within hours or days. Chronic inflammation can last months or years, even after the first trigger is gone. Conditions linked to chronic inflammation include:

Some types of arthritis are the result of inflammation, such as:

Other painful conditions of the joints and musculoskeletal system that may not be related to inflammation include osteoarthritis, fibromyalgia, muscular low back pain, and muscular neck pain.

Scott Brakenridge, M.D., MSCS

Scott Brakenridge, M.D., MSCS., is an assistant professor of surgery on the acute care surgery team at UF Health. Dr. Brakenridge joined the UF faculty in 2013 specifically to work with the multi-disciplinary research team investigating the persistent inflammation/immunosuppression and catabolism syndrome (PICS) that occurs after surgical sepsis. He is currently one of the principal investigators for an NIH-funded P50 center grant for the UF Sepsis and Critical Illness Research Center to study the persistent inflammation, immunosuppression and catabolism syndrome (PICS) following sepsis in surgical intensive care unit patients. His current research projects include:

Persistent inflammation/immunosuppresion and catabolism syndrome (PICS) – Advances in critical care medicine over the past two decades have significantly decreased in-hospital mortality following sepsis in surgical and trauma patients. However, instead of dying from septic shock and sudden multiple organ failure, patients survive with an extended course of chronic critical illness. When we studied the epidemiology of these patients, we recognized that many of the survivors lingered in the intensive care unit with manageable organ dysfunctions. Their clinical course is characterized by recurrent inflammatory insults (e.g., repeat operations and nosocomial infections, which are those that originate in a hospital), a persistent acute-phase response with ongoing loss of lean body mass despite optimal nutritional support, poor wound healing and bedsores. These patients (especially the elderly) are commonly discharged to long-term acute care facilities and skilled nursing facilities with significant cognitive and functional impairments, from which they rarely recover fully. Few ever return to independent function and more than 60 percent of these patients are dead within two years. The researchers in the laboratory of inflammation biology and surgical science at UF Health hypothesize that chronic critical illness, driven by PICS and characterized by morbid long-term outcomes, is now the predominant clinical trajectory in surgical ICU sepsis survivors.

Endothelial C3a receptor mediates vascular inflammation and blood-brain barrier permeability during aging

2 Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA.

3 Institute for Systemic Inflammation Research, Center for Infectiology and Inflammation Research Lübeck, University of Lübeck, Lübeck, Germany.

4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Hui Zheng, Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. Phone: 713.798.1568 Email: [email protected] AL’s present address: Department of Neurology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA.

Find articles by Propson, N. in: JCI | PubMed | Google Scholar | />

1 Department of Molecular and Cellular Biology, and

2 Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA.

3 Institute for Systemic Inflammation Research, Center for Infectiology and Inflammation Research Lübeck, University of Lübeck, Lübeck, Germany.

4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Hui Zheng, Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. Phone: 713.798.1568 Email: [email protected] AL’s present address: Department of Neurology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA.

Find articles by Roy, E. in: JCI | PubMed | Google Scholar | />

1 Department of Molecular and Cellular Biology, and

2 Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA.

3 Institute for Systemic Inflammation Research, Center for Infectiology and Inflammation Research Lübeck, University of Lübeck, Lübeck, Germany.

4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Hui Zheng, Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. Phone: 713.798.1568 Email: [email protected] AL’s present address: Department of Neurology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA.

Find articles by Litvinchuk, A. in: JCI | PubMed | Google Scholar

1 Department of Molecular and Cellular Biology, and

2 Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA.

3 Institute for Systemic Inflammation Research, Center for Infectiology and Inflammation Research Lübeck, University of Lübeck, Lübeck, Germany.

4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Hui Zheng, Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. Phone: 713.798.1568 Email: [email protected] AL’s present address: Department of Neurology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA.

1 Department of Molecular and Cellular Biology, and

2 Huffington Center on Aging, Baylor College of Medicine, Houston, Texas, USA.

3 Institute for Systemic Inflammation Research, Center for Infectiology and Inflammation Research Lübeck, University of Lübeck, Lübeck, Germany.

4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Address correspondence to: Hui Zheng, Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. Phone: 713.798.1568 Email: [email protected] AL’s present address: Department of Neurology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA.

Find articles by Zheng, H. in: JCI | PubMed | Google Scholar | />

Published September 29, 2020 - More info

Related article:

Complement C3a receptor–mediated vascular dysfunction: a complex interplay between aging and neurodegeneration

Complement C3a receptor–mediated vascular dysfunction: a complex interplay between aging and neurodegeneration


Vascular dysfunction resulting in compromised blood-brain barrier (BBB) integrity is evident in aging and disease. Although the complement C3a/C3a receptor (C3a/C3aR) axis influences normal brain aging and disease progression, the mechanisms governing endothelial C3aR–mediated neurovascular inflammation and BBB permeability remain unexplored. In this issue of the JCI, Propson et al. investigated endothelial C3a/C3aR signaling in normal, aged, and neurodegenerative mouse models. Endothelial C3aR signaling modulated age-dependent increases in VCAM1, initiated peripheral lymphocyte infiltration, and enhanced microglial activity. Increased calcium release downstream of C3aR signaling disrupted the vascular endothelial cadherin (VE-cadherin) junctions, increased BBB permeability, and degraded vascular structure and function. Mice lacking C3aR (C3ar1–/–) and mice treated with a C3aR antagonist showed attenuated age-related microglial reactivity and neurodegeneration. These results confirm that complement-mediated signaling impacts vascular health and BBB function in normal aging and neurodegenerative disease, suggesting that complement inhibitors represent a therapeutic option for cerebral microvascular dysfunction.


Kanchan Bhatia, Saif Ahmad, Adam Kindelin, Andrew F. Ducruet

Dysfunction of immune and vascular systems has been implicated in aging and Alzheimer disease however, their interrelatedness remains poorly understood. The complement pathway is a well-established regulator of innate immunity in the brain. Here, we report robust age-dependent increases in vascular inflammation, peripheral lymphocyte infiltration, and blood-brain barrier (BBB) permeability. These phenotypes were subdued by global inactivation and by endothelial cell–specific ablation of C3ar1. Using an in vitro model of the BBB, we identified intracellular Ca 2+ as a downstream effector of C3a/C3aR signaling and a functional mediator of vascular endothelial cadherin junction and barrier integrity. Endothelial C3ar1 inactivation also dampened microglia reactivity and improved hippocampal and cortical volumes in the aging brain, demonstrating a crosstalk between brain vasculature dysfunction and immune cell activation and neurodegeneration. Further, prominent C3aR-dependent vascular inflammation was also observed in a tau-transgenic mouse model. Our studies suggest that heightened C3a/C3aR signaling through endothelial cells promotes vascular inflammation and BBB dysfunction and contributes to overall neuroinflammation in aging and neurodegenerative disease.

The natural aging process includes functional and structural changes within the brain ( 1 , 2 ), and these changes have been shown to play a role in decreased neural stem cell fitness, altered cognition, and increased susceptibility for neurodegenerative disease ( 1 , 3 , 4 ). One such age-dependent change with potentially causal association with both normal decline and disease is dysfunction of the blood-brain barrier (BBB). The BBB is composed of endothelial cells, astrocytes, and pericytes, and an intact BBB is essential for brain health ( 5 ). Loss of vessel integrity is thought to drive BBB dysfunction and can be found in numerous neurological disease conditions, namely traumatic brain injury ( 6 ) and stroke ( 7 ), and often comorbidly in neurodegeneration ( 8 ). However, the mechanisms controlling changes in brain vasculature and their consequence to CNS function, in particular during aging, remain ill-defined.

Recent work has demonstrated a vascular component to age-related changes in the brain, deterioration in cognition, and eventual dementia ( 9 , 10 ). Furthermore, evidence has been reported that vascular inflammation, marked by increased endothelial expression of vascular cell adhesion molecule VCAM1, stokes CNS aging by decreasing neural stem cell numbers and increasing microglial reactivity ( 11 ). Elevated levels of VCAM1 have also been found to correlate with Parkinson disease severity ( 12 ), and, recently, lymphocytes known to bind VCAM1 in brain vasculature were found in aged patients and the brains of patients with Alzheimer disease ( 13 , 14 ). Single-cell transcriptomic analysis of hippocampal brain endothelial cells has shown that age-related changes in these cells are rooted in responses to innate inflammatory cues, hypoxic stimuli, and oxidative stress ( 15 ). Together, these studies suggest a significant inflammatory transition in brain vasculature with age and the potential for a causal connection to CNS diseases.

Some studies of blood plasma components have implicated circulating factors in maintaining or diminishing brain health during aging ( 16 ), and others exploring local inflammatory cues in the CNS have highlighted the inherent capacity of glia to modulate neuroinflammation ( 17 ). One of the primary innate immune-signaling mechanisms involved in neuroinflammation is the complement pathway. Complement components are expressed by cells of the CNS and are reported to influence CNS aging and neurodegenerative disease progression ( 18 ). In particular, the complement component C3 is capable of potentiating age-related and neurodegenerative changes in the CNS ( 19 – 22 ). The active signaling peptide of C3, C3a, is released via cleavage by the extracellular enzyme C3 convertase. Once cleaved, C3a signals through its cognate receptor C3aR, which has been detected on microglia ( 23 ), choroid plexus epithelium ( 24 ), and vascular endothelial cells ( 25 ) in the brain. C3 is upregulated in astrocytes during aging and disease ( 22 , 26 ), and the intimate relationship of astrocytes with the BBB supports the premise that C3 produced by these cells may play a direct role in age-related changes in brain vasculature.

Using in vivo and in vitro models, we identified a mechanism by which the C3a/C3aR signaling axis modulated VCAM1 expression, influenced peripheral immune cell infiltration, altered vascular morphology, increased BBB permeability, and potentiated microglial reactivity and neurodegeneration in aged mice. We further showed that this C3aR-dependent endothelial phenotype was exacerbated in PS19 tau–transgenic mice, a model in which elevated C3/C3aR signaling was shown to modulate CNS inflammation and tau pathology ( 22 ). This study identified complement signaling as a key mediator of vascular dysfunction in brain aging and disease.

C3a/C3aR signaling regulates age-associated endothelial VCAM1 expression and immune cell infiltration. C3 mRNA has been shown to be upregulated in aged astrocytes ( 20 , 26 ). To corroborate this finding, we measured C3 protein in lysates of mouse brains at 2, 12, and 20 months of age by ELISA. We detected a significant increase at 12 months, with a further increase at 20 months, over levels detected in the young mice (Figure 1A). Consistent with our earlier reports in disease models ( 21 , 22 ), coimmunofluorescent labeling of the hippocampus showed C3 expression predominantly colocalized in GFAP + astrocytes, where its levels were elevated during aging (Figure 1, B and C, and Supplemental Figure 1A supplemental material available online with this article Increased C3 mRNA was further validated in FACS-sorted aged astrocytes using our previously published method ( 27 ) (Supplemental Figure 1B). To examine the expression of C3aR in brain vasculature, we isolated vessels from WT and C3ar1 –/– mouse brains ( 28 ) and immunostained them with antibodies against GFAP, VE-cadherin, and C3aR. Positive C3aR staining could be readily detected in VE-cadherin + brain endothelial cells in WT but not C3ar1 –/– vessels (Figure 1D). High-resolution confocal imaging analysis of CD31, Glut1, and C3aR on mouse brain vasculature revealed a greater polarization of C3aR toward the basolateral surface, whereas Glut1 localized predominantly toward the vessel lumen (Figure 1E and Supplemental Figure 1C). In contrast to the endothelial expression, costaining for C3aR and the pericyte marker PDGFR-β did not detect appreciable colocalization (Figure 1E). Flow cytometry analysis of human brain microvascular endothelial cells (HBMECs) showed high levels of positivity for VE-cadherin (80.9%) and Glut1 (94.2%), as expected, and this positivity was also observed for C3aR, although to a lesser degree (34.3%) (Supplemental Figure 1D). Together, these results established C3aR expression in brain vascular endothelial cells and supported a signaling axis involving astroglial C3 and endothelial C3aR at the BBB.

C3a/C3aR signaling regulates age-associated endothelial VCAM1 expression. (A) ELISA measurement of C3 levels in WT mouse brain lysates at 2, 12, and 20 months (n = 8/group). (B) Immunofluorescence staining using anti-GFAP and anti-C3 antibodies demonstrated localization of C3 to astrocytes. (C) Quantification confirming increased C3 staining within GFAP + astrocytes in the hippocampus with age (n = 5/age). (D) Triple immunostaining of isolated vessels from WT and C3ar1 –/– brains using anti-GFAP, anti–VE-cadherin, and anti-C3aR antibodies showing positive C3aR staining along endothelial cell surface that was not present in C3ar1 –/– vessels. (E) Triple immunostaining of brain tissue with anti-Glut1, anti-C3aR, and anti-CD31 or anti–PDGFR-β, anti-C3aR, and anti–Col IV, demonstrating expression of C3aR on brain endothelial cells but not pericytes. (F and G) Immunofluorescence staining and quantification using anti-CD31 and anti-VCAM1 antibodies of WT or C3ar1 –/– mouse cortices at 2, 12, and 20 months demonstrated an increase in VCAM1 with age in WT mice, but VCAM1 was rescued in the absence of C3aR. All data represent the mean ± SEM. Significance was calculated using 1-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Scale bars: 20 μm (B), 10 μm (D), 15 μm (E), and 50 μm (F).

To assess the functional role of this signaling pathway, we first analyzed the expression of VCAM1 because it has been implicated in complement-mediated activation of brain endothelial cells in LPS-induced neuroinflammation ( 29 ) and ischemia models ( 30 ). Coimmunofluorescent labeling of brain cortical vasculature revealed that VCAM1 expression was restricted to CD31 + vasculature, where its levels were increased with age in WT mice but not in C3ar1-null mice (Figure 1, F and G). A similar reduction in vascular VCAM1 was also seen after treating 2-, 12-, and 20-month-old mice with the C3aR antagonist (C3aRA) SB290157 (Supplemental Figure 2, A and B), further confirming our genetic study and validating the inhibitory effect of C3aRA. The same was true when hippocampal samples were analyzed (Supplemental Figure 2C). We then performed deeper analysis by measuring Vcam1 mRNA levels in FACS-sorted mouse brain endothelial cells from 2-, 12-, and 20-month-old cohorts treated with vehicle or C3aRA, which showed increased Vcam1 expression with age in vehicle-treated samples but blunted expression with C3aRA treatment (Supplemental Figure 2D).

To substantiate this signaling pathway in human cells and to test a direct effect of C3a/C3aR signaling, we treated primary HBMECs with recombinant human C3a, with or without C3aRA, and found a robust increase in Vcam1 expression by C3a treatment, which was quelled in the presence of C3aRA (Supplemental Figure 2E). Interestingly, other adhesion molecules, namely Sele and Icam1, were not significantly changed by this treatment (Supplemental Figure 2, F and G). Additionally, similar ICAM1 immuno-intensities (Supplemental Figure 2, H–J) and Icam1 mRNA levels (Supplemental Figure 2K) were detected in 2- and 20-month-old mouse brains. Together, these data support both the necessity and sufficiency of C3a signaling in modulating VCAM1 expression in brain endothelial cells.

Next, we examined the functional consequence of C3-mediated VCAM1 upregulation. It has been previously shown that increased numbers of peripheral lymphocytes are found in the aged brain ( 31 ) and reside in the neural stem cell niche of the subventricular zone ( 13 ). Because adhesion molecules regulate the process of rolling adhesion and extravasation of immune cells, we hypothesized that increased brain VCAM1 expression in vasculature during aging may be associated with increased peripheral immune cell infiltration. Using FACS to discriminate CD45 hi /CD11b – lymphocytes from CD45 hi/mid /CD11b + monocytes and microglia in dissociated brain tissues, we found increasing proportions of CD45 hi /CD11b – infiltrates at 12 and 20 months compared with 2-month-old controls (Figure 2, A and B). In contrast, monocytes were not significantly changed with age (Supplemental Figure 3A). We next asked whether, in addition to reducing VCAM1 expression levels, blocking C3aR signaling could reduce age-related lymphocyte infiltration. Flow cytometry analysis of 12- to 14-month-old C3ar1 –/– mice revealed a reduction in the total percentage of CD45 hi /CD11b – -infiltrating lymphocytes in the brain compared with WT controls (Figure 2, C and D), whereas monocytes were unaffected (Supplemental Figure 3B). Similar reductions in CD45 hi /CD11b – -infiltrating lymphocytes but not monocytes were observed after treating 20-month-old WT mice with C3aRA (Supplemental Figure 3, C–E).

C3aR/VCAM1 axis promotes peripheral lymphocyte infiltration during aging. (A) Representative CD45 and CD11b flow cytometric plots and gating strategy of dissociated mouse brain at 2 months, 12 months, and 20 months. LY, lymphocyte MN, monocyte MG, microglia DN, double-negative. (B) Flow cytometric analysis and quantification of percentage of infiltrating lymphocytes (2 months n = 12, 12 months n = 7, 20 months n = 12) showed age-related increase. (C and D) Flow cytometry analysis and quantification of brain lymphocytes in 12–14-month-old WT (n = 9) and C3ar1 –/– (n = 10) mice showed reduction in aged C3ar1 –/– mice. All data represent the mean ± SEM. Significance was calculated using 1-way ANOVA with Tukey’s post hoc test (**P < 0.01, ***P < 0.001).

Given that acute neuroinflammation induces peripheral immune cell infiltration ( 29 ), we tested the role of C3aR in recruiting these cells after i.c.v. administration of LPS in 3- to 4-month-old WT and C3ar1 –/– mice (Supplemental Figure 4A). As expected, LPS challenge led to increased lymphocyte (Supplemental Figure 4B) and monocyte (Supplemental Figure 4C) infiltration in the brains of WT mice. In contrast, genetic ablation of C3ar1 blunted the infiltration of CD45 hi /CD11b – peripheral lymphocytes (Supplemental Figure 4B), whereas monocyte cell numbers were only marginally affected (Supplemental Figure 4C). Consistent with the specific regulation of VCAM1 by C3a/C3aR signaling, deletion of C3ar1 significantly suppressed Vcam1 gene expression induced by LPS without affecting Sele and Icam1 in FACS-sorted brain endothelial cells (Supplemental Figure 4D).

To assess whether LPS-induced acute neuroinflammation affects BBB permeability, we analyzed each group using a tail-vein injection of a BBB impenetrable TRITC-dextran dye (65–85 kDa). Interestingly, there was no significant increase in BBB permeability under acute neuroinflammatory stimuli that contributed to this infiltration event (Supplemental Figure 4E). To confirm the induction of neuroinflammation by LPS, we analyzed sorted microglia from the vehicle- and LPS-treated animals and found consistent microglial gene response signatures, as we have previously reported ( 27 ) (Supplemental Figure 4F).

Together, these results suggest that C3a acts through endothelial C3aR to promote VCAM1 expression, and that during both aging and acute neuroinflammation, this pathway plays a role in selective infiltration of lymphocytes into the brain.

CD8 + T cells preferentially infiltrate the aged brain. We next examined the lymphocyte subtypes infiltrating the aged brain and compared these with the cells of the aged spleen to better understand the global changes to lymphocyte composition in aged tissues. Dissociated cells from the brain or spleen of 20-month-old WT mice were immunostained using anti-CD45, anti-CD11b, anti-CD3ε, anti-CD19, anti-CD8a, and anti-CD4 antibodies. After antibody staining, cells were analyzed by flow cytometry to differentiate monocytes, microglia, and lymphocytes (Figure 3A, left panels). Further subtyping was carried out to analyze T lymphocyte versus B lymphocyte composition (Figure 3A, middle panels), and T lymphocyte subtyping to differentiate CD8 + versus CD4 + T cells (Figure 3A, right panels). In 20-month-old WT mice, we found that infiltrating lymphocytes were predominantly CD3 + (75%) versus CD19 + (10%), and analysis of matched spleens showed significantly more CD19 + (65%) cells versus CD3 + (25%) cells (Figure 3B), suggesting that the aged brain preferentially recruited CD3 + T cells compared with the aged spleen. Further analysis of brain CD3 + T cells showed that the majority were CD8 + T cells (75%) instead of CD4 + T cells (15%), which also inversely correlated with the peripheral splenic tissue, where CD4 + T cells were significantly more abundant than CD8 + T cells (Figure 3C). This data suggests that CD8 + T cells were preferentially recruited to the aged brain compared with other lymphocyte subsets.

CD8 + T cells are preferentially recruited and infiltrate the aged brain. (A) Schematic for flow cytometry analysis of dissociated infiltrating cells in the 20-month-old brain or spleen using antibody staining against CD45, CD11b, CD3ε, CD19, CD8a, and CD4. TC, T cells BC, B cells. (B) Quantification of flow cytometry analysis showed predominantly CD3 + T cells in the brain compared with the spleen, which showed predominantly CD19 + B cells (n = 7/group). (C) Quantification of T cells showed the predominant subtype enriched in the brain was CD8 + T cells compared with CD4 + T cells in the spleen (n = 7/group). (D) Representative immunostaining of 2-month-old and 20-month-old brain tissue using anti-CD8a and anti–Col IV to determine regional immune cell infiltration within the brain. (E) Magnified image from D highlighting the infiltrated cell types that were counted for analysis. (F) Quantification of regional distribution of CD8 + T cell infiltrates in 4 major brain regions (n = 4/group, 2 tissue sections per mouse). CTX, cortex THAL, thalamus CPu, caudate putamen HPC,hippocampus. (G) Representative images of 3 distinct stages of infiltration observed in all brain regions: perivascular residence (left panel), extravasation (middle panel), and parenchymal surveillance (right panel). Data in B and C represent the mean ± SEM. Analysis was done using 1-way ANOVA with Tukey’s post hoc test (***P < 0.001). Data in F are violin plots displaying medians and quartile ranges. Analysis was done using 2-way ANOVA with Holm-Sidak post hoc test (*P < 0.05, ***P < 0.001). Scale bars: 100 μm (D), 50 μm (E), 10 μm (G).

To further evaluate whether this age-dependent change was specific to brain parenchyma, representing potential infiltration, we isolated the choroid plexus tissue from brain ventricles of 2- and 20-month-old mice prior to dissociation and performed similar analyses as stated above, using the spleen as a control. Using the same flow cytometry gating strategy, we saw increased numbers of CD3 + and CD8 + lymphocytes only in aged brains, but not in aged choroid plexus tissue (Supplemental Figure 5, A–C) or the spleen (Supplemental Figure 5, D–F). These data suggest that the age-related CD8 + lymphocyte infiltration was specific to brain parenchyma and not present in other immune cell–rich regions of the brain or peripheral tissues.

To confirm that peripheral cells are recruited to and infiltrate the aged brain, we employed an mT/mG reporter mouse model, which produces tdTomato under the ROSA26 locus ( 32 ). When crossed with Mx1-Cre mice and activated by peripheral poly I:C (polyinosinic:polycytidylic acid) treatment, Cre-responsive cells in the periphery recombined to express EGFP, generating a chimeric mouse (MXG) (Supplemental Figure 6A). Analysis of PBMCs confirmed that approximately 40% of PBMCs were converted to express EGFP in MXG mice (Supplemental Figure 6, B–D). To assess brain infiltration of EGFP + peripheral cells during aging, we injected mice with poly I:C at 2 months of age and allowed mice to age to 15 months. Flow cytometry analysis of dissociated brains showed that approximately half of infiltrating CD45 hi /CD11b – lymphocytes expressed EGFP in 15-month-old MXG brains (Supplemental Figure 6, E–G), demonstrating that lymphocytes in aged brains were peripherally derived. Confocal imaging of the aged brain tissue confirmed the presence of EGFP + -infiltrated peripheral immune cells along the basolateral surface of tdTomato + EGFP – brain vessels, further indicating the peripheral origin of the cells (Supplemental Figure 6H).

To better understand the regional distribution of these CD8 + infiltrates, we analyzed 2- and 20-month-old mouse brain tissue by immunostaining with antibodies against CD8 to mark the T cells of interest and collagen IV (Col IV) to mark the endothelial basement membrane (Figure 3, D and E). Besides the subventricular zone as previously reported ( 13 ), it was clear that CD8 T cells were present in other brain regions as well. Notably, the cortex, thalamus, caudate putamen, and the hippocampus all had a significant presence of CD8 T cells with age (Figure 3F). These cells were found either residing along the basolateral surface with colocalized Col IV (Figure 3G left panel), extravasating with minimally colocalized Col IV (Figure 3G middle panel), or fully extravasated into the tissue parenchyma (Figure 3G right panel). Overall, these findings demonstrated that during aging, peripherally derived CD8 + T cells preferentially infiltrated and took up residence inside the brain parenchyma in numerous tissue regions.

Inhibition of C3aR rescues age-related changes in vascular morphology and BBB permeability. We next determined the effect of C3a/C3aR signaling on brain vascular morphology at different ages. We performed confocal imaging and 3D reconstruction of the vasculature, visualized by Col IV staining, and measured the average cross-sectional area of hippocampal capillaries by dividing the total Col IV + volume by the total capillary length in each reconstructed image. Capillaries in young mice showed an average cross-sectional area of approximately 60 μm 2 , whereas capillaries in 12- and 20-month-old mice averaged approximately 40 μm 2 (Figure 4A). This reduction was partially but significantly rescued in 20-month-old C3ar1 –/– mice and in mice treated with C3aRA (Figure 4B). Analysis of each component of this measurement demonstrated a decrease in total vascular volume (Supplemental Figure 7A) and an increase in vessel length (Supplemental Figure 7B) during aging, both of which contributed to decreased average cross-sectional area. Additionally, CD31 + vessels showed a higher degree of tortuosity in the aged hippocampus, as previously defined by their corkscrew-like morphology ( 33 , 34 ) (Figure 4C, marked by rectangles). This phenomenon has been linked to decreased hemodynamic flow and hypoxia in affected brain regions ( 33 , 34 ). The overall incidence of tortuous vessel segments was increased approximately 2- and 4-fold by 12 and 20 months, respectively, over that observed in 2-month-old mice (Figure 4, C and D). This vascular phenotype was rescued by both genetic ablation of C3ar1 and C3aRA treatment in 20-month-old mice (Figure 4, C and D). Thus, blocking C3aR significantly improved vascular morphology in aged mice.

Inhibition of C3aR rescues age-related changes in vascular morphology and BBB permeability. (A) Representative collagen IV + (Col IV) staining and IMARIS-aided 3D reconstruction of vasculature in hippocampal sections from 2-, 12-, and 20-month-old WT mice 20-month-old WT mice treated with C3aRA or 20-month-old C3ar1 –/– mice. (B) Quantification of capillary average cross-sectional area in A (n = 5/group, 8 images per mouse). (C) Representative CD31 + staining and 3D reconstruction of hippocampal vasculature in 2-, 12-, and 20-month-old WT mice 20-month-old WT mice treated with C3aRA or 20-month-old C3ar1 –/– mice. Representative tortuous vessels are marked by rectangles. (D) Quantification of number of tortuous vessels per hippocampal areas (n = 5/group, 8 images per mouse). (E) Representative lectin and TRITC-dextran colabeling in 2- and 20-month-old hippocampi. (F) Quantification of TRITC-dextran MFI from brain lysates of 2-month-old (n = 13), 12-month-old (n = 10), and 20-month-old (n = 14) mice. (G) Quantification of TRITC-dextran MFI of 20-month-old mice treated with vehicle or C3aRA (n = 4/group). (H) Representative image of vessels isolated from 2- and 12-month-old mice or 20-month-old mice treated with vehicle or C3aRA and stained with anti-VE-cadherin. (I) Quantification of VE-cadherin staining showed reduced VE-cadherin expression in 12- and 20-month-old mice, which was partially rescued in 20-month-old mice treated with C3aRA (n = 5/group, 5 vessel fragments/mouse). All data represent the mean ± SEM. Significance was calculated using 1-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). NScale bars: 20 μm (A and C) 50 μm (E).

Given the observed age-associated changes in lymphocyte infiltration and vascular morphology, we tested whether BBB integrity was affected by C3a/C3aR signaling. We used a previously reported method allowing detection of peripherally administered fluorescent dextran leakage into the brain ( 35 ) (Figure 4E). At 2 months, there was almost no detection of fluorescent dextran in brain tissues, whereas there was a robust increase in fluorescent signal in 20-month-old brains, localized around lectin + vasculature (Figure 4E). Quantification of dextran levels in brain homogenates of 2-, 12-, and 20-month-old mice showed an age-dependent tracer leakage into the brain at 12 months (

3-fold) (Figure 4F). Treating 20-month-old mice with C3aRA modestly but significantly reduced tracer leakage (Figure 4G).

To further characterize age-associated changes in BBB integrity, we examined VE-cadherin + intercellular junctions by confocal microscopy in vessels isolated from 2-, 12-, and 20-month-old mouse brains ( 28 ) with or without C3aRA treatment (Figure 4H and Supplemental Figure 7C). Quantification of immunoreactivity in large vessels (Figure 4I) and capillaries (Supplemental Figure 7D) revealed age-associated downregulation of VE-cadherin, with modest but significant rescue by C3aRA treatment. Consistent with this, gene expression analysis of FACS-sorted brain endothelial cells showed age-dependent reduction of Cdh5 (encoding VE-cadherin) at 12 and 20 months, along with reductions in Ocln, Tjp1, and Cldn5 mRNAs (Supplemental Figure 7E), reflecting impaired expression of junctional components. These age-dependent reductions were either trending or significantly restored with C3aRA treatment (Supplemental Figure 7E). Together, these data suggest that C3aR inhibition partially restored BBB integrity in aged brains.

C3a-mediated barrier disruption involves Ca 2+ mobilization, cytoskeletal activation, and VE-cadherin disruption. To elucidate the mechanism of C3a-mediated BBB permeability in vitro, we employed the use of transendothelial electrical resistance (TEER) measurements. Using chopstick electrodes and an EVOM2 ohmmeter to measure the resistance of electrical current in an isolated system, we could directly test the effect of various molecules in an in vitro barrier model (Figure 5A). Our model contained a coculture of primary human astrocytes cultured for 2 days on the abluminal surface of a semipermeable membrane prior to the addition of HBMECs, which were cultured on the luminal surface for another 4 days, while TEER was monitored for optimal resistance prior to beginning treatment. We first validated the capability of this system to generate a reproducible barrier. Using the TEER as a readout, we compared resistance of a cell-free membrane with barriers formed by primary astrocytes or endothelial cells, or by endothelial cells cocultured with HeLa cells or primary astrocytes. We found that endothelial cells cocultured with astrocytes, but not with HeLa cells, significantly increased TEER (Supplemental Figure 8, A and B), demonstrating that astrocytes promoted BBB integrity in vitro.

C3a-mediated barrier disruption is dependent on Ca 2+ mobilization and alters VE-cadherin through cytoskeletal activation. (A) Schematic of TEER analysis using a coculture of astrocytes and endothelial cells. (B) TEER values in cocultures treated with vehicle, C3a, C3a with C3aRA, or IL-1β for 0, 1, 4, 12, and 24 hours. (C) Quantification of percentage reduction of TEER at 24 hours from treatments recorded in B. (D) TEER values in cocultures treated with vehicle, C3a, or C3a with C3aRA or BAPTA-AM over 24 hours. (E) Quantification of the percentage of reduction in TEER at 24 hours from the treatments in D. All TEER experiments were performed 2 times with duplicates and normalized to time-point control wells of cell-free membranes. (F) Representative immunofluorescence images of human brain microvascular endothelial cells (HBMECs) treated with vehicle, C3a, or a combination of C3a plus C3aRA (C3a/C3aRA), BAPTA-AM (C3a/BAPTA), or W7 (C3a/W7) for 2 hours and stained with anti-pMLC and anti–VE-cadherin antibodies. (G) Quantification of pMLC or VE-cadherin MFI showed an increase in pMLC but not VE-cadherin (n = 7 areas from 3 replicates of 250–300 cells/condition). (H) Representative immunofluorescence images of HBMECs treated as stated in F using anti-pMLC and anti–VE-cadherin antibodies 24 hours after treatment. (K) Quantification of pMLC or VE-cadherin MFI showed normalized pMLC levels but decreased VE-cadherin (n = 7 areas from 3 replicates of 250–300 cells/condition). All data represent the mean ± SEM. Analysis was performed on average percentage decrease in TEER using 1-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Scale bar: 10 μm.

To test the effect of C3aR signaling, we treated human endothelial cell–astrocyte cocultures with recombinant human C3a, and used IL-1β treatment, known to disrupt barrier integrity, as a positive control ( 36 ). After a 24-hour treatment, we found that C3a treatment resulted in a significant reduction of TEER, similar to IL-1β (Figure 5, B and C). C5a treatment had a marginal but not statistically significant effect (Supplemental Figure 8, C and D). C3a-induced barrier dysfunction was blocked when cultures were cotreated with C3aRA (Figure 5, B and C).

To further elucidate the mechanism of C3aR-mediated BBB permeability, we interrogated intracellular Ca 2+ as a potential second messenger, given previous reports that activation of C3aR triggers Ca 2+ release ( 37 ). Treating the cocultures with ionomycin resulted in a drastic reduction of TEER (Supplemental Figure 8, E and F), suggesting Ca 2+ release was sufficient to increase barrier permeability. Cotreatment with C3a and the calcium chelator BAPTA-AM, to block Ca 2+ signaling, led to a rescue of C3a-mediated TEER reduction to the level observed with C3aRA intervention (Figure 5, D and E), suggesting that calcium release was the primary mechanism of barrier permeability downstream of C3a/C3aR signaling.

Next, we aimed to dissect the connection between changes in VE-cadherin and Ca 2+ that may result in increased barrier permeability downstream of C3aR activation. Previous work has shown that various endothelial cell lines respond to C3a by forming actin stress fibers within the cell ( 25 ). After activation of the calcium-dependent kinase calmodulin, myosin light chain (MLC) kinase is able to phosphorylate MLC motor protein at Ser19 (pMLC), resulting in stress fiber formation ( 38 ). Activation of this pathway is known to initiate a physical, tensile stress at the cell membrane, disrupting VE-cadherin + junctions and BBB integrity ( 39 ). Therefore, we hypothesized that C3a might trigger the calcium release needed to disrupt VE-cadherin + junctions, resulting in BBB permeability.

To test this hypothesis, we treated endothelial cell monolayers with C3a alone or together with C3aRA, BAPTA-AM, or calmodulin inhibitor W7. Immunofluorescence staining of cells treated with C3a alone for 2 hours showed a robust increase in both phalloidin + F-actin stress fibers and overlapping pMLC signal (Supplemental Figure 9A). To quantify the dynamics of this cellular response, we analyzed changes at 2 hours, during the downward slope of barrier integrity, as measured by TEER analysis, after C3a stimulation. We saw a robust increase in pMLC by both immunofluorescence (Figure 5, F and G) and immunoblotting (Supplemental Figure 9, B and C), both of which were blocked by C3aRA, BAPTA, or W7 treatment. When analyzing the effect on VE-cadherin, we did not see overt changes by either immunofluorescence (Figure 5, F and G) or by immunoblotting (Supplemental Figure 9, B and D) under all conditions, suggesting that F-actin stress fiber formation and activation of pMLC, but not VE-cadherin protein alteration, was involved in the early phase of C3a/C3aR signaling. However, after 24 hours of C3a treatment, pMLC levels normalized, but VE-cadherin levels were significantly reduced by both immunofluorescence staining (Figure 5, H and I) and by immunoblotting (Supplemental Figure 9, E and G). Cotreatment with C3aRA, BAPTA, or W7 normalized VE-cadherin without affecting pMLC (Figure 5I and Supplemental Figure 9, E–G).

Overall, these data established intracellular Ca 2+ as a second messenger downstream of C3a/C3aR signaling to mediate pMLC activity and VE-cadherin homeostasis in endothelial cells. These findings suggest that there are 2 phases in endothelial response to C3a. First, a transient phase (2 hours) where endothelial cells respond quickly and rapidly to C3a signaling by forming stress fibers, followed by a failed ability to maintain VE-cadherin protein levels, leading to barrier permeability (24 hours).

Endothelial cell–specific deletion of C3ar1 rescues vascular phenotypes, reduces microglial reactivity, and corrects age-related neurodegeneration. Because C3ar1 genetic ablation and C3aR pharmacological inhibition were able to rescue age-related changes in brain vasculature, we hypothesized that specific endothelial ablation would show similar effects as global targeting, and that such a manipulation would influence age-related neuroinflammation overall. The above in vitro studies support a role of endothelial C3aR in mediating the barrier permeability. To test this hypothesis directly, we produced mice with conditional deletion of C3ar1 in endothelial cells by crossing a C3ar1-floxed allele ( 40 ) with the Tie2-Cre ( 41 ) mice to generate C3ar1 fl/fl Tie2-Cre (T2KO) mice. Littermate C3ar1 +/+ and C3ar1 fl/fl mice were used as controls. The cell type–specific knockout was confirmed by immunofluorescent imaging (Supplemental Figure 10A).

Coimmunofluorescent staining of control and T2KO mice at 3 and 12–14 months of age with anti-VCAM1 and anti-CD31 antibodies revealed significant increases in cortical vascular VCAM1 signal at 12–14 months of age in the control group (Figure 6A). Similar to the germline deletion, age-associated elevation of VCAM1 expression was almost completely attenuated in the T2KO mice (Figure 6, A and B), which was also seen in the hippocampus (Supplemental Figure 10, B and C). Analysis of vessel morphology by CD31 staining and 3D reconstruction showed a significant reduction in vessel cross-sectional area in 12–14-month-old control mice compared with that of 3-month-old mice (Figure 6, C and D). Consistent with the VCAM1 staining, endothelial deletion of C3ar1 was sufficient to rescue the age-associated changes of vessel morphology (Figure 6, C and D). These data demonstrated that specifically ablating C3ar1 in endothelial cells rescued age-related changes in brain vasculature, similar to global ablation and pharmacological inactivation. Thus, endothelial C3aR played a cell-autonomous role in mediating age-dependent changes in vascular inflammation and morphology.

Conditional knockout of C3ar1 in brain endothelial cells rescues age-related vascular phenotypes. (A) Representative VCAM1 and CD31 double-staining images from 3-month-old and 12- to 14-month-old endothelial C3ar1 conditional knockout (T2KO) mice and littermate controls (CTRL) showing increased VCAM1 expression with age in CTRL mice but suppressed expression in T2KO. (B) Quantification of VCAM1 intensity of A. (C) Representative CD31 staining and 3D reconstruction of 3-month-old and 12- to 14-month-old CTRL and T2KO mice. (D) Quantification of average CD31 + cross-sectional areas. All data represent the mean ± SEM of n = 4/group. Analysis for AF was performed using 1-way ANOVA with Tukey’s post hoc test (**P < 0.01, ***P < 0.001). Scale bar: 50 μm.

It was previously reported that reducing VCAM1 expression in endothelial cells can benefit brain function ( 11 ). Thus, we tested whether inhibiting the C3aR/VCAM1 axis at the endothelial cells could influence microglial reactivity. Coimmunostaining of 2- and 12-month-old WT and C3ar1 –/– mice with microglia marker IBA1 and a marker for phagocytic microglia, CD68, followed by colocalization analysis identified a higher percentage of CD68 signal colocalized with IBA1 in 12-month-old WT mice, which was completely normalized by global C3aR inactivation (Figure 7, A and B). Analysis of T2KO mice and their littermate controls at 12–14 months showed a partial but significant reduction of CD68 immunoreactivity (Figure 7, C and D). This result suggests that although C3aR is expressed in other cell types, notably microglia, it also plays a role in mediating neuroinflammation in the brain by modulating the endothelial C3aR/VCAM1 axis and promoting peripheral immune cell interaction at the brain vasculature.

Germline and conditional knockout of C3ar1 rescues age-related microglial reactivity and neurodegenerative phenotypes. (A) Representative IBA1 and CD68 double immunostaining in WT and C3ar1 –/– hippocampus at 2 and 12 months. (B) Quantification of CD68 immunoreactivity within IBA1 + microglia (n = 4/group). (C) Representative IBA1 and CD68 double immunostaining in CTRL and T2KO hippocampus at 3 months and 12–14 months. (D) Quantification of CD68 immunoreactivity within IBA1 + microglia (n = 4/group). (E) Quantification of hippocampal volume through coronal, serially sectioned tissue samples (n = 7–9/group, 9 sections/animal quantified). (F) Quantification of entorhinal cortex volume through coronal, serially sectioned tissue samples (n = 7–9/group, 9 sections/animal quantified). Data for B and E represent the mean ± SEM, and analysis was performed using 1-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Data for D represent the mean ± SEM, and analysis was performed using a 2-tailed Student’s t test (**P < 0.01). Data for F represent the mean ± SEM and analysis was performed using 1-way ANOVA with Holm-Sidak post hoc test (*P < 0.05, **P < 0.01). Scale bar: 50 μm (A and C).

To determine whether the changes of immune cells detected in the middle age (12–14 months) may lead to neuronal loss later in life, we performed Nissl staining of brain sections from young (3 months) and old (20 months) WT control mice and 20-month-old C3ar1 –/– and T2KO mice (Supplemental Figure 11) and quantified hippocampal and piriform/entorhinal cortical volumes (Figure 7, E and F). We found a mild but significant reduction in tissue volumes with age in the control animals. Interestingly, global and endothelial cell–specific ablation of C3aR led to a comparable degree of rescue (Figure 7, E and F). Together, these data suggest that blocking the endothelial C3aR/VCAM1 axis and restoring endothelial vascular morphology could restore microglial function and restore brain health during aging.

C3aR modulates vascular changes in PS19 tau–transgenic mice. Our previous analysis of the widely used PS19 tau–transgenic mouse model reported heightened C3/C3aR signaling associated with hyperphosphorylated tau pathology, such that genetic deletion of C3ar1 effectively reduced tau pathology and neuroinflammation ( 22 ). Examination of the previously reported brain transcriptomes of PS19 and PS19 C3ar1 –/– mice ( 22 ) clearly demonstrated the capacity for C3aR to modulate the innate immune response. Further, overrepresentation analysis of this gene expression data found KEGG and Reactome pathways upregulated in PS19 and downregulated in PS19 C3ar1 –/– , consistent with cytokine activation, leukocyte activation and migration, cell adhesion molecule interactions, and regulation or activation of the cytoskeleton, all of which are consistent with endothelial responses to C3a (Figure 8A). In addition to leukocyte transcripts in our pathway analysis, specific transcripts identifying peripheral immune cell infiltration (Vcam1, Ptpn22, Cd3e, and Cd8a) were also significantly elevated in PS19 brains (Figure 8A and Supplemental Figure 12).

Vascular abnormalities in PS19 tau–transgenic mice and C3aR dependency. (A) RNA-Seq analysis revealed significantly overrepresented pathways in the differentially expressed genes (DEGs) that were increased in 9-month-old PS19 compared with WT animals (red), and that were decreased in PS19 C3ar1 –/– compared with PS19 animals (blue). Terms were selected from results based on their involvement in vascular biology and immune cell infiltration and plotted by P value representative rescued genes contributing to the terms are listed (right). (B) Cortical staining of 9-month-old WT, C3ar1 –/– , PS19, and PS19 C3ar1 –/– hippocampal vasculature with CD31 and VCAM1 demonstrated a significant increase in VCAM1 expression in PS19 mice and a rescue of this phenotype in PS19 mice harboring the C3ar1 deletion. (C) Quantification of VCAM1 immuno-intensity. (D) CD31 staining and IMARIS-aided 3D reconstruction of 9-month-old WT, C3ar1 –/– , PS19, and PS19 C3ar1 –/– hippocampal vasculature. (E) Quantification of the average vessel cross-sectional area. All data represent the mean ± SEM of n = 5/group. Analysis for all results was performed using 1-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Scale bar: 50 μm.

Given the changes in endothelial processes, in addition to the peripheral immune cell response, we hypothesized that the elevated C3a/C3aR/VCAM1 pathway contributes to vascular changes in PS19 mice. Indeed, coimmunofluorescent analysis of cortical vasculature of 9-month-old PS19 mice labeled with CD31 and VCAM1 revealed a significant increase in VCAM1 expression colocalized with CD31 + vasculature, and this phenotype was rescued to control levels by ablating C3ar1 (Figure 8, B and C). Increased VCAM1 expression in PS19 mice was accompanied by drastic reduction in the average cross-sectional area of cortical brain vasculature (Figure 8, D and E). Consistent with aging analysis, C3ar1 ablation in PS19 animals significantly improved vascular morphology (Figure 8, D and E). These data revealed a vascular phenotype associated with tau pathology and suggest that the endothelial C3aR/VCAM1 axis contributes to vascular dysfunction in tauopathy.

Our data provided evidence that activated C3a/C3aR signaling in brain endothelial cells triggered an increase in the cell adhesion molecule VCAM1 and initiated an inflammatory transition affecting brain vascular structure and function during aging. This was associated with lymphocyte infiltration, altered vascular morphology, increased BBB permeability, and, ultimately, age-related neurodegeneration. Genetic or pharmacological C3aR inhibition rescued these age-associated phenotypes. Our in vitro BBB model implicates endothelial C3aR in the barrier disruption and suggests downstream intracellular Ca 2+ signaling and VE-cadherin localization and expression as the underlying mechanisms. A cell-autonomous effect of endothelial C3aR was further validated by our demonstration that endothelial cell–specific ablation of C3ar1 phenocopied germline deletion with regard to vascular phenotypes. Analysis of the endothelial cell–specific C3ar1-knockout mice also supports the notion that morphological and functional changes in vasculature contribute to microglial reactivity and age-related neurodegeneration. Finally, we documented that similar vascular phenotypes and their C3aR dependency were observed in PS19 tau–transgenic mice, supporting a common C3a/C3aR/VCAM1 signaling pathway in the mediation of neuroinflammation in aging and neurodegenerative disease.

To determine the functional consequence of the activated endothelial C3a/C3aR axis during aging, we analyzed VCAM1 expression and peripheral immune cell infiltration in the brain and found a substantial increase in CD8 + T cells, whereas genetic and pharmacological inhibition of the C3a/C3aR pathway reduced these phenotypes. This likely occurred through a VCAM1-regulated process, because the interaction between vascular VCAM1 and its lymphocyte-expressed ligand VLA-4 is well established ( 42 ). These findings point to a strong shift toward vascular inflammation in brain endothelial cells during aging.

A recent report by Yousef et al. concluded that elevated brain VCAM1 expression activated microglia and decreased neural stem cell numbers in aged mice ( 11 ). They also addressed the peripheral immune cell contribution by using αVLA-4–blocking antibodies in 16-month-old mice, showing a rescue of these phenotypes. Work by Dulken et al. also suggested that increased prevalence of clonally expanded CD8 + T cells in the brains of 28- to 29-month-old mice significantly hindered neural stem cell fitness in the subventricular zone ( 13 ). These findings, along with ours, support a model of periodic immune cell infiltration and eventual clonal expansion during aging. Our C3aR inhibition studies and the VLA-4 blockade used by Yousef et al. suggest that blocking peripheral immune cell recruitment during aging may reduce microglial reactivity and dampen the activated neuroimmune environment, whereas allowing this to go unchecked in old mice results in further CD8 + T cell infiltration and clonal expansion (of both early and late recruits). The partial rescue of infiltration in our 20-month-old pharmacological inhibition model supports this supposition of periodic, age-dependent waves of infiltration. Although our study did not address neural stem cell fitness or fate, we showed a C3aR-dependent effect on age-related neurodegeneration in our C3ar1-null and T2KO mouse models. Together, the current findings suggest that, in part, age-related neurodegeneration might be provoked by peripheral immune cell interaction with or signaling to microglia. Future studies should analyze more time points specifically in a later stage of life to determine the exact windows of infiltration that affect brain function and to clearly identify the role of these CD8 + T cell infiltrates. Recent work by Kolev et al. showed that peripheral immune cells elevate intrinsic C3 production after diapedesis into peripheral tissues ( 43 ). They suggest a prominent role for the CD4 + T cell interaction (via LFA-1) with endothelia (via ICAM1), but also identify a similar effect in CD8 + T cells stimulated with VCAM1, showing intrinsic upregulation of IFN-γ and C3 ( 43 ). Further dissection of this mechanism with respect to CD8 + T cells and VCAM1 could shed light on potential feed-forward mechanisms affecting microglial reactivity during aging.

We also analyzed structural and morphological characteristics of brain vasculature in the hippocampus, because this region was previously reported to undergo age-related vascular dysfunction in the form of BBB disruption and reduced cerebral blood flow ( 44 , 45 ). We found that C3a/C3aR signaling induced structural changes in vasculature affecting the vessel cross-sectional area and vessel tortuosity, characteristics previously associated with impaired cerebral blood flow and abnormal angiogenesis ( 34 ). Additional work is needed to understand the exact impact of C3a on hemodynamics. Work by the Zlokovic group has shown that the BBB undergoes age-related permeability in the hippocampus prior to disease ( 10 , 44 – 46 ). We found that aged mouse brain vessels were more permeable to a BBB-impenetrable tracer dye and confirmed the loss of barrier integrity by analyzing FACS-isolated endothelial cells, which showed impaired gene expression of critical BBB genes (Cdh5, Ocln, Tjp1, and Cldn5). Our data showed that C3a/C3aR signaling was partly responsible for these structural and functional changes in BBB integrity during aging.

Our data using dextran indicated that the immune cell infiltration by acute LPS treatment was an active process, rather than the result of a compromised BBB. This interpretation differs from a previous report using heavy isotope– and radio-labeled proteins, which revealed increased BBB permeability by LPS induction ( 47 ). Although the exact cause for this discrepancy is not clear, recent work from the Wyss-Coray laboratory identified potentially novel, age-related, receptor-mediated transcytosis mechanisms for protein transfer across the BBB ( 48 ). It is possible that a similar transcytosis mechanism could underlie the presence of heavy isotope– or radio-labeled proteins in the brain in acute neuroinflammatory models. Given this new development, further work should be done to better understand BBB permeability in acute neuroinflammation.

Mechanistic analysis of the endothelial barrier phenotype suggests a role for calcium-mediated signaling in an in vitro model of the BBB. Further analysis identified a potential phase response, in which initial calcium-mediated signaling induced phosphorylation of MLC protein, resulting in VE-cadherin protein loss at intercellular junctions. These effects were rescued by inhibition of C3aR or calmodulin in endothelial cell cultures, establishing a strong link between C3a/C3aR activation and calcium-mediated effects in brain endothelial cells. Together, these findings showed that changes in brain vessel structure, hemodynamics, and BBB permeability may be directly modulated by glial reactivity and other complement-related changes seen in aged brains.

The major focus of this study addresses the role of age-related vascular changes due to C3a/C3aR signaling however, we observed similar activation in the PS19 model of tauopathy. In our previous study of this model ( 22 ), we identified a key C3aR-dependent microglial activation network and showed that blocking C3aR corrected microglial activation and other transcriptional changes. Activation of the C3a/C3aR network also influenced gene expression signatures consistent with peripheral immune cell activation, a phenomenon previously reported in other Alzheimer disease mouse models ( 49 , 50 ). Given this, we reasoned that impaired vasculature and endothelial cell response may be partly responsible for the presence of peripheral immune cell signatures. Indeed, RNA-Seq analysis revealed increased levels of Vcam1, Cd3e, Cd8a, and Ptpn22 genes in PS19 hippocampi, and histology revealed highly altered vessel morphology. Previous work by Faraco et al. demonstrated a role of hypertension in potentiating accumulation of hyperphosphorylated tau, a finding which corroborates our belief that endothelial structure and function may influence disease pathogenesis ( 51 ). The study by Laurent et al. used a CD3 + T cell depletion strategy showing that Clec7a, Itgax, Cd68, and even astrocytic Gfap mRNA levels are reduced by depleting T cells ( 50 ). Although our current study does not address the downstream effect of these vascular changes as it relates to disease progression, it does highlight a possible role for endothelial cells in potentiating microglial reactivity through vascular function and peripheral immune cell interactions. Together, these studies position endothelial cells as the gatekeepers to disease progression through the C3a/C3aR/VCAM1 axis.

In conclusion, our work identifies a potentially novel complement regulatory axis at the BBB through endothelial C3aR. It implicates a critical role for a C3aR-dependent endothelial inflammatory transition, which results in increased VCAM1 expression in the aged brain. Our data suggest that blocking complement-mediated effects can have a substantial impact on improving vascular health, rescuing BBB permeability, and decreasing neuroinflammation in aging and neurodegeneration. Since the complement pathway is upregulated in both acute inflammatory conditions, such as stroke and traumatic brain injury, and in neurodegenerative diseases, in particular Alzheimer disease, of which age is the greatest risk factor, our findings have direct implications for the pathogenesis and therapeutic targeting of these age-related diseases of the brain.

For additional details, see Supplemental Methods.

Mice and treatment. The aged C57BL/6J mice were obtained from the aging rodent colony of the NIH National Institute on Aging. C3ar1-deficient mice (C3ar1 –/– ) mice were obtained from the Jackson Laboratory and backcrossed to C57BL/6J for 5 generations. Mice were housed 2–4 per cage in a pathogen-free mouse facility with ad libitum access to food and water on a 12-hour light/12-hour dark cycle. Mice for the conditional knockout studies were bred from a mixed background of Tie2 cre mice (C57BL/6J) and C3ar1 fl/+ mice (BALB/c) ( 40 ). The breeding scheme for PS19 studies was previously published by Litvinchuk et al ( 22 ). Males and females in approximately equal numbers were used for all experiments.

Vehicle (0.5% DMSO) or C3aR antagonist (C3aRA 1 mg/kg) were i.p. injected every other day for 4 weeks. For i.c.v. administration of LPS, mice were placed in a Kopf stereotaxic instrument, and glass needles were inserted through bore holes using coordinates to target lateral ventricles (–0.4 mm anteroposterior, ± 1.0 mm mediolateral, and –2.0 mm dorsoventral from the surface of the skull at bregma). LPS (2 μg/mL) or vehicle (PBS) was administered bilaterally (2 μL each side).

BBB analysis was followed as previously described ( 35 ). Briefly, mice were injected via tail vein with 100 μL of 10 mg/mL stock TRITC–dextran (MW 65–85 kDa Sigma-Aldrich, T1162). Dye was allowed to naturally perfuse for 2 hours and was then perfused with PBS. One hemisphere was used to determine TRITC fluorescence signal in tissue homogenates (excitation λ 550 nm, emission λ 580 nm) using a plate reader (Molecular Devices Spectra Max i3x). The other hemisphere was fixed in 4% PFA overnight at 4°C and switched to 30% sucrose. Sagittal brain sections (30 μm) were cut on a sliding microtome, washed in PBS, and stained with lectin-649 (Vector Labs, DL-1178) for 30 minutes at room temperature in PBS containing 0.4% Triton X-100, 4% donkey serum, and 1% BSA to mark brain vasculature. The sections were imaged on a Leica TCS laser confocal microscope at 40× under oil immersion, with a Z-step of 0.5 μm over a total range of 30 μm.

Cell culture. Primary HBMECs were obtained from Cell Systems (ACBRI 376). Cells were thawed and plated into T75 flasks for expansion in Lonza EGM2-MV medium (CC-3202) to reach a P4 culture. Cells were subcultured until confluent, passaged at a 1:4 ratio into T75 flasks for P5 cultures, and allowed to expand until confluent prior to freezing (EGM2-MV plus 10% DMSO). Fresh vials were thawed to obtain P6 cultures, which were used for all further experiments.

Primary human astrocytes were obtained from ScienCell Research Laboratories (ScienCell, 1800). Cells were thawed and plated into a T75 flask for expansion in astrocyte medium (AM) (ScienCell, 1801). Cells were subcultured until near confluence (80%–90%), passaged at a 1:4 ratio into T75 flasks, and allowed to expand until near confluence prior to freezing (AM plus 10% DMSO). Fresh vials were thawed and used for all further experiments.

Primary mouse astrocyte cultures were prepared as described previously ( 20 ) and purified using negative selection by magnetic CD11b beads (Miltenyi Biotec, 130-049-601). Primary HBMECs were cultured on 100 μg/mL fibronectin and Col IV–coated 24-well or 48-well plates. Cells were seeded at a density of 2.5 × 10 5 cells/cm 2 . Confluent cells were treated with IL-1β (10 ng/mL, R&D Systems, 201-LB-005), C3a (500 nM, R&D Systems, 3677-C3-025), C5a (250 nM, R&D Systems, 2037-C5-025), ionomycin (10 μM, Cayman Chemical, 10004974), or in combination with one of the inhibitors SB290157 (5 μM, Calbiochem, 559410), W7 (50 μM, Tocris, 0369) or BAPTA-AM (1 μM, Tocris, 2787). Cells were analyzed after 2 hours or 24 hours of treatment.

TEER analysis was performed using combinations of primary HBMECs and primary human or mouse astrocytes in a coculture. Briefly, semipermeable transwell inserts (Corning, 3470) were coated with 100 μg/mL fibronectin and Col IV on the luminal surface for 2 hours at 37°C in PBS. The remaining coating solution was aspirated, and the transwells were flipped over and placed into 12-well culture plates. The abluminal surface was coated with poly- d -lysine (PDL) at 37°C for 2 hours, and the remaining solution was aspirated. While inverted, primary astrocytes were seeded to the abluminal surface at a density of 1.5 × 10 5 cells/cm 2 and allowed to attach for 4–6 hours at 37°C in 100 μL AM (ScienCell). The membranes were then reverted to normal position in their original culture plate, and the astrocytes were cultured in AM placed into the tissue culture plate (abluminal). The cells were cultured for 48 hours at 37°C and endothelial cells were seeded in the luminal compartment at a density of 1.5 × 10 5 cells/cm 2 in EGM2-MV. All TEER readings were measured using STX2 chopstick electrodes with an EVOM2 volt/ohm meter (World Precision Instruments). Cultures matured over 3–4 days, and when TEER stabilized (

160–180 Ω), treatments were added and TEER was monitored over 24 hours. All TEER readings were normalized to the average reading from 2 cell-free inserts for each time-point recording prior to normalization to the control samples.

Brain vessel preparations. Isolation of mouse brain vessels was carried out as previously described ( 28 ) with minor modifications. Briefly, mice were perfused with PBS, brains were removed, and cerebellum olfactory bulb and brain stem were discarded. They were stripped of dura and meninges, gently sliced with a razor blade, and gently homogenized using a glass Dounce homogenizer (Kontes Glass, 19), all on ice. The homogenate was centrifuged, supernatant was discarded, and the pellet was resuspended in a dextran solution to remove myelin debris. The resulting pellet was then filtered over a 40 μm filter and vessel fragments were retained in the filter. The filter was then turned over, placed on a 50 mL conical tube, and rinsed. Vessel fragments were pelleted at 300g for 5 minutes and fixed in 4% PFA for 30 minutes on ice. Fixed fragments were pelleted at 300g for 5 minutes, washed with PBS, and mounted onto manually gridded slides for staining. Vessels were blocked with PBS containing 0.4% Triton X-100, 4% donkey serum, and 1% BSA for 30 minutes, and incubated in blocking solution with primary antibody overnight at 4°C. Depending on the experiment, primary antibodies were used as follows: rabbit anti-GFAP (MilliporeSigma, G9269), rat anti-C3aR (Hycult, 10130173), and goat anti–mVE-cadherin (R&D Systems, AF1002). Imaging was performed on a Leica TCS laser confocal microscope at 63× under oil immersion, with a Z-step of 0.5 μm over a total range of 10 μm.

Flow cytometry analysis. Flow cytometry analysis of aged brain lymphocytes was performed using CoBrA dissociation strategy as previously described ( 27 ), with slight modifications for myelin/debris removal, antibody staining, and for dissociation to subtype lymphocyte markers. Briefly, adult mice were perfused with PBS, brain tissues were gently minced with sterile razor blades, digested in papain (Worthington Biochemical, LK003172) and DNase (Worthington Biochemical, LK003178), and then triturated 3–4 times using a fire-polished glass Pasteur pipette. After incubation, papain digestion was neutralized with HBSS+ and the suspension was pelleted at 310g for 5 minutes at 4°C. The pellet was resuspended in 1 mL of HBSS+, transferred to an ice-cold 1.7 mL Eppendorf tube and further triturated 3 times, and the supernatant was collected after a brief, low-speed centrifugation. The supernatant at the end of each brief centrifugation was filtered through a prewetted 40 μm cell strainer (BD Biosciences, 352340) into a chilled 50-mL conical tube and centrifuged at 310g for 5 minutes at 4°C. The resulting pellet was depleted of myelin and other debris using a 20% isotonic Percoll PLUS (MilliporeSigma, E0414-250ML) separation. The resulting pellet contained dissociated single cells. For myeloid versus lymphoid discrimination, cells were incubated in 500 μL HBSS+ containing 1:100 Mouse BD Fc Block (BD Biosciences, 553141), 1:500 rat anti–CD45-BV421 (BD Biosciences, 563890), and 1:500 rat anti-CD11b–FITC (BD Biosciences, 553310) on ice for 15–20 minutes. For subtyping the lymphocyte populations, the tissue dissociation strategy was changed using Collagenase/Dispase (MilliporeSigma, 10269638001) in place of papain to preserve the epitopes for CD19, CD8a, and CD4. All other steps of the dissociation strategy remained the same. After tissue dissociation, cells were incubated in 1:500 rat anti–CD45-BUV395 (BD Biosciences, 564279), 1:500 rat anti–CD11b-FITC (BD Biosciences, 553310), hamster anti–CD3ε-BV650 (BD Biosciences, 564378), 1:500 rat anti–CD19-BV480 (BD Biosciences, 566167), 1:500 rat anti–CD4-PE (BD Biosciences, 553730), and 1:500 rat anti–CD8a-APC (BD, 553035) on ice for 15–20 minutes. Cells were washed twice with HBSS+ and resuspended in 500 μL of HBSS+ prior to flow cytometry analysis. Flow analysis was performed using a BD Biosciences LSR Fortessa equipped with 355 nm, 405 nm, 488 nm, 561 nm, and 640 nm lasers to minimize spectral overlap.

For FACS of astrocytes and endothelial cells, see the tissue preparation methods using papain. After tissue dissociation, cells were incubated in 500 μL HBSS+ containing 1:100 Mouse BD Fc Block (BD Biosciences, 553141), LIVE/DEAD Fixable Blue Dead Cell (Thermo Fisher Scientific, L23105), 1:500 rat anti–CD45-BV421 (BD Biosciences, 563890), and 1:500 rat anti–CD11b-FITC (BD Biosciences, 553310), 1:250 anti-CD49a-VioBright PE (Miltenyi Biotec, 130-107-632), and 1:100 anti–ACSA-2-APC (Miltenyi Biotec, 130-116-245) on ice for 15–20 minutes. Endothelial cells were sorted by first excluding CD45 + and CD11b + cells and gating around CD49a + cells. Astrocytes were sorted by gating triple negativity for the former markers and finally gating around ACSA2 + cells. Sorting was performed using a BD Biosciences Aria II on the 100 μm nozzle. Cells were sorted into 1.7 mL Eppendorf tubes containing 200 μL HBSS+, followed by centrifugation and lysis of pellets in Qiagen RLT buffer containing 1% β-mercaptoethanol.

For flow cytometry of HBMECs, cells were singularized with trypsin EDTA (Thermo Fisher Scientific, 25200056) for 5 minutes, and trypsin was neutralized using HBSS+. Cells were pelleted by centrifugation at 300g and washed 3 times with HBSS+. Cells were fixed in 4% PFA for 20–30 minutes at 37˚ C. After fixation, HBSS+ was added to the tube prior to centrifugation to minimize cell loss. Cells were centrifuged at 300g and washed 3 times with HBSS. Antibodies were diluted in HBSS+ and cells were stained with either appropriate IgG controls or a combination of rat anti-C3aR, 1:500 (R&D Systems, MAB10417), mouse anti-Glut1 1:1000 (Thermo Fisher Scientific, MA1-37783), and goat anti–VE-cadherin 1:1000 (R&D Systems, MAB9381). Cells were incubated in antibody solution on a benchtop rotator for 30 minutes, then washed 3 times with HBSS+ and incubated in appropriate secondary antibodies for 30 minutes at room temperature in a benchtop rotator. Cells were washed in HBSS+ 3 times prior to flow cytometry analysis.

Quantitative RT-PCR. RNA was extracted from cells using the RNeasy Micro kit (QIAGEN, 74004). Reverse transcription was performed using the iScript Reverse Transcription Supermix (Bio-Rad, 1708840) according to the manufacturer’s protocol. All RNA isolated from cell pellets was converted into cDNA. Quantitative RT-PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, 172-5120) on a CFX384 Touch Real-Time PCR Detection System.

Immunostaining and image analysis. Cultured cells were fixed with 4% PFA for 20 minutes at 37°C. Samples were washed with PBS and then blocked and permeabilized with PBS containing 0.4% Triton X-100, 4% donkey serum, and 1% BSA for 30 minutes. Samples were incubated in blocking solution containing primary antibody overnight at 4°C. Depending on the experiment, primary antibodies were used as follows: rabbit anti-pMLC2 S19 (Cell Signaling Technology, 3671), goat anti–hVE-cadherin (R&D Systems, AF938), or Phalloidin CruzFluor 555 (Santa Cruz Biotechnology, sc-363794). All images were taken on a Leica TCS laser confocal microscope at 40× or 63× under oil immersion, with a Z-step of 0.5 μm over a total range of 5 μm. MFI was normalized to cell number per image, and each condition consisted of 8–10 images (n = 250–300 cells).

For mouse brain analysis, mice were perfused with 4% PFA, followed by postfixation in 4% PFA overnight at 4°C, and then transferred into 30% sucrose solution until sectioning. Sagittal brain sections (30 mm) were cut on a sliding microtome and stored at –20°C in cryoprotectant. After washing in PBS, sections were blocked with PBS containing 0.4% Triton X-100, 4% donkey serum, and 1% BSA for 30 minutes, and then incubated in blocking solution containing primary antibody overnight at 4°C. Depending on the experiment, primary antibodies were used as follows: rabbit anti-GFAP (MilliporeSigma, G9269), rat anti-C3 (Hycult, 10129042), rat anti-C3aR (Hycult, 10130173), anti-CD106 (BioLegend, 305802), rat anti-CD31 (BD Biosciences, 550274), goat anti–mVE-cadherin (AF1002), goat anti–PDGFR-β (R&D Systems, AF 1042), mouse anti-Glut1 (Thermo Fisher Scientific, MA1-37783), and rabbit anti–Col IV (Abcam, ab6586). After primary antibody staining, sections were washed in 1× PBS 3 times and stained with appropriate secondary antibodies for 1–2 hours and washed again before mounting.

For quantification of VCAM1 in the mouse cortex and hippocampus, sections were stained with CD31 and VCAM1, then fluorescent signal was scanned using an EVOS FL Auto system at 10×. Images were then processed by ImageJ (NIH) and background was subtracted before quantification. Total VCAM1-positive signal was quantified as percentage area for each region, hippocampus or cortex. Colocalization of this signal with CD31 was confirmed for accuracy.

For quantification of percentage occupancy of C3 in astrocytes, Z-stacks (

30 μm thick with 0.5 μm step size) were taken under 40x oil immersion, with labeling for C3, and analyzed using the “Spots” feature of IMARIS 9.2.1 software. Spots were generated automatically for C3 representation. Subsequently, Z-stacks were analyzed using the “Co-loc” feature where the GFAP + signal was used to mask astrocyte outlines and thresholds applied to remove background. Data were then recorded as percentage of region of interest (ROI) (GFAP + signal mask) occupied by the C3 (Spots) signal. Eight images spanning CA1-CA3 were taken across 5 mice per group.

For quantification of average vessel cross-sectional area Z-stacks (

30 μm thick with 0.5 μm step size) were taken under 40× oil immersion, with labeling for Col IV. Images were first analyzed using the “Surfaces” feature of IMARIS 9.2.1 software to generate 3D reconstruction of the vessel and a total vessel volume in μm 3 . Subsequently, using the “Filaments” function, the total vessel length per image was estimated by calculating individual vessel branch measurements in μm. Average cross-sectional area was determined by the proportional measurement of total vessel volume by total vessel length per image. Six images spanning CA1-CA3 were taken across 5 mice per group.

For quantification of tortuous vessel morphology, Z-stacks (

30 μm thick with 0.5 μm step size) were taken under 40× oil immersion with labeling for CD31. Images were manually quantified by counting the number of corkscrew vessels present in projected Z-stack images. Images were projected in Leica LAS X software and manually scrolled through to count the number of corkscrew features in hippocampal vasculature. To represent this morphology, representative images were taken under the same imaging parameters but under 63× oil immersion. The “Surfaces” function of IMARIS 9.2.1 software was used to generate 3D reconstruction of the CD31 vessel, and the animation tab was used to create movies. Six images spanning CA1-CA3 were taken across 5 mice per group for imaging quantification.

For quantification of microglial reactivity, CD68 colocalization in Iba1 signal was quantified using Z-stacks imaged on a Leica confocal microscope with 40× oil objective, a 1.0 digital zoom, a total thickness of 25 μm, and 1 μm step size. Percentage colocalization of CD68 signal within the masked Iba1 ROI was calculated using the “Co-loc” feature in IMARIS and represented as a fold change of this percentage.

To quantify the volume of hippocampus and entorhinal cortex, mouse frozen brain tissues were serially cut at 50 μm. Sections containing hippocampus or entorhinal cortex (every sixth section, 300 μm apart) between bregma +2.1mm and bregma −3.9 mm to the dorsal end of the hippocampus or entorhinal cortex were stained with 0.25% cresyl violet solution, dehydrated in ethanol, and then mounted. Slides were imaged using Nanozoomer 2.0-HT system (Hamamatsu), and areas of interest were traced using NDP Viewer software. The volume of the region of interest was quantified using the following formula: volume = (sum of area) × 0.5 mm.

RNA-Seq analysis. RNA-Seq data containing fold changes and adjusted P values from our previous study ( 22 ) were used (GEO: GSE114910). For pathway analyses, differentially expressed genes with P (adjusted) values of less than 0.05 were uploaded into the InnateDB website, and overrepresentation analysis was used to calculate significant terms from the KEGG and REACTOME databases. Selected significant terms were plotted by P value on the basis of their involvement in vascular biology and immune cell infiltration, and representative genes contributing to the hits were listed. For individual gene plots, FPKM values were used and significance was calculated using 1-way ANOVA.

Statistics. All statistical analysis was performed using GraphPad Prism software, version 8.0.2 (GraphPad Software). All data are presented as the mean ± SEM. Unless otherwise noted, all grouped comparisons were made by 1-way ANOVA with Tukey’s correction, and all pairwise comparisons by 2-sided Student’s t tests, depending on experimental design. For all tests, P values of less than 0.05 were considered significant, and those over 0.05 were considered nonsignificant.

Study approval. All animal procedures were performed in accordance with NIH guidelines and with the approval of the Baylor College of Medicine IACUC.

NEP and HZ conceived of the project and designed the experiments. NEP performed all experiments and data analysis unless otherwise noted. ER provided reagents and technical assistance for IMARIS imaging analysis, performed pathway analysis, and edited the manuscript. AL provided samples and technical assistance and performed brain volumetric tissue analysis. JK provided the C3ar1-floxed mice. NEP wrote the manuscript with input and revision from HZ. All authors read and approved the final manuscript.

We are indebted to D. Holtzman (Washington University) for the generous support with the brain volumetric analysis. We are most appreciative to the National Institute on Aging for offering aged C57BL6/J mice that made this work possible. We thank C. Beeton, J. Sederstrom, and the Baylor College of Medicine Cytometry and Cell Sorting Core supported by grant NCI-CA125123 for FACS analysis. We are grateful to N. Aithmitti and B. Contreras for expert technical support and members of the Zheng laboratory for stimulating discussions. This study was supported by grants from the NIH (R01 NS093652, R01 AG020670, R01 AG057509, RF1 AG054111, and RF1 AG062257 to HZ).

Conflict of interest: The authors have declared that no conflict of interest exists.

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We thank Y. Pan (Fourth Military Medical University) for valuable suggestions, S. Li (Huazhong Agricultural University) for reagents and technical help and members of the Zhong laboratory and the core facilities of the Medical Research Institute for technical help. This study was supported by grants from the National Key Research and Development Program of China (2018TFE0204500 and 2018YFC1004601), Natural Science Foundation of China (31671454 and 31930040), Fundamental Research Funds for Central Universities (2042020kf0207 and 2042020kf0042), Natural Science Foundation of Hubei Province (2018CFA016) and Medical Science Advancement Program (Basic Medical Sciences) of Wuhan University (TFJC2018004).

Prostaglandins are lipid autacoids derived from arachidonic acid. They both sustain homeostatic functions and mediate pathogenic mechanisms, including the inflammatory response. They are generated from arachidonate by the action of cyclooxygenase isoenzymes, and their biosynthesis is blocked by nonsteroidal antiinflammatory drugs, including those selective for inhibition of cyclooxygenase-2. Despite the clinical efficacy of nonsteroidal antiinflammatory drugs, prostaglandins may function in both the promotion and resolution of inflammation. This review summarizes insights into the mechanisms of prostaglandin generation and the roles of individual mediators and their receptors in modulating the inflammatory response. Prostaglandin biology has potential clinical relevance for atherosclerosis, the response to vascular injury and aortic aneurysm.

Inflammation is the immune system's response to infection and injury and has been implicated in the pathogeneses of arthritis, cancer, and stroke, as well as in neurodegenerative and cardiovascular disease. Inflammation is an intrinsically beneficial event that leads to removal of offending factors and restoration of tissue structure and physiological function. The acute phase of inflammation is characterized by the rapid influx of blood granulocytes, typically neutrophils, followed swiftly by monocytes that mature into inflammatory macrophages that subsequently proliferate and thereby affect the functions of resident tissue macrophages. This process causes the cardinal signs of acute inflammation: rubor (redness), calor (heat), tumor (swelling), and dolor (pain). Once the initiating noxious stimulus is removed via phagocytosis, the inflammatory reaction can decrease and resolve. During the resolution of inflammation, granulocytes are eliminated, and macrophages and lymphocytes return to normal preinflammatory numbers and phenotypes. The usual outcome of the acute inflammatory program is successful resolution and repair of tissue damage, rather than persistence and dysfunction of the inflammatory response, which can lead to scarring and loss of organ function. It may be anticipated, therefore, that failure of acute inflammation to resolve may predispose to autoimmunity, chronic dysplastic inflammation, and excessive tissue damage. 1

Prostaglandins (PGs) play a key role in the generation of the inflammatory response. Their biosynthesis is significantly increased in inflamed tissue, and they contribute to the development of the cardinal signs of acute inflammation. Although the proinflammatory properties of individual PGs during the acute inflammatory response are well established, their role in the resolution of inflammation is more controversial.

In this review, we discuss the biosynthesis of and response to PGs and the pharmacology of their blockade in orchestrating the inflammatory response, with particular regard to cardiovascular disease.

Biosynthesis of PGs

PGs and thromboxane A2 (TXA2), collectively termed prostanoids, are formed when arachidonic acid (AA), a 20-carbon unsaturated fatty acid, is released from the plasma membrane by phospholipases and metabolized by the sequential actions of PGG/H synthase or by cyclooxygenase (COX) and their respective synthases.

There are 4 principal bioactive PGs generated in vivo: prostaglandin E2 (PGE2), prostacyclin (PGI2), prostaglandin D2 (PGD2), and prostaglandin F (PGF).

They are ubiquitously produced—usually each cell type generates 1 or 2 dominant products—and act as autocrine and paracrine lipid mediators to maintain local homeostasis in the body. During an inflammatory response, both the level and the profile of PG production change dramatically. PG production is generally very low in uninflamed tissues but increases immediately in acute inflammation before the recruitment of leukocytes and the infiltration of immune cells.

PG production (Figure 1) depends on the activity of PGG/H synthases, colloquially known as COXs , bifunctional enzymes that contain both COX and peroxidase activity and that exist as distinct isoforms referred to as COX-1 and COX-2. 2

Figure 1. Biosynthetic pathway of prostanoids.

COX-1, expressed constitutively in most cells, is the dominant source of prostanoids that subserve housekeeping functions, such as gastric epithelial cytoprotection and homeostasis. 3 COX-2, induced by inflammatory stimuli, hormones, and growth factors, is the more important source of prostanoid formation in inflammation and in proliferative diseases, such as cancer. 3 However, both enzymes contribute to the generation of autoregulatory and homeostatic prostanoids, and both can contribute to prostanoid release during inflammation.

PGH2 is produced by both COX isoforms, and it is the common substrate for a series of specific isomerase and synthase enzymes that produce PGE2, PGI2, PGD2, PGF, and TXA2. COX-1 couples preferentially, but not exclusively, with thromboxane synthase, PGF synthase, and the cytosol (c) PGE synthase (PGES) isozymes. 4 COX-2 prefers prostaglandin I synthase (PGIS) and the microsomal (m) PGES isozymes, both of which are often coinduced along with COX-2 by cytokines and tumor promoters. 4

The profile of prostanoid production is determined by the differential expression of these enzymes within cells present at sites of inflammation. For example, mast cells predominantly generate PGD2, whereas macrophages produce PGE2 and TXA2. 5 In addition, alterations in the profile of prostanoid synthesis can occur on cellular activation. Although resting macrophages produce TXA2 in excess of PGE2, this ratio changes to favor PGE2 production after bacterial lipopolysaccharide (LPS) activation. 5

PG Receptors

PGs exert their effects by activating rhodopsin-like 7-transmembrane-spanning G protein-coupled receptors (Table). The prostanoid receptor subfamily is composed of 8 members: E prostanoid receptor (EP) 1, EP2, EP3, and EP4 subtypes of the PGE receptor PGD receptor (DP1) PGF receptor (FP) PGI receptor (IP) and thromboxane receptor (TP). 6 Two additional isoforms of the human TP (TPα, TPβ) and FP (FPA, FPΒ) and 8 EP3 variants are generated through alternative splicing, which differ only in their C-terminal tails. 7 In addition, there is another G protein-coupled receptor termed chemoattractant receptor-homologous molecule expressed on T helper 2 cells (CRTH2 or DP2) that responds to PGD2 but belongs to the family of chemokine receptors. 8 CRTH2 is a member of the N-formyl-methionyl-leucyl-phenylalanine chemoattractant receptor superfamily (Figure 2).

Table. Signal Transduction of Prostanoid Receptors

Figure 2. Phylogenetic tree of lipid G protein–coupled receptors. Figure modified with permission from Shimizu. 181

Prostanoid receptors couple to a range of intracellular signaling pathways that mediate the effects of receptor activation on cell function. EP2, EP4, IP, and DP1 receptors activate adenylyl cyclase via Gs, increasing intracellular cAMP. EP1 and FP activate phosphatidylinositol metabolism via Gq, leading to the formation of inositol trisphosphate with mobilization of intracellular free calcium. In addition to signaling through Gq, the FP receptor couples to the small G-protein Rho via a Gq-independent mechanism. 9 TP couples mainly to 2 types of G-proteins, the Gq (Gq, G11, G15, G16) and the G13 (G12, G13) families, resulting in the activation of phospholipase C and guanine nucleotide exchange factor of the small G protein Rho, respectively. In addition, TP can also be coupled via Gh to phospholipase C, as well as via Gi and Gs to adenylate cyclase. Both TP isoforms are coupled to phospholipase C activation, but TPα stimulates adenylyl cyclase, whereas TPβ inhibits it. EP3 isoforms can couple via Gi or G12 to elevation of intracellular Ca 2+ , inhibition of cAMP generation, and activation of the small G protein Rho. 10 The DP2/CRTH2 couples to a Gi-type G protein to inhibit cAMP synthesis and elevate intracellular Ca 2+ . However, the effects of prostanoids on these G protein–coupled signaling pathways may change as a function of ligand concentration or structure. 6

Some but not all prostanoid receptors exhibit the capacity to dimerize, which may alter ligand affinity or preference for downstream signaling pathways. Thus, although DP1/DP2 dimers appear not to form, under similar conditions, IP and TP receptors can associate to form homo- and heterodimers. TPα-TPβ heterodimers enhance the response to activation by free radical–catalyzed isoprostanes. 11 Furthermore, dimerization of IP and TPα enables cAMP formation through TP receptor activation, a cellular outcome typically observed with IP activity. 12 Moreover, EP1 receptor activation has been shown to modulate β2-adrenoreceptor function in bronchial airways via formation of a heterodimeric complex. 13

COXs and Inflammation

The 2 COX isoforms, COX-1 and COX-2, are targets of nonsteroidal antiinflammatory drugs (NSAIDs). These drugs are competitive active site inhibitors of both COXs. Although both COXs exist as homodimers, only 1 partner is used at a time for substrate binding. 14 COX-1/COX-2 heterodimers may also exist, but their role in biology remains to be established. 15 NSAIDs bind to and inactivate the COX site at only 1 of the monomers of the COX dimer, and this is sufficient to shut down prostanoid formation. 14 The other monomer appears to play an allosteric function. The peroxidase capacity of both proteins is unaltered by NSAIDs.

The clinical efficacy of structurally distinct NSAIDs, all of which share this capacity for prostanoid inhibition, points to the importance of these mediators in the promotion of pain, fever, and inflammation. 16 The dramatic increase of COX-2 expression on provocation of inflammatory cells, its expression in inflamed tissues, and the assumption that inhibition of COX-1-derived prostanoids in platelets and gastric epithelium explain NSAID-evoked gastrointestinal adverse effects and provide a rationale for development of NSAIDs designed to be selective for inhibition of COX-2 for treating arthritis and other chronic inflammatory diseases. 17

Although COX-2 appears to be the dominant source of PG formation in inflammation, there is some suggestion that both isoforms of the human enzyme may contribute to the acute inflammatory response. COX-1 is constitutively expressed in resident inflammatory cells, and there is evidence for induction of COX-1 during LPS-mediated inflammatory response and cellular differentiation. 18 Both COX isoforms are coexpressed in circulating inflammatory cells ex vivo and in both inflamed rheumatoid arthritis (RA) synovium and atherosclerotic plaques obtained from patients. 19,20 Human data are compatible with COX-1-derived products driving the initial phase of an acute inflammation, with COX-2 upregulation occurring within several hours. 4 However, controlled clinical trials to test the comparative efficacy of NSAIDs that inhibited both COXs versus COX-2 alone were never performed at scale. Such trials were designed to seek divergence in the incidence of gastrointestinal adverse effects rather than to assess comparative clinical efficacy.

Studies in both COX-1- and COX-2-knockout (KO) mice reveal impaired inflammatory responses, although the impacts of gene deletion diverge in time course and intensity.

Mice deficient in COX-1 but not COX-2 exhibit a reduction in AA-induced ear edema, although AA induces an equivalent inflammatory response in wild-type (WT) and COX-2-deficient mice. 21–23 By contrast, the level of edema induced by the tumor promoter tetradecanoyl phorbol acetate was not significantly different among WT, COX-1-deficient, and COX-2-deficient mice. 21–23 The ear inflammation studies indicate that COX-1, as well as COX-2, may contribute to inflammatory responses, and the isoform responsible for the inflammation may depend on the type of inflammatory stimulus or the relative levels of each isoform in the target tissue.

Similarly, arthritis models exhibit a significant reliance on either the COX-1 or COX-2 isoforms for the development of clinical synovitis. This varies, depending on the experimental model used. Thus, in the K/BxN serum–transfer model of arthritis, COX-1-derived PGs, in particular PGI2, make a striking contribution to the initiation and perpetuation of arthritis. 24 In a collagen-induced arthritis model, COX-2 deletion considerably suppresses synovial inflammation and joint destruction, whereas arthritis in COX-1-deficient mice is indistinguishable from that of controls. 25,26

COX-2 deletion also suppresses acute inflammation in the air pouch model. Here, the COX-2 inhibitor NS-398, administered 6 hours after carrageenan treatment, reduced PG production in WT mice to levels comparable to those seen in COX-2-KO mice and was also effective during the early stages of inflammation. 27 Compared with WT mice, the deficiency of COX-2 reduces the level of PGE2 production by approximately 75%, whereas the deficiency of COX-1 reduces the PGE2 level by 25% during this early stage. By day 7 following carrageenan treatment, higher numbers of inflammatory cells were present in the pouch fluid of COX-2-KO mice, and little resolution of inflammation was apparent compared with WT or COX-1-KO mice. 27 These findings indicate that both COX isoforms contribute to PG production during inflammation and also that COX-2-derived PGs appear to be important in both the acute inflammatory process and in the resolution phase. A contribution of COX-2 to both phases of inflammation was also reported in other models. Gilroy et al reported that COX-2 expression and PGE2 levels increased transiently early in the course of carrageenan-induced pleurisy in rats. 28 Later in the response, COX-2 was induced again to even greater levels and generated antiinflammatory PGs, such as PGD2 and 15-deoxy-Δ 12–14- PGJ2 (15d-PGJ2), but only low levels of the proinflammatory PGE2. Further support for an antiinflammatory role of COX-2 in this model was the finding that late administration of the COX-2-selective inhibitor NS-398 exacerbates the inflammatory response. Furthermore, Wallace et al observed that in the paw carrageenan model, the resultant inflammation resolves within 7 days in WT mice but is unaltered over this period in COX-2-deficient mice. 29 An antiinflammatory role of COX-2-derived prostanoids has also been reported in models of inflammatory colitis and allergic airway disease. 30,31 Thus, COX-2 appears to have a dual role in the inflammatory process, initially contributing to the onset of inflammation and later helping to resolve the process. Although COX-2 does play a role in supporting resolution of this process in some models of inflammation, it is unclear which products of the enzyme might contribute in which settings to this effect. This is exemplified by the case of 15d-PGJ2. Long touted as an endogenous ligand to peroxisome proliferator–activated receptor-γ (PPARγ), it remains to be established that endogenous concentration sufficient to subserve this function are formed in any model of resolving inflammation. Erroneously elevated levels have been reported using a variety of immunoassays, but the concentrations of bound and free compound documented to be formed by quantitative mass spectrometry fall far short of the EC50 for PPARγ activation. 32 It is one thing to show that putative proresolution products can be formed in vitro and that the synthetic compounds do exert proresolution actions when administered in vivo and another to document that the concentrations formed in vivo in the setting of inflammation are sufficient and necessary to mediate resolution.

In the case of atherosclerosis, deletion or inhibition of COX-2 has been shown variously to retard, accelerate, or leave unaltered atherogenesis in mouse models. 5 This may reflect an impact postnatally of disruption of the many roles of the enzyme in development in COX-2 KOs, differences in timing of interventions with COX-2 inhibitors, or a failure in most cases to define biochemically the selectivity for inhibition of COX-2 of the drug regimen deployed. In contrast, COX-1 deletion markedly attenuated lesion development in the apolipoprotein E–KO mouse, as does inhibition of COX-1 and COX-2 together in low-density lipoprotein receptor–KO model. 33,34 Thus, products of COX-1, such as TXA2, promote atherogenesis, whereas there is more ambiguity around the role of COX-2. Nevertheless, deletion of the IP, the receptor for the major COX-2 product, PGI2, fosters the initiation and early development of atherosclerosis in hyperlipidemic mice. 35

PGE2 and Inflammation

PGE2 is one of the most abundant PGs produced in the body, is most widely characterized in animal species, and exhibits versatile biological activities. Under physiological conditions, PGE2 is an important mediator of many biological functions, such as regulation of immune responses, blood pressure, gastrointestinal integrity, and fertility. Dysregulated PGE2 synthesis or degradation has been associated with a wide range of pathological conditions. 36 In inflammation, PGE2 is of particular interest because it is involved in all processes leading to the classic signs of inflammation: redness, swelling, and pain. 37 Redness and edema result from increased blood flow into the inflamed tissue through PGE2-mediated augmentation of arterial dilatation and increased microvascular permeability. 37 Pain results from the action of PGE2 on peripheral sensory neurons and on central sites within the spinal cord and the brain. 37

PGE2 is synthesized from PGH2 by cPGES or mPGES-1 and mPGES-2. 38 cPGES is constitutively and abundantly expressed in the cytosol of various tissues and cells and it requires glutathione as a cofactor. 39 The role of cPGES and even its ability to form PGE2 is controversial. cPGES seems capable of converting COX-1-derived but not COX-2-derived PGH2 to PGE2 in cells, particularly during the immediate PGE2-biosynthetic response elicited by Ca 2+ evoked stimuli. Localization of cPGES in the cytosol may allow coupling with proximal COX-1 in the endoplasmic reticulum in preference to distal COX-2 in the perinuclear envelope. 39 Functional coupling of cPGES with COX-1 suggests that the functions of cPGES in vivo overlap significantly, if not entirely, with COX-1. cPGES-deficient mice were developed, but they have not been particularly informative in addressing the importance of cPGES-derived PGE2, because deletion of this enzyme results in perinatal lethality. 40

mPGES-1 is a member of the membrane-associated proteins involved in eicosanoid and glutathione metabolism superfamily, and like cPGES, it requires glutathione as cofactor. 41 mPGES-1 is a perinuclear protein that is markedly induced by cytokines and growth factors and downregulated by antiinflammatory glucocorticoids, as in the case of COX-2. 42,43 It is functionally coupled with COX-2 in marked preference to COX-1. 44 Constitutive expression of mPGES-1 in certain tissues and cell types was also reported.

The generation of mPGES-1-deficient mice has revealed the dominant role of this enzyme in PGE2 generation relevant to promotion of inflammation. In collagen-induced arthritis, a disease model of human RA, mPGES-1-null mice exhibited a reduced incidence and severity of disease compared with WT controls. 45 This difference was not associated with alterations in interleukin (IL)-6 production by peritoneal macrophages or significant differences in circulating IgG2a anticollagen antibodies. Likewise, in collagen antibody–induced arthritis, another model of RA that does not involve the activation of the immune system, mPGES-1-null mice had a similar incidence but a lesser severity of arthritis than WT mice, as well as a 50% reduction in paw levels of PGE2. 46 In the same study, it was also observed that the migration of macrophages following peritoneal injection of thioglycollate was strikingly reduced in mPGES-1-null mice relative to WT mice. 46

The formation of inflammatory granulation tissue and attendant angiogenesis in the dorsum induced by subcutaneous implantation on the paw of a cotton thread was significantly reduced in mPGES-1-KO mice as compared with WT mice. 46 In this model, mPGES-1 deficiency was also associated with reduced induction of vascular endothelial cell growth factor in the granulation tissue. These results indicate that mPGES-1-derived PGE2, in cooperation with vascular endothelial growth factor, may play a critical role in the development of inflammatory granulation and angiogenesis, thus eventually contributing to tissue remodeling.

Together, these findings illustrate that deletion or inhibition of mPGES-1 markedly reduces inflammatory response, in several mouse models. The proinflammatory effect of mPGES-1-derived PGE2 was also observed in atherosclerosis. Deletion of mPGES-1 retarded atherogenesis in fat-fed hyperlipidemic mice, in both sexes. 47 Interestingly, in addition to the expected depression of PGE2 production, deletion of mPGES-1 permits rediversion of the PGH2 substrate to other PG synthases, with, for example, augmented formation of PGI2 and PGD2. 47 This complicates the selection of mPGES-1 as a drug target. Thus, elevated PGI2 may contribute to the more benign cardiovascular profile of mPGES-1 deletion compared with COX-2 deletion or inhibition: less predisposition to hypertension and thrombogenesis. However, this same effect may attenuate relief of pain, in the case of augmented PGI2, or may mediate adverse effects on bronchial tone, allergic inflammatory disease, or the sleep-wakefulness cycle, in case of elevated PGD2. This may account for the less impressive effectiveness of disrupting mPGES-1 versus COX-2 in some models of pain. 48 Recently, Brenneis et al have provided evidence that mPGES-1 derived PGE2 may contribute both to promotion and resolution of neuroinflammation in mice. 49 Finally, the major substrate products of rediversion will be influenced by the dominant cell type in a particular setting. For example, augmented PGI2 may contribute to the restraint of atherogenesis in hyperlipidemic mice consequent to mPGES-1 deletion. 47 However, it remains to be seen whether mPGES-1 inhibition in the setting of established atherosclerosis causes regression or possibly accelerates further progression of disease because of endoperoxide rediversion to TXA2 in macrophage-rich plaques.

mPGES-2 is synthesized as a Golgi membrane–associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. 37 mPGES-2-deficient mice showed no specific phenotype and no alteration in PGE2 levels in several tissues or in LPS-stimulated macrophages. 50 Studies with PGES-null mice have revealed that cross-regulation between the different PGES isoforms may function, on occasion, as a compensatory mechanism. For example, mPGES-1-null mice exhibit a delayed increase in urinary PGE2 excretion in response to acute water loading, coincident with enhanced renal medullary expression of cPGES but not of mPGES-2. 51 Similar evidence suggestive of cross-regulation was observed with the COXs. Thus, deletion of COX-2 in macrophages is associated with upregulated expression of COX-2 in vascular smooth muscle cells (VSMCs) in atherosclerotic plaque. 52

After PGE2 is formed, it is actively transported through the membrane by the ATP-dependent multidrug resistance protein-4 or diffuses across the plasma membrane to act at or near its site of secretion. 53

PGE2 then acts locally through binding of 1 or more of its 4 cognate receptors (Table), termed EP1–EP4. 45 Among the 4 EPs, EP3 and EP4 receptors are the most widely distributed, with their mRNAs being expressed in almost all mouse tissues, and have the highest affinity for binding PGE2. In contrast, the distribution of EP1 mRNA is restricted to several organs, such as the kidney, lung, and stomach, and EP2 is the least abundant of the EP receptors. Both EP1 and EP2 bind PGE2 with lower affinity. 54 Each EP subtype shows a distinct cellular localization within tissues. 54

One of the lessons learned from the KO mouse studies is that PGE2 can exert both proinflammatory and antiinflammatory responses, and these actions are often produced through regulation of receptor gene expression in relevant tissues. For example, hyperalgesia, a classic sign of inflammation, is mediated mainly by PGE2 through EP1 receptor signaling that acts on peripheral sensory neurons at the site of inflammation, as well as on central neuronal sites. 55 Other studies have also implicated the EP3 receptor in the inflammatory pain response mediated by low doses of PGE2. 56

EP2 and EP4 redundantly mediate development of paw swelling associated with collagen-induced arthritis. 57 Likewise, studies of carrageenan-induced paw edema and carrageenan-induced pleurisy both revealed participation of EP2 and EP3 in inflammatory exudation. 58 The EP4 receptor also appears to play a proinflammatory role in the pathogenesis of RA. PGE2 produced by rheumatoid synovium has been implicated in IL-6 production and joint destruction. 59,60 Mice deficient in the EP4 but not in the EP1, EP2, or EP3 receptors exhibit an attenuated response in the collagen antibody–induced arthritis model, with significantly lower levels of the inflammatory cytokines IL-6 and IL-1 and a dramatic reduction in the clinical signs of disease. 61 Antiinflammatory actions of PGs are seen typically in allergic or immune inflammation and are usually balanced by proinflammatory actions of other PGs. Such contrasting biology is evident between the PGI2-IP and TXA2-TP pathways in cardiovascular disease and between the PGD2-DP and the PGE2-EP3 pathways in elicitation of allergic asthma. 62,63

PGE2, binding to different EP receptors, can regulate the function of many cell types including macrophages, dendritic cells (DCs) and T and B lymphocytes, leading to both pro- and antiinflammatory effects. As proinflammatory mediator, PGE2 contributes to the regulation of the cytokine expression profile of DCs and has been reported to bias T cell differentiation toward a T helper (Th) 1 or Th2 response. 35 A recent study showed that PGE2-EP4 signaling in DCs and T cells facilitates Th1 and IL-23-dependent Th17 differentiation. 64 In addition, PGE2 is fundamental to induction of a migratory DC phenotype permitting their homing to draining lymph nodes. 65,66 Simultaneously, PGE2 stimulation early during maturation induces the expression of costimulatory molecules of the tumor necrosis factor superfamily on DCs resulting in an enhanced T-cell activation. 67 In contrast, PGE2 has also been demonstrated to suppress Th1 differentiation, B-cell functions, and allergic reactions. 68 Moreover, PGE2 can exert antiinflammatory actions on innate immune cells, such as neutrophils, monocytes, and natural killer cells. 68

PGE2 can thus modulate various steps of inflammation in a context-dependent manner and coordinate the whole process in both proinflammatory and antiinflammatory directions.

This dual role of PGE2 and its receptors in modulating the inflammatory response has been observed in several disorders. In atherosclerosis, EP4 deficiency promotes macrophage apoptosis and suppresses early atherosclerosis in low-density lipoprotein receptor −/− mice chimeric for EP4 −/− in hematopoietic cells after 8 weeks on a Western diet. 69 EP2 deficiency in hematopoietic cells revealed a trend for similar, but modest, effects on atherosclerosis. 69 In the same study macrophage EP4 appeared to play a proinflammatory role in the early stages of atherosclerosis by regulating production of inflammatory cytokines ,such as IL-1β, IL-6, and monocyte chemotactic protein-1. 69 In contrast, EP4 deletion in bone marrow-derived cells enhanced local inflammation (increased expression of chemotactic proteins, including monocyte chemotactic protein-1 and IL-10, and increased inflammatory cells, such as macrophages and T cells) and altered lesion composition (increased smooth muscle cells within plaque) but did not alter plaque size or morphology in established atherosclerosis (after 10 weeks of high-fat diet). 70

PGE2 also plays contrasting roles during neuroinflammation. LPS-induced PGE2 synthesis causes deleterious effects in neurons resulting in lesions or enhanced pain transmission. 71–73

However, PGE2 also has antiinflammatory properties. It mediates bradykinin-induced neuroprotection and blocks LPS and ATP-induced cytokine synthesis in cultured microglia and in neuron-glia cocultures. 74,75 The antiinflammatory and neuroprotective effects of PGE2 are mediated via microglial EP2 and EP4 receptors. Recently, it has been reported that PGE2 limits cytokine and PG synthesis mainly through EP2 activation in a model of LPS-induced neuroinflammation and that mPGES-1 is a critical enzyme in this negative feedback regulation. 49

PGI2 and Inflammation

PGI2 is one of the most important prostanoids that regulates cardiovascular homeostasis. Vascular cells, including endothelial cells, VSMCs, and endothelial progenitor cells, are the major source of PGI2. 76

PGI2 is generated by the sequential action of COX and PGIS, a member of the cytochrome P450 superfamily that specifically converts PGH2 to PGI2. PGIS colocalizes with COX in the endoplasmic reticulum, plasma membrane, and nuclear membrane. 77 PGIS is constitutively expressed in endothelial cells, where it couples with COX-1, 78 although COX-2-dependent PGI2 production by endothelial cells has been reported to be modulated in vitro by thrombin, shear stress, oxidized low-density lipoprotein, hypoxia, and inflammatory cytokines, and it is synchronized by upregulation of COX-2. 79,80 In vivo studies in mice and humans showed that COX-2 was the dominant source of PGI2. 81

Once generated, PGI2 is released to act on neighboring VSMCs, as well as circulating platelets. Indeed, PGI2 exerts its effects locally, is not stored, and is rapidly converted by nonenzymatic processes to an inactive hydrolysis product, 6-keto-PGF. 82

PGI2 is a potent vasodilator and an inhibitor of platelet aggregation, leukocyte adhesion, and VSMC proliferation. 75 PGI2 is also antimitogenic and inhibits DNA synthesis in VSMC. 83

These actions of PGI2 are mediated through specific IP receptors (Table). This receptor is expressed in kidney, liver, lung, platelets, heart, and aorta. 84 There is inconclusive evidence that some effects of PGI2 on the vasculature might be mediated by the PPARδ pathway, in addition to the classical IP-cAMP signaling pathway. 85 PGI2 can indeed activate PPARδ however, just as with 15d-PGD2 and PPARγ, it is unclear that it represents a biological target at concentrations of the ligand attained in vivo. 86

Although IP-deficient mice mature normally without experiencing spontaneous thrombosis, both the response to thrombogenic stimuli and VSMC proliferation in response to vascular injury are enhanced compared with control littermates. 87 Mice lacking IP are also sensitive to dietary salt induced hypertension 88 and exhibit accelerated atherogenesis with enhanced platelet activation and increased adhesion of leukocytes on the vessel walls in both the low-density lipoprotein receptor and apolipoprotein E KO models. 35,89

In addition to its cardiovascular effects, PGI2 is an important mediator of the edema and pain that accompany acute inflammation. PGI2 is rapidly produced following tissue injury or inflammation and it is present at high concentrations in inflammatory milieus. 90 PGI2 is the most abundant prostanoid in synovial fluid in human arthritic knee joints, as well as in peritoneal cavity fluid from mice injected with irritants. 91,92 In IP receptor–deficient mice, potentiation of bradykinin-induced microvascular permeability by PGI2 is abolished in addition, these mice have substantially reduced carrageenan-induced paw edema. 93 The level of paw edema observed in IP-deficient mice was equivalent to that of indomethacin-treated controls, and indomethacin treatment of IP-deficient animals did not induce a further decrease in swelling, indicating that PGI2-IP receptor signaling is the major prostanoid pathway that mediates the acute inflammatory response in this model. It has also been suggested that bradykinin induces PGI2 formation leading to enhancement of microvascular permeability and edema. 94 The IP receptor has been shown to mediate nociceptive pain during acute inflammation. IP receptor mRNA is present in dorsal root ganglion neurons including those that express substance P, a marker for nociceptive sensory neurons. 95 IP receptor-deficient mice have an attenuated writhing response following intraperitoneal injection of either acetic acid or PGI2, indicating that the IP receptor plays a role in mediating peripheral nociceptive sensitization to inflammatory stimuli. 93 The IP receptor is also expressed in the spinal cord and has been implicated in spinal pain transmission in response to peripheral inflammation. 96 IP antagonists were shown to reduce pain responses in several models, including acetic acid–induced abdominal constriction, mechanical hyperalgesia produced by carrageenan, and pain associated with models of osteoarthritis and inflammatory arthritis. 97,98 In contrast to the proinflammatory effects of IP receptor activation in nonallergic acute inflammation, some studies have suggested that IP receptor signaling suppresses Th2-mediated allergic inflammatory responses. 99 IP receptor mRNA is upregulated in CD4+ Th2 cells, and inhibition of PGI2 formation by the COX-2 inhibitor NS-398 during antigen-induced airway inflammation results in greater lung Th2-mediated lung inflammation. 99 PGI2 has been suggested to exert this effect in part by enhancing Th2 cell production of the antiinflammatory cytokine IL-10. This immunosuppressive role for the IP receptor in Th2-mediated inflammation is supported by the observation that in ovalbumin (OVA)-induced asthma, IP deletion results in increased antigen-induced leukocyte accumulation in bronchoalveolar lavage fluid and peribronchiolar and perivascular inflammatory infiltration. 100 Thus, PGI2 may shift the balance within the immune system away from a Th2 dominant response and inhibit allergic inflammation.

PGD2 and Inflammation

PGD2 is a major eicosanoid that is synthesized in both the central nervous system and peripheral tissues and appears to function in both an inflammatory and homeostatic capacity. 101 In the brain, PGD2 is involved in the regulation of sleep and other central nervous system activities, including pain perception. 102,103 In peripheral tissues, PGD2 is produced mainly by mast cells but also by other leukocytes, such as DCs and Th2 cells. 104–106 Two genetically distinct PGD2-synthesizing enzymes have been identified, including hematopoietic- and lipocalin-type PGD synthases (H-PGDS and L-PGDS, respectively). H-PGDS is generally localized to the cytosol of immune and inflammatory cells, whereas L-PGDS is more restrained to tissue-based expression. 107

PGD2 can be further metabolized to PGF, 9α,11β-PGF2 (the stereoisomer of PGF) and the J series of cyclopentanone PGs, including PGJ2, Δ 12 -PGJ2, and 15d-PGJ2. 108 Synthesis of J series PGs involves PGD2 undergoing an initial dehydration reaction to produce PGJ2 and 15d-PGJ2, after which PGJ2 is converted to 15d-PGJ2 and Δ 12 -PGJ2 via albumin-dependent and albumin-independent reactions, respectively. 109

PGD2 activity is principally mediated through DP or DP1 and CRTH2 or DP2, as described previously (Table). Also, 15d-PGJ2 binds with low affinity the nuclear PPARγ. 110

PGD2 has long been associated with inflammatory and atopic conditions, although it might exert an array of immunologically relevant antiinflammatory functions as well.

PGD2 is the predominant prostanoid produced by activated mast cells, which initiate IgE-mediated type I acute allergic responses. 104,111 It is well established that the presence of an allergen triggers the production of PGD2 in sensitized individuals. In asthmatics, PGD2, which can be detected in the bronchoalveolar lavage fluid within minutes, reaches biologically active levels at least 150-fold higher than preallergen levels. 112 PGD2 is produced also by other immune cells, such as antigen-presenting DCs and Th2 cells, suggesting a modulatory role for PGD2 in the development of antigen-specific immune system responses. 104,105 PGD2 challenge elicits several hallmarks of allergic asthma, such as bronchoconstriction and airway eosinophil infiltration, 113,114 and mice that overexpress L-PGDS have elevated PGD2 levels and an increased allergic response in the OVA-induced model of airway hyperreactivity. 115

The proinflammatory effects of PGD2 appear to be mediated by both DP1 and DP2/CRTH2 receptors. Because both receptors bind PGD2 with similar high affinity, PGD2 produced by activated mast cells or T cells would be capable of activating multiple signaling pathways leading to different effects, depending on whether the DP1 or DP2/CRTH2 receptors or both are locally expressed.

The DP1 receptor is expressed on bronchial epithelium and has been proposed to mediate production of chemokines and cytokines that recruit inflammatory lymphocytes and eosinophils, leading to airway inflammation and hyperreactivity seen in asthma. 116 Mice deficient in DP1 exhibit reduced airway hypersensitivity and Th2-mediated lung inflammation in the OVA-induced asthma model, suggesting that the DP1 plays a key role in mediating the effects of PGD2 released by mast cells during an asthmatic response. 62 Furthermore, PGD2 may inhibit eosinophil apoptosis via the DP1 receptor. 117

DP1 antagonists exert antiinflammatory properties in several experimental models, including inhibition of antigen-induced conjunctival microvascular permeability in guinea pigs and OVA-induced airway hyperreactivity in mice. 118,119

DP2/CRTH2 receptors contribute largely to pathogenic responses by mediating inflammatory cell trafficking and by modulating effector functions. PGD2 released from mast cells may mediate recruitment of Th2 lymphocytes and eosinophils directly via the DP2/CRTH2 receptor. In humans, the DP2/CRTH2 receptor is expressed on Th2 lymphocytes, eosinophils, and basophils, 8,120,121 and an increase in DP2/CRTH2 + T cells has been positively associated with certain forms of atopic dermatitis. 122 The DP2/CRTH2 receptor has been demonstrated to mediate PGD2-stimulated chemotaxis of these cells in vitro and leukocyte mobilization in vivo. 123

In contrast to the proinflammatory role of PGD2 in allergic inflammation, PGD2 may act to inhibit inflammation in other contexts. The DP1 receptor is expressed on DCs that play a key role in initiating an adaptive immune response to foreign antigens. PGD2 activation of the DP1 receptor inhibits DC migration from lung to lymph nodes following OVA challenge, leading to reduced proliferation and cytokine production by antigen specific T cells. 124 DP1 activation also reduces eosinophilia in allergic inflammation in mice and mediates inhibition of antigen-presenting Langerhans cell function by PGD2. 125,126 As mentioned, PGD2 and its degradation product 15d-PGJ2 have been suggested as the COX-2 products involved in the resolution of inflammation. 28,127 Administration of a COX-2 inhibitor during the resolution phase exacerbated inflammation in a carrageenan-induced pleurisy model 28 . In a zymosan-induced peritonitis model, deletion of H-PGDS induced a more aggressive inflammatory response and compromised resolution, findings that were moderated by addition of a DP1 agonist or 15d-PGJ2. 123 Although these data appear to implicate PGD2 and 15d-PGJ2 in resolution, there is a large disparity between the nanomolar to picomolar amounts of 15d-PGJ2 detected by physicochemical methodology in in vivo settings and the amount needed to have a biological effect in vitro on PPARγ or nuclear factor-κB (micromolar amounts). 32,128,129 This discrepancy is supported by recent data reported in zymosan-induced peritonitis, where we observed evoked biosynthesis of PGD2 only during the proinflammatory phase and not during resolution. Consistent with this observation, DP2/CRTH2 deletion reduced the severity of acute inflammation, but neither DP1 or DP2/CRTH2 deletion affected resolution. Although 15d-PGJ2 is readily detected when synthetic PGD2 is infused into rodents, 130 endogenous biosynthesis of 15d-PGJ2 was not detected during promotion or resolution of inflammation (J. Mehta et al, unpublished data, 2010).

PGD2 may play a role in the evolution of atherosclerosis. In the context of inflamed intima, PGD2 in part derives from H-PGDS-producing inflammatory cells that are chemotactically compelled to permeate the vasculature. 131,132 Additionally, L-PGDS expression is induced by laminar sheer stress in vascular endothelial cells and is actively expressed in synthetic smooth muscle cells of atherosclerotic intima and coronary plaques of arteries with severe stenosis. 133–135 PGD2 has been shown to inhibit expression of proinflammatory genes, such as inducible nitric oxide synthase and plasminogen activator inhibitor. 136,137 Indeed, L-PGDS deficiency accelerates atherogenesis. 138

In summary, studies with COX-2 inhibitors suggest that products of this enzyme may play a role in resolution in several models of inflammation. However, the identity of such products, whether formed directly by COX-2 or because of substrate diversion consequent to COX-2 inhibition, remains, in many cases, to be established.

PGF2α and Inflammation

PGF is synthesized from PGH2 via PGF synthase, and it acts via the FP, which couples with Gq protein to elevate the intracellular free calcium concentration (Table). Two differentially spliced variants of the sheep FP receptor ortholog have been reported: FPA and FPB, which differ from each other in the length of their C-terminal tails. 139 The FP receptor is the least selective of the prostanoid receptors in binding the principal endogenous PGs both PGD2 and PGE2 ligate the FP with EC50 values in the nanomolar range. 140 PGF ring compounds can be formed as minor products from other PGs. For example, enzymatic reduction of 9-keto group of PGE compounds by 9-ketoreductases results in either 9a-hydroxyl, yielding PGFα compounds, or more rarely a 9b-hydroxyl, yielding PGFβ compounds. 141 PGF ring metabolites may also be formed from PGD ring compounds by 11-keto reductases. 142

15-Keto-dihydro-PGF, a major stable metabolite of PGF that reflects in vivo PGF biosynthesis, is found in larger quantities first in the peripheral plasma and later on in the urine both in basal physiological conditions and in certain physiological and pathophysiological situations, such as acute and chronic inflammation. 143

PGF, derived mainly from COX-1 in the female reproductive system, plays an important role in ovulation, luteolysis, contraction of uterine smooth muscle, and initiation of parturition. 144,145 Recent studies have shown that PGF also plays a significant role in renal function, 146 contraction of arteries, 147 myocardial dysfunction, 148,149 brain injury, 150 and pain. 151 Analogs of PGF have previously been developed for estrus synchronization and abortion in domestic animals 152,153 and to influence human reproductive function. 154 FP agonists are being widely used worldwide to reduce intraocular pressure in the treatment of glaucoma. 155

Administration of PGF leads to acute inflammation, and NSAIDs inhibit PGF biosynthesis both in vitro and in vivo. 144 In models of acute inflammation, evoked biosynthesis of PGF may coincide with free radical catalyzed generation of F ring isoprostanes, indices of lipid peroxidation. 156,157 The tachycardia induced in WT mice by injection of LPS is greatly attenuated in FP-deficient or TP-deficient mice and is completely absent in mice lacking both of these receptors. 148 A recent study reported that deletion of FP selectively attenuates pulmonary fibrosis without a change in alveolar inflammation after microbial invasion. 158

Elevated biosynthesis of PGF has been reported in patients experiencing RA, psoriatic arthritis, reactive arthritis, and osteoarthritis. 159

Cardiovascular risk factors, such as diabetes, obesity, smoking, and thickening of intima-media ratio in the carotid artery, have been variably associated with elevations in PGF metabolites, together with IL-6 and acute phase proteins in body fluids. 160,161 Deletion of the FP reduces blood pressure and retards the attendant atherogenesis in hyperlipidemic mice despite the absence of detectable FP in large blood vessels and their atherosclerotic plaques. 162 PGF is the most abundant prostanoid formed by human umbilical cord endothelial cells in response to laminar shear stress that upregulates expression of COX-2. 163

The emerging role of PGF in acute and chronic inflammation opens opportunities for the design of new antiinflammatory drugs.

Thromboxane and Inflammation

TXA2, an unstable AA metabolite with a half-life of about 30 seconds, is synthesized from PGH2 via thromboxane synthase, and it is nonenzymatically degraded into biologically inactive TXB2. TXA2 is predominantly derived from platelet COX-1, but it can also be produced by other cell types, including macrophage COX-2. 164,165

TXA2 activity is principally mediated through the TP, which couples with Gq, G12/13, and multiple small G proteins, which in turn regulate several effectors, including phospholipase C, small G protein Rho, and adenylyl cyclase (Table). 166 TPα and TPβ, 2 spliced isoforms of TP in humans, communicate with different G proteins and undergo heterodimerization, resulting in changes in intracellular traffic and receptor protein conformations. Only the TPα protein is expressed in mice.

TP activation mediates several physiological and pathophysiological responses, including platelet adhesion and aggregation, smooth muscle contraction and proliferation, and activation of endothelial inflammatory responses. 167 TP function is regulated by several factors, such as oligomerization, desensitization and internalization, glycosylation, and cross-talk with receptor tyrosine kinases. 167

Although TXA2 is the preferential physiological ligand of the TP receptor, PGH2, in particular, can also activate this receptor. 168 In addition, isoprostanes (nonenzymatic free radical-catalyzed peroxidative products of polyunsaturated fatty acids) and hydroxyeicosatetraenoic acids (generated by lipoxygenases and cytochrome P450 monooxygenases or formed by nonenzymatic lipid peroxidation) are also potent agonists at TP receptors. 169,170 Epoxyeicosatrienoic acid (cytochrome P450 metabolites of AA) dihydro derivatives, in contrast, are selective endogenous antagonists of TP. 171 However, whether PGH2, isoprostanes, or hydroxyeicosatetraenoic acids significantly contribute to the responses attributed to TP activation in vivo is still to be investigated. For example, TP activation by isoprostanes may play an important role in clinical settings of oxidative stress, such as during reperfusion after organ transplant.

TP-deficient mice are normotensive but have blunted vascular responses to TP agonists and show a tendency to bleeding. 172

The deletion of TP decreases vascular proliferation and platelet activation in response to vascular injury, delays atherogenesis, and prevents angiotensin II– and l -NAME-induced hypertension and the associated cardiac hypertrophy. 87,89,173,174

In septic shock models, TP deletion or TP antagonism protected against various LPS-induced responses, such as the increase in inducible nitric oxide synthase expression, acute renal failure, and inflammatory tachycardia, 175,176 suggesting a potential role of TXA2 as proinflammatory mediator.

The phenotype of thromboxane synthase–deficient mice is much less pronounced, perhaps because TXA2 is only 1 of the endogenous ligands of TP and more likely because the deletion of this enzyme may redirect the PGH2 toward other countervailing synthases. 177

Prostanoids in Translation

This review has described a stunning complexity of evidence about the role of prostanoids in inflammation. Different products have conflicting effects on both the promotion and resolution of inflammation. The same product formed by different enzymes—COX-1 or COX-2—may either promote or resolve inflammation. Products of the same enzyme may promote or resolve inflammation in different models. Different cell types that predominate at varying stages of disease evolution generate prostanoids that have contrasting effects on inflammation. Individual prostanoids overlap considerably in their biological effects with other mediators.

These observations prompt several questions.

First, given this complex array of biological effects mediated by prostanoids, how does their general inhibition, high up in the cascade, result in drugs that are reasonably well tolerated and reasonably effective? Aspirin and the myriad NSAIDs, including acetaminophen and those developed to inhibit selectively COX-2, are among the most commonly consumed drugs on the planet. Hard evidence with which to address this question is in short supply, but let us speculate. Aspirin at doses less than 100 mg per day or greater than 1 g per day have equivalent effects on platelet COX-1-derived TXA2. They both suppress it completely. However, increasing daily doses of aspirin increasingly inhibit PGI2 coincidentally with this effect. We do not have direct randomized comparisons across doses, but indirect comparisons suggest that the cardioprotective efficacy of aspirin may be progressively attenuated as the dose is increased. Similarly, locally formed, COX-1-derived PGE2 and PGI2 are protective of gastroduodenal epithelial integrity, and platelet COX-1-derived TXA2 contributes to hemostatic competence. Disruption of these pathways by NSAIDs that inhibit COX-1 is thought to account for NSAID induced ulcers. However, NSAIDs selective for inhibition of COX-2 only halve the comparative incidence of serious gastrointestinal events. This may in part reflect their impact on gastroduodenal epithelial COX-2-dependent prostanoids that accelerate ulcer healing. In these examples, inhibitors high up in the pathway confer benefit, but it is a net benefit, and of course the margin of that benefit may vary substantially between individuals. We only poorly understand interindividual differences in antiinflammatory efficacy among the reversibly acting NSAIDs.

Prostanoids tend to be relatively weak agonists in systems where their blockade has resulted in clinical efficacy. For example, TXA2 is a relatively weak platelet agonist compared with thrombin, and there is a considerable amount of redundancy in the system.

Why does blockade of just 1 of the many pathways of platelet activation result in an effect so great that its impact can be detected by as crude and instrument as a randomized clinical trial? Similarly, why does blockade of sulfidopeptide leukotrienes alone among many bronchoconstrictors result in clinical efficacy in asthma, or why do other mediators, such as NO, not substitute for the cardioprotective effects of PGI2, suppressed by NSAIDs selective for inhibition of COX-2? Perhaps drug efficacy is realized because eicosanoids often tend to function as amplifying signals for other, more potent agonists. Activate platelets with thrombin, ADP, or collagen and release of TXA2 amplifies and sustains the aggregation response. Perhaps this is why aspirin is so effective in the secondary prevention of myocardial infarction or stroke. As for why other mediators do not step in to substitute for suppressed prostanoids, this may speak to their singular importance in circumstances of phenotypic perturbations, as discussed next.

Given the myriad biological effects of these compounds, how are drugs that shut down their synthesis even tolerated? NSAIDs may indeed result in life-threatening gastrointestinal or cardiovascular adverse events, but only in a small minority (perhaps 1% to 2%) of patients exposed. This may reflect the fact that prostanoid formation is a homeostatic response system. Under physiological conditions, trivial amounts of these compounds are formed, and their biological importance is, in many cases, marginal. However, when a system is stressed, they may become pivotal. Examples include their essential role in the maintenance of renal blood flow under renoprival conditions, their antihypertensive effects in patients infused with vasopressors, or their antithrombotic effects in patients at increased risk of thrombogenesis. For example, deletion of the IP does not result in spontaneous thrombosis but rather accentuates the response to thrombogeneic stimuli. Similarly, although preexisting cardiovascular disease was often used to dilute the legal liability of the sponsor in cases where patients experienced myocardial infarctions while taking coxibs, the relative risk of myocardial infarctions on celecoxib relates to the underlying burden of cardiovascular disease, an expected consequence of suppression of COX-2 derived PGI2. 164

Given the contrasting effects of prostanoids, should we move down the pathway to get a more targeted and safer response? Presently, we have almost no data that address this question. In the case of downstream PG synthase versus COX-2 inhibition, the experience with mPGES-1 deletion highlights the complexity of the comparison. Here, substrate rediversion to PGIS may attenuate the cardiovascular risk of COX-2 inhibition but dilute analgesic efficacy. Another example is the comparison between low-dose aspirin for cardioprotection and TP antagonism. The latter strategy avoids PGI2 suppression, but this is very modest, on average ≈15%, with low-dose aspirin. One would need an enormous clinical trial to detect that theoretical benefit. Alternatively, the antagonist, unlike aspirin, might block TP activation by unconventional ligands, such as isoprostanes and hydroxyeicosatetraenoic acids. However, although these compounds can activate the TP, their relevance in vivo, even in settings of tissue reperfusion where they are formed in excess, remains speculative. One might of course model this comparison and use COX-1 knockdown mice, which mimic the asymmetrical impact on platelet COX-1 of low-dose aspirin, to address the question. Finally, as the overlap in the biological consequences of activating several prostanoid receptors—the IP, EP2, EP4, and DP1 group or the FP, TP, and EP1 group, for example—hint at the possibility that efficacy might be diluted as one moves downstream to target just 1 prostanoid in the pathway. Our poor understanding of such potential functional redundancy at the receptor level, never mind insight into the implications of receptor dimerization, leave these as open questions for drug development.

Surely, this complexity suggests that experiments in animals are going to be of virtually no value when it comes to predicting drug effects in vivo. Certainly the limitations of standard experimental paradigms apply in this pathway, as in others. We study mouse models of atherosclerosis that fail to undergo spontaneous plaque fissure and thrombotic occlusion of vital arteries to reach conclusions about prevention or provocation of myocardial infarction. We extrapolate from drug action on murine tolerance of a hotplate to patients undergoing molar extraction in an effort to divine therapies for elderly women with osteoarthritis of the knees. These and many such examples inspire caution in the field of translational therapeutics. However, consistency of evidence from different model systems across multiple species can, especially when integrated with independent lines of evidence, predict with some confidence the outcome in randomized clinical trials of drug action in this pathway. Such was the case in the prediction and mechanistic elucidation of the cardiovascular hazard from NSAIDs. 178

Finally, is it likely that this pathway will yield more therapeutic opportunities? Besides drugs already approved for various indications (like aspirin and the myriad NSAIDs analogues of PGE2, PGF2a, and PGI2 TP and leukotriene antagonists) currently inhibitors of mPGES-1, 5–lipoxygenase and its activating factor (five lipoxygenase activating protein) and antagonists of DP1, DP2, and EP4 are all undergoing clinical evaluation. Many other targets in the eicosanoid pathway are undergoing preclinical evaluation, just as others, such as the multiple secretory phospholipases and the soluble epoxide hydrolase, emerge. This coincides with new information about old targets.

Why do we need to inhibit both COXs, not just COX-1, to see gastrointestinal injury in model systems? 29 Why is a form of aspirin confined to platelet inhibition in the presystemic circulation associated with a reduction in the incidence of colon cancer? 179,180 Much remains to be discovered about the biology of the eicosanoids, and from this is likely to come new therapeutic opportunity.


Prostanoids can promote or restrain acute inflammation. Products of COX-2 in particular may also contribute to resolution of inflammation in certain settings. Presently, we have little information on which products of COX-2 might subserve this role or indeed whether the dominant factors reflect rediversion of the AA substrate to other metabolic pathways consequent to deletion or inhibition of COX-2. As with cyclopentanone prostanoids, many arachidonate derivatives, including transcellular products, when synthesized and administered as exogenous compounds, can promote resolution in models of inflammation. However, rigorous physicochemical evidence for the formation of the endogenous species in relevant quantities to subserve this role in vivo is limited. Elucidation of whether and how prostanoids might restrain inflammation and how substrate modification, such as with fish oils, might exploit this understanding is currently a focus of much research from which novel therapeutic strategies are likely to emerge.

Sources of Funding

The work that formed the basis for opinions expressed in this review was supported by National Institutes of Health Grants HL062250 , HL083799 , and HL054500 (to G.A.F). Dr FitzGerald is the McNeil Professor of Translational Medicine and Therapeutics.


Dr FitzGerald has consulted in the past year for Astra Zeneca, Daiichi Sankyo, Logical Therapeutics, Lilly, and Nicox on NSAIDs and related compounds. The other authors report no conflicts.


Neuroinflammation is widely regarded as chronic, as opposed to acute, inflammation of the central nervous system. [5] Acute inflammation usually follows injury to the central nervous system immediately, and is characterized by inflammatory molecules, endothelial cell activation, platelet deposition, and tissue edema. [6] Chronic inflammation is the sustained activation of glial cells and recruitment of other immune cells into the brain. It is chronic inflammation that is typically associated with neurodegenerative diseases. Common causes of chronic neuroinflammation include:

  • Toxic metabolites
  • Autoimmunity
  • Aging
  • Microbes
  • Viruses
  • Traumatic brain injury
  • Spinal cord injury

Glial cells Edit

Microglia are recognized as the innate immune cells of the central nervous system. [2] Microglia actively survey their environment through, and change their cell morphology significantly in response to neural injury. [7] Acute inflammation in the brain is typically characterized by rapid activation of microglia. [5] During this period, there is no peripheral immune response. Over time, however, chronic inflammation causes the degradation of tissue and of the blood–brain barrier. During this time, microglia generate reactive oxygen species and release signals to recruit peripheral immune cells for an inflammatory response. [7]

Astrocytes are glial cells that are the most abundant cells in the brain. They are involved in maintenance and support of neurons and compose a significant component of the blood–brain barrier. After insult to the brain, such as traumatic brain injury, astrocytes may become activated in response to signals released by injured neurons or activated microglia. [6] [1] Once activated, astrocytes may release various growth factors and undergo morphological changes. For example, after injury astrocytes form the glial scar composed of a proteoglycan matrix that hinders axonal regeneration. [6] However more recent studies revealed that glia scar is not detrimental but beneficial for axonal regeneration. [8]

Cytokines Edit

Cytokines are a class of proteins that regulates inflammation, cell signaling, and various cell processes such as growth and survival. [9] Chemokines are a subset of cytokines that regulate cell migration, such as attracting immune cells to a site of infection or injury. [9] Various cell types in the brain may produce cytokines and chemokines such as microglia, astrocytes, endothelial cells, and other glial cells. Physiologically, chemokines and cytokines function as neuromodulators that regulate inflammation and development. In the healthy brain, cells secrete cytokines to produce a local inflammatory environment to recruit microglia and clear the infection or injury. However, in neuroinflammation, cells may have sustained release of cytokines and chemokines which may compromise the blood–brain barrier. [10] Peripheral immune cells are called to the site of injury via these cytokines and may now migrate across the compromised blood brain barrier into the brain. Common cytokines produced in response to brain injury include: interleukin-6 (IL-6), which is produced during astrogliosis, and interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), which can induce neuronal cytotoxicity. Although the pro-inflammatory cytokines may cause cell death and secondary tissue damage, they are necessary to repair the damaged tissue. [11] For example, TNF-α causes neurotoxicity at early stages of neuroinflammation, but contributes to tissue growth at later stages of inflammation.

The blood–brain barrier is a structure composed of endothelial cells and astrocytes that forms a barrier between the brain and circulating blood. Physiologically, this enables the brain to be protected from potentially toxic molecules and cells in the blood. Astrocytes form tight junctions and therefore may strictly regulate what may pass the blood–brain barrier and enter the interstitial space. [6] After injury and sustained release of inflammatory factors such as chemokines, the blood–brain barrier may be compromised, becoming permeable to circulating blood components and peripheral immune cells. Cells involved in the innate and adaptive immune responses, such as macrophages, T Cells, and B Cells, may then enter into the brain. This exacerbates the inflammatory environment of the brain and contributes to chronic neuroinflammation and neurodegeneration.

Traumatic brain injury (TBI) is brain trauma caused by significant force to the head. [6] Following TBI, there are both reparative and degenerative mechanisms that lead to an inflammatory environment. Within minutes of injury, pro-inflammatory cytokines are released. The pro-inflammatory cytokine Il-1β is one such cytokine that exacerbates the tissue damage caused by TBI. TBI may cause significant damage to vital components to the brain, including the blood–brain barrier. Il-1β causes DNA fragmentation and apoptosis, and together with TNF-α may cause damage to the blood–brain barrier and infiltration of leukocytes. (). Increased density of activated immune cells have been found in the human brain after concussion. [1]

Spinal Cord Injury (SCI) can be divided into three separate phases. The primary or acute phase occurs from seconds to minutes post injury, the secondary phase occurs from minutes to weeks after injury, and the chronic phase occurs from months to years following injury. [12] A primary SCI is caused by spinal cord compression or transection, leading to glutamate excitotoxicity, sodium and calcium ion imbalances, and free radical damage. [13] Neurodegeneration via apoptosis and demyelination of neuronal cells causes inflammation at the injury site. [12] This leads to a secondary SCI, whose symptoms include edema, cavitation of spinal parenchyma, reactive gliosis, and potentially permanent loss of function. [12]

During the SCI induced inflammatory response, several pro-inflammatory cytokines including interleukin 1β (IL-1β), inducible Nitric Oxide Synthase (iNOS), Interferon-γ (IFN-γ), IL-6, IL-23, and tumor necrosis factor α (TNFα) are secreted, activating local microglia and attracting various immune cells such as naive bone-marrow derived macrophages. [14] These activated microglia and macrophages play a role in the pathogenesis of SCI.

Upon infiltration of the injury site's epicenter, macrophages will undergo phenotype switching from an M2 phenotype to an M1-like phenotype. The M2 phenotype is associated with anti-inflammatory factors such as IL-10, IL-4, and IL-13 and contributes to wound healing and tissue repair. However, the M1-like phenotype is associated with pro-inflammatory cytokines and reactive oxygen species that contribute to increased damage and inflammation. [15] Factors such as myelin debris, which is formed by the injury at the damage site, has been shown to induce the phenotype shift from M2 to M1. [16] A decreased population of M2 macrophages and an increased population of M1 macrophages is associated with chronic inflammation. [16] Short term inflammation is important in clearing cell debris from the site of injury, but it is this chronic, long-term inflammation that will lead to further cell death and damage radiating from the site of injury. [17]

Aging is often associated with cognitive impairment and increased propensity for developing neurodegenerative diseases, such as Alzheimer's disease. [18] Elevated inflammatory markers seemed to accelerate the brain aging process [19] In the aged brain alone, without any evident disease, there are chronically increased levels of pro-inflammatory cytokines and reduced levels of anti-inflammatory cytokines. The homeostatic imbalance between anti-inflammatory and pro-inflammatory cytokines in aging is one factor that increases the risk for neurodegenerative disease. Additionally, there is an increased number of activated microglia in aged brains, which have increased expression of major histocompatibility complex II (MHC II), ionized calcium binding adaptor-1 (IBA1), CD86, ED1 macrophage antigen, CD4, and leukocyte common antigen. [20] These activated microglia decrease the ability for neurons to undergo long term potentiation (LTP) in the hippocampus and thereby reduce the ability to form memories. [21]

Alzheimer's disease Edit

Alzheimer's disease (AD) has historically been characterized by two major hallmarks: neurofibrillary tangles and amyloid-beta plaques. [22] Neurofibrillary tangles are insoluble aggregates of tau proteins, and amyloid-beta plaques are extracellular deposits of the amyloid-beta protein. Current thinking in AD pathology goes beyond these two typical hallmarks to suggest that a significant portion of neurodegeneration in Alzheimer's is due to neuroinflammation. [22] [23] Activated microglia are seen in abundance in post-mortem AD brains. Current thought is that inflammatory cytokine-activated microglia cannot phagocytose amyloid-beta, which may contribute to plaque accumulation as opposed to clearance. [24] Additionally, the inflammatory cytokine IL-1β is upregulated in AD and is associated with decreases of synaptophysin and consequent synaptic loss. Further evidence that inflammation is associated with disease progression in AD is that persons that take non-steroidal anti-inflammatory drugs (NSAIDs) regularly have been associated with reduced AD later in life. [ citation needed ] Elevated inflammatory markers showed an association with accelerated brain aging, which might explain the link to neurodegeneration in AD-related brain regions. [19]

Parkinson's disease Edit

The leading hypothesis of Parkinson's disease progression includes neuroinflammation as a major component. [25] This hypothesis stipulates that Stage 1 of Parkinson's disease begins in the gut, as evidenced by a large number of cases that begin with constipation [ citation needed ] . The inflammatory response in the gut may play a role [ citation needed ] in alpha-synuclein (α-Syn) aggregation and misfolding, a characteristic of Parkinson's disease pathology. If there is a balance between good bacteria and bad bacteria in the gut, the bacteria may remain contained to the gut. However, dysbiosis of good bacteria and bad bacteria may cause a “leaky” gut, creating an inflammatory response. This response aids α-Syn misfolding and transfer across neurons, as the protein works its way up to the CNS. [ citation needed ] The brainstem is vulnerable to inflammation, which would explain Stage 2, including sleep disturbances and depression. In Stage 3 of the hypothesis, the inflammation affects the substantia nigra, the dopamine producing cells of the brain, beginning the characteristic motor deficits of Parkinson's disease. Stage 4 of Parkinson's disease includes deficits caused by inflammation in key regions of the brain that regulate executive function and memory. As evidence supporting this hypothesis, patients in Stage 3 (motor deficits) that are not experiencing cognitive deficits already show that there is neuroinflammation of the cortex. This suggests that neuroinflammation may be a precursor to the deficits seen in Parkinson's disease. [25]

Multiple sclerosis Edit

Multiple sclerosis is the most common disabling neurological disease of young adults. [26] It is characterized by demyelination and neurodegeneration, which contribute to the common symptoms of cognitive deficits, limb weakness, and fatigue. [27] In multiple sclerosis, inflammatory cytokines disrupt the blood–brain barrier and allow for the migration of peripheral immune cells into the central nervous system. When they have migrated into the central nervous system, B cells and plasma cells produce antibodies against the myelin sheath that insulates neurons, degrading the myelin and slowing conduction in the neurons. Additionally, T cells may enter through the blood–brain barrier, be activated by local antigen presenting cells, and attack the myelin sheath. This has the same effect of degrading the myelin and slowing conduction. As in other neurodegenerative diseases, activated microglia produce inflammatory cytokines that contribute to widespread inflammation. It has been shown that inhibiting microglia decreases the severity of multiple sclerosis. [25]

Drug therapy Edit

Because neuroinflammation has been associated with a variety of neurodegenerative diseases, there is increasing interest to determine whether reducing inflammation will reverse neurodegeneration. Inhibiting inflammatory cytokines, such as IL-1β, decreases neuronal loss seen in neurodegenerative diseases. Current treatments for multiple sclerosis include interferon-B, Glatiramer acetate, and Mitoxantrone, which function by reducing or inhibiting T Cell activation, but have the side effect of systemic immunosuppression [28] In Alzheimer's disease, the use of non-steroidal anti-inflammatory drugs decreases the risk of developing the disease. Current treatments for Alzheimer's disease include NSAIDs and glucocorticoids. NSAIDs function by blocking conversion of prostaglandin H2 into other prostaglandins (PGs) and thromboxane (TX). Prostoglandins and thromboxane act as inflammatory mediators and increase microvascular permeability.

Exercise Edit

Exercise is a promising mechanism of prevention and treatment for various diseases characterized by neuroinflammation. [20] Aerobic exercise is used widely to reduce inflammation in the periphery. Exercise has been shown to decrease proliferation of microglia in the brain, decrease hippocampal expression of immune-related genes, and reduce expression of inflammatory cytokines such as TNF-α.

Inflammatory mediators

The inflammatory response is a combination of diverse chemical mediators from blood circulation, immune cells, and wounded tissue. These include vasoactive amines (histamine), peptides (bradykinin), and eicosanoids (leukotrienes).

Vasoactive amines

These are a group of compounds contains an amino acid to modify the pervasiveness of blood vessels. There are two effective molecules are histamine and serotonin. They are known as beginning inflammatory mediators stored in mast cells.

Histamine is made by mast cells generally. Basophils and platelets also produced it by the decarboxylation of histidine amino acid. It releases due to the number of stimulators such as tissue damage, Fc receptor binding of IgE antibody to the mast cell, C3a and C5a complement, etc. It widens blood vessels by tempting endothelial contraction and the development of interendothelial gaps.

It is a derivative of tryptophan present in platelets. It is also present in neurons and enterochromaffin cells. Platelets accumulation allow its release and function to narrow down blood vessels, neurotransmitter, etc. It functions in inflammation, opposite to histamine.

Bradykinin are nonapeptides, vasoactive in nature. They produced by the activity of the proteases enzyme. They are significant in blood pressure control and inflammatory reaction. It causes vasodilation of arteries and veins of gut, aorta, uterus, and urethra. It is now also known for its role in neuro-signaling and a controller of some kidney and vascular functions.


Eicosanoids obtain from polyunsaturated fatty acids by oxygenation of biolipid. They function in various physiological mechanisms and pathological responses such as allergy, fever, inhibition of inflammation, etc. Its signaling mechanism is alike to cytokines in the inflammatory response and pro-inflammatory component formation. Eicosanoids have significant subgroups of diverse compounds most noticeably prostaglandins, leukotrienes, lipoxin, thromboxane, eoxins, etc.


They have a significant role in inflammatory response generation. Their production increased in injured inflamed tissues and significant inflammation signs indicated by their output. Prostaglandin E2 is most plentifully made in the body with various functioning such as blood pressure control, immune reaction, fertility, etc. in the inflammatory process, they have an important character for generating basic signs of inflammation: redness, swelling, and pain. Erythema and swelling occur due to rapid blood flow by enlargement and increased pervasiveness of vessels. Pain is mediated due to the action of PGE2 on sensory neurons.


Leukotrienes belong to the eicosanoids family and use lipid signaling as autocrine or paracrine signaling. Their synthesis is complemented with histamine and prostaglandin production. They stimulate smooth muscle contraction and their excess amount causes inflammation in the bronchial lining and in allergic reactions. They have also chemotactic influence on neutrophils.


Thromboxane are also produced from arachidonic acid metabolism. They obtain this name due to their clot formation activity known as thrombosis. The chief thromboxane types are thromboxane A2 and thromboxane B2. They are discharged from platelets but their secretion mechanism not understood. It has hypertensive nature and possesses vasoconstriction activity. And they promote platelet accumulation and formation of a clot.

Inflammatory cytokine

Cytokine are small protein released from cells having particular activity on the interaction between cells and it is a communicating module of cells. They self-action or neighboring action molecules and in some cases they function on secluded cells also. These cytokines include tumor necrotic factor, interferon-gamma, interleukin 1 (IL-1), IL-12, IL-8, and granulocyte-macrophage colony-stimulating factor.

Acute-phase protein

Acute-phase proteins (APP) generated as a component of innate immune response with variable serum concentration. These acute-phase proteins classified as negative and positive plasma concentrations.

Positive acute-phase proteins

Positive acute-phase protein is a sign of high inflammatory reaction. They are sub-grouped according to their level of raising concentration in the blood are major, moderate, and minor. Promptness and extent of these proteins contrast with different species.

Negative acute phase proteins

These proteins reduce in concentration by 25% in inflammatory reaction. The two chief negative acute-phase proteins are transferrin and albumin. The concentration of protein decreases slowly. The mechanism of reduction involves a number of responses such as the production of these proteins reduced, inflammatory cytokines induce less production, etc.


Comparison between acute and chronic inflammation:
Acute Chronic
Causative agent Bacterial pathogens, injured tissues Persistent acute inflammation due to non-degradable pathogens, viral infection, persistent foreign bodies, or autoimmune reactions
Major cells involved neutrophils (primarily), basophils (inflammatory response), and eosinophils (response to helminth worms and parasites), mononuclear cells (monocytes, macrophages) Mononuclear cells (monocytes, macrophages, lymphocytes, plasma cells), fibroblasts
Primary mediators Vasoactive amines, eicosanoids IFN-γ and other cytokines, growth factors, reactive oxygen species, hydrolytic enzymes
Onset Immediate Delayed
Duration Few days Up to many months, or years
Outcomes Resolution, abscess formation, chronic inflammation Tissue destruction, fibrosis, necrosis

Acute inflammation Edit

Acute inflammation occurs immediately upon injury, lasting only a few days. [7] Cytokines and chemokines promote the migration of neutrophils and macrophages to the site of inflammation. [7] Pathogens, allergens, toxins, burns, and frostbite are some of the causes of acute inflammation. [7] Toll-like receptors (TLRs) recognize microbial pathogens. [7] Acute inflammation can be a way tissues are protected from injury. [7] Inflammation lasting 2–6 weeks is designated subacute inflammation. [7] [8]

Chronic inflammation Edit

Chronic inflammation is inflammation that lasts for months or years. [8] Macrophages, lymphocytes, and plasma cells predominate in chronic inflammation, in contrast to the neutrophils that predominate in acute inflammation. [8] Diabetes, cardiovascular disease, allergies, and chronic obstructive pulmonary disease (COPD) are examples of diseases mediated by chronic inflammation. [8] Obesity, smoking, stress, and poor diet are some of the factors that promote chronic inflammation. [8] A 2014 study reported that 60% of Americans had at least one chronic inflammatory condition, whereas 42% had more than one. [8]

Cardinal signs Edit

The classic signs and symptoms of acute inflammation:
English Latin
Redness Rubor*
Swelling Tumor*
Heat Calor*
Pain Dolor*
Loss of function Functio laesa**
All the above signs may be observed in specific instances, but no single sign must, as a matter of course, be present. [9]

These are the original, or cardinal signs of inflammation. [9] *

Functio laesa is an antiquated notion, as it is not unique to inflammation and is a characteristic of many disease states. [10] **

Acute inflammation is a short-term process, usually appearing within a few minutes or hours and begins to cease upon the removal of the injurious stimulus. [11] It involves a coordinated and systemic mobilization response locally of various immune, endocrine and neurological mediators of acute inflammation. In a normal healthy response, it becomes activated, clears the pathogen and begins a repair process and then ceases. [12] It is characterized by five cardinal signs: [13]

The traditional names for signs of inflammation come from Latin:

The first four (classical signs) were described by Celsus (ca. 30 BC–38 AD), [15] while loss of function was probably added later by Galen. [16] However, the addition of this fifth sign has also been ascribed to Thomas Sydenham [17] and Virchow. [11] [13]

Redness and heat are due to increased blood flow at body core temperature to the inflamed site swelling is caused by accumulation of fluid pain is due to the release of chemicals such as bradykinin and histamine that stimulate nerve endings. Loss of function has multiple causes. [13]

Acute inflammation of the lung (usually caused in response to pneumonia) does not cause pain unless the inflammation involves the parietal pleura, which does have pain-sensitive nerve endings. [13]

Process of acute inflammation Edit

The process of acute inflammation is initiated by resident immune cells already present in the involved tissue, mainly resident macrophages, dendritic cells, histiocytes, Kupffer cells and mast cells. These cells possess surface receptors known as pattern recognition receptors (PRRs), which recognize (i.e., bind) two subclasses of molecules: pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PAMPs are compounds that are associated with various pathogens, but which are distinguishable from host molecules. DAMPs are compounds that are associated with host-related injury and cell damage.

At the onset of an infection, burn, or other injuries, these cells undergo activation (one of the PRRs recognize a PAMP or DAMP) and release inflammatory mediators responsible for the clinical signs of inflammation. Vasodilation and its resulting increased blood flow causes the redness (rubor) and increased heat (calor). Increased permeability of the blood vessels results in an exudation (leakage) of plasma proteins and fluid into the tissue (edema), which manifests itself as swelling (tumor). Some of the released mediators such as bradykinin increase the sensitivity to pain (hyperalgesia, dolor). The mediator molecules also alter the blood vessels to permit the migration of leukocytes, mainly neutrophils and macrophages, to flow out of the blood vessels (extravasation) and into the tissue. The neutrophils migrate along a chemotactic gradient created by the local cells to reach the site of injury. [11] The loss of function (functio laesa) is probably the result of a neurological reflex in response to pain.

In addition to cell-derived mediators, several acellular biochemical cascade systems consisting of preformed plasma proteins act in parallel to initiate and propagate the inflammatory response. These include the complement system activated by bacteria and the coagulation and fibrinolysis systems activated by necrosis, e.g. a burn or a trauma. [11]

Acute inflammation may be regarded as the first line of defense against injury. Acute inflammatory response requires constant stimulation to be sustained. Inflammatory mediators are short-lived and are quickly degraded in the tissue. Hence, acute inflammation begins to cease once the stimulus has been removed. [11]

Vasodilation and increased permeability Edit

As defined, acute inflammation is an immunovascular response to an inflammatory stimulus. This means acute inflammation can be broadly divided into a vascular phase that occurs first, followed by a cellular phase involving immune cells (more specifically myeloid granulocytes in the acute setting). The vascular component of acute inflammation involves the movement of plasma fluid, containing important proteins such as fibrin and immunoglobulins (antibodies), into inflamed tissue.

Upon contact with PAMPs, tissue macrophages and mastocytes release vasoactive amines such as histamine and serotonin, as well as eicosanoids such as prostaglandin E2 and leukotriene B4 to remodel the local vasculature. Macrophages and endothelial cells release nitric oxide. These mediators vasodilate and permeabilize the blood vessels, which results in the net distribution of blood plasma from the vessel into the tissue space. The increased collection of fluid into the tissue causes it to swell (edema). This exuded tissue fluid contains various antimicrobial mediators from the plasma such as complement, lysozyme, antibodies, which can immediately deal damage to microbes, and opsonise the microbes in preparation for the cellular phase. If the inflammatory stimulus is a lacerating wound, exuded platelets, coagulants, plasmin and kinins can clot the wounded area and provide haemostasis in the first instance. These clotting mediators also provide a structural staging framework at the inflammatory tissue site in the form of a fibrin lattice – as would construction scaffolding at a construction site – for the purpose of aiding phagocytic debridement and wound repair later on. Some of the exuded tissue fluid is also funnelled by lymphatics to the regional lymph nodes, flushing bacteria along to start the recognition and attack phase of the adaptive immune system.

Acute inflammation is characterized by marked vascular changes, including vasodilation, increased permeability and increased blood flow, which are induced by the actions of various inflammatory mediators. Vasodilation occurs first at the arteriole level, progressing to the capillary level, and brings about a net increase in the amount of blood present, causing the redness and heat of inflammation. Increased permeability of the vessels results in the movement of plasma into the tissues, with resultant stasis due to the increase in the concentration of the cells within blood – a condition characterized by enlarged vessels packed with cells. Stasis allows leukocytes to marginate (move) along the endothelium, a process critical to their recruitment into the tissues. Normal flowing blood prevents this, as the shearing force along the periphery of the vessels moves cells in the blood into the middle of the vessel.

Plasma cascade systems Edit

  • The complement system, when activated, creates a cascade of chemical reactions that promotes opsonization, chemotaxis, and agglutination, and produces the MAC.
  • The kinin system generates proteins capable of sustaining vasodilation and other physical inflammatory effects.
  • The coagulation system or clotting cascade, which forms a protective protein mesh over sites of injury.
  • The fibrinolysis system, which acts in opposition to the coagulation system, to counterbalance clotting and generate several other inflammatory mediators.

Plasma-derived mediators Edit

Name Produced by Description
Bradykinin Kinin system A vasoactive protein that is able to induce vasodilation, increase vascular permeability, cause smooth muscle contraction, and induce pain.
C3 Complement system Cleaves to produce C3a and C3b. C3a stimulates histamine release by mast cells, thereby producing vasodilation. C3b is able to bind to bacterial cell walls and act as an opsonin, which marks the invader as a target for phagocytosis.
C5a Complement system Stimulates histamine release by mast cells, thereby producing vasodilation. It is also able to act as a chemoattractant to direct cells via chemotaxis to the site of inflammation.
Factor XII (Hageman Factor) Liver A protein that circulates inactively, until activated by collagen, platelets, or exposed basement membranes via conformational change. When activated, it in turn is able to activate three plasma systems involved in inflammation: the kinin system, fibrinolysis system, and coagulation system.
Membrane attack complex Complement system A complex of the complement proteins C5b, C6, C7, C8, and multiple units of C9. The combination and activation of this range of complement proteins forms the membrane attack complex, which is able to insert into bacterial cell walls and causes cell lysis with ensuing bacterial death.
Plasmin Fibrinolysis system Able to break down fibrin clots, cleave complement protein C3, and activate Factor XII.
Thrombin Coagulation system Cleaves the soluble plasma protein fibrinogen to produce insoluble fibrin, which aggregates to form a blood clot. Thrombin can also bind to cells via the PAR1 receptor to trigger several other inflammatory responses, such as production of chemokines and nitric oxide.

The cellular component involves leukocytes, which normally reside in blood and must move into the inflamed tissue via extravasation to aid in inflammation. Some act as phagocytes, ingesting bacteria, viruses, and cellular debris. Others release enzymatic granules that damage pathogenic invaders. Leukocytes also release inflammatory mediators that develop and maintain the inflammatory response. In general, acute inflammation is mediated by granulocytes, whereas chronic inflammation is mediated by mononuclear cells such as monocytes and lymphocytes.

Leukocyte extravasation Edit

Various leukocytes, particularly neutrophils, are critically involved in the initiation and maintenance of inflammation. These cells must be able to move to the site of injury from their usual location in the blood, therefore mechanisms exist to recruit and direct leukocytes to the appropriate place. The process of leukocyte movement from the blood to the tissues through the blood vessels is known as extravasation and can be broadly divided up into a number of steps:

  1. Leukocyte margination and endothelial adhesion: The white blood cells within the vessels which are generally centrally located move peripherally towards the walls of the vessels. [19] Activated macrophages in the tissue release cytokines such as IL-1 and TNFα, which in turn leads to production of chemokines that bind to proteoglycans forming gradient in the inflamed tissue and along the endothelial wall. Inflammatory cytokines induce the immediate expression of P-selectin on endothelial cell surfaces and P-selectin binds weakly to carbohydrate ligands on the surface of leukocytes and causes them to "roll" along the endothelial surface as bonds are made and broken. Cytokines released from injured cells induce the expression of E-selectin on endothelial cells, which functions similarly to P-selectin. Cytokines also induce the expression of integrin ligands such as ICAM-1 and VCAM-1 on endothelial cells, which mediate the adhesion and further slow leukocytes down. These weakly bound leukocytes are free to detach if not activated by chemokines produced in injured tissue after signal transduction via respective G protein-coupled receptors that activates integrins on the leukocyte surface for firm adhesion. Such activation increases the affinity of bound integrin receptors for ICAM-1 and VCAM-1 on the endothelial cell surface, firmly binding the leukocytes to the endothelium.
  2. Migration across the endothelium, known as transmigration, via the process of diapedesis: Chemokine gradients stimulate the adhered leukocytes to move between adjacent endothelial cells. The endothelial cells retract and the leukocytes pass through the basement membrane into the surrounding tissue using adhesion molecules such as ICAM-1. [19]
  3. Movement of leukocytes within the tissue via chemotaxis: Leukocytes reaching the tissue interstitium bind to extracellular matrix proteins via expressed integrins and CD44 to prevent them from leaving the site. A variety of molecules behave as chemoattractants, for example, C3a or C5, and cause the leukocytes to move along a chemotactic gradient towards the source of inflammation.

Phagocytosis Edit

Extravasated neutrophils in the cellular phase come into contact with microbes at the inflamed tissue. Phagocytes express cell-surface endocytic pattern recognition receptors (PRRs) that have affinity and efficacy against non-specific microbe-associated molecular patterns (PAMPs). Most PAMPs that bind to endocytic PRRs and initiate phagocytosis are cell wall components, including complex carbohydrates such as mannans and β-glucans, lipopolysaccharides (LPS), peptidoglycans, and surface proteins. Endocytic PRRs on phagocytes reflect these molecular patterns, with C-type lectin receptors binding to mannans and β-glucans, and scavenger receptors binding to LPS.

Upon endocytic PRR binding, actin-myosin cytoskeletal rearrangement adjacent to the plasma membrane occurs in a way that endocytoses the plasma membrane containing the PRR-PAMP complex, and the microbe. Phosphatidylinositol and Vps34-Vps15-Beclin1 signalling pathways have been implicated to traffic the endocytosed phagosome to intracellular lysosomes, where fusion of the phagosome and the lysosome produces a phagolysosome. The reactive oxygen species, superoxides and hypochlorite bleach within the phagolysosomes then kill microbes inside the phagocyte.

Phagocytic efficacy can be enhanced by opsonization. Plasma derived complement C3b and antibodies that exude into the inflamed tissue during the vascular phase bind to and coat the microbial antigens. As well as endocytic PRRs, phagocytes also express opsonin receptors Fc receptor and complement receptor 1 (CR1), which bind to antibodies and C3b, respectively. The co-stimulation of endocytic PRR and opsonin receptor increases the efficacy of the phagocytic process, enhancing the lysosomal elimination of the infective agent.

Cell-derived mediators Edit

Name Type Source Description
Lysosome granules Enzymes Granulocytes These cells contain a large variety of enzymes that perform a number of functions. Granules can be classified as either specific or azurophilic depending upon the contents, and are able to break down a number of substances, some of which may be plasma-derived proteins that allow these enzymes to act as inflammatory mediators.
GM-CSF Glycoprotein Macrophages, monocytes, T-cells, B-cells, and tissue-resident cells Elevated GM-CSF has been shown to contribute to inflammation in inflammatory arthritis, osteoarthritis, colitis asthma, obesity, and COVID-19.
Histamine Monoamine Mast cells and basophils Stored in preformed granules, histamine is released in response to a number of stimuli. It causes arteriole dilation, increased venous permeability, and a wide variety of organ-specific effects.
IFN-γ Cytokine T-cells, NK cells Antiviral, immunoregulatory, and anti-tumour properties. This interferon was originally called macrophage-activating factor, and is especially important in the maintenance of chronic inflammation.
IL-6 Cytokine and Myokine Macrophages, osteoblasts, adipocytes, and smooth muscle cells (cytokine) Skeletal muscle cells (myokine) Pro-inflammatory cytokine secreted by macrophages in response to pathogen-associated molecular patterns (PAMPs) pro-inflammatory cytokine secreted by adipocytes, especially in obesity anti-inflammatory myokine secreted by skeletal muscle cells in response to exercise.
IL-8 Chemokine Primarily macrophages Activation and chemoattraction of neutrophils, with a weak effect on monocytes and eosinophils.
Leukotriene B4 Eicosanoid Leukocytes, cancer cells Able to mediate leukocyte adhesion and activation, allowing them to bind to the endothelium and migrate across it. In neutrophils, it is also a potent chemoattractant, and is able to induce the formation of reactive oxygen species and the release of lysosomal enzymes by these cells.
LTC4, LTD4 Eicosanoid eosinophils, mast cells, macrophages These three Cysteine-containing leukotrienes contract lung airways, increase micro-vascular permeability, stimulate mucus secretion, and promote eosinophil-based inflammation in the lung, skin, nose, eye, and other tissues.
5-oxo-eicosatetraenoic acid Eicosanoid leukocytes, cancer cells Potent stimulator of neutrophil chemotaxis, lysosome enzyme release, and reactive oxygen species formation monocyte chemotaxis and with even greater potency eosinophil chemotaxis, lysosome enzyme release, and reactive oxygen species formation.
5-HETE Eicosanoid Leukocytes Metabolic precursor to 5-Oxo-eicosatetraenoic acid, it is a less potent stimulator of neutrophil chemotaxis, lysosome enzyme release, and reactive oxygen species formation monocyte chemotaxis and eosinophil chemotaxis, lysosome enzyme release, and reactive oxygen species formation.
Prostaglandins Eicosanoid Mast cells A group of lipids that can cause vasodilation, fever, and pain.
Nitric oxide Soluble gas Macrophages, endothelial cells, some neurons Potent vasodilator, relaxes smooth muscle, reduces platelet aggregation, aids in leukocyte recruitment, direct antimicrobial activity in high concentrations.
TNF-α and IL-1 Cytokines Primarily macrophages Both affect a wide variety of cells to induce many similar inflammatory reactions: fever, production of cytokines, endothelial gene regulation, chemotaxis, leukocyte adherence, activation of fibroblasts. Responsible for the systemic effects of inflammation, such as loss of appetite and increased heart rate. TNF-α inhibits osteoblast differentiation.
Tryptase Enzymes Mast Cells This serine protease is believed to be exclusively stored in mast cells and secreted, along with histamine, during mast cell activation. [20] [21] [22]

Specific patterns of acute and chronic inflammation are seen during particular situations that arise in the body, such as when inflammation occurs on an epithelial surface, or pyogenic bacteria are involved.

  • Granulomatous inflammation: Characterised by the formation of granulomas, they are the result of a limited but diverse number of diseases, which include among others tuberculosis, leprosy, sarcoidosis, and syphilis.
  • Fibrinous inflammation: Inflammation resulting in a large increase in vascular permeability allows fibrin to pass through the blood vessels. If an appropriate procoagulative stimulus is present, such as cancer cells, [11] a fibrinous exudate is deposited. This is commonly seen in serous cavities, where the conversion of fibrinous exudate into a scar can occur between serous membranes, limiting their function. The deposit sometimes forms a pseudomembrane sheet. During inflammation of the intestine (Pseudomembranous colitis), pseudomembranous tubes can be formed.
  • Purulent inflammation: Inflammation resulting in large amount of pus, which consists of neutrophils, dead cells, and fluid. Infection by pyogenic bacteria such as staphylococci is characteristic of this kind of inflammation. Large, localised collections of pus enclosed by surrounding tissues are called abscesses.
  • Serous inflammation: Characterised by the copious effusion of non-viscous serous fluid, commonly produced by mesothelial cells of serous membranes, but may be derived from blood plasma. Skin blisters exemplify this pattern of inflammation.
  • Ulcerative inflammation: Inflammation occurring near an epithelium can result in the necrotic loss of tissue from the surface, exposing lower layers. The subsequent excavation in the epithelium is known as an ulcer.

Inflammatory abnormalities are a large group of disorders that underlie a vast variety of human diseases. The immune system is often involved with inflammatory disorders, demonstrated in both allergic reactions and some myopathies, with many immune system disorders resulting in abnormal inflammation. Non-immune diseases with causal origins in inflammatory processes include cancer, atherosclerosis, and ischemic heart disease. [11]

Examples of disorders associated with inflammation include:

Atherosclerosis Edit

Atherosclerosis, formerly considered a bland lipid storage disease, actually involves an ongoing inflammatory response. Recent advances in basic science have established a fundamental role for inflammation in mediating all stages of this disease from initiation through progression and, ultimately, the thrombotic complications of atherosclerosis. These new findings provide important links between risk factors and the mechanisms of atherogenesis. Clinical studies have shown that this emerging biology of inflammation in atherosclerosis applies directly to human patients. Elevation in markers of inflammation predicts outcomes of patients with acute coronary syndromes, independently of myocardial damage. In addition, low-grade chronic inflammation, as indicated by levels of the inflammatory marker C-reactive protein, prospectively defines risk of atherosclerotic complications, thus adding to prognostic information provided by traditional risk factors. Moreover, certain treatments that reduce coronary risk also limit inflammation. In the case of lipid lowering with statins, this anti-inflammatory effect does not appear to correlate with reduction in low-density lipoprotein levels. These new insights into inflammation in atherosclerosis not only increase our understanding of this disease but also have practical clinical applications in risk stratification and targeting of therapy for this scourge of growing worldwide importance. [23]

Allergy Edit

An allergic reaction, formally known as type 1 hypersensitivity, is the result of an inappropriate immune response triggering inflammation, vasodilation, and nerve irritation. A common example is hay fever, which is caused by a hypersensitive response by mast cells to allergens. Pre-sensitised mast cells respond by degranulating, releasing vasoactive chemicals such as histamine. These chemicals propagate an excessive inflammatory response characterised by blood vessel dilation, production of pro-inflammatory molecules, cytokine release, and recruitment of leukocytes. [11] Severe inflammatory response may mature into a systemic response known as anaphylaxis.

Myopathies Edit

Inflammatory myopathies are caused by the immune system inappropriately attacking components of muscle, leading to signs of muscle inflammation. They may occur in conjunction with other immune disorders, such as systemic sclerosis, and include dermatomyositis, polymyositis, and inclusion body myositis. [11]

Leukocyte defects Edit

Due to the central role of leukocytes in the development and propagation of inflammation, defects in leukocyte functionality often result in a decreased capacity for inflammatory defense with subsequent vulnerability to infection. [11] Dysfunctional leukocytes may be unable to correctly bind to blood vessels due to surface receptor mutations, digest bacteria (Chédiak–Higashi syndrome), or produce microbicides (chronic granulomatous disease). In addition, diseases affecting the bone marrow may result in abnormal or few leukocytes.

Pharmacological Edit

Certain drugs or exogenous chemical compounds are known to affect inflammation. Vitamin A deficiency causes an increase in inflammatory responses, [24] and anti-inflammatory drugs work specifically by inhibiting the enzymes that produce inflammatory eicosanoids. Certain illicit drugs such as cocaine and ecstasy may exert some of their detrimental effects by activating transcription factors intimately involved with inflammation (e.g. NF-κB). [25] [26]

Cancer Edit

Inflammation orchestrates the microenvironment around tumours, contributing to proliferation, survival and migration. [27] Cancer cells use selectins, chemokines and their receptors for invasion, migration and metastasis. [28] On the other hand, many cells of the immune system contribute to cancer immunology, suppressing cancer. [29] Molecular intersection between receptors of steroid hormones, which have important effects on cellular development, and transcription factors that play key roles in inflammation, such as NF-κB, may mediate some of the most critical effects of inflammatory stimuli on cancer cells. [30] This capacity of a mediator of inflammation to influence the effects of steroid hormones in cells, is very likely to affect carcinogenesis on the one hand on the other hand, due to the modular nature of many steroid hormone receptors, this interaction may offer ways to interfere with cancer progression, through targeting of a specific protein domain in a specific cell type. Such an approach may limit side effects that are unrelated to the tumor of interest and may help preserve vital homeostatic functions and developmental processes in the organism.

According to a review of 2009, recent data suggests that cancer-related inflammation (CRI) may lead to accumulation of random genetic alterations in cancer cells. [31]

Role in cancer Edit

In 1863, Rudolf Virchow hypothesized that the origin of cancer was at sites of chronic inflammation. [32] [33] At present, chronic inflammation is estimated to contribute to approximately 15% to 25% of human cancers. [33] [34]

Mediators and DNA damage in cancer Edit

An inflammatory mediator is a messenger that acts on blood vessels and/or cells to promote an inflammatory response. [35] Inflammatory mediators that contribute to neoplasia include prostaglandins, inflammatory cytokines such as IL-1β, TNF-α, IL-6 and IL-15 and chemokines such as IL-8 and GRO-alpha. [36] [33] These inflammatory mediators, and others, orchestrate an environment that fosters proliferation and survival. [32] [36]

Inflammation also causes DNA damages due to the induction of reactive oxygen species (ROS) by various intracellular inflammatory mediators. [32] [36] [33] In addition, leukocytes and other phagocytic cells attracted to the site of inflammation induce DNA damages in proliferating cells through their generation of ROS and reactive nitrogen species (RNS). ROS and RNS are normally produced by these cells to fight infection. [32] ROS, alone, cause more than 20 types of DNA damage. [37] Oxidative DNA damages cause both mutations [38] and epigenetic alterations. [39] [33] [40] RNS also cause mutagenic DNA damages. [41]

A normal cell may undergo carcinogenesis to become a cancer cell if it is frequently subjected to DNA damage during long periods of chronic inflammation. DNA damages may cause genetic mutations due to inaccurate repair. In addition, mistakes in the DNA repair process may cause epigenetic alterations. [33] [36] [40] Mutations and epigenetic alterations that are replicated and provide a selective advantage during somatic cell proliferation may be carcinogenic.

Genome-wide analyses of human cancer tissues reveal that a single typical cancer cell may possess roughly 100 mutations in coding regions, 10-20 of which are “driver mutations” that contribute to cancer development. [33] However, chronic inflammation also causes epigenetic changes such as DNA methylations, that are often more common than mutations. Typically, several hundreds to thousands of genes are methylated in a cancer cell (see DNA methylation in cancer). Sites of oxidative damage in chromatin can recruit complexes that contain DNA methyltransferases (DNMTs), a histone deacetylase (SIRT1), and a histone methyltransferase (EZH2), and thus induce DNA methylation. [33] [42] [43] DNA methylation of a CpG island in a promoter region may cause silencing of its downstream gene (see CpG site and regulation of transcription in cancer). DNA repair genes, in particular, are frequently inactivated by methylation in various cancers (see hypermethylation of DNA repair genes in cancer). A 2018 report [44] evaluated the relative importance of mutations and epigenetic alterations in progression to two different types of cancer. This report showed that epigenetic alterations were much more important than mutations in generating gastric cancers (associated with inflammation). [45] However, mutations and epigenetic alterations were of roughly equal importance in generating esophageal squamous cell cancers (associated with tobacco chemicals and acetaldehyde, a product of alcohol metabolism).

HIV and AIDS Edit

It has long been recognized that infection with HIV is characterized not only by development of profound immunodeficiency but also by sustained inflammation and immune activation. [46] [47] [48] A substantial body of evidence implicates chronic inflammation as a critical driver of immune dysfunction, premature appearance of aging-related diseases, and immune deficiency. [46] [49] Many now regard HIV infection not only as an evolving virus-induced immunodeficiency but also as chronic inflammatory disease. [50] Even after the introduction of effective antiretroviral therapy (ART) and effective suppression of viremia in HIV-infected individuals, chronic inflammation persists. Animal studies also support the relationship between immune activation and progressive cellular immune deficiency: SIVsm infection of its natural nonhuman primate hosts, the sooty mangabey, causes high-level viral replication but limited evidence of disease. [51] [52] This lack of pathogenicity is accompanied by a lack of inflammation, immune activation and cellular proliferation. In sharp contrast, experimental SIVsm infection of rhesus macaque produces immune activation and AIDS-like disease with many parallels to human HIV infection. [53]

Delineating how CD4 T cells are depleted and how chronic inflammation and immune activation are induced lies at the heart of understanding HIV pathogenesis––one of the top priorities for HIV research by the Office of AIDS Research, National Institutes of Health. Recent studies demonstrated that caspase-1-mediated pyroptosis, a highly inflammatory form of programmed cell death, drives CD4 T-cell depletion and inflammation by HIV. [54] [55] [56] These are the two signature events that propel HIV disease progression to AIDS. Pyroptosis appears to create a pathogenic vicious cycle in which dying CD4 T cells and other immune cells (including macrophages and neutrophils) release inflammatory signals that recruit more cells into the infected lymphoid tissues to die. The feed-forward nature of this inflammatory response produces chronic inflammation and tissue injury. [57] Identifying pyroptosis as the predominant mechanism that causes CD4 T-cell depletion and chronic inflammation, provides novel therapeutic opportunities, namely caspase-1 which controls the pyroptotic pathway. In this regard, pyroptosis of CD4 T cells and secretion of pro-inflammatory cytokines such as IL-1β and IL-18 can be blocked in HIV-infected human lymphoid tissues by addition of the caspase-1 inhibitor VX-765, [54] which has already proven to be safe and well tolerated in phase II human clinical trials. [58] These findings could propel development of an entirely new class of “anti-AIDS” therapies that act by targeting the host rather than the virus. Such agents would almost certainly be used in combination with ART. By promoting “tolerance” of the virus instead of suppressing its replication, VX-765 or related drugs may mimic the evolutionary solutions occurring in multiple monkey hosts (e.g. the sooty mangabey) infected with species-specific lentiviruses that have led to a lack of disease, no decline in CD4 T-cell counts, and no chronic inflammation.

Resolution of inflammation Edit

The inflammatory response must be actively terminated when no longer needed to prevent unnecessary "bystander" damage to tissues. [11] Failure to do so results in chronic inflammation, and cellular destruction. Resolution of inflammation occurs by different mechanisms in different tissues. Mechanisms that serve to terminate inflammation include: [11] [59]

  • Short half-life of inflammatory mediatorsin vivo.
  • Production and release of transforming growth factor (TGF) beta from macrophages[60][61][62]
  • Production and release of interleukin 10 (IL-10) [63]
  • Production of anti-inflammatory specialized proresolving mediators, i.e. lipoxins, resolvins, maresins, and neuroprotectins[64][65]
  • Downregulation of pro-inflammatory molecules, such as leukotrienes.
  • Upregulation of anti-inflammatory molecules such as the interleukin 1 receptor antagonist or the soluble tumor necrosis factor receptor (TNFR) of pro-inflammatory cells [66]
  • Desensitization of receptors.
  • Increased survival of cells in regions of inflammation due to their interaction with the extracellular matrix (ECM) [67][68]
  • Downregulation of receptor activity by high concentrations of ligands
  • Cleavage of chemokines by matrix metalloproteinases (MMPs) might lead to production of anti-inflammatory factors. [69]

Acute inflammation normally resolves by mechanisms that have remained somewhat elusive. Emerging evidence now suggests that an active, coordinated program of resolution initiates in the first few hours after an inflammatory response begins. After entering tissues, granulocytes promote the switch of arachidonic acid–derived prostaglandins and leukotrienes to lipoxins, which initiate the termination sequence. Neutrophil recruitment thus ceases and programmed death by apoptosis is engaged. These events coincide with the biosynthesis, from omega-3 polyunsaturated fatty acids, of resolvins and protectins, which critically shorten the period of neutrophil infiltration by initiating apoptosis. As a consequence, apoptotic neutrophils undergo phagocytosis by macrophages, leading to neutrophil clearance and release of anti-inflammatory and reparative cytokines such as transforming growth factor-β1. The anti-inflammatory program ends with the departure of macrophages through the lymphatics. [70]

Connection to depression Edit

There is evidence for a link between inflammation and depression. [71] Inflammatory processes can be triggered by negative cognitions or their consequences, such as stress, violence, or deprivation. Thus, negative cognitions can cause inflammation that can, in turn, lead to depression. [72] [73] [ dubious – discuss ] In addition there is increasing evidence that inflammation can cause depression because of the increase of cytokines, setting the brain into a "sickness mode". [74] Classical symptoms of being physically sick like lethargy show a large overlap in behaviors that characterize depression. Levels of cytokines tend to increase sharply during the depressive episodes of people with bipolar disorder and drop off during remission. [75] Furthermore, it has been shown in clinical trials that anti-inflammatory medicines taken in addition to antidepressants not only significantly improves symptoms but also increases the proportion of subjects positively responding to treatment. [76] Inflammations that lead to serious depression could be caused by common infections such as those caused by a virus, bacteria or even parasites. [77]

An infectious organism can escape the confines of the immediate tissue via the circulatory system or lymphatic system, where it may spread to other parts of the body. If an organism is not contained by the actions of acute inflammation it may gain access to the lymphatic system via nearby lymph vessels. An infection of the lymph vessels is known as lymphangitis, and infection of a lymph node is known as lymphadenitis. When lymph nodes cannot destroy all pathogens, the infection spreads further. A pathogen can gain access to the bloodstream through lymphatic drainage into the circulatory system.

When inflammation overwhelms the host, systemic inflammatory response syndrome is diagnosed. When it is due to infection, the term sepsis is applied, with the terms bacteremia being applied specifically for bacterial sepsis and viremia specifically to viral sepsis. Vasodilation and organ dysfunction are serious problems associated with widespread infection that may lead to septic shock and death.

Acute-phase proteins Edit

Inflammation also induces high systemic levels of acute-phase proteins. In acute inflammation, these proteins prove beneficial however, in chronic inflammation they can contribute to amyloidosis. [11] These proteins include C-reactive protein, serum amyloid A, and serum amyloid P, which cause a range of systemic effects including: [11]

Leukocyte numbers Edit

Inflammation often affects the numbers of leukocytes present in the body:

    is often seen during inflammation induced by infection, where it results in a large increase in the amount of leukocytes in the blood, especially immature cells. Leukocyte numbers usually increase to between 15 000 and 20 000 cells per microliter, but extreme cases can see it approach 100 000 cells per microliter. [11] Bacterial infection usually results in an increase of neutrophils, creating neutrophilia, whereas diseases such as asthma, hay fever, and parasite infestation result in an increase in eosinophils, creating eosinophilia. [11] can be induced by certain infections and diseases, including viral infection, Rickettsia infection, some protozoa, tuberculosis, and some cancers. [11]

Systemic inflammation and obesity Edit

With the discovery of interleukins (IL), the concept of systemic inflammation developed. Although the processes involved are identical to tissue inflammation, systemic inflammation is not confined to a particular tissue but involves the endothelium and other organ systems.

Chronic inflammation is widely observed in obesity. [78] [79] Obese people commonly have many elevated markers of inflammation, including: [80] [81]

Low-grade chronic inflammation is characterized by a two- to threefold increase in the systemic concentrations of cytokines such as TNF-α, IL-6, and CRP. [84] Waist circumference correlates significantly with systemic inflammatory response. [85]

Loss of white adipose tissue reduces levels of inflammation markers. [78] The association of systemic inflammation with insulin resistance and type 2 diabetes, and with atherosclerosis is under preliminary research, although rigorous clinical trials have not been conducted to confirm such relationships. [86]

C-reactive protein (CRP) is generated at a higher level in obese people, and may increase the risk for cardiovascular diseases. [87]

The outcome in a particular circumstance will be determined by the tissue in which the injury has occurred and the injurious agent that is causing it. Here are the possible outcomes to inflammation: [11]

  1. Resolution
    The complete restoration of the inflamed tissue back to a normal status. Inflammatory measures such as vasodilation, chemical production, and leukocyte infiltration cease, and damaged parenchymal cells regenerate. In situations where limited or short-lived inflammation has occurred this is usually the outcome.
  2. Fibrosis
    Large amounts of tissue destruction, or damage in tissues unable to regenerate, cannot be regenerated completely by the body. Fibrous scarring occurs in these areas of damage, forming a scar composed primarily of collagen. The scar will not contain any specialized structures, such as parenchymal cells, hence functional impairment may occur.
  3. Abscess formation
    A cavity is formed containing pus, an opaque liquid containing dead white blood cells and bacteria with general debris from destroyed cells.
  4. Chronic inflammation
    In acute inflammation, if the injurious agent persists then chronic inflammation will ensue. This process, marked by inflammation lasting many days, months or even years, may lead to the formation of a chronic wound. Chronic inflammation is characterised by the dominating presence of macrophages in the injured tissue. These cells are powerful defensive agents of the body, but the toxins they release (including reactive oxygen species) are injurious to the organism's own tissues as well as invading agents. As a consequence, chronic inflammation is almost always accompanied by tissue destruction.

Inflammation is usually indicated by adding the suffix "itis", as shown below. However, some conditions such as asthma and pneumonia do not follow this convention. More examples are available at list of types of inflammation.


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