Publications by authors named "Israel Charo"

80 Publications

CCR2 inhibition reduces tumor myeloid cells and unmasks a checkpoint inhibitor effect to slow progression of resistant murine gliomas.

Proc Natl Acad Sci U S A 2020 01 26;117(2):1129-1138. Epub 2019 Dec 26.

Department of Pharmacology & Therapeutics, College of Medicine, University of Florida, Gainesville, FL 32610;

Immunotherapy directed at the PD-L1/PD-1 axis has produced treatment advances in various human cancers. Unfortunately, progress has not extended to glioblastoma (GBM), with phase III clinical trials assessing anti-PD-1 monotherapy failing to show efficacy in newly diagnosed and recurrent tumors. Myeloid-derived suppressor cells (MDSCs), a subset of immunosuppressive myeloid derived cells, are known to infiltrate the tumor microenvironment of GBM. Growing evidence suggests the CCL2-CCR2 axis is important for this process. This study evaluated the combination of PD-1 blockade and CCR2 inhibition in anti-PD-1-resistant gliomas. CCR2 deficiency unmasked an anti-PD-1 survival benefit in KR158 glioma-bearing mice. CD11b/Ly6C/PD-L1 MDSCs within established gliomas decreased with a concomitant increase in overall CCR2 cells and MDSCs within bone marrow of CCR2-deficient mice. The CCR2 antagonist CCX872 increased median survival as a monotherapy in KR158 glioma-bearing animals and further increased median and overall survival when combined with anti-PD-1. Additionally, combination of CCX872 and anti-PD-1 prolonged median survival time in 005 GSC GBM-bearing mice. In both models, CCX872 decreased tumor associated MDSCs and increased these cells within the bone marrow. Examination of tumor-infiltrating lymphocytes revealed an elevated population, increased IFNγ expression, indicating enhanced cytolytic activity, as well as decreased expression of exhaustion markers in CD4 and CD8 T cells following combination treatment. These data establish that combining CCR2 and PD-1 blockade extends survival in clinically relevant murine glioma models and provides the basis on which to advance this combinatorial treatment toward early-phase human trials.
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http://dx.doi.org/10.1073/pnas.1910856117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969504PMC
January 2020

CCR2-Mediated Uptake of Constitutively Produced CCL2: A Mechanism for Regulating Chemokine Levels in the Blood.

J Immunol 2019 12 1;203(12):3157-3165. Epub 2019 Nov 1.

ChemoCentryx, Inc., Mountain View, CA 94043; and.

C-C chemokine receptor 2 (CCR2) is a key driver of monocyte/macrophage trafficking to sites of inflammation and has long been considered a target for intervention in autoimmune disease. However, systemic administration of CCR2 antagonists is associated with marked increases in CCL2, a CCR2 ligand, in the blood. This heretofore unexplained phenomenon complicates interpretation of in vivo responses to CCR2 antagonism. We report that CCL2 elevation after pharmacological CCR2 blockade is due to interruption in a balance between CCL2 secretion by a variety of cells and its uptake by constitutive internalization and recycling of CCR2. We observed this phenomenon in response to structurally diverse CCR2 antagonists in wild-type mice, and also found substantially higher CCL2 plasma levels in mice lacking the CCR2 gene. Our findings suggest that CCL2 is cleared from blood in a CCR2-dependent but G protein (Gα, Gα or Gα)-independent manner. This constitutive internalization is rapid: on a given monocyte, the entire cell surface CCR2 population is turned over in <30 minutes. We also found that constitutive receptor internalization/recycling and ligand uptake are not universal across monocyte-expressed chemokine receptors. For example, CXCR4 does not internalize constitutively. In summary, we describe a mechanism that explains the numerous preclinical and clinical reports of increased CCL2 plasma levels following in vivo administration of CCR2 antagonists. These findings suggest that constitutive CCL2 secretion by monocytes and other cell types is counteracted by constant uptake and internalization by CCR2-expressing cells. The effectiveness of CCR2 antagonists in disease settings may be dependent upon this critical equilibrium.
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http://dx.doi.org/10.4049/jimmunol.1900961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028331PMC
December 2019

Efficacy of Chemokine Receptor Inhibition in Treating IL-36α-Induced Psoriasiform Inflammation.

J Immunol 2019 03 4;202(6):1687-1692. Epub 2019 Feb 4.

ChemoCentryx, Inc., Mountain View, CA 94043.

Several types of psoriasiform dermatitis are associated with increased IL-36 cytokine activity in the skin. A rare, but severe, psoriasis-like disorder, generalized pustular psoriasis (GPP), is linked to loss-of-function mutations in the gene encoding IL-36RA, an important negative regulator of IL-36 signaling. To understand the effects of IL-36 dysregulation in a mouse model, we studied skin inflammation induced by intradermal injections of preactivated IL-36α. We found the immune cells infiltrating IL-36α-injected mouse skin to be of dramatically different composition than those infiltrating imiquimod-treated skin. The IL-36α-induced leukocyte population comprised nearly equal numbers of CD4 αβ T cells, neutrophils, and inflammatory dendritic cells, whereas the imiquimod-induced population comprised γδ T cells and neutrophils. Ligands for chemokine receptors CCR6 and CXCR2 are increased in both GPP and IL-36α-treated skin, which led us to test an optimized small-molecule antagonist (CCX624) targeting CCR6 and CXCR2 in the IL-36α model. CCX624 significantly reduced the T cell, neutrophil, and inflammatory dendritic cell infiltrates and was more effective than saturating levels of an anti-IL-17RA mAb at reducing inflammatory symptoms. These findings put CCR6 and CXCR2 forward as novel targets for a mechanistically distinct therapeutic approach for inflammatory skin diseases involving dysregulated IL-36 signaling, such as GPP.
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http://dx.doi.org/10.4049/jimmunol.1801519DOI Listing
March 2019

CCL7 Is a Negative Regulator of Cutaneous Inflammation Following Infection.

Front Immunol 2018 8;9:3063. Epub 2019 Jan 8.

Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, University of Rochester, Rochester, NY, United States.

The chemokine CCL7 (MCP3) is known to promote the recruitment of many innate immune cell types including monocytes and neutrophils to sites of bacterial and viral infection and eosinophils and basophils to sites of allergic inflammation. CCL7 upregulation has been associated with many inflammatory settings including infection, cardiovascular disease, and the tumor microenvironment. CCL7's pleotropic effects are due in part to its ability to bind numerous chemokine receptors, namely CCR1, CCR2, CCR3, CCR5, and CCR10. CCL7-blockade or CCL7-deficiency is often marked by decreased inflammation and poor pathogen control. In the context of infection, CCL7 is specifically upregulated in the skin one-2 weeks after infection but its role in control is unclear. To determine CCL7's impact on the response to we infected WT and CCL7 C57BL/6 mice. infection of CCL7-deficient mice led to an unexpected in inflammation in the infected skin 2 weeks post-infection. A broad increase in immune cell subsets was observed but was dominated by enhanced neutrophilic infiltration. Increased neutrophil recruitment was associated with an enhanced IL-17 gene profile in the infected skin. CCL7 was shown to directly antagonize neutrophil migration and CCL7 add-back specifically reduced neutrophil influx into the infected skin revealing an unexpected role for CCL7 in limiting neutrophil recruitment during infection. Enhanced neutrophilic infiltration in CCL7-deficient mice changed the balance of infected host cells with an increase in the ratio of infected neutrophils over monocytes/macrophages. To determine the consequence of CCL7 deficiency on control we analyzed parasite load cutaneously at the site of infection and viscerally in the draining LN and spleen. The CCL7 mice supported robust cutaneous parasite control similar to their WT C57BL/6 counterparts. In contrast, CCL7-deficiency led to greater parasite dissemination and poor parasite control in the spleen. Our studies reveal a novel role for CCL7 in negatively regulating cutaneous inflammation, specifically neutrophils, early during infection. We propose that CCL7-mediated dampening of the early immune response in the skin may limit the ability of the parasite to disseminate without compromising cutaneous control.
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http://dx.doi.org/10.3389/fimmu.2018.03063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331479PMC
October 2019

CCR2 antagonism leads to marked reduction in proteinuria and glomerular injury in murine models of focal segmental glomerulosclerosis (FSGS).

PLoS One 2018 21;13(3):e0192405. Epub 2018 Mar 21.

ChemoCentryx, Inc., Mountain View, CA, United States of America.

Focal segmental glomerulosclerosis (FSGS) comprises a group of uncommon disorders that present with marked proteinuria, nephrotic syndrome, progressive renal failure and characteristic glomerular lesions on histopathology. The current standard of care for patients with FSGS include immunosuppressive drugs such as glucocorticoids followed by calcineurin inhibitors, if needed for intolerance or inadequate response to glucocorticoids. Renin-angiotensin-aldosterone (RAAS) blockers are also used to control proteinuria, an important signature of FSGS. Existing treatments, however, achieved only limited success. Despite best care, treatment failure is common and FSGS is causal in a significant proportion of end stage renal disease. Thus, an unmet need exists for novel disease modifying treatments for FSGS. We employed two widely-used murine models of FSGS to test the hypothesis that systemic inhibition of chemokine receptor CCR2 would have therapeutic benefit. Here we report that administration CCX872, a potent and selective small molecule antagonist of CCR2, achieved rapid and sustained attenuation of renal damage as determined by urine albumin excretion and improved histopathological outcome. Therapeutic benefit was present when CCX872 was used as a single therapy, and moreover, the combination of CCX872 and RAAS blockade was statistically more effective than RAAS blockade alone. In addition, the combination of CCR2 and RAAS blockade was equally as effective as endothelin receptor inhibition. We conclude that specific inhibition of CCR2 is effective in the Adriamycin-induced and 5/6 nephrectomy murine models of FSGS, and thus holds promise as a mechanistically distinct therapeutic addition to the treatment of human FSGS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192405PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862408PMC
June 2018

CC chemokine receptor 2 promotes recruitment of myeloid cells associated with insulin resistance in nonalcoholic fatty liver disease.

Am J Physiol Gastrointest Liver Physiol 2018 04 8;314(4):G483-G493. Epub 2018 Feb 8.

National Institute for Health Research Biomedical Research Unit and Centre for Liver Research, University of Birmingham , Birmingham , United Kingdom.

Nonalcoholic fatty liver disease (NAFLD) is a common disease, closely associated with obesity and insulin resistance. We investigated the presence of a subset of myeloid cells associated with metabolic disturbance in the liver of patients with NAFLD and a murine model of obesity-induced liver disease. Gene and protein expression in liver and serum was investigated with RT-PCR or ELISA and correlated to clinical disease. Liver-infiltrating immune cells were isolated from normal or diseased human liver for flow cytometric analysis. In animal experiments, mice were fed a high-fat diet (60% of calories from fat) for 16 wk, or high-fat diet with 30% fructose for 32 wk to induce steatohepatitis and fibrosis. A small molecule inhibitor of CC chemokine receptor 2 (CCR2), CCX872, was administered to some mice. A subset of CD11cCD206 immune cells was enriched in human liver tissue, and greater infiltration was observed in NAFLD. The presence of CD11cCD206 myeloid cells correlated with systemic insulin resistance. CD11cCD206 cells expressed high levels of CCR2, and liver CC chemokine ligand 2 (CCL2) expression was increased in nonalcoholic steatohepatitis and correlated with disease activity. In mice, CCR2 inhibition reduced infiltration of liver CD11bCD11cF4/80 monocytes, which are functional homologs of human CD11cCD206 cells, and improved liver injury and glycemic control. A role for CCR2/CCL2 in human NAFLD has long been postulated. These data confirm a role for this chemokine/receptor axis, through mediating adipose and hepatic infiltration of myeloid cells. Inhibition of CCR2 improved hepatic inflammation and fibrosis in murine models of NAFLD. These data confirm the rationale for targeting CCR2 to treat NAFLD. NEW & NOTEWORTHY These data show for the first time that CD11cCD206 myeloid cells, previously associated with human adipose tissue inflammation, infiltrate into liver tissue in nonalcoholic fatty liver disease. These cells express CCR2. Inhibition of CCR2 in mice inhibits hepatic inflammation caused by a murine homolog of these myeloid cells and improves experimental liver disease.
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http://dx.doi.org/10.1152/ajpgi.00213.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966749PMC
April 2018

IL-17-Secreting γδ T Cells Are Completely Dependent upon CCR6 for Homing to Inflamed Skin.

J Immunol 2017 11 29;199(9):3129-3136. Epub 2017 Sep 29.

ChemoCentryx Inc., Mountain View, CA 94043.

mAbs that neutralize IL-17 or its receptor have proven efficacious in treating moderate-to-severe psoriasis, confirming IL-17 as an important driver of this disease. In mice, a rare population of T cells, γδT17 cells, appears to be a dominant source of IL-17 in experimental psoriasis. These cells traffic between lymph nodes and the skin, and are identified by their coexpression of the TCR variable regions γ4 and δ4. These cells are homologous to the Vγ9Vδ2 T cell population identified in human psoriatic plaques. In this study we report that a potent and specific small molecule antagonist of the CCR6 chemokine receptor, CCX2553, was efficacious in reducing multiple aspects of psoriasis in two different murine models of the disease. Administration of CCX2553 ameliorated skin inflammation in both the IL-23-induced ear swelling model and the topical imiquimod model, and significantly reduced the number of γδT17 cells in inflamed skin. γδT17 cells were greatly reduced in imiquimod-treated skin of CCR6 mice, but adoptively transferred wild-type (CCR6) γδT17 cells homed normally to the skin of imiquimod-treated CCR6 mice. Our data suggest that γδT17 cells are completely dependent on CCR6 for homing to psoriasiform skin. Thus, CCR6 may constitute a novel target for a mechanistically distinct therapeutic approach to treating psoriasis.
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http://dx.doi.org/10.4049/jimmunol.1700826DOI Listing
November 2017

Characterization of Pharmacologic and Pharmacokinetic Properties of CCX168, a Potent and Selective Orally Administered Complement 5a Receptor Inhibitor, Based on Preclinical Evaluation and Randomized Phase 1 Clinical Study.

PLoS One 2016 21;11(10):e0164646. Epub 2016 Oct 21.

Department of Discovery and Research, ChemoCentryx, Inc., 850 Maude Avenue, Mountain View, California, United States of America.

The complement 5a receptor has been an attractive therapeutic target for many autoimmune and inflammatory disorders. However, development of a selective and potent C5aR antagonist has been challenging. Here we describe the characterization of CCX168 (avacopan), an orally administered selective and potent C5aR inhibitor. CCX168 blocked the C5a binding, C5a-mediated migration, calcium mobilization, and CD11b upregulation in U937 cells as well as in freshly isolated human neutrophils. CCX168 retains high potency when present in human blood. A transgenic human C5aR knock-in mouse model allowed comparison of the in vitro and in vivo efficacy of the molecule. CCX168 effectively blocked migration in in vitro and ex vivo chemotaxis assays, and it blocked the C5a-mediated neutrophil vascular endothelial margination. CCX168 was effective in migration and neutrophil margination assays in cynomolgus monkeys. This thorough in vitro and preclinical characterization enabled progression of CCX168 into the clinic and testing of its safety, tolerability, pharmacokinetic, and pharmacodynamic profiles in a Phase 1 clinical trial in 48 healthy volunteers. CCX168 was shown to be well tolerated across a broad dose range (1 to 100 mg) and it showed dose-dependent pharmacokinetics. An oral dose of 30 mg CCX168 given twice daily blocked the C5a-induced upregulation of CD11b in circulating neutrophils by 94% or greater throughout the entire day, demonstrating essentially complete target coverage. This dose regimen is being tested in clinical trials in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Trial Registration ISRCTN registry with trial ID ISRCTN13564773.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164646PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5074546PMC
June 2017

Differential Roles of Chemokines CCL2 and CCL7 in Monocytosis and Leukocyte Migration during West Nile Virus Infection.

J Immunol 2015 Nov 23;195(9):4306-18. Epub 2015 Sep 23.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029;

West Nile virus (WNV) is a re-emerging pathogen and the leading cause of epidemic encephalitis in the United States. Inflammatory monocytes are a critical component of the cellular infiltrate found in the CNS during WNV encephalitis, although the molecular cues involved in their migration are not fully understood. In mice, we previously showed that WNV infection induces a CCR2-dependent monocytosis that precedes monocyte migration into the CNS. Currently, the relative contribution of the CCR2 ligands, chemokines CCL2 and CCL7, in directing monocyte mobilization and leukocyte migration into the CNS is unclear. In this study, we demonstrate that, although both CCL2 and CCL7 are required for efficient monocytosis and monocyte accumulation in the CNS, only CCL7 deficiency resulted in increased viral burden in the brain and enhanced mortality. The enhanced susceptibility in the absence of CCL7 was associated with the delayed migration of neutrophils and CD8(+) T cells into the CNS compared with WT or Ccl2(-/-) mice. To determine whether CCL7 reconstitution could therapeutically alter the survival outcome of WNV infection, we administered exogenous CCL7 i.v. to WNV-infected Ccl7(-/-) mice and observed a significant increase in monocytes and neutrophils, but not CD8(+) T cells, within the CNS, as well as an enhancement in survival compared with Ccl7(-/-) mice treated with a linear CCL7 control peptide. Our experiments suggest that CCL7 is an important protective signal involved in leukocyte trafficking during WNV infection, and it may have therapeutic potential for the treatment of acute viral infections of the CNS.
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http://dx.doi.org/10.4049/jimmunol.1500352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610864PMC
November 2015

Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation.

Nat Commun 2015 Sep 10;6:8164. Epub 2015 Sep 10.

Gladstone Institute of Neurological Disease, University of California, San Francisco, California 94158, USA.

Autoimmunity and macrophage recruitment into the central nervous system (CNS) are critical determinants of neuroinflammatory diseases. However, the mechanisms that drive immunological responses targeted to the CNS remain largely unknown. Here we show that fibrinogen, a central blood coagulation protein deposited in the CNS after blood-brain barrier disruption, induces encephalitogenic adaptive immune responses and peripheral macrophage recruitment into the CNS leading to demyelination. Fibrinogen stimulates a unique transcriptional signature in CD11b(+) antigen-presenting cells inducing the recruitment and local CNS activation of myelin antigen-specific Th1 cells. Fibrinogen depletion reduces Th1 cells in the multiple sclerosis model, experimental autoimmune encephalomyelitis. Major histocompatibility complex (MHC) II-dependent antigen presentation, CXCL10- and CCL2-mediated recruitment of T cells and macrophages, respectively, are required for fibrinogen-induced encephalomyelitis. Inhibition of the fibrinogen receptor CD11b/CD18 protects from all immune and neuropathologic effects. Our results show that the final product of the coagulation cascade is a key determinant of CNS autoimmunity.
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http://dx.doi.org/10.1038/ncomms9164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4579523PMC
September 2015

A dynamic spectrum of monocytes arising from the in situ reprogramming of CCR2+ monocytes at a site of sterile injury.

J Exp Med 2015 Apr 23;212(4):447-56. Epub 2015 Mar 23.

Immunology Research Group, Snyder Institute for Chronic Diseases; Department of Microbiology, Immunology, and Infectious Diseases; and Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta T2N 4N1, Canada Immunology Research Group, Snyder Institute for Chronic Diseases; Department of Microbiology, Immunology, and Infectious Diseases; and Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Monocytes are recruited from the blood to sites of inflammation, where they contribute to wound healing and tissue repair. There are at least two subsets of monocytes: classical or proinflammatory (CCR2(hi)CX3CR1(low)) and nonclassical, patrolling, or alternative (CCR2(low)CX3CR1(hi)) monocytes. Using spinning-disk confocal intravital microscopy and mice with fluorescent reporters for each of these subsets, we were able to track the dynamic spectrum of monocytes that enter a site of sterile hepatic injury in vivo. We observed that the CCR2(hi)CX3CR1(low) monocytes were recruited early and persisted for at least 48 h, forming a ringlike structure around the injured area. These monocytes transitioned, in situ, from CCR2(hi)Cx3CR1(low) to CX3CR1(hi)CCR2(low) within the ringlike structure and then entered the injury site. This phenotypic conversion was essential for optimal repair. These results demonstrate a local, cytokine driven reprogramming of classic, proinflammatory monocytes into nonclassical or alternative monocytes to facilitate proper wound-healing.
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http://dx.doi.org/10.1084/jem.20141539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4387291PMC
April 2015

Differential roles of microglia and monocytes in the inflamed central nervous system.

J Exp Med 2014 Jul 7;211(8):1533-49. Epub 2014 Jul 7.

Neuroinflammation Research Center and Department of Neurosciences, Lerner Research Institute; Department of Quantitative Health Sciences; and Mellen Center for Multiple Sclerosis Treatment and Research, Neurological Institute, Cleveland Clinic, Cleveland, OH 44106Neuroinflammation Research Center and Department of Neurosciences, Lerner Research Institute; Department of Quantitative Health Sciences; and Mellen Center for Multiple Sclerosis Treatment and Research, Neurological Institute, Cleveland Clinic, Cleveland, OH 44106 Cleveland Clinic Lerner College of Medicine, Cleveland, OH 44106

In the human disorder multiple sclerosis (MS) and in the model experimental autoimmune encephalomyelitis (EAE), macrophages predominate in demyelinated areas and their numbers correlate to tissue damage. Macrophages may be derived from infiltrating monocytes or resident microglia, yet are indistinguishable by light microscopy and surface phenotype. It is axiomatic that T cell-mediated macrophage activation is critical for inflammatory demyelination in EAE, yet the precise details by which tissue injury takes place remain poorly understood. In the present study, we addressed the cellular basis of autoimmune demyelination by discriminating microglial versus monocyte origins of effector macrophages. Using serial block-face scanning electron microscopy (SBF-SEM), we show that monocyte-derived macrophages associate with nodes of Ranvier and initiate demyelination, whereas microglia appear to clear debris. Gene expression profiles confirm that monocyte-derived macrophages are highly phagocytic and inflammatory, whereas those arising from microglia demonstrate an unexpected signature of globally suppressed cellular metabolism at disease onset. Distinguishing tissue-resident macrophages from infiltrating monocytes will point toward new strategies to treat disease and promote repair in diverse inflammatory pathologies in varied organs.
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http://dx.doi.org/10.1084/jem.20132477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113947PMC
July 2014

CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury.

J Neurotrauma 2014 Oct 21;31(20):1677-88. Epub 2014 Jul 21.

1 Immunology Section, San Francisco VA Medical Center , San Francisco, California.

Traumatic brain injury (TBI) provokes inflammatory responses, including a dramatic rise in brain macrophages in the area of injury. The pathway(s) responsible for macrophage infiltration of the traumatically injured brain and the effects of macrophages on functional outcomes are not well understood. C-C-chemokine receptor 2 (CCR2) is known for directing monocytes to inflamed tissues. To assess the role of macrophages and CCR2 in TBI, we determined outcomes in CCR2-deficient (Ccr2(-/-)) mice in a controlled cortical impact model. We quantified brain myeloid cell numbers post-TBI by flow cytometry and found that Ccr2(-/-) mice had greatly reduced macrophage numbers (∼80-90% reduction) early post-TBI, compared with wild-type mice. Motor, locomotor, and cognitive outcomes were assessed. Lack of Ccr2 improved locomotor activity with less hyperactivity in open field testing, but did not affect anxiety levels or motor coordination on the rotarod three weeks after TBI. Importantly, Ccr2(-/-) mice demonstrated greater spatial learning and memory, compared with wild-type mice eight weeks after TBI. Although there was no difference in the volume of tissue loss, Ccr2(-/-) mice had significantly increased neuronal density in the CA1-CA3 regions of the hippocampus after TBI, compared with wild-type mice. These data demonstrate that Ccr2 directs the majority of macrophage homing to the brain early after TBI and indicates that Ccr2 may facilitate harmful responses. Lack of Ccr2 improves functional recovery and neuronal survival. These results suggest that therapeutic blockade of CCR2-dependent responses may improve outcomes following TBI.
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http://dx.doi.org/10.1089/neu.2013.3252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545982PMC
October 2014

Alkylsulfone-containing trisubstituted cyclohexanes as potent and bioavailable chemokine receptor 2 (CCR2) antagonists.

Bioorg Med Chem Lett 2014 Apr 21;24(7):1843-5. Epub 2014 Feb 21.

Research and Development, Bristol-Myers Squibb Company, Princeton, NJ 08543-4000, United States.

We describe novel alkylsulfones as potent CCR2 antagonists with reduced hERG channel activity and improved pharmacokinetics over our previously described antagonists. Several of these new alkylsulfones have a profile that includes functional antagonism of CCR2, in vitro microsomal stability, and oral bioavailability. With this improved profile, we demonstrate that two of these antagonists, 2 and 12, are orally efficacious in an animal model of inflammatory recruitment.
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http://dx.doi.org/10.1016/j.bmcl.2014.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539253PMC
April 2014

The transcriptional repressor BLIMP1 curbs host defenses by suppressing expression of the chemokine CCL8.

J Immunol 2014 Mar 29;192(5):2291-304. Epub 2014 Jan 29.

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605;

The transcriptional repressor B lymphocyte-induced maturation protein 1 (BLIMP1) is a master regulator of B and T cell differentiation. To examine the role of BLIMP1 in innate immunity, we used a conditional knockout (CKO) of Blimp1 in myeloid cells and found that Blimp1 CKO mice were protected from lethal infection induced by Listeria monocytogenes. Transcriptome analysis of Blimp1 CKO macrophages identified the murine chemokine (C-C motif) ligand 8, CCL8, as a direct target of Blimp1-mediated transcriptional repression in these cells. BLIMP1-deficient macrophages expressed elevated levels of Ccl8, and consequently Blimp1 CKO mice had higher levels of circulating CCL8, resulting in increased neutrophils in the peripheral blood, promoting a more aggressive antibacterial response. Mice lacking the Ccl8 gene were more susceptible to L. monocytogenes infection than were wild-type mice. Although CCL8 failed to recruit neutrophils directly, it was chemotactic for γ/δ T cells, and CCL8-responsive γ/δ T cells were enriched for IL-17F. Finally, CCL8-mediated enhanced clearance of L. monocytogenes was dependent on γ/δ T cells. Collectively, these data reveal an important role for BLIMP1 in modulating host defenses by suppressing expression of the chemokine CCL8.
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http://dx.doi.org/10.4049/jimmunol.1301799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943885PMC
March 2014

CCR2(+) monocytes infiltrate atrophic lesions in age-related macular disease and mediate photoreceptor degeneration in experimental subretinal inflammation in Cx3cr1 deficient mice.

EMBO Mol Med 2013 11 21;5(11):1775-93. Epub 2013 Oct 21.

Inserm, U 968, Paris, France; UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, France; Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, Paris, France; Hôtel Dieu, Service d'Ophtalmologie, Centre de Recherche Ophtalmologique, Paris, France.

Atrophic age-related macular degeneration (AMD) is associated with the subretinal accumulation of mononuclear phagocytes (MPs). Their role in promoting or inhibiting retinal degeneration is unknown. We here show that atrophic AMD is associated with increased intraocular CCL2 levels and subretinal CCR2(+) inflammatory monocyte infiltration in patients. Using age- and light-induced subretinal inflammation and photoreceptor degeneration in Cx3cr1 knockout mice, we show that subretinal Cx3cr1 deficient MPs overexpress CCL2 and that both the genetic deletion of CCL2 or CCR2 and the pharmacological inhibition of CCR2 prevent inflammatory monocyte recruitment, MP accumulation and photoreceptor degeneration in vivo. Our study shows that contrary to CCR2 and CCL2, CX3CR1 is constitutively expressed in the retina where it represses the expression of CCL2 and the recruitment of neurotoxic inflammatory CCR2(+) monocytes. CCL2/CCR2 inhibition might represent a powerful tool for controlling inflammation and neurodegeneration in AMD.
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http://dx.doi.org/10.1002/emmm.201302692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840491PMC
November 2013

B lymphocytes trigger monocyte mobilization and impair heart function after acute myocardial infarction.

Nat Med 2013 Oct 15;19(10):1273-80. Epub 2013 Sep 15.

1] Institut National de la Santé et de la Recherche Médicale (INSERM), Unit 970, Paris Cardiovascular Research Center, Paris, France. [2] Université Paris-Descartes, Paris, France. [3].

Acute myocardial infarction is a severe ischemic disease responsible for heart failure and sudden death. Here, we show that after acute myocardial infarction in mice, mature B lymphocytes selectively produce Ccl7 and induce Ly6C(hi) monocyte mobilization and recruitment to the heart, leading to enhanced tissue injury and deterioration of myocardial function. Genetic (Baff receptor deficiency) or antibody-mediated (CD20- or Baff-specific antibody) depletion of mature B lymphocytes impeded Ccl7 production and monocyte mobilization, limited myocardial injury and improved heart function. These effects were recapitulated in mice with B cell-selective Ccl7 deficiency. We also show that high circulating concentrations of CCL7 and BAFF in patients with acute myocardial infarction predict increased risk of death or recurrent myocardial infarction. This work identifies a crucial interaction between mature B lymphocytes and monocytes after acute myocardial ischemia and identifies new therapeutic targets for acute myocardial infarction.
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http://dx.doi.org/10.1038/nm.3284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042928PMC
October 2013

CCR2 antagonist CCX140-B provides renal and glycemic benefits in diabetic transgenic human CCR2 knockin mice.

Am J Physiol Renal Physiol 2013 Nov 28;305(9):F1288-97. Epub 2013 Aug 28.

850 Maude Ave., Mountain View, CA 94043.

Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene (db/db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function.
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http://dx.doi.org/10.1152/ajprenal.00316.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073927PMC
November 2013

Dual effect of chemokine CCL7/MCP-3 in the development of renal tubulointerstitial fibrosis.

Biochem Biophys Res Commun 2013 Aug 19;438(2):257-63. Epub 2013 Jul 19.

Institut National de la Santé et de la Recherche Médicale (INSERM) U1048, Institute of Cardiovascular and Metabolic Disease, Toulouse, France.

Most end-stage renal disease kidneys display accumulation of extracellular matrix (ECM) in the renal tubular compartment (tubular interstitial fibrosis - TIF) which is strongly correlated with the future loss of renal function. Although inflammation is a key event in the development of TIF, it can also have a beneficial anti-fibrotic role depending in particular on the stage of the pathology. Chemokines play an important role in monocyte extravasation in the inflammatory process. CCL2 has already been shown to be involved in the development of TIF but CCL7, a close relative of CCL2 and able to bind to similar receptors, has not been studied in renal disease. We therefore studied chemokine CCL7 in a model of unilateral ureteral obstruction (UUO)-induced TIF. We observed that the role of CCL7 differs depending on the stage of the pathology. In early stages (0-8 days), CCL7 deficient (CCL7-KO) mice displayed attenuated TIF potentially involving two mechanisms: an early (0-3 days) decrease of inflammatory cell infiltration followed (3-8 days) by a decrease in tubular ECM production independent of inflammation. In contrast, during later stages of obstruction (10-14 days), CCL7-KO mice displayed increased TIF which was again associated with reduced inflammation. Interestingly, the switch between this anti- to profibrotic effect was accompanied by an increased influx of immunosuppressive regulatory T cells. In conclusion, these results highlight for the first time a dual role for CCL7 in the development of renal TIF, deleterious in early stages but beneficial during later stages.
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http://dx.doi.org/10.1016/j.bbrc.2013.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4043121PMC
August 2013

Regulation of adaptive immunity by the fractalkine receptor during autoimmune inflammation.

J Immunol 2013 Aug 1;191(3):1063-72. Epub 2013 Jul 1.

Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA.

Fractalkine, a chemokine anchored to neurons or peripheral endothelial cells, serves as an adhesion molecule or as a soluble chemoattractant. Fractalkine binds CX3CR1 on microglia and circulating monocytes, dendritic cells, and NK cells. The aim of this study is to determine the role of CX3CR1 in the trafficking and function of myeloid cells to the CNS during experimental autoimmune encephalomyelitis (EAE). Our results show that, in models of active EAE, Cx3cr1(-/-) mice exhibited more severe neurologic deficiencies. Bone marrow chimeric mice confirmed that CX3CR1 deficiency in bone marrow enhanced EAE severity. Notably, CX3CR1 deficiency was associated with an increased accumulation of CD115(+)Ly6C(-)CD11c(+) dendritic cells into EAE-affected brains that correlated with enhanced demyelination and neuronal damage. Furthermore, higher IFN-γ and IL-17 levels were detected in cerebellar and spinal cord tissues of CX3CR1-deficient mice. Analyses of peripheral responses during disease initiation revealed a higher frequency of IFN-γ- and IL-17-producing T cells in lymphoid tissues of CX3CR1-deficient as well as enhanced T cell proliferation induced by CX3CR1-deficient dendritic cells. In addition, adoptive transfer of myelin oligodendrocyte glycoprotein35-55-reactive wild-type T cells induced substantially more severe EAE in CX3CR1-deficient recipients when compared with wild-type recipients. Collectively, the data demonstrate that besides its role in chemoattraction, CX3CR1 is a key regulator of myeloid cell activation contributing to the establishment of adaptive immune responses.
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http://dx.doi.org/10.4049/jimmunol.1300040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720756PMC
August 2013

Microglial repopulation model reveals a robust homeostatic process for replacing CNS myeloid cells.

Proc Natl Acad Sci U S A 2012 Oct 15;109(44):18150-5. Epub 2012 Oct 15.

German Center for Neurodegenerative Diseases, DZNE, and Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, 72076 Tuebingen, Germany.

Under most physiological circumstances, monocytes are excluded from parenchymal CNS tissues. When widespread monocyte entry occurs, their numbers decrease shortly after engraftment in the presence of microglia. However, some disease processes lead to focal and selective loss, or dysfunction, of microglia, and microglial senescence typifies the aged brain. In this regard, the long-term engraftment of monocytes in the microglia-depleted brain remains unknown. Here, we report a model in which a niche for myeloid cells was created through microglia depletion. We show that microglia-depleted brain regions of CD11b-HSVTK transgenic mice are repopulated with new Iba-1-positive cells within 2 wk. The engrafted cells expressed high levels of CD45 and CCR2 and appeared in a wave-like pattern frequently associated with blood vessels, suggesting the engrafted cells were peripheral monocytes. Although two times more numerous and morphologically distinct from resident microglia up to 27 wk after initial engraftment, the overall distribution of the engrafted cells was remarkably similar to that of microglia. Two-photon in vivo imaging revealed that the engrafted myeloid cells extended their processes toward an ATP source and displayed intracellular calcium transients. Moreover, the engrafted cells migrated toward areas of kainic acid-induced neuronal death. These data provide evidence that circulating monocytes have the potential to occupy the adult CNS myeloid niche normally inhabited by microglia and identify a strong homeostatic drive to maintain the myeloid component in the mature brain.
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http://dx.doi.org/10.1073/pnas.1210150109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497743PMC
October 2012

Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications.

J Pharmacol Exp Ther 2012 Jun 5. Epub 2012 Jun 5.

The following manuscript was published as a Fast Forward article on February 29, 2012: Sullivan TJ, Dairaghi DJ, Krasinski A, Miao Z, Wang Y, Zhao BN, Baumgart T, Berahovich R, Ertl LS, Pennell A, Seitz L, Miao S, Ungashe S, Wei Z, Johnson D, Boring L, Tsou C-L, Charo IF, Bekker P, Schall TJ, and Jaen JC, Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications. J Pharmacol Exp Ther jpet.111.190918; doi:10.1124/jpet.111.190918 It was later found that the chemical identity of a compound cited in the article, CCX140-B, was not sufficiently disclosed. The authors are unable, at this time, to provide the chemical identity of CCX140-B in accordance with the editorial policies of The Journal of Pharmacology and Experimental Therapeutics. As a result, the authors have voluntarily withdrawn this manuscript from publication. We apologize for any inconvenience this may cause JPET's readers.
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http://dx.doi.org/10.1124/jpet.111.190918DOI Listing
June 2012

Resolving postoperative neuroinflammation and cognitive decline.

Ann Neurol 2011 Dec;70(6):986-995

Department of Anesthesia and Perioperative Care, University of California, San Francisco, CA 94143-0648.

Objective: Cognitive decline accompanies acute illness and surgery, especially in the elderly. Surgery engages the innate immune system that launches a systemic inflammatory response that, if unchecked, can cause multiple organ dysfunction. We sought to understand the mechanisms whereby the brain is targeted by the inflammatory response and how this can be resolved.

Methods: C57BL/6J, Ccr2(RFP/+)Cx3cr1(GFP/+), Ikk(F/F) mice and LysM-Cre/Ikk(F/F) mice underwent stabilized tibial fracture operation under analgesia and general anesthesia. Separate cohorts of mice were tested for systemic and hippocampal inflammation, integrity of the blood-brain barrier (BBB), and cognition. The putative resolving effects of the cholinergic pathway on these postoperative responses were also studied.

Results: Peripheral surgery disrupts the BBB via release of tumor necrosis factor-alpha (TNFα), which facilitates the migration of macrophages into the hippocampus. Macrophage-specific deletion of Ikappa B kinase (IKK)β, a central coordinator of TNFα signaling through activation of nuclear factor (NF) κB, prevents BBB disruption and macrophage infiltration in the hippocampus following surgery. Activation of the α7 subtype of nicotinic acetylcholine receptors, an endogenous inflammation-resolving pathway, prevents TNFα-induced NF-κB activation, macrophage migration into the hippocampus, and cognitive decline following surgery.

Interpretation: These data reveal the mechanisms for bidirectional communication between the brain and immune system following aseptic trauma. Pivotal molecular mechanisms can be targeted to prevent and/or resolve postoperative neuroinflammation and cognitive decline.
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http://dx.doi.org/10.1002/ana.22664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556354PMC
December 2011

The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.

J Immunol 2012 Jan 11;188(1):29-36. Epub 2011 Nov 11.

Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.

Microglial cells are difficult to track during development because of the lack of specific reagents for myeloid subpopulations. To further understand how myeloid lineages differentiate during development to create microglial cells, we investigated CX3CR1 and CCR2 transcription unit activation in Cx3cr1(+/GFP)CCR2(+/RFP) knockin fluorescent protein reporter mice. The principal findings include: 1) CX3CR1(+) cells localized to the aorta-gonad-mesonephros region, and visualized at embryonic day (E)9.0 in the yolk sac and neuroectoderm; 2) at E10.5, CX3CR1 single-positive microglial cells were visualized penetrating the neuroepithelium; and 3) CX3CR1 and CCR2 distinguished infiltrating macrophages from resident surveillant or activated microglia within tissue sections and by flow cytometric analyses. Our results support the contribution of the yolk sac as a source of microglial precursors. We provide a novel model to monitor chemokine receptor expression changes in microglia and myeloid cells early (E8.0-E10.5) in development and during inflammatory conditions, which have been challenging to visualize in mammalian tissues.
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http://dx.doi.org/10.4049/jimmunol.1100421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3244524PMC
January 2012

Notch2 receptor signaling controls functional differentiation of dendritic cells in the spleen and intestine.

Immunity 2011 Nov 20;35(5):780-91. Epub 2011 Oct 20.

Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032, USA.

Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b(+) DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esam(hi) DC subset required lymphotoxin beta receptor signaling, proliferated in situ, and facilitated CD4(+) T cell priming. The Notch-independent Esam(lo) DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b(+)CD103(+) DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4(+) T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b(+) DC subsets in the spleen and intestine.
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http://dx.doi.org/10.1016/j.immuni.2011.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225703PMC
November 2011

Anti-inflammatory therapeutics for the treatment of atherosclerosis.

Nat Rev Drug Discov 2011 May;10(5):365-76

Gladstone Institute of Cardiovascular Disease, 1650 Owens Street #149, San Francisco, California 94158, USA.

Atherosclerosis is the primary cause of heart disease and stroke and is thus the underlying pathology of the leading causes of death in the western world. Although risk can be reduced by lowering lipid levels, the equally important contribution of inflammation to the development of cardiovascular disease is not adequately addressed by existing therapies. Here, we summarize the evidence supporting a role for inflammation in the pathogenesis of atherosclerosis, discuss agents that are currently in the clinic and provide a perspective on the challenges faced in the development of drugs that target vascular inflammation.
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http://dx.doi.org/10.1038/nrd3444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947588PMC
May 2011

Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+ T(H)2 cells.

Nat Immunol 2011 Feb 9;12(2):167-77. Epub 2011 Jan 9.

Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

Mouse CCL8 is a CC chemokine of the monocyte chemoattractant protein (MCP) family whose biological activity and receptor usage have remained elusive. Here we show that CCL8 is highly expressed in the skin, where it serves as an agonist for the chemokine receptor CCR8 but not for CCR2. This distinguishes CCL8 from all other MCP chemokines. CCL8 responsiveness defined a population of highly differentiated, CCR8-expressing inflammatory T helper type 2 (T(H)2) cells enriched for interleukin (IL)-5. Ccr8- and Ccl8-deficient mice had markedly less eosinophilic inflammation than wild-type or Ccr4-deficient mice in a model of chronic atopic dermatitis. Adoptive transfer studies established CCR8 as a key regulator of T(H)2 cell recruitment into allergen-inflamed skin. In humans, CCR8 expression also defined an IL-5-enriched T(H)2 cell subset. The CCL8-CCR8 chemokine axis is therefore a crucial regulator of T(H)2 cell homing that drives IL-5-mediated chronic allergic inflammation.
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http://dx.doi.org/10.1038/ni.1984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3863381PMC
February 2011