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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(hi)/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.

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.

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 (Gαi, Gαs or Gαq/11)–independent manner. This constitutive internalization is rapid: on a given monocyte, the entire cell surface CCR2 population is turned over in

SUMMARY In traumatic brain injury (TBI), a diversity of brain resident and peripherally derived myeloid cells have the potential to worsen damage and/or to assist in healing. We define the heterogeneity of microglia and macrophage phenotypes during TBI in wild-type (WT) mice and Ccr2−/− mice, which lack macrophage influx following TBI and are resistant to brain damage. We use unbiased single-cell RNA sequencing methods to uncover 25 microglia, monocyte/macrophage, and dendritic cell subsets in acute TBI and normal brains. We find alterations in transcriptional profiles of microglia subsets in Ccr2−/− TBI mice compared to WT TBI mice indicating that infiltrating monocytes/macrophages influence microglia activation to promote a type I IFN response. Preclinical pharmacological blockade of hCCR2 after injury reduces expression of IFN-responsive gene, Irf7, and improves outcomes. These data extend our understanding of myeloid cell diversity and crosstalk in brain trauma and identify therapeutic targets in myeloid subsets., Graphical Abstract, In brief By single-cell RNA sequencing of traumatically injured and normal brains from wild-type and Ccr2−/− mice, Somebang et al. define microglia, macrophage, and dendritic cell phenotypes in TBI. Targeting mouse and/or human CCR2 reduces specific TBI brain CNS myeloid compartments, dampens type I interferon responses, and improves cognition after TBI.

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 Leishmania major infection, CCL7 is specifically upregulated in the skin one-2 weeks after infection but its role in L. major control is unclear. To determine CCL7's impact on the response to L. major we infected WT and CCL7−/− C57BL/6 mice. L. major infection of CCL7-deficient mice led to an unexpected increase 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 in vitro and CCL7 add-back in vivo specifically reduced neutrophil influx into the infected skin revealing an unexpected role for CCL7 in limiting neutrophil recruitment during L. major infection. Enhanced neutrophilic infiltration in CCL7-deficient mice changed the balance of L. major infected host cells with an increase in the ratio of infected neutrophils over monocytes/macrophages. To determine the consequence of CCL7 deficiency on L. major 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 L. major 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.

In response to sterile liver injury, CCR2hiCX3CR1low inflammatory monocytes infiltrate the liver and form a ringlike structure around the injury site. The cells then transition into CCR2lowCX3CR1hi alternative monocytes that enter the injury site; this phenotypic transition was required for optimal repair., 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 (CCR2hiCX3CR1low) and nonclassical, patrolling, or alternative (CCR2lowCX3CR1hi) 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 CCR2hiCX3CR1low 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 CCR2hiCx3CR1low to CX3CR1hiCCR2low 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.

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.

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.

International audience; 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.

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.

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.

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.

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.

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.

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. CCR2

CC chemokine receptor 2 (CCR2) is essential to acute skeletal muscle injury repair. We studied the subpopulation of inflammatory cells recruited via CCR2 signaling and their cellular functions with respect to muscle regeneration. Mobilization of monocytes/macrophages (MOs/MPs), but not lymphocytes or neutrophils, was impaired from bone marrow to blood and from blood to injured muscle in Ccr2−/− mice. While the Ly-6C+ but not the Ly-6C− subset of MOs/MPs was significantly reduced in blood, both subsets were drastically reduced in injured muscle of Ccr2−/− mice. Expression of insulin-like growth factor-1 (IGF-I) was markedly up-regulated in injured muscle of wild-type but not Ccr2−/− mice. IGF-I was strongly expressed by macrophages within injured muscle, more prominently by the Ly-6C− subset. A single injection of IGF-I, but not PBS, into injured muscle to replace IGF-I remarkably improved muscle regeneration in Ccr2−/− mice. CCR2 was not detected in myogenic cells or capillary endothelial cells in injured muscle to suggest its direct involvement in muscle regeneration or angiogenesis. We conclude that CCR2 is essential to acute skeletal muscle injury repair primarily by recruiting Ly-6C+ MOs/MPs. Within injured muscle, these cells conduct phagocytosis, contribute to accumulation of intramuscular Ly-6C− macrophages, and produce a high level of IGF-I to promote muscle regeneration.—Lu, H., Huang, D., Saederupm, N., Charo, I. F., Ransohoff, R. M., Zhou, L. Macrophages recruited via CCR2 produce insulin-like growth factor-1 to repair acute skeletal muscle injury.

Spermatogenesis is a complex process through which male germ line stem cells undergo a multi-step differentiation program and sequentially become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. In this process, transcription factors act as switches that precisely regulate the expression of genes that in turn control the developmental program of male germ cells. Transcription factors identified to be essential for normal haploid gene expression all display transcription-activating effects and thus serve as the "on" switch for haploid gene expression. Here, we report that ZMYND15 acts as a histone deacetylase-dependent transcriptional repressor and controls normal temporal expression of haploid cell genes during spermiogenesis. Inactivation of Zmynd15 results in early activation of transcription of numerous important haploid genes including Prm1, Tnp1, Spem1, and Catpser3; depletion of late spermatids; and male infertility. ZMYND15 represents the first transcriptional repressor identified to be essential for sperm production and male fertility.

HSCs are BM-derived, self-renewing multipotent cells that develop into circulating blood cells. They have been implicated in the repair of inflamed parenchymal tissue, but the signals that regulate their trafficking to sites of inflammation are unknown. As monocytes are recruited to sites of inflammation via chemoattractants that activate CCR2 on their surface, we investigated whether HSCs are also recruited to sites of inflammation through CCR2. Initial analysis indicated that in mice, CCR2 was expressed on subsets of HSCs and hematopoietic progenitor cells (HPCs) and that freshly isolated primitive hematopoietic cells (Lin–c-Kit+ cells) responded to CCR2 ligands in vitro. In vivo analysis indicated that after instillation of thioglycollate to cause aseptic inflammation and after administration of acetaminophen to induce liver damage, endogenous HSCs/HPCs were actively recruited to the peritoneum and liver, respectively, in WT but not Ccr2–/– mice. HSCs/HPCs recovered from the peritoneum successfully engrafted into the BM of irradiated primary and secondary recipients, confirming their self renewal and multipotency. Importantly, administration of exogenous WT, but not Ccr2–/–, HSCs/HPCs accelerated resolution of acetaminophen-induced liver damage and triggered the expression of genes characteristic of the macrophage M2 or repair phenotype. These findings reveal what we believe to be a novel role for CCR2 in the homing of HSCs/HPCs to sites of inflammation and suggest new functions for chemokines in promoting tissue repair and regeneration.

OBJECTIVE—To define the mechanisms underlying the accumulation of monocytes/macrophages in the islets of Langerhans.RESEARCH DESIGN AND METHODS—We tested the hypothesis that macrophage accumulation into the islets is caused by overexpression of the chemokine CCL2. To test this hypothesis, we generated transgenic mice and evaluated the cellular composition of the islets by immunohistochemistry and flow cytometry. We determined serum levels of CCL2 by enzyme-linked immunosorbent assay, determined numbers of circulating monocytes, and tested whether CCL2 could mobilize monocytes from the bone marrow directly. We examined development of diabetes over time and tested whether CCL2 effects could be eliminated by deletion of its receptor, CCR2.RESULTS—Expression of CCL2 by β-cells was associated with increased numbers of monocytes in circulation and accumulation of macrophages in the islets of transgenic mice. These changes were promoted by combined actions of CCL2 at the level of the bone marrow and the islets and were not seen in animals in which the CCL2 receptor (CCR2) was inactivated. Mice expressing higher levels of CCL2 in the islets developed diabetes spontaneously. The development of diabetes was correlated with the accumulation of large numbers of monocytes in the islets and did not depend on T- and B-cells. Diabetes could also be induced in normoglycemic mice expressing low levels of CCL2 by increasing the number of circulating myeloid cells.CONCLUSIONS—These results indicate that CCL2 promotes monocyte recruitment by acting both locally and remotely and that expression of CCL2 by insulin-producing cells can lead to insulitis and islet destruction.

Background— Monocyte-derived foam cells are the hallmark of early atherosclerosis, and recent evidence indicates that chemokines play important roles in directing monocyte migration from the blood to the vessel wall. Genetic deletions of monocyte chemoattractant protein-1 (MCP-1, CCL2), fractalkine (CX3CL1), or their cognate receptors, CCR2 and CX3CR1, markedly reduce atherosclerotic lesion size in murine models of atherosclerosis. The aim of this study was to determine whether these 2 chemokines act independently or redundantly in promoting atherogenesis. Methods and Results— We crossed CX3CL1 −/− ApoE −/− and CCR2 −/− ApoE −/− mice to create CX3CL1 −/− CCR2 −/− ApoE −/− triple knockouts and performed a 4-arm atherosclerosis study. Here, we report that deletion of CX3CL1 in CCR2 −/− mice dramatically reduced macrophage accumulation in the artery wall and the subsequent development of atherosclerosis. Deletion of CX3CL1 did not reduce the number of circulating monocytes in either “wild-type” ApoE −/− mice or CCR2 −/− ApoE −/− mice, which suggests a role for CX3CL1 in the direct recruitment and/or capture of CCR2-deficient monocytes. Conclusions— These data provide the first in vivo evidence for independent roles for CCR2 and CX3CL1 in macrophage accumulation and atherosclerotic lesion formation and suggest that successful therapeutic strategies may need to target multiple chemokines or chemokine receptors.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a murine model for multiple sclerosis, involves recruitment of T cells and macrophages to the CNS after infection. We hypothesized that CCR2, the only known receptor for CCL2, would be required for TMEV-induced demyelinating disease development because of its role in macrophage recruitment. TMEV-infected SJL CCR2 knockout (KO) mice showed decreased long-term clinical disease severity and less demyelination compared with controls. Flow cytometric data indicated that macrophages (CD45(high) CD11b(+) ) in the CNS of TMEV-infected CCR2 KO mice were decreased compared with control mice throughout disease. CD4(+) and CD8(+) T cell percentages in the CNS of TMEV-infected control and CCR2 KO mice were similar over the course of disease. There were no apparent differences between CCR2 KO and control peripheral immune responses. The frequency of interferon-gamma-producing T cells in response to proteolipid protein 139-151 in the CNS was also similar during the autoimmunity stage of TMEV-induced demyelinating disease. These data suggest that CCR2 is important for development of clinical disease by regulating macrophage accumulation after TMEV infection.

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., Autoimmune brain inflammation is associated with activation of macrophages and microglia. Here the authors show that fibrinogen induces encephalitogenic T-cell activation and macrophage recruitment to the central nervous system, and promotes demyelination in a mouse model of multiple sclerosis.

We examined the effects of the chemokine fractalkine (CX3CL1) on EPSCs evoked by electrical stimulation of Schaffer collaterals in patch-clamped CA1 pyramidal neurons from rat hippocampal slices. Acute application of CX3CL1 caused a sustained reduction of EPSC amplitude, with partial recovery after washout. CX3CL1-induced EPSC depression is postsynaptic in nature, because paired-pulse ratio was maintained, amplitude distribution of spontaneous excitatory postsynaptic currents shifted to lower values, and whole-cell current responses to AMPA were reversibly inhibited. EPSC depression by CX3CL1 is mediated by CX3CL1 receptor (CX3CR1), because CX3CL1 was unable to influence EPSC amplitude in CA1 pyramidal neurons from CX3CR1 knock-out mice. CX3CL1-induced depression of both EPSC and AMPA current was not observed in the absence of afferent fiber stimulation or AMPA receptor activation, respectively, indicating the requirement of sustained receptor activity for its development. Findings obtained from hippocampal slices, cultured hippocampal neurons, and transfected human embryonic kidney cells indicate that a Ca2+-, cAMP-, and phosphatase-dependent process is likely to modulate CX3CL1 effects because of the following: (1) CX3CL1-induced depression was antagonized by intracellular BAPTA, 8Br-cAMP, phosphatase inhibitors, and pertussis toxin (PTX); (2) CX3CL1 inhibited forskolin-induced cAMP formation sensitive to PTX; and (3) CX3CL1 inhibited forskolin-induced Ser845 GluR1 phosphorylation, which was sensitive to PTX and dependent on Ca2+and phosphatase activity. Together, these findings indicate that CX3CL1 negatively modulates AMPA receptor function at active glutamatergic synapses through cell-signaling pathways by influencing the balance between kinase and phosphatase activity.

Background— The uptake of oxidized low-density lipoprotein (OxLDL) by macrophage scavenger receptors is thought to be a key process in the formation of foam cells, the hallmark of early atherosclerotic lesions. CXCL16/scavenger receptor for phosphatidylserine and OxLDL is a multifunctional chemokine that exhibits scavenger receptor activity toward oxidized lipids in a membrane-bound configuration and may be shed to serve as a chemoattractant for T helper 1–polarized T lymphocytes. These properties, as well as the expression of CXCL16 in human and mouse atheroma, suggest that CXCL16 plays a role in atherosclerosis. Methods and Results— To examine the role of CXCL16 in plaque formation, we created CXCL16-deficient mice ( CXCL16 −/− ) and bred them with mice deficient in the LDL receptor ( LDLR −/− ). In vitro, macrophages from CXCL16 −/− mice have a significant reduction in the capacity to bind and internalize OxLDL. We found that CXCL16 −/− / LDLR −/− mice have accelerated atherosclerosis, enhanced macrophage recruitment to the aortic arch, and more abundant mRNA for monocyte chemotactic protein-1 and tumor necrosis factor-α. Conclusions— These data suggest that scavenger receptor activity mediated by CXCL16 in vivo is atheroprotective, and they contrast with studies that document protection from atherosclerosis in scavenger receptor class A- and CD36-deficient mice.

The membrane-anchored form of CX3CL1 has been proposed as a novel adhesion protein for leukocytes. This functional property of CX3CL1 is mediated through CX3CR1, a chemokine receptor expressed predominantly on circulating white blood cells. Thus far, it is still uncertain at what stage of the trafficking process CX3CR1 becomes importantly involved and how the CX3CR1-dependent adhesion of leukocytes is regulated during inflammation. The objective of this study was to examine the functional effects of chemokine stimulation on CX3CR1-mediated adhesion of human monocytes. Consistent with previous reports, our data indicate that the activity of CX3CR1 on resting monocytes is sufficient to mediate cell adhesion to CX3CL1. However, the basal, nonstimulated adhesion activity is low, and we hypothesized that like the integrins, CX3CR1 may require a preceding activation step to trigger firm leukocyte adhesion. Compatible with this hypothesis, stimulation of monocytes with MCP-1 significantly increased their adhesion to immobilized CX3CL1, under both static and physiological flow conditions. The increase of the adhesion activity was mediated through CCR2-dependent signaling and obligatory activation of the p38 MAPK pathway. Stimulation with MCP-1 also induced a rapid increase of CX3CR1 protein on the cell surface. Inhibition of the p38 MAPK pathway prevented this increase of CX3CR1 surface expression and blunted the effect of MCP-1 on cell adhesion, indicating a causal link between receptor surface density and adhesion activity. Together, our data suggest that a chemokine signal is required for firm CX3CR1-dependent adhesion and demonstrate that CCR2 is an important regulator of CX3CL1-dependent leukocyte adhesion.

The C-C motif chemokine receptor-2 (CCR2) regulates monocyte and macrophage recruitment and is necessary for macrophage-dependent inflammatory responses and the development of atherosclerosis. Although adipose tissue expression and circulating concentrations of CCL2 (also known as MCP1), a high-affinity ligand for CCR2, are elevated in obesity, the role of CCR2 in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity, has not been studied. To determine what role CCR2 plays in the development of metabolic phenotypes, we studied the effects of Ccr2 genotype on the development of obesity and its associated phenotypes. Genetic deficiency in Ccr2 reduced food intake and attenuated the development of obesity in mice fed a high-fat diet. In obese mice matched for adiposity, Ccr2 deficiency reduced macrophage content and the inflammatory profile of adipose tissue, increased adiponectin expression, ameliorated hepatic steatosis, and improved systemic glucose homeostasis and insulin sensitivity. In mice with established obesity, short-term treatment with a pharmacological antagonist of CCR2 lowered macrophage content of adipose tissue and improved insulin sensitivity without significantly altering body mass or improving hepatic steatosis. These data suggest that CCR2 influences the development of obesity and associated adipose tissue inflammation and systemic insulin resistance and plays a role in the maintenance of adipose tissue macrophages and insulin resistance once obesity and its metabolic consequences are established.

Background— Recruitment of mononuclear leukocytes within atherosclerotic lesions is a critical step in atherogenesis. Mice lacking the chemokine receptor CCR2, highly expressed on macrophages but also on T lymphocytes, show a striking reduction of atherosclerotic lesion formation. The chemokine receptor CXCR3 is a marker of activated T helper type 1 lymphocytes, the principal T lymphocyte type detected within atheroma. We investigated whether the deletion of both of these 2 important receptors expressed on the principal inflammatory cells present in atheroma would further affect atherogenesis in vivo. Methods and Results— We crossed ApoE −/− mice with either CCR2 −/− or CXCR3 − mice and crossed ApoE −/− CCR2 −/− mice with the ApoE −/− CXCR3 − mice to generate a triple knockout strain. Analysis of atherosclerosis development after 10 weeks of high-cholesterol diet revealed differential effects on early atherosclerotic lesions in the abdominal aorta and on advanced lesions in aortic roots. ApoE −/− CXCR3 − mice, but not the triple knockout mice, displayed significantly reduced atherosclerotic lesion development within abdominal aortas compared with ApoE −/− CCR2 −/− and ApoE −/− mice. This reduction of lesion formation correlated with an upregulation of antiinflammatory molecules such as interleukin-10, interleukin-18BP, and endothelial nitric oxide synthase and with an increased number of regulatory T lymphocytes within atherosclerotic lesions. In contrast, lesion size development within the aortic roots was more enhanced in ApoE −/− and ApoE −/− CXCR3 − mice compared with ApoE −/− CCR2 −/− and triple knockout mice. Conclusions— Blocking chemokine signaling in vivo through deletion of the chemokine receptors CCR2 and CXCR3 has differential effects during atherogenesis. In addition, our results point to an important role of regulatory T lymphocytes during early atherogenesis.

Objective—CD1d-restricted natural killer T (NKT) cells are reported to play a proatherogenic role in the development of atherosclerosis. However, the contribution of NKT cells to mature lesion formation and the effector mechanisms through which they act are unknown.Methods and Results—We measured lesion size in CD1d-null (CD1d−/−) mice on the low-density lipoprotein (LDL) receptor–deficient (LDLR−/−) genetic background after 4, 8, and 12 weeks of feeding on a Western diet. Lesions inCD1d−/−LDLR−/−mice were 47% smaller at 4 weeks thanCD1d+/+LDLR−/−controls; however, there were no differences in lesion size betweenCD1d−/−LDLR−/−andCD1d+/+LDLR−/−mice at 8 or 12 weeks. We found that although NKT cells were present in the aortic arch ofCD1d+/+LDLR−/−mice on the Western diet, no differences in mRNA abundance for Th1 or Th2 cytokines were observed betweenCD1d−/−LDLR−/−andCD1d+/+LDLR−/−mice.Conclusions—CD1d-restricted NKT cells contribute to the formation of fatty streaks; however, their influence on lesion progression is transient, and they do not significantly affect the inflammatory cytokine milieu of mature lesions.

Our increasing appreciation of the importance of inflammation in vascular disease has focused attention on the molecules that direct the migration of leukocytes from the blood stream to the vessel wall. In this review, we summarize roles of the chemokines, a family of small secreted proteins that selectively recruit monocytes, neutrophils, and lymphocytes to sites of vascular injury, inflammation, and developing atherosclerosis. Chemokines induce chemotaxis through the activation of G-protein-coupled receptors, and the receptors that a given leukocyte expresses determines the chemokines to which it will respond. Monocyte chemoattractant protein 1 (MCP-1), acting through its receptor CCR2, appears to play an early and important role in the recruitment of monocytes to atherosclerotic lesions and in the formation of intimal hyperplasia after arterial injury. Acute thrombosis is an often fatal complication of atherosclerotic plaque rupture, and recent evidence suggests that MCP-1 contributes to thrombin generation and thrombus formation by generating tissue factor. Because of their critical roles in monocyte recruitment in vascular and nonvascular diseases, MCP-1 and CCR2 have become important therapeutic targets, and efforts are underway to develop potent and specific antagonists of these and related chemokines.

Dendritic cell (DC) recruitment is a hallmark event in antigen (Ag)-challenged lungs. We previously reported models for analyzing DC migration and activation in the lung after Th1- or Th2-eliciting pathogen Ag-bead challenge. To determine the role of chemokines in DC mobilization, we applied this analysis to CCR1, CCR2, CCR5, and CCR6 chemokine receptor knockout mice. Both Mycobacteria bovis protein Ags and helminthic, Schistosoma mansoni egg Ags elicited multiple chemokines, including CCR1, CCR2, CCR5, and to a lesser extent CCR6 ligands. DCs from wild-type lungs expressed transcripts for chemokine receptors, CCR1, CCR2, CCR5, and CXCR4. In all knockout strains, CD11c+ cells were recruited to Ag-beads likely because of receptor redundancy. However, DCs in CCR2−/− mice had significantly decreased MHCII and CD40 expression. This was associated with abrogated cytokine production in draining lymph node cultures. Analysis of local innate inflammation revealed a 50% reduction in macrophage recruitment in CCR2−/− mice. Bone marrow chimeras of mixed CCR2+/+ green fluorescent protein transgenic and CCR2−/− green fluorescent protein-negative cells confirmed the DC maturation defect was only among the latter population. In conclusion, CCR2 knockout confers an intrinsic DC activation defect and CCR2 ligands likely promote the local activation/maturation of inflammatory DCs.

We previously reported that CCR2−/− mice are susceptible to Mycobacterium tuberculosis infection. Susceptibility was associated with an early and sustained macrophage trafficking defect, followed by delayed recruitment of dendritic cells (DCs) and T cells to the lungs. However, the relative importance of the lack of CCR2 expression by macrophages and DCs vs T cells in susceptibility to infection was unclear. In this study, we used mixed bone marrow transplantation to create mice in which the genotype of the T cells was either CCR2+/+ or CCR2−/− while maintaining the genotype of the myeloid cells as CCR2+/+. After infection with M. tuberculosis, we found that the genotype of the macrophages and/or DCs, but not that of the T cells, was critical for both T cell and myeloid cell migration to the lungs. Further investigation revealed a critical role for CCR2 in the recruitment of F4/80dim macrophages and CD11cdim/intermediate DCs to the infected lung.

Idiopathic pneumonia syndrome (IPS) is a significant cause of mortality post-bone marrow transplant (BMT) in humans. In our murine model, lethal pre-BMT conditioning and allogeneic T cells result in the recruitment of host antigen-presenting cells (APC) and donor T cells into the lung post-BMT concomitant with development of severe lung dysfunction. CCL2 induction is found in bronchoalveolar lavage fluid (BALF) before host monocyte influx. The major receptor for CCL2 is CCR2 present on monocytes; this interaction can play a crucial role in monocyte recruitment in inflammation. To determine whether blockade of the CCL2/CCR2 pathway could hinder host monocyte influx, lethally conditioned wild-type (WT), CCL2-/-, or CCR2-/-mice were transplanted with allogeneic marrow and spleen cells. WT and-/-recipients exhibited equivalent lung dysfunction post-BMT. The frequencies of host macrophages as well as donor CD4+and CD8+T cells in lungs post-BMT did not differ between WT and-/-recipients. However, the T cell dependency of the host CD11b+major histocompatibility complex class II+cell influx was lost in CCR2-/-recipients. In CCR2-/-mice, this influx was accompanied by elevated levels of CCL20. Post-BMT BALF and sera of-/-mice did not reveal any decrease in cytokines or chemokines compared with WT mice. CCL2-/-mice had a deficiency of CCL2 in their BALF and sera post-BMT, confirming our hypothesis that CCL2 is predominantly host derived. Therefore, IPS can occur independently of host expression of CCL2 or CCR2, and compensatory mechanisms exist for regulating APC recruitment into the lung during the early post-BMT period.

Choroidal neovascularization (CNV) is directly related to visual loss in some eye diseases, such as age-related macular degeneration. Although several human histological studies have suggested the participation of macrophages in CNV formation, the precise mechanisms are still not fully understood. In this study, we elucidated the role of ocular-infiltrating macrophages in experimental CNV using CCR2 knockout (KO) mice, wild-type mice, and C57BL/6 (B6) mice. CCR2 is the receptor of monocyte chemoattractant protein-1, and the number of infiltrating macrophage and the area of CNV were significantly reduced in CCR2 KO mice. Enriched ocular-infiltrating macrophages from B6 mice actually showed angiogenic ability in a dorsal air sac assay. Moreover, their expression of class II, CD40, B7-1 and B7-2 molecules, and the mRNA for potential angiogenic factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor α, was also observed. Collectively, we conclude that ocular-infiltrating macrophages play an important role in CNV generation.

Langerhans cells (LCs) are bone marrow (BM)–derived epidermal dendritic cells (DCs) that represent a critical immunologic barrier to the external environment, but little is known about their life cycle. Here, we show that in lethally irradiated mice that had received BM transplants, LCs of host origin remained for at least 18 months, whereas DCs in other organs were almost completely replaced by donor cells within 2 months. In parabiotic mice with separate organs, but a shared blood circulation, there was no mixing of LCs. However, in skin exposed to ultraviolet light, LCs rapidly disappeared and were replaced by circulating LC precursors within 2 weeks. The recruitment of new LCs was dependent on their expression of the CCR2 chemokine receptor and on the secretion of CCR2-binding chemokines by inflamed skin. These data indicate that under steady-state conditions, LCs are maintained locally, but inflammatory changes in the skin result in their replacement by blood-borne LC progenitors.

Monocyte chemoattractant protein (MCP)-1 is upregulated in atherosclerotic plaques and in the media and intima of injured arteries. CC chemokine receptor 2 (CCR2) is the only known functional receptor for MCP-1. Mice deficient in MCP-1 or CCR2 have marked reductions in atherosclerosis. This study examines the effect of CCR2 deficiency in a murine model of femoral arterial injury. Four weeks after injury, arteries from CCR2−/−mice showed a 61.4% reduction (PP+/+littermates. The response of CCR2+/−mice was not significantly different from that of CCR2+/+mice. Five days after injury, the medial proliferation index, determined by bromodeoxyuridine incorporation, was decreased by 59.8% in CCR2−/−mice when compared with CCR2+/+littermates (P−/−or CCR2+/+mice 5 and 28 days after injury. These results demonstrate that CCR2 plays an important role in mediating smooth muscle cell proliferation and intimal hyperplasia in a non-hyperlipidemic model of acute arterial injury. CCR2 may thus be an important target for inhibiting the response to acute arterial injury.

IL-13 stimulates inflammatory and remodeling responses and contributes to the pathogenesis of human airways disorders. To further understand the cellular and molecular events that mediate these responses, we characterized the effects of IL-13 on monocyte chemotactic proteins (MCPs) and compared the tissue effects of transgenic IL-13 in mice with wild-type (+/+) and null (−/−) CCR2 loci. Transgenic IL-13 was a potent stimulator of MCP-1, -2, -3, and -5. This stimulation was not specific for MCPs because macrophage-inflammatory protein (MIP)-1α, MIP-1β, MIP-2, MIP-3α, thymus- and activation-regulated chemokine, thymus-expressed chemokine, eotaxin, eotaxin 2, macrophage-derived chemokines, and C10 were also induced. The ability of IL-13 to increase lung size, alveolar size, and lung compliance, to stimulate pulmonary inflammation, hyaluronic acid accumulation, and tissue fibrosis, and to cause respiratory failure and death were markedly decreased, whereas mucus metaplasia was not altered in CCR2−/− mice. CCR2 deficiency did not decrease the basal or IL-13-stimulated expression of target matrix metalloproteinases or cathepsins but did increase the levels of mRNA encoding α1-antitrypsin, tissue inhibitor of metalloproteinase-1, -2, and -4, and secretory leukocyte proteinase inhibitor. In addition, the levels of bioactive and total TGF-β1 were decreased in lavage fluids from IL-13 transgenic mice with −/− CCR2 loci. These studies demonstrate that IL-13 is a potent stimulator of MCPs and other CC chemokines and document the importance of MCP-CCR2 signaling in the pathogenesis of the IL-13-induced pulmonary phenotype.