CCX559 is a potent, orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity.

The interaction of PD-L1 with PD-1 is a major immune checkpoint that limits effector T cell function against cancer cells; monoclonal antibodies that block this pathway have been approved in multiple tumor indications. As a next generation therapy, small molecule inhibitors of PD-L1 have inherent drug properties that may be advantageous for certain patient populations compared to antibody therapies. In this report we present the pharmacology of the orally-available, small molecule PD-L1 inhibitor CCX559 for cancer immunotherapy. CCX559 potently and selectively inhibited PD-L1 binding to PD-1 and CD80 in vitro, and increased activation of primary human T cells in a T cell receptor-dependent fashion. Oral administration of CCX559 demonstrated anti-tumor activity similar to an anti-human PD-L1 antibody in two murine tumor models. Treatment of cells with CCX559 induced PD-L1 dimer formation and internalization, which prevented interaction with PD-1. Cell surface PD-L1 expression recovered in MC38 tumors upon CCX559 clearance post dosing. In a cynomolgus monkey pharmacodynamic study, CCX559 increased plasma levels of soluble PD-L1. These results support the clinical development of CCX559 for solid tumors; CCX559 is currently in a Phase 1, first in patient, multicenter, open-label, dose-escalation study (ACTRN12621001342808).

Chromatin Remodeling Drives Immune-Fibroblast Crosstalk in Heart Failure Pathogenesis.

Chronic inflammation and tissue fibrosis are common stress responses that worsen organ function, yet the molecular mechanisms governing their crosstalk are poorly understood. In diseased organs, stress-induced changes in gene expression fuel maladaptive cell state transitions and pathological interaction between diverse cellular compartments. Although chronic fibroblast activation worsens dysfunction of lung, liver, kidney, and heart, and exacerbates many cancers, the stress-sensing mechanisms initiating the transcriptional activation of fibroblasts are not well understood. Here, we show that conditional deletion of the transcription co-activator Brd4 in Cx3cr1-positive myeloid cells ameliorates heart failure and is associated with a dramatic reduction in fibroblast activation. Analysis of single-cell chromatin accessibility and BRD4 occupancy in vivo in Cx3cr1-positive cells identified a large enhancer proximal to Interleukin-1 beta (Il1b), and a series of CRISPR deletions revealed the precise stress-dependent regulatory element that controlled expression of Il1b in disease. Secreted IL1B functioned non-cell autonomously to activate a p65/RELA-dependent enhancer near the transcription factor MEOX1, resulting in a profibrotic response in human cardiac fibroblasts. In vivo, antibody-mediated IL1B neutralization prevented stress-induced expression of MEOX1, inhibited fibroblast activation, and improved cardiac function in heart failure. The elucidation of BRD4-dependent crosstalk between a specific immune cell subset and fibroblasts through IL1B provides new therapeutic strategies for heart disease and other disorders of chronic inflammation and maladaptive tissue remodeling.

CCR2 deficiency alters activation of microglia subsets in traumatic brain injury.

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.

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

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+/Ly6Chi/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.

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

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 <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.

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

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+ αß 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.

Correction: 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.

[This corrects the article DOI: 10.1371/journal.pone.0164646.].

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

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.

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

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 CD11c+CD206+ immune cells was enriched in human liver tissue, and greater infiltration was observed in NAFLD. The presence of CD11c+CD206+ myeloid cells correlated with systemic insulin resistance. CD11c+CD206+ 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 CD11b+CD11c+F4/80+ monocytes, which are functional homologs of human CD11c+CD206+ 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 CD11c+CD206+ 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.

CCL7 Is a Negative Regulator of Cutaneous Inflammation Following Leishmania major Infection.

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.

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

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.

Correction: Selective Chemokine Receptor Usage by Central Nervous System Myeloid Cells in CCR2-Red Fluorescent Protein Knock-In Mice.

[This corrects the article DOI: 10.1371/journal.pone.0013693.].

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.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.