CCR5 Inhibition Research Articles

CCR2 mediates increased susceptibility to post-H1N1 bacterial pneumonia by limiting dendritic cell induction of IL-17.

Post Influenza bacterial pneumonia is associated with significant mortality and morbidity. Dendritic cells (DCs) play a crucial role in host defense against bacterial pneumonia, but their contribution to post influenza-susceptibility to secondary bacterial pneumonia is incompletely understood. WT and CCR2−/− mice were infected with 100 plaque forming units (pfu) H1N1 intranasally alone or were challenged on day 5 with 7×107 colony forming units (cfu) methicillin-resistant Staphylococcus aureus intratracheally. WT mice express abundant CCL2 mRNA and protein post-H1N1 alone or dual infection. CCR2−/− mice had significantly higher survival as compared to WT mice, associated with significantly improved bacterial clearance at 24 and 48 hours (10 fold and 14 fold, respectively) post-bacterial challenge. There was robust upregulation of IL-23 and IL-17 as well as down-regulation of IL-27 expression in CCR2−/− mice following sequential infection as compared to WT mice, which was also associated with significantly greater accumulation of CD103+ DC. Finally, WT mice treated with a CCR2 inhibitor showed improved bacterial clearance in association with similar cytokine profiles as CCR2−/− mice. Thus, CCR2 significantly contributes to increased susceptibility to bacterial infection after influenza pneumonia likely via altered dendritic cell responses and thus, CCR2 antagonism represents a potential therapeutic strategy.

CCR2 and CCR5 promote diclofenac-induced hepatotoxicity in mice.

Liver injury, one of the major side effects of diclofenac (DIC), plagues thousands of patients who treated with it. Although involvements of metabolic factors, oxidative stress, and mitochondrial injury have been characterized, the exact immunomolecular mechanism of the hepatotoxicity of DIC still remains ambiguous. In this study, we investigated the role of chemokine receptors CCR2 and CCR5 in this progression. Ccr2, Ccr5, and Tnfr1/2-deficient mice, as well as wild type littermates, were administrated DIC or vehicle for 24h, receptively. Hepatic expression of CCR2, CCR5, and their ligands were upregulated after DIC treatment. DIC-induced liver injury was augmented in Ccr2+/+ mice than Ccr2-/- mice, a similar phenotype was observed in Ccr5-deficient mice. In addition, antagonists of CCR2 or CCR5 protected liver damage caused by diclofenac. Besides, the number of neutrophils present in the liver was gradually increased from 0 to 12h after drug administration. However, the recruitment of neutrophils was dramatically lessened after blocking CCR2 or CCR5 signaling. Furthermore, TNF-α level in the liver was decreased in Ccr2-/- mice compared with Ccr2+/+ mice. Intriguingly, in line with this, TNF receptor 1 and 2 double knockout mice showed markedly attenuated hepatotoxicity of DIC. These suggested that CCR2 and CCR5 mediated hepatotoxicity induced by diclofenac, TNF-α was responsible, at least in part, for it, and the pharmacological inhibition of CCR2 or CCR5 might serve as a novel therapeutic approach for DIC-induced hepatotoxicity.

Abstract B56: CXCR2 blockade reduces granulocytic myeloid cell compensation in response to macrophage targeted therapy and further enhances the efficacy of chemotherapy in pancreatic ductal adenocarcinoma

Abstract Background: The incidence of pancreatic ductal adenocarcinoma (PDAC) is rising, yet patient survival rates following conventional treatment remain abysmal. A unique feature of PDAC is the presence of a prominent tumor stroma with a dense network of fibrotic material and abundant inflammatory immune infiltrate. Bone marrow derived myeloid cells of both monocytic and granulocytic origin, including tumor associated macrophages (TAM) and neutrophils (TAN) respectively, are the most prevalent immune cells in the tumor microenvironment (TME) of PDAC. PDAC tumors co-opt chemokine signaling pathways to attract myeloid cells from the bone marrow to the TME where they suppress anti-tumor immune responses, support tumor progression, and confer resistance to therapy. In a previous clinical study, we demonstrated that targeted blockade of CCR2+ TAM with a small molecule CCR2 inhibitor (PF-04136309) nearly doubled responses to the FOLFIRINOX chemotherapy regimen in patients with locally advanced PDAC. Here, we show that tumor infiltrating CXCR2+ TAN compensate for TAM depletion in response to CCR2 blockade, so we hypothesized that dual targeting of both CCR2+ TAM and CXCR2+ TAN would further enhance chemotherapy efficacy for treating PDAC. Methods: Human blood, bone marrow, and tumor samples were acquired under an IRB approved protocol. PDAC cell lines (KCKO or KP) derived from spontaneous tumors were subcutaneously or orthotopically implanted into C57BL/6 mice. Tumor bearing mice were randomized and treated with vehicle, FOLFIRINOX, CCR2i (PF-04136309 or RS504393), CXCR2i (SB225002), CCR2i+CXCR2i, CCR2i+FOLFIRINOX, CXCR2i+FOLFIRINOX, or CCR2i+CXCR2i+FOLFIRINOX. Mice were sacrificed at fixed time points for tissue collection or followed for survival. Resected tissues were snap frozen for RNA extraction to perform qRT-PCR, fixed in formalin for histological staining and immunohistochemistry, or digested into single cell suspensions for flow cytometry analysis. Results: The prevalence of peripheral blood and bone marrow CXCR2+ TAN is prognostic in patients with PDAC. Tumors expressed elevated levels of the ELR+ chemokines including CXCL1, CXCL2, CXCL5, and CXCL8 and were infiltrated with significantly more CXCR2+ TAN compared to normal pancreas. Tumor expression of the ELR+ chemokines correlated with tumor infiltrating TAN and their levels were prognostic in human disease. Analysis of longitudinally paired tumor biopsies from patients treated with PF-04136309 had significantly increased levels of CXCR2+ TAN (p < 0.05). In mice, orthotopically implanted PDAC tumors significantly upregulated the ELR+ chemokines resulting in increased levels of CXCR2+ TAN in the peripheral blood, bone marrow, and TME compared to normal pancreas controls, recapitulating human disease. CXCR2i significantly decreased tumor infiltrating CXCR2+ TAN and reduced overall tumor burden. Single agent targeting of either TAM or TAN, using CCR2i or CXCR2i respectively, resulted in a compensatory influx of the other myeloid cell subset. However, dual targeting of both CCR2+ TAM and CXCR2+ TAN blocked myeloid compensation and further enhanced the efficacy of FOLFIRINOX. Dual CCR2/CXCR2 blockade reprogrammed the TME and generated a more robust anti-tumor T cell response compared to either agent alone. Conclusions: The ELR+ CXC/CXCR2 chemokine pathway plays a key role in PDAC tumor progression and CXCR2+ TAN compensation is observed with depletion of TAM in response to CCR2 blockade. Duel targeting of CCR2+ TAM and CXCR2+ TAN prevents myeloid plasticity to single agent therapy and further enhances the efficacy of chemotherapy. Thus, further studies exploring complementary immune targeting strategies are warranted in PDAC. Citation Format: Booyeon J. Han, Tim M. Nywening, Brian A. Belt, Jian Ye, William G. Hawkins, David G. DeNardo, David C. Linehan. CXCR2 blockade reduces granulocytic myeloid cell compensation in response to macrophage targeted therapy and further enhances the efficacy of chemotherapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B56.

Recruitment of CCR2+ tumor associated macrophage to sites of liver metastasis confers a poor prognosis in human colorectal cancer

ABSTRACTThe tumor microenvironment (TME) represents a significant barrier to creating effective therapies for metastatic colorectal cancer (mCRC). In several malignancies, bone marrow derived CCR2+ inflammatory monocytes (IM) are recruited to the TME by neoplastic cells, where they become immunosuppressive tumor associated macrophages (TAM). Here we report that mCRC expression of the chemokine CCL2 facilitates recruitment of CCR2+ IM from the bone marrow to the peripheral blood. Immune monitoring of circulating monocytes in patients with mCRC found this influx was a prognostic biomarker and correlated with worse clinical outcomes. At the metastatic site, mCRC liver tumors were heavily infiltrated by TAM, which displayed a robust ability to dampen endogenous anti-tumor lymphocyte activity. Using a murine model of mCRC that recapitulates these findings from human disease, we show that targeting CCR2 reduces TAM accumulation in liver metastasis and restores anti-tumor immunity. Additional quantitative analysis of hepatic metastatic tumor burden and treatment efficacy found that administration of a small molecule CCR2 inhibitor (CCR2i) improves chemotherapeutic responses and increases overall survival in mice with mCRC liver tumors. Our study suggests that targeting the CCL2/CCR2 chemokine axis decreases TAM at the metastatic site, disrupting the immunosuppressive TME and rendering mCRC susceptible to anti-tumor T-cell responses.

Characterization of C-C motif chemokine ligand 4 in the porcine endometrium during the presence of the maternal–fetal interface

Chemokines and their receptors play a crucial role in embryo implantation at the maternal–fetal interface during pregnancy. In this study, we investigated the role of CCL4 in development of the porcine endometrium in the early gestational period. Porcine CCL4 showed high similarity with the human counterpart, and mRNA expression of CCL4 and its receptor (CCR5) was predominantly present in the endometrium during early pregnancy. Treatment with CCL4 increased proliferation of porcine uterine luminal epithelial (pLE) cells by activation of PI3K and MAPK signal transduction. In addition, CCL4 recovered the endoplasmic-reticulum stress–reduced proliferation and decreased the unfolded protein response in pLE cells. Besides, the lipopolysaccharide-activated NF-κB pathway was suppressed in response to CCL4 in pLE cells. Inhibition of CCR5 decreased the proliferation of pLE cells and activation of the PI3K and MAPK pathways by CCL4. Furthermore, CCL4 enhanced conceptus-maternal interactions between porcine trophectoderm (pTr) cells and pLE cells during early pregnancy by activating expression of migration and implantation-related genes. Collectively, the results suggest that CCL4 may improve successful implantation in early pregnancy in pigs.

Abstract 5790: TG-1601 is a novel BET inhibitor with strong binding affinity and long-lasting effect in pre-clinical models

Abstract BET (bromodomain and extra-terminal) proteins bind to acetylated lysine residues on chromatin and participate in the regulation of gene transcription. Inhibition of BET protein binding to chromatin with small molecules selectively suppresses the transcription of a set of oncogenes, including MYC and BCL-2. TG-1601 is a novel, selective and potent small molecule inhibitor of BET bromodomains. TG-1601 binds to the first and second bromodomains (BD1, BD2) of the BET protein family, BRD2, BRD3, BRD4, and BRDT, with Kd values ranging from 0.5 nM to 9.1 nM. MYC protein expression is strongly inhibited in the MV4-11 cancer cell line with an EC50 of 5 nM, with GI50 comprised between 15 nM and 85 nM in a variety of leukemia and myeloma cancer cell lines, indicating potent inhibition of cell proliferation. Time course and dose-response studies conducted in mice bearing subcutaneous MV4-11 xenografts showed that MYC protein was undetectable 3 hours following a single 25 mg/kg oral dose, with a TG-1601 tumor concentration of 6uM achieved. Interestingly, at 24h post-dose, while TG-1601 is cleared from the tumor, MYC protein level remains below 40% of its initial level, indicating a long-lasting effect pharmacodynamic of TG-1601, potentially attributable to enhanced binding affinity compared to earlier generation molecules. In agreement with this long-lasting effect, efficacy studies in MV4-11 tumor-bearing mice, dosed with a 20 mg/kg/day PO regimen interrupted by increasing drug holiday periods, showed that drug holidays of 2, 3 and 4 days per week only modestly affected efficacy (3%, 15% and 12% TGI respectively), suggesting discontinuous dosing of TG-1601 in clinic may not significantly impact efficacy. Anti-cancer agents have been shown - in vitro or in vivo - to synergize with BET inhibitors. Here we show that TG-1601 and anti PD-1 antibody demonstrated synergistic anti-tumor activity when combined in the B16F10 model, an aggressive syngeneic model of melanoma. TG-1601 inhibition of MYC, CCR-2 and IL1RN gene expression was tested in a whole blood ex-vivo experiment, and the genes were validated as pharmacodynamic markers to monitor TG-1601 activity in clinic. In conclusion, TG-1601 is a novel BET inhibitor with remarkably strong affinity and a potent ability to inhibit MYC expression and cell growth, with favorable pharmacokinetic properties supporting clinical development. Its properties in vivo provide an opportunity for the rational development of TG-1601 as an anti-cancer agent, taken alone or in combination with other small molecules or antibodies. IND enabling studies are underway, with clinical evaluation expected to commence in the first half of 2018. Citation Format: Emmanuel Normant, Leonid Gorelik, Rama Shmeis, Henry Le, Robert Nisch, Karen TenHuisen, Teja Turpuseema, James Oliviero, Hari P. Miskin, Peter Sportelli, Michael S. Weiss. TG-1601 is a novel BET inhibitor with strong binding affinity and long-lasting effect in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5790.

Abstract CT124: A phase Ib/II study of BMS-813160, a CC chemokine receptor (CCR) 2/5 dual antagonist, in combination with chemotherapy or nivolumab in patients (pts) with advanced pancreatic or colorectal cancer

Abstract Background: During cancer progression, immunosuppressive cells, such as those of myeloid and lymphoid origin, are recruited to the tumor microenvironment (TME) through interactions between chemokines and chemokine receptors (CKRs). CCR2 and CCR5 are CKRs expressed on both myeloid and T-cell infiltrates in the TME and have been shown to mediate the migration of monocyte/myeloid-derived suppressor cells (MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs), respectively, from bone marrow to the blood and TME. CCR5 can also recruit regulatory T cells to the TME, potentiate PMN-MDSC immunosuppressive function via arginase-1, and polarize tumor-associated macrophages into an immunosuppressive M2 phenotype. Engagement of CCR2 or CCR5 promotes an immunosuppressive environment and prevents antitumor immunity; this has been preclinically validated in multiple tumor types, including pancreatic and colorectal. Preliminary data also support the utility of combination with PD-(L)1 inhibition. Clinically, CCR2- and CCR5-selective antagonists have separately shown proof of mechanism in combination with chemotherapy in pts with pancreatic and colorectal cancers, respectively (1, 2). These encouraging studies support further investigation of CCR2/5 dual antagonism in providing greater benefit to pts than CCR2- or CCR5-selective inhibition when combined with chemotherapy. Here we describe the design of a phase 1b/2, nonrandomized, open-label study of BMS-813160 (small-molecule CCR2/5 dual antagonist) as monotherapy or in combination with chemotherapy or nivolumab in pts with advanced pancreatic or colorectal cancer (NCT03184870). Methods: Approximately 260 pts aged ≥ 18 years with an ECOG PS of ≤ 1, adequate marrow and organ function, and the ability to undergo pre- and on-treatment biopsies will be enrolled. Exclusion criteria include prior treatment with CCR2 and/or CCR5 inhibitors, requirement for corticosteroids/other immunosuppressive medications, CNS metastases, autoimmune diseases, and symptomatic interstitial lung disease. In part 1, pts will receive BMS-813160 monotherapy safety lead-in for 2 weeks followed by combination with chemotherapy (5-fluorouracil + leucovorin + irinotecan [colorectal cancer] or nab-paclitaxel + gemcitabine [pancreatic cancer]) or nivolumab. In part 2, pts will receive either the same regimens (without a monotherapy lead-in) or BMS-813160 monotherapy. Primary outcomes are safety, tolerability, objective response rate, median duration of response, and progression-free survival rate at 24 weeks. Secondary outcomes are pharmacokinetics and pharmacodynamics of BMS-813160 and immunogenicity of nivolumab.

Chemokine receptor CCR2 contributes to neuropathic pain and the associated depression via increasing NR2B-mediated currents in both D1 and D2 dopamine receptor-containing medium spiny neurons in the nucleus accumbens shell.

Patients with neuropathic pain are usually accompanied by depression. Chemokine-mediated neuroinflammation is involved in a variety of diseases, including neurodegenerative diseases, depression, and chronic pain. The nucleus accumbens (NAc) is an important area in mediating pain sensation and depression. Here we report that spinal nerve ligation (SNL)induced upregulation of chemokine CCL2 and its major receptor CCR2 in both dopamine D1 and D2 receptor (D1R and D2R)-containing neurons in the NAc. Inhibition of CCR2 by shRNA lentivirus in the NAc shell attenuated SNL-induced pain hypersensitivity and depressive behaviors. Conversely, intra-NAc injection of CCL2-expressing lentivirus-induced nociceptive and depressive behaviors in naïve mice. Whole-cell patch clamp recording of D1R-positive or D2R-positive medium spiny neurons (MSNs) showed that SNL increased NMDA receptor (NMDAR)-mediated currents that are induced by stimulation of prefrontal cortical afferents to MSNs, which was inhibited by a CCR2 antagonist. Furthermore, Ccr2 shRNA also reduced NMDAR-mediated currents, and this reduction was mainly mediated via NR2B subunit. Consistently, NR2B, colocalized with CCR2 in the NAc, was phosphorylated after SNL and was inhibited by intra-NAc injection of Ccr2 shRNA. Furthermore, SNL or CCL2 induced ERK activation in the NAc. Inhibition of ERK by a MEK inhibitor reduced NR2B phosphorylation induced by SNL or CCL2. Finally, intra-NAc injection of NR2B antagonist or MEK inhibitor attenuated SNL-induced pain hypersensitivity and depressive behaviors. Collectively, these results suggest that CCL2/CCR2 signaling in the NAc shell is important in mediating neuropathic pain and depression via regulating NR2B-mediated NMDAR function in D1R- and D2R-containing neurons following peripheral nerve injury.

Modulating Macrophage Polarization through CCR2 Inhibition and Multivalent Engagement.

Excessive or prolonged recruitment of inflammatory monocytes to damaged tissue can significantly worsen patient outcomes. Monocytes migrate to sites of tissue inflammation in response to high local concentrations of CCL2, a chemokine that binds to and signals through the CCR2 receptor. While the role of CCR2 in cellular migration is well studied, it is unclear how CCR2 inhibition affects macrophage polarization and if multivalency can increase downstream signaling effects. Using affinity selection with a phage library, we identified a novel single-chain variable fragment (scFv) (58C) that binds specifically and with high affinity to the N-terminal domain of CCR2 ( KD = 59.8 nM). The newly identified 58C-scFv bound to native CCR2 expressed on macrophages and MDA-MB-231 cells, inhibited migration, and induced a pro-inflammatory M1-phenotype in macrophages. The M1/M2 macrophage phenotype ratio for monomeric 58C-scFv was significantly increased over the negative control by 1.0 × 104-fold (iNOS/Arg-1), 5.1 × 104-fold (iNOS/Mgl2), 3.4 × 105-fold (IL-6/Arg-1), and 1.7 × 106-fold (IL-6/Mgl2). The multivalent display of 58C-scFv on liposomes further reduced migration of both cell types by 25-40% and enhanced M1 polarization by 200% over monomeric 58C-scFv. These studies demonstrate that CCR2 inhibition polarizes macrophages toward an inflammatory M1 phenotype, and that multivalent engagement of CCR2 increases the effects of 58C-scFv on polarization and migration. These data provide important insights into the role of multivalency in modulating binding, downstream signaling, and cellular fate.

Highlights of This Issue

The efficacy of PD-1/L1 checkpoint blockade in cancer patients is limited by inadequate T-cell invigoration. Buchan and colleagues report synergism between PD-1/L1 blockade and anti-CD27 mAb for CD8+ T-cell activation and tumor therapy in multiple models, including in human CD27 transgenic mice. They find that combination therapy triggers a convergent program largely dependent on synergistic IL-2 signalling and Myc, but a division of labor such that PD-1/L1 blockade promotes a cytotoxic gene-expression profile, whereas anti-CD27 preferentially drives proliferation. These data provide a mechanistic basis for pursuing the combination of checkpoint blockade and anti-CD27 in the clinic.The central nervous system (CNS) is a sanctuary site from targeted drugs. While the multiple kinase inhibitor, entrectinib, is effective in the treatment of NTRK1-fusion positive cancers with CNS metastasis, resistance usually develops. Nishiyama and colleagues found that the NTRK1-G667C mutation, which induces moderate resistance to entrectinib, caused acquired entrectinib resistance of brain metastasis. Importantly, foretinib inhibited TRK-A with G667C mutation and overcame entrectinib resistance in the liver and brain metastasis models. These findings provide a rationale for clinical trials with foretinib in cancer patients with entrectinib-resistant tumors harboring the NTRK1-G667C mutation, including patients with brain metastases.Preoperative chemotherapy is used in certain groups of breast cancer patients, but many patients do not respond and/or suffer disease progression after treatment. Liu and colleagues show that cytotoxic chemotherapy induces monocytosis in the bone marrow, resulting in increased blood levels of monocyte chemoattractant proteins, which stimulate the cancer stem-like cell phenotype to promote tumor malignancy. The study suggests that pharmacologic inhibition of CCR2 may serve as a potential cotreatment during conventional chemotherapy to target therapy-induced cancer stemness.The prognostic value of the androgen receptor (AR) in breast cancer is controversial, due in part to differences in cohorts and methodology, including the cut-point used for AR positivity. To resolve this controversy, Ricciardelli and colleagues assessed AR positivity in two independent primary breast cancer cohorts and determined the optimal cut-point by ROC analysis. An AR cut-point of 78% positivity, approximating the median staining, provided optimal sensitivity and specificity to predict breast cancer survival. Testing this cut-point in prospective cohorts is now warranted for clinical implementation of AR as a prognostic factor in the management of breast cancer.

Chemotherapy Induces Breast Cancer Stemness in Association with Dysregulated Monocytosis.

Purpose: Preoperative or neoadjuvant therapy (NT) is increasingly used in patients with locally advanced or inflammatory breast cancer to allow optimal surgery and aim for pathologic response. However, many breast cancers are resistant or relapse after treatment. Here, we investigated conjunctive chemotherapy-triggered events occurring systemically and locally, potentially promoting a cancer stem-like cell (CSC) phenotype and contributing to tumor relapse.Experimental Design: We started by comparing the effect of paired pre- and post-NT patient sera on the CSC properties of breast cancer cells. Using cell lines, patient-derived xenograft models, and primary tumors, we investigated the regulation of CSCs and tumor progression by chemotherapy-induced factors.Results: In human patients and mice, we detected a therapy-induced CSC-stimulatory activity in serum, which was attributed to therapy-associated monocytosis leading to systemic elevation of monocyte chemoattractant proteins (MCP). The post-NT hematopoietic regeneration in the bone marrow highlighted both altered monocyte-macrophage differentiation and biased commitment of stimulated hematopoietic stem cells toward monocytosis. Chemotherapeutic agents also induce monocyte expression of MCPs through a JNK-dependent mechanism. Genetic and pharmacologic inhibitions of the MCP-CCR2 pathway blocked chemotherapy's adverse effect on CSCs. Levels of nuclear Notch and ALDH1 were significantly elevated in primary breast cancers following NT, whereas higher levels of CCR2 in pre-NT tumors were associated with a poor response to NT.Conclusions: Our data establish a mechanism of chemotherapy-induced cancer stemness by linking the cellular events in the bone marrow and tumors, and suggest pharmacologic inhibition of CCR2 as a potential cotreatment during conventional chemotherapy in neoadjuvant and adjuvant settings. Clin Cancer Res; 24(10); 2370-82. ©2018 AACR.

Sphingosine kinase 1 promotes liver fibrosis by preventing miR-19b-3p-mediated inhibition of CCR2.

Chronic liver disease mediated by activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs) leads to liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the sphingosine kinase (SphK)1‐dependent effect on liver fibrosis. The levels of expression and activity of SphK1 were significantly increased in fibrotic livers compared with the normal livers in human. SphK1 was coexpressed with a range of HSC/KC markers including desmin, α‐smooth muscle actin (α‐SMA) and F4/80 in fibrotic liver. Deficiency of SphK1 (SphK1−/−) resulted in a marked amelioration of hepatic injury, including transaminase activities, histology, collagen deposition, α‐SMA and inflammation, in CCl4 or bile duct ligation (BDL)‐induced mice. Likewise, treatment with a specific inhibitor of SphK1, 5C, also significantly prevented liver injury and fibrosis in mice induced by CCl4 or BDL. In cellular levels, inhibition of SphK1 significantly blocked the activation and migration of HSCs and KCs. Moreover, SphK1 knockout in KCs reduced the secretion of CCL2, and SphK1 knockout in HSCs reduced C‐C motif chemokine receptor 2 ([CCR2] CCL2 receptor) expression in HSCs. CCL2 in SphK1−/− mice was lower whereas microRNA‐19b‐3p in SphK1−/− mice was higher compared with wild‐type (WT) mice. Furthermore, microRNA‐19b‐3p downregulated CCR2 in HSCs. The functional effect of SphK1 in HSCs on liver fibrosis was further strengthened by the results of animal experiments using a bone marrow transplantation (BMT) method. Conclusion: SphK1 has distinct roles in the activation of KCs and HSCs in liver fibrosis. Mechanistically, SphK1 in KCs mediates CCL2 secretion, and SphK1 in HSCs upregulates CCR2 by downregulation of miR‐19b‐3p. (Hepatology 2018).

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.

Local Delivery Is Critical for Monocyte Chemotactic Protein-1 Mediated Site-Specific Murine Aneurysm Healing.

Local delivery of monocyte chemotactic protein-1 (MCP-1/CCL2) via our drug-eluting coil has been shown to promote intrasaccular aneurysm healing via an inflammatory pathway. In this study, we validate the importance of local MCP-1 in murine aneurysm healing. Whether systemic, rather than local, delivery of MCP-1 can direct site-specific aneurysm healing has significant translational implications. If systemic MCP-1 is effective, then MCP-1 could be administered as a pill rather than by endovascular procedure. Furthermore, we confirm that MCP-1 is the primary effector in our MCP-1 eluting coil-mediated murine aneurysm healing model. We compare aneurysm healing with repeated intraperitoneal MCP-1 versus vehicle injection, in animals with control poly(lactic-co-glycolic) acid (PLGA)-coated coils. We demonstrate elimination of the MCP-1-associated tissue-healing response by knockout of MCP-1 or CCR2 (MCP-1 receptor) and by selectively inhibiting MCP-1 or CCR2. Using immunofluorescent probing, we explore the cell populations found in healed aneurysm tissue following each intervention. Systemically administered MCP-1 with PLGA coil control does not produce comparable aneurysm healing, as seen with MCP-1 eluting coils. MCP-1-directed aneurysm healing is eliminated by selective inhibition of MCP-1 or CCR2 and in MCP-1-deficient or CCR2-deficient mice. No difference was detected in M2 macrophage and myofibroblast/smooth muscle cell staining with systemic MCP-1 versus vehicle in aneurysm wall, but a significant increase in these cell types was observed with MCP-1 eluting coil implant and attenuated by MCP-1/CCR2 blockade or deficiency. We show that systemic MCP-1 concurrent with PLGA-coated platinum coil implant is not sufficient to produce site-specific aneurysm healing. MCP-1 is a critical, not merely complementary, actor in the aneurysm healing pathway.

MT4-MMP deficiency increases patrolling monocyte recruitment to early lesions and accelerates atherosclerosis

Matrix metalloproteinases are involved in vascular remodeling. Little is known about their immune regulatory role in atherosclerosis. Here we show that mice deficient for MT4-MMP have increased adherence of macrophages to inflamed peritonea, and larger lipid deposits and macrophage burden in atherosclerotic plaques. We also demonstrate that MT4-MMP deficiency results in higher numbers of patrolling monocytes crawling and adhered to inflamed endothelia, and the accumulation of Mafb+ apoptosis inhibitor of macrophage (AIM)+ macrophages at incipient atherosclerotic lesions in mice. Functionally, MT4-MMP-null Mafb+AIM+ peritoneal macrophages express higher AIM and scavenger receptor CD36, are more resistant to apoptosis, and bind acLDL avidly, all of which contribute to atherosclerosis. CCR5 inhibition alleviates these effects by hindering the enhanced recruitment of MT4-MMP-null patrolling monocytes to early atherosclerotic lesions, thus blocking Mafb+AIM+ macrophage accumulation and atherosclerosis acceleration. Our results suggest that MT4-MMP targeting may constitute a novel strategy to boost patrolling monocyte activity in early inflammation.

Overall survival in a trial of orally administered CCR2 inhibitor CCX872 in locally advanced/metastatic pancreatic cancer: Correlation with blood monocyte counts.

92 Background: The CCL2-CCR2 signaling axis may facilitate migration of myeloid derived suppressor cells to pancreatic cancer resulting in an immune suppressive tumor microenvironment. CCR2 inhibition in patients with non-metastatic pancreatic cancer was previously reported to decrease tumor-infiltrating macrophages/Treg cells and increase effector T cells (Nywening et al, 2016). Here, a CCR2 specific antagonist CCX872 was used in combination with FOLFIRINOX to treat subjects with locally advanced or metastatic, non-resectable pancreatic cancer in a multi-center study. Methods: Fifty subjects (ECOG score ≤ 2) were enrolled, receiving FOLFIRINOX (fluorouracil [5-FU], leucovorin, irinotecan, oxaliplatin) once every two weeks (maximum 12 cycles) plus 150 mg CCX872 QD or BID for 12 weeks. Subjects showing at least stable disease at the end of the 12-week treatment period were eligible to CCX872 treatment until disease progression. In this ongoing study, all subjects are followed for overall survival (OS). Blood samples were taken at baseline and at intervals throughout the active treatment period for hematologic and flow cytometric analysis of circulating immune cell populations. Results: The all-subjects OS at 18 months is 29%. This compares favorably with previously published data: i.e., OS of only 18.6% at 18 months for FOLFIRINOX regimen alone (Conroy et al, 2011). Peripheral blood monocyte counts at baseline inversely correlate with OS (p = 0.0071, Hazard ratio = 1.169) with CCX872 and FOLFIRINOX combination therapy. Overall, circulating monocytes, inflammatory monocytes and monocytic myeloid derived suppressor cells were reduced by treatment. Conclusions: CCX872-B plus FOLFIRINOX resulted in an OS of 29% at 18 months with no safety issues. Better OS was associated with lower peripheral blood monocyte counts at baseline. Clinical trial information: NCT02345408.

P669 Human β-defensin 2 suppresses TNF-α secretion in human and mouse dendritic cells mediated by chemokine receptor-2

Defensins are small endogenous peptide antibiotics and are part of the innate immune system. They possess broad-spectrum antimicrobial activity against viruses, bacteria, and fungi. Human β-defensins are expressed by epithelial cells in the entire body. Especially human β-defensin-2 (hBD2) has strong antimicrobial and immune modulatory functions and is induced by inflammatory stimuli or exogenous microbial substances. HBD2 promotes intestinal wound healing and angiogenesis in vitro and can act as a chemoattractant for dendritic cells (DCs), monocytes, and T cells through interaction with the chemokine receptor-2 (CCR2) and -6 (CCR6). PBMCs were isolated and stimulated with various bacterial cell wall components such as LPS and were additionally stimulated with or without hBD2. In the next step, we generated human monocyte-derived dendritic cells (mo-DCs) out of PBMCs and pretreated them with a CCR2 inhibitor prior to the stimulation. Furthermore, bone marrow-derived dendritic cells (BM-DCs) were generated out of C57BL/6 or CCR2−/− mice and were treated equally like the human mo-DCs. HBD2 dose-dependently downregulates the pro-inflammatory cytokines TNF-α (p < 0.0001) and IL-1β (p < 0.0001) in LPS stimulated PBMCs while in contrast inducing the secretion of the anti-inflammatory IL-10 (p < 0.001). In a next step, dendritic cells were identified as the probably responsible cell fraction in PBMCs modulated by hBD2. Human mo-DCs (p < 0.0001) as well as murine BM-DCs (p < 0.0001) showed decreasing effects of TNF-α after hBD2 and LPS stimulation. Inhibition of dendritic cell TNF-α production was depending on the presence of CCR2. This effect was completely abolished in CCR2−/− BM-DCs. The data in this study provide evidence that hBD2 acts like an anti-TNF agent. It is a highly efficient immunomodulatory substance which will be further developed to treat inflammatory diseases in man. Herein we describe hBD2 highly efficient as a therapeutic anti-TNF agent.

Sphingosine kinase 1 promotes liver fibrosis by preventing miR-19b-mediated inhibition of CCR2

Sphingosine kinase 1 promotes liver fibrosis by preventing miR-19b-mediated inhibition of CCR2

Targeting both tumour-associated CXCR2+ neutrophils and CCR2+ macrophages disrupts myeloid recruitment and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma

ObjectiveChemokine pathways are co-opted by pancreatic adenocarcinoma (PDAC) to facilitate myeloid cell recruitment from the bone marrow to establish an immunosuppressive tumour microenvironment (TME). Targeting tumour-associated CXCR2+neutrophils (TAN) or tumour-associated...

CCR5-Dependent Homing of T Regulatory Cells to the Tumor Microenvironment Contributes to Skin Squamous Cell Carcinoma Development.

Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. Recent studies show that regulatory T cells (Treg) have a critical role in the modulation of an antitumor immune response, and consequently the SCC development. Because the accumulation of Tregs at the tumor site is, in part, due to selective recruitment through CCR5- and CCR5-associated chemokines, we investigated the role of CCR5 in the SCC development. Our findings showed that CCR5-deficient mice (CCR5KO) were efficient in controlling papilloma's incidence when compared with wild-type mice. Analysis of tumor lesions in wild-type (WT) and CCR5KO mice revealed that lack of CCR5 lead to significant reduction in frequency of Tregs and increased of CD4 T cells into the tumors. Moreover, the adoptive transfer of naturally occurring Tregs CD4+CD25+CCR5+, CD4+CD25-CCR5+ or CD8+CCR5+ conventional T cells to CCR5KO mice resulted in an increased papilloma incidence. Interestingly, adoptive transfer of WT CD4+CD25+CCR5+ cells to CCR5KO mice induced more undifferentiated SCC lesions, characterized by higher infiltration of macrophages and dendritic cells. In this study, we also demonstrated that Treg migration to the tumor microenvironment is mediated by CCR5, and these cells are promoting tumor growth via inhibition of antitumor cells such as cytotoxic CD8+ T cells. Our findings reinforce the therapeutic potential of CCR5 inhibition for cancer treatment, and indicate an attractive approach for SCC treatment. Mol Cancer Ther; 16(12); 2871-80. ©2017 AACR.