Differential impact of the dual CCR2/CCR5 inhibitor cenicriviroc on migration of monocyte and lymphocyte subsets in acute liver injury.
ABSTRACT: A hallmark of acute hepatic injury is the recruitment of neutrophils, monocytes and lymphocytes, including natural killer (NK) or T cells, towards areas of inflammation. The recruitment of leukocytes from their reservoirs bone marrow or spleen into the liver is directed by chemokines such as CCL2 (for monocytes) and CCL5 (for lymphocytes). We herein elucidated the impact of chemokine receptor inhibition by the dual CCR2 and CCR5 inhibitor cenicriviroc (CVC) on the composition of myeloid and lymphoid immune cell populations in acute liver injury. CVC treatment effectively inhibited the migration of bone marrow monocytes and splenic lymphocytes (NK, CD4 T-cells) towards CCL2 or CCL5 in vitro. When liver injury was induced by an intraperitoneal injection of carbon tetrachloride (CCl4) in mice, followed by repetitive oral application of CVC, flow cytometric and unbiased t-SNE analysis of intrahepatic leukocytes demonstrated that dual CCR2/CCR5 inhibition in vivo significantly decreased numbers of monocyte derived macrophages in acutely injured livers. CVC also reduced numbers of Kupffer cells (KC) or monocyte derived macrophages with a KC-like phenotype, respectively, after injury. In contrast to the inhibitory effects in vitro, CVC had no impact on the composition of hepatic lymphoid cell populations in vivo. Effective inhibition of monocyte recruitment was associated with reduced inflammatory macrophage markers and moderately ameliorated hepatic necroses at 36h after CCl4. In conclusion, dual CCR2/CCR5 inhibition primarily translates into reduced monocyte recruitment in acute liver injury in vivo, suggesting that this strategy will be effective in reducing inflammatory macrophages in conditions of liver disease.
Project description:The CCR2/CCR5 inhibitor cenicriviroc blocks infiltration of inflammatory monocytes into the liver, thereby reducing NASH progression and fibrosis. Overall design: The study demonstrates the hepatic accumulation of inflammatory CCR2+ macrophages in patients with NASH fibrosis and reveals that the dual CCR2/CCR5 inhibitor CVC effectively ameliorates steatohepatitis and fibrosis in mouse models by inhibiting the infiltration of monocytes during chronic liver injury. Thus, this study provides functional evidence for the successful therapeutic targeting of monocyte recruitment in steatohepatitis and fibrosis, mandating the clinical development of CCR2/CCR5 inhibitors in patients with NASH.
Project description:Interactions between C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC) is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC's anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis.Monocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC's antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH) and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses.CVC significantly reduced monocyte/macrophage recruitment in vivo at doses ?20 mg/kg/day (p < 0.05). At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05), and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls). CVC treatment had no notable effect on body or liver/kidney weight.CVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC's antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475).
Project description:BACKGROUND & AIMS:Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC. METHODS:Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2-/- mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2-/- mice) were determined in BV6-injected mice. RESULTS:We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC. CONCLUSIONS:Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target. LAY SUMMARY:Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.
Project description:<h4>Background & aims</h4>Macrophages are key regulators of inflammation and cancer promotion in the liver, and their recruitment and activation is linked to chemokine receptor signaling. However, the exact roles of the chemokine receptors CCR2 and CCR5 for macrophage functions in the liver is obscure.<h4>Methods</h4>To study CCR2 and CCR5 in inflammatory liver injury, we used mice with a hepatocyte-specific knock-out of the nuclear factor ?B (NF-?B) essential modulator (NEMO), termed NEMO<sup>LPC-KO</sup> mice, and generated NEMO<sup>LPC-KO</sup>Ccr2<sup>-/-</sup> and NEMO<sup>LPC-KO</sup>Ccr5<sup>-/-</sup> mice. NEMO<sup>LPC-KO</sup> mice develop hepatitis and fibrosis after two and liver tumors after six months.<h4>Results</h4>We found that both CCR2 and CCR5 deficiency led to reduced fibrosis, while CCR5 deficiency increased steatosis and tumor burden in NEMO<sup>LPC-KO</sup> mice. CCR2 was required for recruitment of hepatic macrophages, whereas CCR5 promoted stellate cell activation. The reduction of monocytes and macrophages by either anti-Gr1 antibody or clodronate-loaded liposomes (CLL), but not of CD8<sup>+</sup> T cells or NK cells, significantly aggravated liver injury in NEMO<sup>LPC-KO</sup> mice and was further increased in NEMO<sup>LPC-KO</sup>Ccr5<sup>-/-</sup> mice. CLL-induced liver injury was dampened by the adoptive transfer of ex vivo generated macrophages, whereas the adoptive transfer of control CD115<sup>+</sup> immature monocytes or B cells did not reduce liver injury.<h4>Conclusions</h4>Although CCR2 and CCR5 principally promote liver fibrosis, they exert differential functions on hepatic macrophages during liver disease progression in NEMO<sup>LPC-KO</sup> mice. While CCR2 controls the recruitment of monocytes to injured livers, CCR5-dependent functions of liver macrophages limit hepatic injury, thereby reducing steatosis and hepatocarcinogenesis.
Project description:OBJECTIVE:To evaluate changes in neuropsychological (NP) performance and in plasma and cell surface markers of peripheral monocyte activation/migration after treatment with cenicriviroc (CVC), a dual C-C chemokine receptor type 2 (CCR2) and type 5 (CCR5) antagonist, in treatment-experienced, HIV-infected individuals. SETTING:Single-arm, 24-week, open-label clinical trial. METHODS:HIV-infected individuals on antiretroviral therapy ?1 year with plasma HIV RNA ?50 copies per milliliter and below-normal cognitive performance [defined as age-, sex-, and education-adjusted NP performance (NPZ) <-0.5 in a single cognitive domain or in global performance] were enrolled. Changes over 24 weeks were assessed for global and domain-specific NPZ scores, plasma markers of monocyte/macrophage activation [neopterin, soluble (s)CD14, and sCD163] quantified by ELISA, and CCR2 and CCR5 expression on monocytes, and T cells measured by flow cytometry. RESULTS:Seventeen of 20 enrolled participants completed the study. Improvements over 24 weeks were observed in global NPZ [median change (?) = 0.24; P = 0.008], and in cognitive domains of attention (?0.23; P = 0.011) and working memory (?0.44; P = 0.017). Plasma levels of sCD163, sCD14 and neopterin decreased significantly (P's < 0.01). CCR2 and CCR5 monocyte expression remained unchanged; however, CCR5 levels on CD4 and CD8 T cells and CCR2 expression on CD4 T cells increased (P's < 0.01). CONCLUSIONS:CVC given over 24 weeks was associated with improved NP test performance and decreased plasma markers of monocyte immune activation in virally suppressed, HIV-infected participants. These data potentially link changes in monocyte activation to cognitive performance. Further study of CVC for HIV cognitive impairment in a randomized controlled study is warranted.
Project description:BACKGROUND: Monocyte-derived macrophages critically perpetuate inflammatory responses after liver injury as a prerequisite for organ fibrosis. Experimental murine models identified an essential role for the CCR2-dependent infiltration of classical Gr1/Ly6C(+) monocytes in hepatic fibrosis. Moreover, the monocyte-related chemokine receptors CCR1 and CCR5 were recently recognized as important fibrosis modulators in mice. In humans, monocytes consist of classical CD14(+)CD16(-) and non-classical CD14(+)CD16(+) cells. We aimed at investigating the relevance of monocyte subpopulations for human liver fibrosis, and hypothesized that 'non-classical' monocytes critically exert inflammatory as well as profibrogenic functions in patients during liver disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed circulating monocyte subsets from freshly drawn blood samples of 226 patients with chronic liver disease (CLD) and 184 healthy controls by FACS analysis. Circulating monocytes were significantly expanded in CLD-patients compared to controls with a marked increase of the non-classical CD14(+)CD16(+) subset that showed an activated phenotype in patients and correlated with proinflammatory cytokines and clinical progression. Correspondingly, CD14(+)CD16(+) macrophages massively accumulated in fibrotic/cirrhotic livers, as evidenced by immunofluorescence and FACS. Ligands of monocyte-related chemokine receptors CCR2, CCR1 and CCR5 were expressed at higher levels in fibrotic and cirrhotic livers, while CCL3 and CCL4 were also systemically elevated in CLD-patients. Isolated monocyte/macrophage subpopulations were functionally characterized regarding cytokine/chemokine expression and interactions with primary human hepatic stellate cells (HSC) in vitro. CD14(+)CD16(+) monocytes released abundant proinflammatory cytokines. Furthermore, CD14(+)CD16(+), but not CD14(+)CD16(-) monocytes could directly activate collagen-producing HSC. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate the expansion of CD14(+)CD16(+) monocytes in the circulation and liver of CLD-patients upon disease progression and suggest their functional contribution to the perpetuation of intrahepatic inflammation and profibrogenic HSC activation in liver cirrhosis. The modulation of monocyte-subset recruitment into the liver via chemokines/chemokine receptors and their subsequent differentiation may represent promising approaches for therapeutic interventions in human liver fibrosis.
Project description:Molecular profiling of infiltrating monocyte-derived macrophages versus resident kupffer cells following acute liver injury The liver has a remarkable capacity to regenerate after injury; yet, the role of macrophages (MF) in this process remains controversial mainly due to difficulties in distinguishing between different MF-subsets. Here, we utilized a murine model of acute liver injury caused by overdose of acetaminophen (APAP) and defined three distinct MF subsets that populate the liver following injury. Accordingly, resident Kupffer cells (KC) were significantly reduced upon APAP-challenge and started recovering by self-renewal at resolution phase without contribution of circulating Ly6Chi monocytes. The latter were recruited in a CCR2 and M-CSF mediated pathway at the necro-inflammatory phase and differentiated into ephemeral Ly6Clo MF subset at resolution phase. Moreover, their inducible ablation resulted in impaired recovery. Microarray based molecular profiling uncovered high similarity between steady state KC and those recovered at the resolution phase. In contrast, KC and monocyte-derived MF displayed distinct pro-restorative genetic signature at the resolution phase. Finally, we show that infiltrating monocytes acquire a pro-restorative polarization manifested by unique expression of pro-angiogenesis mediators and genes involved with inhibition of neutrophil activity and recruitment and promotion of their clearance. Collectively, our results present a novel phenotypic, ontogenic and molecular definition of liver-MF compartment following acute injury. 11 Samples (arrays) were performed. We generated pairwise comparison between all the different macrophages stages, using Partek Genomics Suite. Genes with p?5%[FDR] and a fold-change difference of ?2 or <-2 were selected.
Project description:The therapy of primary biliary cholangitis (PBC) has lagged behind other autoimmune diseases despite significant improvements in our understanding of both immunological and molecular events that lead to loss of tolerance to the E2 component of pyruvate dehydrogenase, the immunodominant autoepitope of PBC. It is well known that Ly6Chi monocytes are innate immune cells infiltrating inflammatory sites that are dependent on the expression of C-C motif chemokine receptor 2 (CCR2) for emigration from bone marrow. Importantly, humans with PBC have a circulating monocyte pro-inflammatory phenotype with macrophage accumulation in portal tracts. We have taken advantage of an inducible chemical xenobiotic model of PBC and recapitulated the massive infiltration of monocytes to portal areas. To determine the clinical significance, we immunized both CCR2-deficient mice and controls with 2OA-BSA and noted that CCR2 deficiency is protective for the development of autoimmune cholangitis. Importantly, because of the therapeutic potential, we focused on inhibiting monocyte infiltration through the use of cenicriviroc (CVC), a dual chemokine receptor CCR2/CCR5 antagonist shown to be safe in human trials. Importantly, treatment with CVC resulted in amelioration of all aspects of disease severity including serum total bile acids, histological severity score, and fibrosis stage. In conclusion, our results indicate a major role for Ly6Chi monocytes and for CCR2 in PBC pathogenesis and suggest that inhibition of this axis by CVC should be explored in humans through the use of clinical trials.
Project description:Incidences of cardiovascular diseases (CVD) are high among virologically suppressed HIV-infected individuals. Monocyte activation and trafficking are key mechanisms in the evolution of CVD. We studied the ability of cenicriviroc (CVC), a dual C-C chemokine receptor type 2 (CCR2) and CCR5 antagonist, to influence the migration of monocytes from HIV-infected individuals on antiretroviral therapy (ART). Monocytes were derived from 23 ART-suppressed HIV-infected and 16 HIV-uninfected donors. In a trans-endothelial migration model, monocytes, and human aortic endothelial cells (HAoECs) were exposed to cenicriviroc and migrated monocytes, quantified. Expression of CCR2 and CCR5 on monocytes and adhesion molecules (E-selectin, ICAM-1, VCAM-1, PECAM-1, and CD99) on HAoECs were measured. The single antagonists, BMS-22 (CCR2), and maraviroc (CCR5), served as controls. When both HAoECs and monocytes together were exposed to the antagonists, cenicriviroc led to a greater decrease in monocyte migration compared to BMS-22 or vehicle in both HIV-infected and HIV-uninfected groups (P < 0.05), with maraviroc having no inhibitory effect. Cenicriviroc treatment of HAoECs alone decreased monocyte migration in the HIV-infected group when compared to vehicle (P < 0.01). Inhibition of migration was not evident when monocytes alone were exposed to cenicriviroc, BMS-22 or maraviroc. Incubation of HAoECs with cenicriviroc decreased E-selectin expression (P = 0.045) but had limited effects on the other adhesion molecules. Cenicriviroc inhibits monocyte trans-endothelial migration more effectively than single chemokine receptor blockade, which may be mediated via disruption of monocyte-endothelial tethering through reduced E-selectin expression. Cenicriviroc should be considered as a therapeutic intervention to reduce detrimental monocyte trafficking.
Project description:BACKGROUND AND AIMS:Liver and systemic inflammatory factors influence monocyte phenotype and function, which has implications for hepatic recruitment and subsequent inflammatory and fibrogenic responses, as well as host defence. METHODS:Peripheral blood monocyte surface marker (CD14, CD16, CD163, CSF1R, CCR2, CCR4, CCR5, CXCR3, CXCR4, CX3CR1, HLA-DR, CD62L, SIGLEC-1) expression and capacity for phagocytosis, oxidative burst and LPS-stimulated TNF production were assessed in patients with hepatitis C (HCV) (n = 39) or non-alcoholic fatty liver disease (NAFLD) (n = 34) (classified as non-advanced disease, compensated cirrhosis and decompensated cirrhosis) and healthy controls (n = 11) by flow cytometry. RESULTS:The selected markers exhibited similar monocyte-subset-specific expression patterns between patients and controls. Monocyte phenotypic signatures differed between NAFLD and HCV patients, with an increased proportion of CD16+ non-classical monocytes in NAFLD, but increased expression of CXCR3 and CXCR4 in HCV. In both cohorts, monocyte CCR2 expression was reduced and CCR4 elevated over controls. CD62L expression was specifically elevated in patients with decompensated cirrhosis and positively correlated with the model-for-end-stage-liver-disease score. Functionally, monocytes from patients with decompensated cirrhosis had equal phagocytic capacity, but displayed features of dysfunction, characterised by lower HLA-DR expression and blunted oxidative responses. Lower monocyte TNF production in response to LPS stimulation correlated with time to death in 7 (46%) of the decompensated patients who died within 8 months of recruitment. CONCLUSIONS:Chronic HCV and NAFLD differentially affect circulating monocyte phenotype, suggesting specific injury-induced signals may contribute to hepatic monocyte recruitment and systemic activation state. Monocyte function, however, was similarly impaired in patients with both HCV and NAFLD, particularly in advanced disease, which likely contributes to the increased susceptibility to infection in these patients.