Therapy for steatohepatitis: Do macrophages hold the clue?
Authors: Reben Raeman, Frank A. Anania
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. See Article On Page 1270 Supported by the National Institutes of Health (DK062092, DK111678, and DK113147, to F.A.A.; DK110264, to R.R.) and the Emory University School of Medicine (to F.A.A.). Monocytes and macrophages are key regulators of tissue injury, regeneration, and fibrosis.1 While their role in nonalcoholic steatohepatitis (NASH) pathogenesis is not disputed,3 their functional ambiguity and phenotypical diversity complicate our ability to target such cells from a therapeutic perspective. In this issue of hepatology, Krenkel and colleagues advance our understanding of the functional differences in macrophage heterogeneity in NASH pathogenesis.4 Using human tissue and conventional mouse models for NASH, these investigators elegantly demonstrate involvement of a specific subset of bone marrow–derived macrophages, chemokine (C‐C motif) receptor 2 (CCR2+) macrophages, that are summoned to the liver by chemokine (C‐C motif) ligand 2 secreted by Kupffer cells (KCs), hepatic stellate cells, and hepatocytes. Liver‐resident macrophages or KCs (CD11b+F4/80hiLy6C–), monocyte‐derived macrophages (MoMFs; CD11b+F4/80loLy6CLo), and bone marrow–derived monocytes are primary mediators of hepatic inflammation and fibrosis in NASH.3 KCs arise from embryonic progenitors and are sustained in the liver through self‐renewal.5 Monocytes, however, are recruited from the circulation into the injured liver. Two major subsets of monocytes are recruited into the injured liver in NASH, classical or inflammatory monocytes (Ly6ChiCCR2+CX3CRllo in mice, CD14+CD16− in humans) and nonclassical, tissue‐resident, or repair monocytes (Ly6CloCCR2−CX3CR1hi in mice, CD14+CD16++ in humans).3 Ly6Chi classical monocytes are recruited predominantly in a chemokine (C‐C motif) ligand 2/CCR2‐dependent manner, while the CX3CL1/CX3CR1 axis drives recruitment of Ly6Clo nonclassical monocytes.6 Ly6Chi classical monocytes give rise to MoMFs as well as Ly6Clo monocytes, but this is dependent on the local inflammatory milieu (Fig. 1).1 Because KCs, MoMFs, and monocytes are central to the duration of the inflammatory response and its resolution, there is substantial interest in delineating the specific tasks for these players in hepatic inflammation, fibrosis, and repair in NASH.Figure 1: MoMFs perpetuate hepatic fibrosis in NASH. In NASH, damage‐associated molecular patterns released by fat‐laden hepatocytes, pathogen‐associated molecular patterns from the gut, as well as free fatty acids activate liver‐resident KCs (CD11b+F4/80hiLy6C–). Activated KCs recruit circulating Ly6ChiCCR2+ monocytes to the liver in a chemokine (C‐C motif) ligand 2/CCR2‐dependent manner. Ly6ChiCCR2+ monocytes differentiate into proinflammatory, profibrotic MoMFs (CD11b+F4/80loLy6Clo) and, along with activated KCs, promote activation and survival of hepatic stellate cells, resulting in fibrosis. CVC, a CCR2/CCR5 dual antagonist, specifically blocks CCR2/CCR5‐mediated infiltration of Ly6ChiCCR2+ monocytes but does not influence resident KCs. The consequent decrease in MoMF‐mediated hepatic stellate cell activation resolves fibrosis. Abbreviations: CCL2, chemokine (C‐C motif) ligand 2; DAMP, damage‐associated molecular pattern; FFA, free fatty acid; HSC, hepatic stellate cell; PAMP, pathogen‐associated molecular pattern.The scientific premise of the current study by Krenkel et al.4 is based on human data that demonstrate a significantly greater number of CCR2+ macrophages in patients with NASH and fibrosis (stage 3) as well as in patients diagnosed with NASH and cirrhosis (stage 4). These data support the authors' rationale to prevent hepatic infiltration of CCR2+ MoMFs using cenicriviroc (CVC; Tobira), an oral, once‐daily dual CCR2/CCR5 antagonist with nanomolar potency and a long half‐life in humans.8 A phase 2b clinical trial (NCT02217475) is now nearing completion with CVC,8 and results of the study were recently reported in this journal.9 The phase 2b data reveal improvement in the fibrosis score by one or more stage with no worsening of steatohepatitis after 1 year of CVC treatment. While CVC reduced systemic markers of inflammation including interleukins 1 and 6, fibrinogen, and C‐reactive protein, no difference in the inflammatory scores on liver biopsy between patients receiving placebo or CVC was observed.9 Thus, the trial failed to meet either of the primary study endpoints, resolution of inflammation and steatosis; however, as noted by study investigators a phase 3 trial is under way.9 The current work by Krenkel et al. therefore, picks up where the clinical data concerning CVC left off, by providing key insights into how a dual CCR2/CCR5 antagonist would be beneficial in attenuating fibrosis in NASH. The investigators wisely choose two feeding strategies to study the mechanisms of CVC‐mediated protection from NASH—a western diet (WD) and the methionine/choline‐deficient (MCD) diet. The former diet administered to mice produces all of the features of metabolic syndrome including hepatic steatosis. While the MCD diet does not produce significant metabolic derangements, its use results in robust steatofibrosis. The treatment strategy—to gavage mice with CVC—was also wise because it mimics the clinical trial and circumvents the short half‐life of parenterally administered CVC in mice. Additionally, parenteral administration detracts from translational relevance because approved drugs for NASH and fibrosis would optimally be administered orally. In the WD model, CVC treatment reduced infiltration of MoMFs identified as CD11b++F4/80+ cells in the liver but did not affect KCs identified as CD11b+F4/80++ cells. The authors importantly demonstrated that CVC specifically inhibited infiltration of MoMFs but not other immune cells including T cells, B cells, and natural killer T cells. In the WD‐fed mice, CVC treatment decreased hepatic triglyceride content and the liver weight/body weight ratio and improved glucose tolerance but did not affect body weight or fat weight. However, the metabolic improvements in CVC‐treated mice were marginal and not robust. In the MCD diet–fed mice, CVC inhibited both F4/80+ macrophage and MoMF infiltration in the liver. It is not clear, however, whether CVC also affected the KC population because no KC data were provided. As anticipated, CVC treatment attenuated hepatic fibrosis and resulted in one‐point improvement in the overall nonalcoholic fatty liver disease activity score but failed to improve steatosis and lobular inflammation scores. While the investigators did not provide NASH histology scoring for the WD‐fed mice, the histology scores presented from the CVC‐gavaged, MCD diet–fed mice are similar to the human data reported from the phase 2b clinical trial. To further investigate the profibrotic role of MoMFs, the authors performed RNA sequencing on isolated hepatic MoMFs and KCs from MCD diet–fed mice. The gene expression profile revealed that both KCs and MoMFs are involved in driving hepatic inflammation in NASH. Both cellular subsets also express growth factors involved in angiogenesis and anti‐inflammatory cytokines; however, expression of these factors was significantly up‐regulated in the MoMFs suggesting a distinguishing role for MoMFs in injury resolution compared to resident KCs. Finally, based on their RNA‐sequencing data, the investigators performed a follow‐up experiment to determine whether MoMF depletion during resolution of hepatic injury and fibrosis in mice fed the MCD diet would affect tissue repair. Histological data demonstrated significantly greater resolution of fibrosis in the CVC‐treated, MCD diet–fed mice compared to vehicle‐treated mice, suggesting that the absence of MoMFs did not hamper tissue repair or fibrosis resolution. While no data on the infiltrating immune cells or resident KCs are reported in this experiment, we presume that KCs and Ly6Clo monocytes compensate for the lack of MoMF and promote resolution of injury. Together, the data from the WD and MCD diet models strongly suggest that MoMFs are critical to the genesis of hepatic fibrosis in NASH. In conclusion, the article by Krenkel et al. not only provides challenges to existing paradigms concerning macrophage polarization but foreshadows the molecular and cellular bases for what is likely to be an avalanche of human clinical studies for both nonalcoholic fatty liver disease and its sequelae (e.g., NASH‐related fibrosis). This study, together with the outcome of the early‐phase clinical trial, provides compelling evidence that MoMFs are involved in driving hepatic fibrosis but that they do not play a major role in hepatic inflammation and steatosis. Despite these exciting data related to NASH fibrosis, many questions remain unanswered. The inability of CVC to block infiltration of lymphocytes including natural killer cells and T cells, despite a decrease in CCR5 expression in CVC‐treated mice, may explain the lack of improvement in inflammation. This, however, also raises the question of whether CVC failed to inhibit T‐cell infiltration in the NASH patients and consequently failed to improve their inflammatory scores on liver biopsy. Clearly, substantial work is still needed to understand the effect of CVC on immune cells other than KCs and MoMFs in NASH. Lastly, these studies point to the complexity of NASH pathogenesis in patients and raise the need for more thorough studies with human tissue samples to facilitate a greater understanding of the role of multiple immune cell types involved in human disease. Author names in bold designate shared co‐first authorship.