Novel Mechanisms for Resolution of Liver Inflammation: Therapeutic Implications.

Traditional concepts have classically viewed resolution of inflammation as a passive process yet insight into the pathways by which inflammation is resolved has challenged this idea. Resolution has been revealed as a highly dynamic and active event that is essential to counteract the dysregulated inflammatory response that drives diverse disease states. Abrogation of the hepatic inflammatory response through the stimulation of proresolving mechanisms represents a new paradigm in the setting of chronic inflammatory-driven liver diseases. Elucidation of the role of different cells of the innate and adaptive immune system has highlighted the interplay between them as an important orchestrator of liver repair. A finely tuned interaction between neutrophils and macrophages has risen as revolutionary mechanism that drives the restoration of hepatic function and architecture. Specialized proresolving mediators have also been shown to act as stop signals of the inflammatory response and promote resolution as well as tissue regeneration. In this review, we discuss the discovery and understanding of the mechanisms by which inflammation is resolved and highlight novel proresolving pathways that represent promising therapeutic strategies.

BRP39 Regulates Neutrophil Recruitment in NLRP3 Inflammasome-Induced Liver Inflammation.

BACKGROUND & AIMS: Breast regression protein 39 (BRP39) (Chi3L1) and its human homolog YKL-40, is an established biomarker of liver fibrosis in nonalcoholic steatohepatitis (NASH) patients, but its role in NASH pathogenesis remains unclear. We recently identified Chi3L1 as one of the top up-regulated genes in mice with inducible gain-of-function NOD-like receptor protein 3 (NLRP3) activation that mimics several liver features of NASH. This study aimed to investigate the effects of BRP39 deficiency on NLRP3-induced liver inflammation using tamoxifen-inducible Nlrp3 knockin mice sufficient (Nlrp3A350V CRT) and deficient for BRP39 (Nlrp3A350V/BRP-/- CRT). METHODS: Using Nlrp3A350V CRT mice and Nlrp3A350V BRP-/- CRT, we investigated the consequences of BRP39 deficiency influencing NLRP3-induced liver inflammation. RESULTS: Our results showed that BRP39 deficiency in NLRP3-induced inflammation improved body weight and liver weight. Moreover, liver inflammation, fibrosis, and hepatic stellate cell activation were reduced significantly, corresponding to significantly decreased Ly6C+ infiltrating macrophages, CD68+ osteopontin-positive hepatic lipid-associated macrophages, and activated Lymphocyte antigen 6 complex locus G6D positive (Ly6G+) and citrullinated histone H3 postivie (H3Cit+) neutrophil accumulation in the liver. Further investigation showed that circulatory neutrophils from NLRP3-induced BRP39-deficient mice have impaired chemotaxis and migration ability, and this was confirmed by RNA bulk sequencing showing reduced immune activation, migration, and signaling responses in neutrophils. CONCLUSIONS: These data showcase the importance of BRP39 in regulating the NLRP3 inflammasome during liver inflammation and fibrotic NASH by altering cellular activation, recruitment, and infiltration during disease progression, and revealing BRP39 to be a potential therapeutic target for future treatment of inflammatory NASH and its associated diseases.

Myeloid-specific deletion of chitinase-3-like 1 protein ameliorates murine diet-induced steatohepatitis progression.

Chitinase-3-like 1 protein (CHI3L1) is a secreted glycoprotein, strongly correlated with fibrosis severity in chronic liver diseases including non-alcoholic steatohepatitis (NASH). However, the mechanisms by which CHI3L1 contributes to fibrogenesis remain undefined. Here, we showed that infiltrating monocyte-derived liver macrophages represent the main source of CHI3L1 in murine NASH. We developed a floxed CHI3L1 knock-out (KO) mouse to further study the cell-specific role of CHI3L1 ablation. Wildtype (WT) and myeloid cell-specific CHI3L1 KO mice (CreLyz) were challenged with a highly inflammatory and fibrotic dietary model of NASH by administering choline-deficient high-fat diet for 10 weeks. Macrophage accumulation and inflammatory cell recruitment were significantly ameliorated in the CreLyz group compared to WT (F4/80 IHC p < 0.0001, CD11b IHC p < 0.0001). Additionally, hepatic stellate cell (HSC) activation and fibrosis were strongly decreased in this group (α-SMA IHC p < 0.0001, picrosirius red staining p < 0.0001). In vitro studies were performed stimulating bone marrow derived macrophages, THP-1 (human monocytes) and LX2 (human HSCs) cells with recombinant CHI3L1 to dissect its relationship with fibrosis development. Results showed an important role of CHI3L1 regulating fibrosis-promoting factors by macrophages (TGFB1 p < 0.05, CTGF p < 0.01) while directly activating HSCs (ACTA2 p < 0.01, COL1A1 p < 0.01), involving IL13Rα2 as the potential mediator. Our findings uncovered a novel role of CHI3L1 derived from liver macrophages in NASH progression and identifies this protein as a potential anti-fibrotic therapeutic target. KEY MESSAGES: We showed that CHI3L1 expression is increased in murine CDAA-HFAT diet NASH model, and that infiltrating macrophages are a key source of CHI3L1 production. Myeloid cell-specific CreLyz CHI3L1 knock-out in mice fed with CDAA-HFAT diet improved the NASH phenotype, with significantly reduced accumulation of pro-inflammatory macrophages and neutrophils compared with WT group. DEG and qPCR analysis of genes in CreLyz CHI3L1 knock-out mouse liver showed the mechanistic role of CHI3L1 in cellular chemotaxis. HSC is directly activated by CHI3L1 via receptor IL13Rα2, leading to upregulation of collagen deposition and pro-fibrotic gene, TIMP-1 and TIMP-2 release in whole liver. Direct stimulation of macrophages with CHI3L1 leads to upregulated expression of HSC-activation factors, suggesting its role in modulating macrophage-HSC crosstalk.

Cell-specific Deletion of NLRP3 Inflammasome Identifies Myeloid Cells as Key Drivers of Liver Inflammation and Fibrosis in Murine Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. The NLRP3 inflammasome, a platform for caspase-1 activation and release of interleukin 1ß, is increasingly recognized in the induction of inflammation and liver fibrosis during NAFLD. However, the cell-specific contribution of NLRP3 inflammasome activation in NAFLD remains unknown. METHODS: To investigate the role of NLRP3 inflammasome activation in hepatocytes, hepatic stellate cells (HSCs) and myeloid cells, a conditional Nlrp3 knock-out mouse was generated and bred to cell-specific Cre mice. Both acute and chronic liver injury models were used: lipopolysaccharide/adenosine-triphosphate to induce in vivo NLRP3 activation, choline-deficient, L-amino acid-defined high-fat diet, and Western-type diet to induce fibrotic nonalcoholic steatohepatitis (NASH). In vitro co-culture studies were performed to dissect the crosstalk between myeloid cells and HSCs. RESULTS: Myeloid-specific deletion of Nlrp3 blunted the systemic and hepatic increase in interleukin 1ß induced by lipopolysaccharide/adenosine-triphosphate injection. In the choline-deficient, L-amino acid-defined high-fat diet model of fibrotic NASH, myeloid-specific Nlrp3 knock-out but not hepatocyte- or HSC-specific knock-out mice showed significant reduction in inflammation independent of steatosis development. Moreover, myeloid-specific Nlrp3 knock-out mice showed ameliorated liver fibrosis and decreased HSC activation. These results were validated in the Western-type diet model. In vitro co-cultured studies with human cell lines demonstrated that HSC can be activated by inflammasome stimulation in monocytes, and this effect was significantly reduced if NLRP3 was downregulated in monocytes. CONCLUSIONS: The study provides new insights in the cell-specific role of NLRP3 in liver inflammation and fibrosis. NLRP3 inflammasome activation in myeloid cells was identified as crucial for the progression of NAFLD to fibrotic NASH. These results may have implications for the development of cell-specific strategies for modulation of NLRP3 activation for treatment of fibrotic NASH.

Dual role of neutrophils in modulating liver injury and fibrosis during development and resolution of diet-induced murine steatohepatitis.

Inflammatory changes in the liver represent a key feature of non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD). Innate immune activation including hepatic neutrophilic infiltration acts as an important inflammatory trigger as well as a potential mediator of inflammation resolution. In this study, we dissected the effects of neutrophil depletion via anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies administration during ongoing high fat-fructose-cholesterol (FFC) diet-induced murine NASH and during inflammation resolution by switching into a low-fat control diet. During NASH progression, protective effects were shown as HSC activation, cell infiltration and activation of pro-inflammatory macrophages were ameliorated. Furthermore, these changes were contrasted with the effects observed when neutrophil depletion was performed during the resolution phase. Impaired resolving mechanisms, such as a failure to balance the pro and anti-inflammatory cytokines ratio, deficient macrophage phenotypic switch into a pro-restorative profile, and defective repair and remodeling processes were observed when neutrophils were depleted in this scenario. This study described phase-dependent contrasting roles of neutrophils as triggers and pro-resolutive mediators of liver injury and fibrosis associated with diet-induced NASH in mice. These findings have important translational implications at the time of designing NASH therapeutic strategies.