Bile acids (BA) as important signaling molecules are considered crucial in development of cholestatic liver injury, but there is limited understanding on the involved cell types and signaling pathways. The aim of this study was to evaluate the inflammatory and fibrotic potential of key BA and the role of distinct liver cell subsets focusing on the NLRP3 inflammasome. C57BL/6 wild-type (WT) and Nlrp3−/− mice were fed with a diet supplemented with cholic (CA), deoxycholic (DCA) or lithocholic acid (LCA) for 7 days. Additionally, primary hepatocytes, Kupffer cells (KC) and hepatic stellate cells (HSC) from WT and Nlrp3−/− mice were stimulated with aforementioned BA ex vivo. LCA feeding led to strong liver damage and activation of NLRP3 inflammasome. Ex vivo KC were the most affected cells by LCA, resulting in a pro-inflammatory phenotype. Liver damage and primary KC activation was both ameliorated in Nlrp3-deficient mice or cells. DCA feeding induced fibrotic alterations. Primary HSC upregulated the NLRP3 inflammasome and early fibrotic markers when stimulated with DCA, but not LCA. Pro-fibrogenic signals in liver and primary HSC were attenuated in Nlrp3−/− mice or cells. The data shows that distinct BA induce NLRP3 inflammasome activation in HSC or KC, promoting fibrosis or inflammation.

Financial support for this work was received from the National Natural Science Foundation of China under grant no. 81400586 and Zhejiang Provincial Natural Science Foundation of China under grant no. LQ14H030002. Potential conflict of interest: Nothing to report. Author names in bold designate shared co‐first authorship. To the Editor: Emerging evidence underscores the importance of the gut‐liver axis in the pathogenesis of nonalcoholic fatty liver disease.1 A key role in the maintenance of gut‐liver axis health has been attributed to intestinal microbiota. Intestinal dysbiosis disrupts intestinal barrier integrity, which facilitates intestinal leakiness and translocation of intestinal‐derived bacteria or bacterial products to the liver. These foreign antigens trigger an inflammatory response that contributes to the development and progression of steatohepatitis. The intestinal immune system recognizes antigens specifically and compartmentalizes intestinal immune response to avoid inflammation. Key participants in intestinal innate immunity are intestinal mononuclear phagocytes (MNPs), which comprise dendritic cells (DCs) and macrophages (Mφs). MNPs not only kill pathogens directly, but also present antigens to immune effector cells, which elicit innate and adaptive immune responses that limit microbial translocation to the portal vein. MNPs can be subdivided based on expression of CD103 or CX3CR1, whereas CX3CR1+ MNPs migrate to draining lymph nodes and initiate immune responses under conditions of dysbiosis.2 Schneider et al. recently recognized the central role of CX3CR1 in microbiota‐mediated activation of innate immune responses, which participated in the procession of diet‐induced steatohepatitis.3 Using Cx3cr1‐deficient mice, they concluded that CX3CR1 signaling was crucial in maintaining intestinal barrier integrity. Lack of Cx3cr1 expression was associated with reduced numbers of intestinal Mφs and increased bacterial translocation. It is noteworthy that the roles of CX3CR1+MHCIIhiCD11b+CD11c+F4/80+ Mφs were determined in their study whereas CX3CR1+MHCIIhiCD11b–CD11c+F4/80– DCs were not evaluated. CX3CR1+ DCs are also key regulators of intestinal immune homeostasis and inflammation as Mφs. CX3CR1+ DCs are on the front line in maintaining intestinal integrity, which can form transepithelial dendrites to phagocytose luminal pathogenic bacteria directly.4 CX3CR1+ DCs display poor T‐cell stimulatory capacity under steady‐state conditions while they shuttle harmful pathogens to mesenteric lymph nodes in the case of dysbiosis, where they trigger specific immune response through a MyD88‐dependent signaling pathway. Niess et al. suggested that CX3CR1‐dependent processes, which controlled host interactions of specialized DCs with commensal and pathogenic bacteria, might regulate immunological tolerance and inflammation.4 It was recently reported that CX3CR1+ DCs could up‐regulate CCR7 and migrate to secondary lymphoid organs, suggesting a broader role in orchestrating immunity.2 Therefore, CX3CR1+ DCs are essential for both induction of active immunity and maintenance of barrier homeostasis. Despite phagocytosing bacteria or presenting antigens to immune effector cells similar to Mφs, CX3CR1+ DCs may play different roles in recognizing and responding to different microorganisms by producing distinct patterns of inflammatory cytokines. CX3CR1+ DCs may participate in maintaining intestinal integrity alone or in combination with Mφs to limit bacterial translocation to the liver by multiple mechanisms. Their specific functions can be studied in vivo by generating novel mouse strains, allowing depletion of each cell population. Without a thorough understanding of the activities of CX3CR1+ DCs within the intestinal immune system, it is not possible to decipher the role of CX3CR1 in the maintenance of gut‐liver axis health. The incomplete evidence might lead to unreliable conclusions. Because the specific roles of CX3CR1+ DCs were not examined in their study, can the authors explain the validity of their conclusions in the absence of studying such DCs?

Non-alcoholic steatohepatitis (NASH) is characterized by liver lipid accumulation and inflammation. The mechanisms that trigger hepatic inflammation are poorly understood and subsequently, no specific non-invasive markers exist. We previously demonstrated a reduction in the plasma lysosomal enzyme, cathepsin D (CatD), in children with NASH compared to children without NASH. Recent studies have raised the concept that non-alcoholic fatty liver disease (NAFLD) in adults is distinct from children due to a different histological pattern in the liver. Yet, the link between plasma CatD to adult NASH was not examined. In the current manuscript, we investigated whether plasma CatD in adults correlates with NASH development and regression. Biopsies were histologically evaluated for inflammation and NAFLD in three complementary cohorts of adults (total n = 248). CatD and alanine aminotransferase (ALT) were measured in plasma. Opposite to our previous observations with childhood NASH, we observed increased levels of plasma CatD in patients with NASH compared to adults without hepatic inflammation. Furthermore, after surgical intervention, we found a reduction of plasma CatD compared to baseline. Our observations highlight a distinct pathophysiology between NASH in children and adults. The observation that plasma CatD correlated with NASH development and regression is promising for NASH diagnosis.

Background & Aims: Non-alcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation and inflammation. Currently, the underlying mechanisms, leading to hepatic inflammation, are still unknown. The breakdown of free cholesterol inside Kupffer cells (KCs) by the mitochondrial enzyme CYP27A1 produces 27-hydroxycholesterol (27HC). We recently demonstrated that administration of 27HC to hyperlipidemic mice reduced hepatic inflammation. In line, hematopoietic deletion of Cyp27a1 resulted in increased hepatic inflammation. In the current manuscript, the effect of hematopoietic overexpression of Cyp27a1 on the development of NASH and cholesterol trafficking was investigated. We hypothesized that Cyp27a1 overexpression in KCs will lead to reduced hepatic inflammation.Methods: Irradiated Ldlr(-/-) mice were transplanted (tp) with bone marrow from mice overexpressing Cyp27a1 (Cyp27a1(over)) and wild type (Wt) mice and fed either chow or a high-fat, high-cholesterol (HFC) diet for 3 months. Additionally, gene expression was assessed in bone marrow-derived macrophages (BMDM) from Cyp27a1(over) and Wt mice.Results: In line with our hypothesis, hepatic inflammation in HFC-fed Cyp27a1(over)-tp mice was reduced and KCs were less foamy compared to Wt-tp mice. Remarkably, these changes occurred even though plasma and liver levels of 27HC did not differ between both groups. BMDM from Cyp27a1(over) mice revealed reduced inflammatory gene expression and increased expression of cholesterol transporters compared to Wt BMDM after lipopolysaccharide (LPS) stimulation.Conclusions: Our data suggest that overexpression of Cyp27a1 in KCs reduces hepatic inflammation independently of 27HC levels in plasma and liver, further pointing towards KCs as specific target for improving the therapy of NASH. (C) 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Nonalcoholic fatty liver disease is seen as the hepatic manifestation of the metabolic syndrome and represents the most common liver disease in Western societies. The G protein–coupled chemokine receptor CX3CR1 plays a central role in several metabolic syndrome–related disease manifestations and is involved in maintaining intestinal homeostasis. Because diet‐induced intestinal dysbiosis is a driver for nonalcoholic fatty liver disease, we hypothesized that CX3CR1 may influence the development of steatohepatitis. In two independent models of diet‐induced steatohepatitis (high‐fat diet and methionine/choline‐deficient diet), CX3CR1 protected mice from excessive hepatic steatosis and inflammation, as well as systemic glucose intolerance. Lack of Cx3cr1 expression was associated with significantly altered intestinal microbiota composition, which was linked to an impaired intestinal barrier. Concomitantly, endotoxin levels in portal serum and inflammatory macrophages in liver were increased in Cx3cr1 –/– mice, indicating an increased inflammatory response. Depletion of intestinal microbiota by administration of broad‐spectrum antibiotics suppressed the number of infiltrating macrophages and promoted macrophage polarization in liver. Consequently, antibiotic‐treated mice demonstrated a marked improvement of steatohepatitis. Conclusion: Microbiota‐mediated activation of the innate immune responses through CX3CR1 is crucial for controlling steatohepatitis progression, which recognizes CX3CR1 as an essential gatekeeper in this scenario. (Hepatology 2015;62:1405–1416)

The inflammatory component of non-alcoholic steatohepatitis (NASH) can lead to irreversible liver damage. Therefore there is an urgent need to identify novel interventions to combat hepatic inflammation. In mice, omitting cholesterol from the diet reduced hepatic inflammation. Considering the effects of plant sterol/stanol esters on cholesterol metabolism, we hypothesized that plant sterol/stanol esters reduces hepatic inflammation. Indeed, adding plant sterol/stanol esters to a high-fat-diet reduced hepatic inflammation as indicated by immunohistochemical stainings and gene expression for inflammatory markers. Finally, adding sterol/stanol esters lowered hepatic concentrations of cholesterol precursors lathosterol and desmosterol in mice, which were highly elevated in the HFD group similarly as observed in severely obese patients with NASH. In vitro, in isolated LPS stimulated bone marrow derived macrophages desmosterol activated cholesterol efflux whereas sitostanol reduced inflammation. This highly interesting observation that plant sterol/stanol ester consumption leads to complete inhibition of HFD-induced liver inflammation opens new venues in the treatment and prevention of hepatic inflammation.

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome and covers a disease spectrum ranging from steatosis to inflammation, fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The innate immune response in the liver plays an important role during NAFLD progression. In addition, changes in the intestinal microbial balance and bacterial translocation can further affect disease progression. Immune cells in the liver recognize cell damage or pathogen invasion with intracellular or surface-expressed pattern recognition receptors (PRRs), subsequently initiating signaling cascades that trigger the release of factors promoting the inflammatory response during NAFLD progression. Therefore, mechanisms by which cells of the immune system are activated and recruited into the liver and how these cells cause injury and stress are important for understanding the inflammatory response during NAFLD.

The role of the inflammatory response is to combat tissue injury and infection. Innate immune cells recognize cell damage or pathogen invasion with intracellular or surface-expressed pattern recognition receptors (PRRs). Activated PRRs subsequently initiate signaling cascades that trigger the release of factors promoting the inflammatory response. Because the liver is a site where foreign antigens from the gastrointestinal tract encounter the immune system, it is particularly enriched with innate immune cells. These cells can modify and disrupt critical processes implicated in metabolic disease. As such, metabolic stress initiates a feedforward cycle of inflammatory responses, resulting in a state of unresolved chronic inflammation in the liver. Accordingly, the crosstalk between these innate immune cells and the resident parenchymal cells plays an important role in the development of acute and chronic liver disease.

Background & Aims While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation. Methods Ldlr -/- mice were transplanted (tp) with wild-type (Wt) or caspase-1/11-/- (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11-/- mice were incubated with oxLDL for 24h and autophagy was assessed. Results In line with our hypothesis, caspase-1/11-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11-/- mice had normal autophagy activity. Conclusion Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from HDL to LDL and high CETP activity results in an atherogenic lipid phenotype. Thus, inhibiting CETP is a target for dyslipidemia treatment. However, the true origin of CETP in humans is uncertain. We aimed to elucidate the cellular origin of CETP and the relative contribution of adipose tissue and liver to plasma CETP levels.