BaCKgRoUND aND aIMS: Nonalcoholic steatohepatitis is rapidly becoming the leading cause of liver failure and indication for liver transplantation. Hepatic inflammation is a key feature of NASH but the immune pathways involved in this process are poorly understood. B lymphocytes are cells of the adaptive immune system that are critical regulators of immune responses. However, the role of B cells in the pathogenesis of NASH and the potential mechanisms leading to their activation in the liver are unclear.appRoaCH aND ReSUltS: In this study, we report that NASH livers accumulate B cells with elevated proinflammatory cytokine secretion and antigen-presentation ability. Single-cell and bulk RNA sequencing of intrahepatic B cells from mice with NASH unveiled a transcriptional landscape that reflects their pro-inflammatory function. Accordingly, B-cell deficiency ameliorated NASH progression, and adoptively transferring B cells from NASH livers recapitulates the disease. Mechanistically, B-cell activation during NASH involves signaling through the innate adaptor myeloid differentiation primary response protein 88 (MyD88) as B cell-specific deletion of MyD88 reduced hepatic T cell-mediated inflammation and fibrosis, but not steatosis. In addition, activation of intrahepatic B cells implicates B cellreceptor signaling, delineating a synergy between innate and adaptive mechanisms of antigen recognition. Furthermore, fecal microbiota transplantation of human NAFLD gut microbiotas into recipient mice promoted the progression of NASH by increasing the accumulation and activation of intrahepatic B cells, suggesting that gut microbial factors drive the pathogenic function of B cells during NASH. CoNClUSIoN:Our findings reveal that a gut microbiotadriven activation of intrahepatic B cells leads to hepatic inflammation and fibrosis during the progression of NASH through innate and adaptive immune mechanisms. (Hepatology 2021;74:704-722). NAFLD is estimated to affect 30% of the population and is now recognized as the most prevalent chronic liver disease worldwide. (1) The disease covers a wide spectrum of liver pathology, ranging from simple lipid accumulation to the development of NASH, defined by hepatic steatosis, local inflammation, hepatocellular injury, and fibrosis. (2) NASH-associated inflammation is driven by innate and adaptive immune mechanisms comprising macrophages, dendritic cells, neutrophils, and lymphocytes. (3) Recent single-cell transcriptome analyses have uncovered the heterogeneity of intrahepatic

Highlights d Environmental stiffness promotes DC inflammatory function d Tension primes DC metabolism, even without pattern recognition receptor input d TAZ bridges mechanosensory signals to DC metabolism and function d Tension directs phenotypes of human monocyte-derived DCs

Rationale: The initial hypertrophy response to cardiac pressure overload is considered compensatory, but with sustained stress, it eventually leads to heart failure. Recently, a role for recruited macrophages (mψs) in determining the transition from compensated to decompensated hypertrophy has been established. However, whether cardiac-resident immune cells influence the early phase of hypertrophy development has not been established. Objective: To assess the role of cardiac immune cells in the early hypertrophy response to cardiac pressure overload-induced by transverse aortic constriction (TAC). Methods and Results: We performed cytometry-by-time-of-flight to determine the identity and abundance of immune cells in the heart at 1 and 4 weeks after TAC. We observed a substantial increase in cardiac mψs 1 week after TAC. We then conducted Cite-Seq single-cell RNA sequencing of cardiac immune cells isolated from 4 sham and 6 TAC hearts. We identified 12 clusters of monocytes and mψs, categorized as either resident or recruited mψs, that showed remarkable changes in their abundance between sham and TAC conditions. To determine the role of cardiac-resident mψs early in the response to a hypertrophic stimulus, we used a blocking antibody against macrophage colony-stimulating factor 1 receptor (CD115). As blocking CD115 initially depletes all macrophages, we allowed the replenishment of recruited mψs by monocytes before performing TAC. This preferential depletion of resident mψs resulted in enhanced fibrosis and a blunted angiogenesis response to TAC. Mψ-depletion in CCR2 knockout mice showed that aggravated fibrosis was primarily caused by the recruitment of monocyte-derived mψs. Finally, 6 weeks after TAC these early events lead to depressed cardiac function and enhanced fibrosis, despite complete restoration of cardiac immune cells. Conclusions: Cardiac resident mψs are a heterogeneous population of immune cells with key roles in stimulating angiogenesis and inhibiting fibrosis in response to cardiac pressure overload.

NAFLD is one of the leading causes of abnormal liver function worldwide. NAFLD refers to a group of liver conditions ranging from nonalcoholic fatty liver to NASH, which involves inflammation, hepatocellular damage, and fibrosis. Triggering of inflammation in NASH is a key event in the progression of the disease, and identifying the factors that initiate or dysregulate this process is needed to develop strategies for its prevention or treatment. B cells have been implicated in several autoimmune and inflammatory diseases. However, their role in the pathogenesis of NAFLD and NASH is less clear. This review discusses the emerging evidence implicating intrahepatic B cells in the progression of NAFLD. We highlight the potential mechanisms of B-cell activation during NAFLD, such as increased hepatic expression of B-cell-activating factor, augmented oxidative stress, and translocation of gut-derived microbial products. We discuss the possible effector functions by which B cells promote NAFLD, including the production of proinflammatory cytokines and regulation of intrahepatic T cells and macrophages. Finally, we highlight the role of regulatory and IgA + B cells in the pathogenesis of NASH-associated HCC. In this review, we make the case that future research is needed to investigate the potential of B-cell-targeting strategies for the treatment of NAFLD. (Hepatology 2021;0:1-10). The NAFLD Inflammatory Environment at a Glance Role oF INNate IMMUNe CellSThe liver receives a constant influx of gut-derived products and acts as a firewall for the antigen-rich

Objective Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology ranging from simple fatty liver to non-alcoholic steatohepatitis (NASH). Notably, immune cell-driven inflammation is a key mechanism in the transition from fatty liver to the more serious NASH. Although exercise training is effective in ameliorating obesity-related diseases, the underlying mechanisms of the beneficial effects of exercise remain unclear. It is unknown whether there is an optimal modality and intensity of exercise to treat NAFLD. The objective of this study was to determine whether high-intensity interval training (HIIT) or moderate-intensity continuous training (MIT) is more effective at ameliorating the progression of NASH. Methods Wild-type mice were fed a high-fat, high-carbohydrate (HFHC) diet for 6 weeks and left sedentary (SED) or assigned to either an MIT or HIIT regimen using treadmill running for an additional 16 weeks. MIT and HIIT groups were pair-fed to ensure that energy intake was similar between the exercise cohorts. To determine changes in whole-body metabolism, we performed insulin and glucose tolerance tests, indirect calorimetry, and magnetic resonance imaging. NASH progression was determined by triglyceride accumulation, expression of inflammatory genes, and histological assessment of fibrosis. Immune cell populations in the liver were characterized by cytometry by time-of-flight mass spectrometry, and progenitor populations within the bone marrow were assessed by flow cytometry. Finally, we analyzed the transcriptional profile of the liver by bulk RNA sequencing. Results Compared with SED mice, both HIIT and MIT suppressed weight gain, improved whole-body metabolic parameters, and ameliorated the progression of NASH by reducing hepatic triglyceride levels, inflammation, and fibrosis. However, HIIT was superior to MIT at reducing adiposity, improving whole-body glucose tolerance, and ameliorating liver steatosis, inflammation, and fibrosis, without any changes in body weight. Improved NASH progression in HIIT mice was accompanied by a substantial decrease in the frequency of pro-inflammatory infiltrating, monocyte-derived macrophages in the liver and reduced myeloid progenitor populations in the bone marrow. Notably, an acute bout of MIT or HIIT exercise had no effect on the intrahepatic and splenic immune cell populations. In addition, bulk mRNA sequencing of the entire liver tissue showed a pattern of gene expression confirming that HIIT was more effective than MIT in improving liver inflammation and lipid biosynthesis. Conclusions Our data suggest that exercise lessens hepatic inflammation during NASH by reducing the accumulation of hepatic monocyte-derived inflammatory macrophages and bone marrow precursor cells. Our findings also indicate that HIIT is superior to MIT in ameliorating the disease in a dietary mouse model of NASH.

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Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid associated with nonalcoholic steatohepatitis (NASH). Immune cell-driven inflammation is a key determinant of NASH progression. Macrophages, monocytes, NK cells, T cells, NKT cells, and B cells variably express S1P receptors from a repertoire of 5 receptors termed S1P1 – S1P5. We have previously demonstrated that non-specific S1P receptor antagonism ameliorates NASH and attenuates hepatic macrophage accumulation. However, the effect of S1P receptor antagonism on additional immune cell populations in NASH remains unknown. We hypothesized that S1P receptor specific modulation may ameliorate NASH by altering leukocyte recruitment. A murine NASH model was established by dietary feeding of C57BL/6 male mice with a diet high in fructose, saturated fat, and cholesterol (FFC) for 24 weeks. In the last 4 weeks of dietary feeding, the mice received the S1P1,4,5 modulator Etrasimod or the S1P1 modulator Amiselimod, daily by oral gavage. Liver injury and inflammation were determined by histological and gene expression analyses. Intrahepatic leukocyte populations were analyzed by flow cytometry, immunohistochemistry, and mRNA expression. Alanine aminotransferase, a sensitive circulating marker for liver injury, was reduced in response to Etrasimod and Amiselimod treatment. Liver histology showed a reduction in inflammatory foci in Etrasimod-treated mice. Etrasimod treatment substantially altered the intrahepatic leukocyte populations through a reduction in the frequency of T cells, B cells, and NKT cells and a proportional increase in CD11b+ myeloid cells, polymorphonuclear cells, and double negative T cells in FFC-fed and control standard chow diet (CD)-fed mice. In contrast, FFC-fed Amiselimod-treated mice showed no changes in the frequencies of intrahepatic leukocytes. Consistent with the improvement in liver injury and inflammation, hepatic macrophage accumulation and the gene expression of proinflammatory markers such as Lgals3 and Mcp-1 were decreased in Etrasimod-treated FFC-fed mice. Etrasimod treated mouse livers demonstrated an increase in non-inflammatory (Marco) and lipid associated (Trem2) macrophage markers. Thus, S1P1,4,5 modulation by Etrasimod is more effective than S1P1 antagonism by Amiselimod, at the dose tested, in ameliorating NASH, likely due to the alteration of leukocyte trafficking and recruitment. Etrasimod treatment results in a substantial attenuation of liver injury and inflammation in murine NASH.

In the past few decades, higher rates of obesity have coincided with a rising number of individuals affected by allergic disease. Our study objective was to determine if obesity alters the immune cell landscape of the small intestine towards an inflammatory environment that aggravates the development of food allergy. Obesity was induced by feeding male BALB/c mice a western diet for 8 weeks. To induce food allergy, mice were epicutaneously sensitized and orally-challenged with OVA. Compared with lean controls, sensitized obese mice showed a lower body temperature after challenge and increased OVA-specific IgE in serum, suggesting an exacerbated allergic response. Mass cytometry analysis of the intestinal lamina propria revealed that OVA-sensitized obese mice had decreased T regulatory cells, which are critical for the oral tolerance to food allergens. In the mesenteric lymph node, obese mice showed increased frequencies of T follicular helper cells (Tfh) compared with lean controls. Tfh cells are required for the production of allergen-specific IgE, and implicated in allergic disease pathogenesis. To further define the transcriptional mechanisms underpinning the increased inflammatory and allergic responses during obesity, we performed single-cell RNA sequencing of more than 28,000 barcoded CD45+ enriched lamina propria cells from lean and obese mice, with and without OVA sensitization. We provide a transcriptional map of inflammatory cells and define a module of gene expression that distinguishes immune cell-driven allergic responses in the obese intestine. Overall, our findings highlight obesity-induced inflammatory changes in the intestine that may mediate the increased food allergic sensitization during obesity.