BaCKgRoUND aND aIMS: Mounting evidence supports an association between cholestatic liver disease and changes in the composition of the microbiome. Still, the role of the microbiome in the pathogenesis of this condition remains largely undefined. appRoaCH aND ReSUltS: To address this, we have used two experimental models, administering alpha-naphtylisocyanate or feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet, to induce cholestatic liver disease in germ-free mice and germ-free mice conventionalized with the microbiome from wild-type, specific pathogen-free animals. Next, we have inhibited macrophage activation by depleting these cells using clodronate liposomes and inhibiting the inflammasome with a specific inhibitor of NOD-, LRR-, and pyrin domain-containing protein 3. Our results demonstrate that cholestasis, the accumulation of bile acids in the liver, fails to promote liver injury in the absence of the microbiome in vivo. Additional in vitro studies supported that endotoxin sensitizes hepatocytes to bile-acid-induced cell death. We also demonstrate that during cholestasis, macrophages contribute to promoting intestinal permeability and to altered microbiome composition through activation of the inflammasome, overall leading to increased endotoxin flux into the cholestatic liver. CoNClUSIoNS: We demonstrate that the intestinal microbiome contributes to cholestasis-mediated cell death and inflammation through mechanisms involving activation of the inflammasome in macrophages. (Hepatology 2020;72:2090-2108). T he intestine is a selective barrier that prevents pathogenic bacteria translocating to the systemic circulation, while simultaneously allowing nutrient absorption. In the intestine, crosstalk regulation among the microbiome, the immune system, and epithelial cells is essential to preserve barrier function. (1,2) Intestinal permeability is tightly regulated by the immune system as inflammatory cytokines (e.g., tumor necrosis factor [TNF] and interferon gamma) modulate the expression of tight junction (TJ) proteins. (3) In the intestine, the microbiome shapes the immune system, rendering a tolerant environment where microbes and host cells can coexist. (1,2) Reciprocally, inflammation can determine the composition of the intestinal microbiome, (4,5) adding another layer of complexity to the regulation of intestinal permeability and barrier function. The "leaky gut" hypothesis proposes that chronic liver disease is associated with breaching of the

Acute infection is known to induce rapid expansion of hematopoietic stem cells (HSCs), but the mechanisms supporting this expansion remain incomplete. Using mouse models, we show that inducible CD36 is required for free fatty acid uptake by HSCs during acute infection, allowing the metabolic transition from glycolysis towards β-oxidation. Mechanistically, high CD36 levels promote FFA uptake, which enables CPT1A to transport fatty acyl chains from the cytosol into the mitochondria. Without CD36-mediated FFA uptake, the HSCs are unable to enter the cell cycle, subsequently enhancing mortality in response to bacterial infection. These findings enhance our understanding of HSC metabolism in the bone marrow microenvironment, which supports the expansion of HSCs during pathogenic challenge.

Hepatocellular carcinoma (HCC) is the most common malignancy occuring in the context of chronic liver disease and is one of the main causes of cancer-derived death worldwide. The lack of effective treatments, together with the poor prognosis, underlines the urge to develop novel and multidisciplinary therapeutics. An increasing body of evidence shows that HCC associates with changes in intestinal microbiota abundance and composition as well as with impaired barrier function, leading to the release of bacteria and their metabolites to the liver. These factors trigger a cascade of inflammatory responses contributing to liver cirrhosis and constituting an ideal environment for the progression of HCC. Interestingly, the use of bacteriotherapy in human and preclinical studies of chronic liver disease and HCC has been shown to successfully modify the microbiota composition, reducing overall inflammation and fibrosis. In this review, we explore the existing knowledge on the characterisation of the intestinal microbial composition in humans and experimental murine chronic liver disease and HCC, as well as the use of antibiotics and bacteriotherapy as therapeutic options.

Here, we describe that the SIRT1/mTOR axis regulates metabolic rewiring, inflammasome activation, and autophagy in macrophages, in which SIRT1 overexpression actively contributes to aggravate cholestatic liver disease progression in mice.

No abstract

Acute infection is known to promote rapid differentiation of hematopoietic stem cells (HSC) and expansion of leukocytes. Metabolic changes in the HSC underpin the mammalian response to pathogenic stimuli however, knowledge of how this occurs is not fully understood. Here we investigate the immunometabolic changes which facilitate HSC expansion in response to acute infection. We created a novel in vivo transplant model to understand if the immune response to infection involves the acquisition of free fatty acids (FFA) by HSCs in the bone marrow (BM). We transduced CD45.1 lineage negative, c-kit positive cells with firefly luciferase and transplanted into CD45.2 animals. Post engraftment animals were treated with lipopolysaccharide (LPS). Live animal imaging confirmed activation of luciferase in the BM, demonstrating in vivo, uptake of long chain FFA by haematopoietic cells in response to LPS. To confirm the specific haematopoietic stem and/or progenitor cells (HSPC) with increased lipids during infection, mice were treated with S.typhimurium for 72h then sacrificed Analysis of LSK, MPP and HSC populations (LN-, CD117+, Sca1+, CD48-, CD150+ and CD34+) showed increased intracellular BODIPY 493/503 neutral lipid dye and uptake of Bodipy FL-C12 (FFA linked to bodipy) at 72 h compared to control non-infected animals. Together, these experiments show that HSC, multipotent progenitor (MPP) and LSK (LN-, CD117+, Sca1+) cells all acquire FFA in response to bacterial infection. Seahorse mitochondrial stress test confirmed increased oxygen consumption levels in HSC from LPS (16 hours) and S.typhimurium (72 hours) treated C57BL/6. LSK metabolic activity for different substrates was assessed LSKs from LPS treated animals had an increased dependency on β-oxidation when compared to control cells. Moreover, the β-oxidation inhibitor etomoxir blocks LSK basal metabolism and reduced HSC expansion as measured by Ki-67 staining in LPS and S.typhimurium treated animals. To understand the importance of β-oxidation in HSC expansion in response to infection we first measured CPT1A expression in HSC. RNA analysis confirmed CPT1A was increased in sorted HSCs in response to LPS. Next we transplanted WTor CPT1A KD cells into WT animals, and treated with LPS. HSC expansion as measured by Ki-67 staining was inhibited in the CPT1A KD cells in response to LPS, thus confirming the importance of β-oxidation based metabolism in HSC expansion. To determine how FFA are transported into the HSC we analysed the expression of known genes to be involved in lipid uptake. HSC sorted from LPS and S.typhimurium treated animals had increased CD36 mRNA expression compared to control. C57BL/6 mice were pre-treated with the CD36 inhibitor sulfosuccinimidyl oleate (SSO) before injection with LPS. HSCs had had less lipids, lower maximal respiration and reduced cycling when compared to animals treated with LPS alone. In a similar way CD36 KO animals were treated with LPS for 16 h, no increased FFA uptake or increased lipid content or HSC cycling was observed. Seahorse mitochondrial stress test confirmed CD36-/- LSK have increased basal ECAR but no change in basal OCR in response to S.typhimurium infection. Moreover, CD36-/- LSK have reduced dependency on β-oxidation. To determine if the uptake of FFA response is specific to HSC we transplanted CD36+/+ (CD45.1) LK into CD36-/- (CD45.2) animals. We found that CD36 expression was elevated in CD36+/+ HSC transplanted into CD36-/- treated with LPS, normal uptake of FFA, an increase in lipid content and cycling of HSCs. In addition, transplanted WT CD36+/+ (CD45.1) LSK into CD36-/- (CD45.2) animals reversed the metabolic phenotype observed in CD36-/- in response to LPS. We transplanted WT(CD36+/+) or CD36KO (WT(CD36-/-)) LSK into WT animals infected with S. typhimurium for 4 days. WT(CD36-/-) transplanted animals showed enhanced mortality, increased weight loss and increased liver injury. We also observed less HSC cycling and a reduced lipid content in HSC from WT(CD36-/-) compared to WT(CD36+/+). These findings expand the mechanistic understanding of the interplay between HSC and the bone marrow microenvironment, and in doing so explains how immunometabolic reprogramming of HSCs during infection supports the metabolic demands of HSCs in response to a pathogenic challenge without which results in increased susceptibility to infection. Disclosures Bowles: AbbVie: Research Funding; Janssen: Research Funding. Rushworth:AbbVie: Research Funding; Janssen: Research Funding.