Intestinal Microbiome-Macrophage Crosstalk Contributes to Cholestatic Liver Disease by Promoting Intestinal Permeability in Mice.

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.

Fine-Tuning of Sirtuin 1 Expression Is Essential to Protect the Liver From Cholestatic Liver Disease.

Cholestasis comprises aetiologically heterogeneous conditions characterized by accumulation of bile acids in the liver that actively contribute to liver damage. Sirtuin 1 (SIRT1) regulates liver regeneration and bile acid metabolism by modulating farnesoid X receptor (FXR); we here investigate its role in cholestatic liver disease. We determined SIRT1 expression in livers from patients with cholestatic disease, in two experimental models of cholestasis, as well as in human and murine liver cells in response to bile acid loading. SIRT1-overexpressing (SIRToe ) and hepatocyte-specific SIRT1-KO (knockout) mice (SIRThep-/- ) were subjected to bile duct ligation (BDL) and were fed with a 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) diet to determine the biological relevance of SIRT1 during cholestasis. The effect of NorUDCA (24-norursodeoxycholic acid) was tested in BDL/SIRToe mice. We found that SIRT1 was highly expressed in livers from cholestatic patients, mice after BDL, and Mdr2 knockout mice (Mdr2-/- ) animals. The detrimental effects of SIRT1 during cholestasis were validated in vivo and in vitro. SIRToe mice showed exacerbated parenchymal injury whereas SIRThep-/- mice evidenced a moderate improvement after BDL and 0.1% DDC feeding. Likewise, hepatocytes isolated from SIRToe mice showed increased apoptosis in response to bile acids, whereas a significant reduction was observed in SIRThep-/- hepatocytes. Importantly, the decrease, but not complete inhibition, of SIRT1 exerted by norUDCA treatment correlated with pronounced improvement in liver parenchyma in BDL/SIRToe mice. Interestingly, both SIRT1 overexpression and hepatocyte-specific SIRT1 depletion correlated with inhibition of FXR, whereas modulation of SIRT1 by NorUDCA associated with restored FXR signaling. Conclusion: SIRT1 expression is increased during human and murine cholestasis. Fine-tuning expression of SIRT1 is essential to protect the liver from cholestatic liver damage.

Metabolic Regulation of Macrophages by SIRT1 Determines Activation During Cholestatic Liver Disease in Mice.

BACKGROUND & AIMS: Inflammation is the hallmark of chronic liver disease. Metabolism is a key determinant to regulate the activation of immune cells. Here, we define the role of sirtuin 1 (SIRT1), a main metabolic regulator, in controlling the activation of macrophages during cholestatic liver disease and in response to endotoxin. METHODS: We have used mice overexpressing SIRT1, which we treated with intraperitoneal lipopolysaccharides or induced cholestasis by bile duct ligation. Bone marrow-derived macrophages were used for mechanistic in vitro studies. Finally, PEPC-Boy mice were used for adoptive transfer experiments to elucidate the impact of SIRT1-overexpressing macrophages in contributing to cholestatic liver disease. RESULTS: We found that SIRT1 overexpression promotes increased liver inflammation and liver injury after lipopolysaccharide/GalN and bile duct ligation; this was associated with an increased activation of the inflammasome in macrophages. Mechanistically, SIRT1 overexpression associated with the activation of the mammalian target of rapamycin (mTOR) pathway that led to increased activation of macrophages, which showed metabolic rewiring with increased glycolysis and broken tricarboxylic acid cycle in response to endotoxin in vitro. Activation of the SIRT1/mTOR axis in macrophages associated with the activation of the inflammasome and the attenuation of autophagy. Ultimately, in an in vivo model of cholestatic disease, the transplantation of SIRT1-overexpressing myeloid cells contributed to liver injury and fibrosis. CONCLUSIONS: Our study provides novel mechanistic insights into the regulation of macrophages during cholestatic disease and the response to endotoxin, in which the SIRT1/mTOR crosstalk regulates macrophage activation controlling the inflammasome, autophagy and metabolic rewiring.