The Mechanisms Of Gut Microbiota Promote Liver Regeneration In Mice

The large numbers of resident microbial communities inhabit in the intestine of mammalian and build-up a host-microbial relationship with each other. Gut microbiota could shape the mammalian immune system during health and disease while the interaction between resident microbe and the immune system could also effect on the composition of gut commensal bacteria. The dysbiosis of gut commensal bacteria could cause not only intestinal-associated diseases but also lung and skin inflammation. The food antigen, microbiota products from intestine continuously flow to the liver from portal vein and take part in the progression of chronic liver disease. Commensal bacteria have also been proposed to play a role in liver repair after partial (67%) hepatectomy, however, the underlying immune mechanisms remain elusive.In our research, we treated mice with antibiotics water, including ampicillin, vancomycin, metronidazole and neomycin, over a4-week period before performing PHx and control mice with normal drinking water. By observing the liver body ratio, the rate of hepatocytes proliferation, we compare the rate of liver regeneration of mice with different water treatment. We tested the effect of bacterial load in the colon and proliferation of hepatocytes in mice subjected to1,2,3,4weeks of oral antibiotic treatment. Then we separated mice into groups that received each individual antibiotic alone and narrow down which bacterial class was responsible for promoting liver regeneration. We investigated whether immune cells play a critical role in microbiota-mediated promotion of liver regeneration by evaluating the total number, percentage and activation of total hepatic lymphocytes.Here, we show that liver regeneration was impaired in antibiotic (Atb) water-treated mice and antibiotic water treatment could neither increase the ALT and AST level in serum nor impair the intact structure of the intestine or colon. The impairment of liver regeneration is strongly correlated with commensal bacterial load. Among the various Atbs used in our cocktail, ampicillin-sensitive commensal bacterial was associated with normal liver regeneration.The percentage, number and activation of CD3+NK1.1+natural killer T (NKT) cells in Atb-treated hepatectomized mice was markedly increased at various time points after PHx compared to their water-treated littermates, and these NKT cells were CD Id-dependent and functionally overactivated to produce higher interferon-y and up-regulate the expression of activated receptor NKG2D, Fas and CD69, down-regulated the inhibitory receptor NKG2A after a-Galcer stimulation. The accumulation of NKT cells in liver after antibiotic water treatment was due to the up-regulated expression of chemokine receptor CXCR6and inability to apoptosis. Deficiency of NKT cells or antibody blockade of the CDld-NKT interaction increased hepatocyte proliferation, which improved liver regeneration while T cells or NK cells depletion had no effect on the rate of liver regeneration. Transferring purified CDld-dependent NKT cells could aggravate the impaied liver regeneration in atb-water treated mice.We found that F4/80+Kupffer cells expanded in the liver of Atb-treated mice before and after PHx. This increase was due to enhanced self-proliferation of tissue resident F4/80+Kupffer cells rather than the differentiation of Ly6Chhi circulating monocytes. The activation markers CD80, CD86, CD68as well as Toll-like receptor TLR2, TLR4were up-regulated on expanded Kupffer cells and proinflammatory cytokine secretion (IL-6, TNFa, IL-10, IL-12) was also up-regulated after in vitro LPS stimulation. Injection of TLR3agonist poly I:C could accelerate liver regeneration in Atb-treated mice while single injection of TLR2agonist Pamcsk4and TLR4agonist LPS impaired the rate of liver regeneration. Injection low dose of LPS continuously for three days to induce the LPS tolerance in Kupffer cells down-regulated the CD80, CD86and TLR receptor on the surface of Kupffer cells and at the same time enhance hepatocytes proliferation in Atb-treated mice.To determine whether Kupffer cells played a role in activating hepatic NKT cells in the absence of the microbiota in the PHx model, we depleted Kupffer cells by intravenously injecting clodronate liposome48hours before PHx. Indeed, Kupffer cells depletion decreased CD Id-dependent NKT-cell activation as assessed by CD69, IFNy, CD25and NKG2D expression. These Kupffer cells produced higher interleukin-12, which then functioned to activate hepatic NKT cells. Interleukin-12p40deficiency or treatment with an anti-interleukin-12antibody significantly inhibited NKT cell overactivation and recovered liver regeneration in Atb-treated mice. Conclusion:Altogether, we propose that commensal bacteria play a critical role in maintaining Kupffer cells in a tolerant state, preventing subsequent NKT cell overactivation during liver regeneration; moreover, our data suggest that long-term Atb use, which can impair the gut microbiota, may influence liver function by retarding liver regeneration.