Synergistic Inhibitory Effect of the Gut Microbiome and Lithocholic Acid on Liver Fibrosis

Background: Lithocholic acid are essential signaling molecules that mediate the relationship between the gut microbiome and liver function by regulating inammation. The purpose of this study is to investigate the role of lithocholic acid in liver brosis. Methods: A liver brosis mouse model was induced by carbon tetrachloride followed by gavage of lithocholic acid, and the effects of lithocholic acid were evaluated by serum biochemical analysis and liver histology. Plasma cytokine levels and the number of immune cells were determined by cytometric bead array and ow cytometry, respectively. Results: Lithocholic acid treatment increased the recruitment of NK cells and reduced the activation of NKT cells, and reduced M1 macrophages differentiation and increased M2 macrophages differentiation. Furthermore, the lithocholic acid prevented inammatory liver disease by reducing TNF-α and IL-22 secretion. However, the effect of lithocholic acid disappeared when the host gut microbiome was treated with antibiotics. Conclusions: It showed that the activation of lithocholic acid-mediated signaling was linked to the inhibition of inammation and improvement of liver brosis. The role of lithocholic acid in liver brosis is mediated by the gut microbiome. The association between the gut microbiome, lithocholic acid, and liver function can serve as a therapeutic target for liver brosis.


Introduction
The enterohepatic circulation of bile acids (BAs) and blood circulation of the hepatic portal vein closely connect the gut microbiome with liver diseases, and form the gut microbiome-bile acids-liver triangle.
However, the relationship between bile acids, the gut microbiome and liver diseases is incompletely understood. We speculate that the in ammatory response and innate immunity play an essential role between them.
Previous studies have suggested that bile acids are tissue-damaging agents that promote in ammation 1 .
In liver diseases, the accumulation of hydrophobic bile acids disrupts the mitochondrial membrane and the intestinal mucosal barrier and causes cell necrosis, allowing the gut microbiome and its metabolites to enter the liver 2,3 . However, in recent years, studies have fully con rmed the role of bile acids in host metabolism and cancer progression, especially the effect of lithocholic acid (LCA) in innate immunity. As the strongest natural agonist of transmembrane G protein-coupled receptor (TGR5), LCA can reduce the phagocytic activity and the production of pro-in ammatory cytokine by activating TGR5 on monocytes and macrophages 4 . In addition, it can also inhibit the secretion of IL-6, IL-1A, and IL-1B induced by LPS and TNF secretion by Kupffer cells through a TGR5-cAMP-dependent pathway, ultimately inhibiting liver in ammation 5 . Because of the special anatomic relationship between the intestine and liver and the bidirectional interaction between bile acids and the gut microbiome. This study investigates whether the gut microbiome can affect the liver in ammatory response and the progress of liver brosis through LCA.

Materials And Methods
Murine studies C57BL/6 male mice aged 6-7 weeks were purchased from Shanghai SLAC Laboratory Animal Co. Ltd.
The CCl4+ LCA + VM group received CCl4 and LCA as described above and both ampicillin (MCE, 1g/L) and vancomycin (Sigma, 0.5 g/L) in the drinking water 6 . All mice were treated for 8 weeks and were sacri ced 48 hours after the last injection. The study conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the research ethics committee of the Zhejiang University School of Medicine.

16S rRNA sequencing and analysis
The methods for analyzing the gut microbiome in mouse stool samples were described previously 7 .

Statistical analysis
Statistical analysis was performed using SPSS software version 21.0. Data were expressed as means and ± standard deviations. The differences between groups were assessed by two-tailed unpaired Student's t-test or ANOVA. A p-value smaller than 0.05 was considered statistically signi cant.

Lithocholic acid inhibits liver in ammation and improves liver brosis
The healthy liver is known for an active innate immune response and a weaker adaptive immune response 8 . Viruses, toxins, insulin resistance and hypoxia induce a chronic low-grade in ammation in the liver, and essentially creates a brotic environment 9 . Studies have shown that compensated cirrhosis is reversible after treating hepatitis B virus (HBV) and hepatitis C virus infections, which eliminates longterm stimulation of the liver by in ammation, demonstrating that controlling liver in ammation improves liver brosis and may lead to brinolysis 10,11 .
The regulation of in ammation by LCA was investigated in vivo. For this purpose, LCA was fed to mice with CCl4-induced liver brosis. The mRNA expression of liver brosis makers, including matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinases 1 (Timp-1), α-smooth muscle actin (α-SMA), collagens I and III was measured. LCA treatment signi cantly reduced the mRNA expression of the markers in mice, expect for the anti-brotic MMP-2 (Fig. 1A). Liver sections were stained with hematoxylin-eosin and Masson trichrome to evaluate the function of LCA on liver injury and brosis. The results showed that collagen deposition and in ammatory cell in ltration were lower in the CCl4+LCA group than in the CCl4 group. However, the anti-brotic effect of LCA was reduced in the CCl4+LCA+VM group (Figs.1B and C). These data indicate that LCA can improve liver brosis progression by controling liver in ammation, and that the anti-brotic effect of LCA is mediated by the gut microbiome.

Anti-brotic activity of hepatic immune cells
To investigate the mechanism underlying liver brosis suppression and in ammation, we analyzed the subsets of intrahepatic immune cells in mice. Macrophages are classi ed into two types: M1 with a proin ammatory effect; and M2 with anti-in ammatory and immunosuppressive effects 12 . The results of multicolor ow cytometric analyses showed that the number of M1 macrophages decreased and the number of M2 macrophages increased in the CCl4+LCA group compared with the CCl4 group ( Figs.2A and B). However, after the LCA treated mice add antibiotics to destroy the homeostasis of the gut microbiome, the number of M1 and M2 macrophages not signi cantly different between the CCl4+LCA and CCl4+LCA+VM groups. The results suggest that LCA has anti-in ammatory activity in the liver and can modulate innate immunity by regulating macrophage differentiation. Nonetheless, the effect of LCA on macrophage differentiation was not mediated by the gut microbiome.
NKT cells are abundant in the liver, representing approximately 30% of hepatic lymphocytes 13 . Our data showed that NKT cells were depleted during liver brosis progression (Figs.3A and B). Consistent with other previous studies, the number of intrahepatic NKT cells was reduced in chronic liver injury, and this phenomenon is most signi cant in CCl4-induced toxic brosis 14 . This result is due to either activationinduced NKT cell death or loss of cell markers such as NK1.1 15,16 . Our results suggest that gut microbiome can improve liver injury by suppressed NKT cells activation via LCA. However, the protective effect of LCA disappeared after treatment with antibiotics to reduce the gut commensal bacteria (Figs.1A, B, and C).
The human liver contains more NK cells than NKT cells, corresponding to 30-50% of total lymphocytes 17 . In our results, the number of NK cells was signi cantly higher in the CCl4 + LCA group than in the CCl4 group, and this number decreased after antibiotics treatment (Fig.2C). Consistent with our previous ndings, the effect of LCA disappeared in the absence of gut commensal bacteria. The hepatoprotective

effect of NK cells has been well documented in chronic liver diseases. NK cells can suppress in ammation by secreting anti-in ammatory factors. Moreover, activated NK cells have anti-brotic activity by killing pro-brotic hepatic stellate cells (HSCs), and this activity mediated by NKp46-NCR1
and NKG2D-MIC-A or MIC-B interactions, thereby reducing liver brosis in vivo 18 .
To connect these ndings, several cytokines were measured in the peripheral blood of mice. Serum TNFα levels of were signi cantly higher in the CCl4 group than in the CCl4+LCA group. However, TNF-α levels signi cantly increased after disrupting the gut microbiome with antibiotics (Fig.4A). M1 macrophage in ltration induces the production of in ammatory factors, including TNF-α, with strong pro-in ammatory or brotic effects on myo broblasts and HSCs 19,20 . Previous studies have shown that NKT cells release pro-in ammatory cytokines, including TNF-α, IL-4, and IFN-g, which promote brosis 19 . In other words, LCA can inhibits TNF-α secretion by reducing the activation of NKT cells and M1 macrophages, and this effect is mediated by the gut microbiome.
As expected, we also found that LCA can decreased the serum levels of IL-22, whereas changing the gut microbiome through antibiotics, which had little effect on IL-22 levels (Fig.4B). It is known that multiple intrahepatic immune cells, including macrophages, NK and NKT cells, and CD4 and CD8 T cells, secrete IL-22 21 . Interestingly, there are many articles con rming that IL-22 mediates liver protection functions in acute liver injury 22,23 . However, increasing evidence has shown that IL-22 aggravates liver cell damage and in ammation in chronic liver disease 24 . Many advances pointed out that the expression of IL-22 was up-regulated in the liver of patients with chronic HBV infection, and promoted liver brosis by the inducing the migration of intrahepatic Th17 cells 21,25 .

The gut microbiome mediates the anti-in ammatory effects of lithocholic acid
The liver is exposed to the gut microbiome and its metabolites through the intestinal circulation, which accounts for 70% of the blood supply to the liver 26 . Changes in the gut microbiome can affect the function of hepatic immune cells, enterohepatic circulation, and LCA metabolism. Our results showed that antibiotics markedly reduced the abundance of gut commensal bacteria, increased the serum levels of ALT and AST (Fig.4C), and promoted the activation of NKT cells and M1 macrophages in mouse liver.
Furthermore, antibiotic treatment aggravated liver in ammation and brosis, demonstrating that the antiin ammatory and anti-brotic effects of LCA are reduced in the liver in the absence of a balanced gut microbiome.
Our study found that compared with the CCl4 group, the Simpson's diversity index increased after LCA treatment (Fig.4D). Antibiotics treatment decreased microbial diversity and the relative abundance of Firmicutes. However, the Proteobacteria show excessive proliferation (especially Enterobacteriales) in CCl4+LCA+VM group (Figs.4E and F). Studies commonly supported this concept that the excessive proliferation of Proteobacteria means that gut microbiome dysbiosis or an unstable gut microbial community structure 27 . Generally, the human gut microbiome contains only a minor proportion of the phylum Proteobacteria under healthy conditions 28 . However, this bacterial group causes colitis and in ammation under pathological conditions 29,30 .
The relative abundance of Proteobacteria is positively correlated with intestinal in ammation, which may be because his bacterial taxon affects metabolism and immunity 31,32 . In this respect, the oral administration of Helicobacter typhlonius, one of the Proteobacteria species also triggered colitis in mice lacking the recombinase-activating gene and the transcription factor T-bet 33 . Antibiotics indreasd serum TNF-a levels and abolished the anti-brotic effect of LCA ( Fig. 4A and Figs.1A, B and C), which may be related to the increased relative abundance of Escherichia coli and Firmicutes, leading to local or systemic metabolic dysfunction 34,35 . However, we suspect that LCA needs to be modi ed and/or transformed by the gut microbiome before it can play an anti-in ammatory and anti-brotic role in the liver.

Discussion
Some studies demonstrated the anti-in ammatory effect of bile acids 4,36,37 . In this respect, LCA can reduce the phagocytic activity and the production of pro-in ammatory cytokines in monocytes, macrophages, and other immune cells in the liver by activating TGR5 4 . With regard to the effect of LCA on liver in ammation and brosis, our results showed that (1) LCA regulated multiple intrahepatic immune cells, including NKT/NK cells and M1 and M2 macrophages; (2) LCA increased TNF-a and IL-22 and improved chronic hepatic in ammation and brosis by activating NKT cells and M1 macrophages; and (3) The anti-in ammatory and anti-brotic effects of LCA required the participation of the gut microbiome.
Although, gut commensal bacteria are involved in immune regulation and in ammatory response mediated by LCA in the liver, how the gut microbiome affects the role of LCA in liver in ammation, the mechanism of action of LCA, and the immune pathways regulated by LCA in NKT/NK cells remain to be explored.
In summary, our study identi ed a mechanism by which gut commensal bacteria use LCA as messengers to regulate intrahepatic immune cells and improve brosis in the mouse liver. Therefore, maintaining the gut microbiome homeostasis while administering LCA is a potential therapeutic strategy to improve liver brosis even reverse it. Furthermore, a better understanding of the interactions of gut microbiome-bile acids-liver triangle in liver brosis may help develop effective microecological and immune interventions. The study conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the research ethics committee of the Zhejiang University School of Medicine.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated and/or analyzed during the current study are not publicly available due to their con dentiality, but are available from the corresponding author upon reasonable request.

Con icts of Interest
The authors declare that there is no con ict of interest regarding the publication of this paper.

Source of Funding
The