Gastroenterology

Gastroenterology

Volume 139, Issue 1, July 2010, Pages 323-334.e7
Gastroenterology

Basic—Liver, Pancreas, and Biliary Tract
Toll-Like Receptor 9 Promotes Steatohepatitis by Induction of Interleukin-1β in Mice

https://doi.org/10.1053/j.gastro.2010.03.052Get rights and content

Background & Aims

Development of nonalcoholic steatohepatitis (NASH) involves the innate immune system and is mediated by Kupffer cells and hepatic stellate cells (HSCs). Toll-like receptor 9 (TLR9) is a pattern recognition receptor that recognizes bacteria-derived cytosine phosphate guanine (CpG)–containing DNA and activates innate immunity. We investigated the role of TLR9 signaling and the inflammatory cytokine interleukin-1β (IL-1β) in steatohepatitis, fibrosis, and insulin resistance.

Methods

Wild-type (WT), TLR9−/−, IL-1 receptor (IL-1R)−/−, and MyD88−/− mice were fed a choline-deficient amino acid-defined (CDAA) diet for 22 weeks and then assessed for steatohepatitis, fibrosis, and insulin resistance. Lipid accumulation and cell death were assessed in isolated hepatocytes. Kupffer cells and HSCs were isolated to assess inflammatory and fibrogenic responses, respectively.

Results

The CDAA diet induced NASH in WT mice, characterized by steatosis, inflammation, fibrosis, and insulin resistance. TLR9−/− mice showed less steatohepatitis and liver fibrosis than WT mice. Among inflammatory cytokines, IL-1β production was suppressed in TLR9−/− mice. Kupffer cells produced IL-1β in response to CpG oligodeoxynucleotide. IL-1β but not CpG-oligodeoxynucleotides, increased lipid accumulation in hepatocytes. Lipid accumulation in hepatocytes led to nuclear factor-κB inactivation, resulting in cell death in response to IL-1β. IL-1β induced fibrogenic responses in HSCs, including secretion of tissue inhibitor of metalloproteinase-1. IL-1R−/− mice had reduced steatohepatitis and fibrosis, compared with WT mice. Mice deficient in MyD88, an adaptor molecule for TLR9 and IL-1R signaling, also had reduced steatohepatitis and fibrosis. TLR9−/−, IL-1R−/−, and MyD88−/− mice had less insulin resistance than WT mice on the CDAA diet.

Conclusions

In a mouse model of NASH, TLR9 signaling induces production of IL-1β by Kupffer cells, leading to steatosis, inflammation, and fibrosis.

Section snippets

Animals

Wild-type (WT) C57BL/6 mice and IL-1 receptor (IL-1R)−/− mice were purchased from Jackson Laboratories (Bar Harbor, ME). TLR9−/− and MyD88−/− mice backcrossed at least 10 generations onto the C57BL/6 background were a gift from Dr Akira (Osaka University, Japan).12, 18 These null mice exhibited similar hepatobiliary phenotypes and hepatic lipid contents when fed standard laboratory chow (Supplementary Table 1). Male mice were divided into 3 groups at 8 weeks old; standard chow group (PicoLab

TLR9 Signaling Induces Steatohepatitis and Fibrosis

We investigated the contribution of TLR9 in a murine model of NASH. WT and TLR9−/− mice were fed a CDAA or control CSAA diet for 22 weeks. WT mice had marked lipid accumulation with inflammatory cell infiltration, hepatocyte death, and liver fibrosis (Figure 1A–C;Supplementary Figure 1) after CDAA diet feeding. In contrast, TLR9−/− mice had a significant reduction of steatosis, inflammation, and fibrosis compared with WT mice (Figure 1A–C). No significant differences were observed in food

Discussion

The present study shows that steatohepatitis is diminished in TLR9−/−, IL-1R−/−, and MyD88−/− mice. Kupffer cells, but not hepatocytes and HSCs, respond to TLR9 ligands to produce IL-1β. This IL-1β induces lipid accumulation and cell death in hepatocytes and the expression of fibrogenic mediators in HSCs, resulting in steatosis, hepatocyte injury, and fibrosis (Figure 7B).

Two possible mechanisms activate innate immune systems in NASH. First, translocated bacteria and their products activate the

Acknowledgments

The authors thank Dr Shizuo Akira (Osaka University, Japan) for the generous gift of TLR9−/− and MyD88−/− mice and Dr Katsumi Miyai (Department of Pathology at UCSD), Dr Wuqiang Fan, Dr Saswata Talukdar, Rie Seki, and Karin Diggle (Department of Medicine at UCSD) for excellent technical assistance.

References (52)

  • M. Imamura et al.

    Contribution of TIR domain-containing adapter inducing IFN-beta-mediated IL-18 release to LPS-induced liver injury in mice

    J Hepatol

    (2009)
  • S. Chitturi et al.

    NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome

    Hepatology

    (2002)
  • S. Chitturi et al.

    Serum leptin in NASH correlates with hepatic steatosis but not fibrosis: a manifestation of lipotoxicity?

    Hepatology

    (2002)
  • T. Ota et al.

    Insulin resistance accelerates a dietary rat model of nonalcoholic steatohepatitis

    Gastroenterology

    (2007)
  • S. He et al.

    A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis

    J Biol Chem

    (2010)
  • J.D. Browning et al.

    Molecular mediators of hepatic steatosis and liver injury

    J Clin Invest

    (2004)
  • J.J. Maher et al.

    Beyond insulin resistance: innate immunity in nonalcoholic steatohepatitis

    Hepatology

    (2008)
  • R. Bataller et al.

    Liver fibrosis

    J Clin Invest

    (2005)
  • T. Kawai et al.

    The roles of TLRs, RLRs and NLRs in pathogen recognition

    Int Immunol

    (2009)
  • E. Seki et al.

    Toll-like receptors and adaptor molecules in liver disease: update

    Hepatology

    (2008)
  • G. Szabo et al.

    Pattern recognition receptors: a contemporary view on liver diseases

    Hepatology

    (2006)
  • W.E. Naugler et al.

    Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production

    Science

    (2007)
  • E. Seki et al.

    TLR4 enhances TGF-beta signaling and hepatic fibrosis

    Nat Med

    (2007)
  • H. Hemmi et al.

    A Toll-like receptor recognizes bacterial DNA

    Nature

    (2000)
  • R. Frances et al.

    Bacterial DNA in patients with cirrhosis and noninfected ascites mimics the soluble immune response established in patients with spontaneous bacterial peritonitis

    Hepatology

    (2008)
  • C. Guarner et al.

    The detection of bacterial DNA in blood of rats with CCl4-induced cirrhosis with ascites represents episodes of bacterial translocation

    Hepatology

    (2006)
  • Cited by (611)

    View all citing articles on Scopus

    Conflicts of interest The authors disclose no conflicts.

    Funding This study was supported by a Liver Scholar Award from the American Association for the Study of Liver Diseases/American Liver Foundation and by a pilot project from the Southern California Research Center for ALPD and Cirrhosis (P50 AA11999) funded by NIAAA (both to E.S.), NIH grants 5R01GM041804 and 5R01DK072237 (D.A.B.), and Takeda Science Foundation (K.M.).

    View full text