Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jul;139(1):323-34.e7.
doi: 10.1053/j.gastro.2010.03.052. Epub 2010 Mar 27.

Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice

Kouichi Miura  1 , Yuzo Kodama, Sayaka Inokuchi, Bernd Schnabl, Tomonori Aoyama, Hirohide Ohnishi, Jerrold M Olefsky, David A Brenner, Ekihiro Seki
Affiliations

Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice

Kouichi Miura et al. Gastroenterology. 2010 Jul.

Abstract

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-1beta (IL-1beta) 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-1beta production was suppressed in TLR9(-/-) mice. Kupffer cells produced IL-1beta in response to CpG oligodeoxynucleotide. IL-1beta but not CpG-oligodeoxynucleotides, increased lipid accumulation in hepatocytes. Lipid accumulation in hepatocytes led to nuclear factor-kappaB inactivation, resulting in cell death in response to IL-1beta. IL-1beta 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-1beta by Kupffer cells, leading to steatosis, inflammation, and fibrosis.

PubMed Disclaimer

Conflict of interest statement

Conflicts of interest

The authors disclose no conflicts.

Figures

Figure 1
Figure 1
TLR9 deficiency attenuates steatohepatitis. WT and TLR9−/− mice were fed a CSAA (CS; n = 4) or CDAA (CD; n = 8) diet for 22 weeks. Closed bar, WT mice; open bar, TLR9−/− mice. (A) Liver sections were stained with H&E (arrowheads, inflammatory cells; arrows, ballooning hepatocytes), Oil-Red O, TUNEL (arrows; apoptotic cells), and Sirius red. Original magnification, ×200 for H&E and TUNEL staining, ×100 for Oil-Red O and Sirius red stainings. (B) Number of TUNEL-positive cells, (C) Sirius red–positive area and (D) NAFLD activity score were calculated. N.D., not detected. (E) Hepatic triglyceride and serum ALT levels were measured. (F) Hepatic mRNA levels of fibrogenic markers were determined by quantitative real-time PCR. Data represent mean ± SD; *P < .05; n.s., not significant.
Figure 2
Figure 2
TLR9 induces IL-1β production in Kupffer cells. (A and B) The livers and sera were harvested from 22-week-old CSAA (CS) or CDAA (CD) diet-fed WT, TLR9−/−, and MyD88−/− mice. (A) Hepatic mRNA levels of inflammatory cytokines were measured by quantitative real-time PCR (qPCR). (B) Serum IL-1β levels were measured by enzyme-linked immunoabsorbent assay (ELISA). (C) WT mice (21 weeks old) fed the CDAA diet were injected control (Veh; n = 6) or clodronate liposome (Clo; n = 6) to deplete Kupffer cells. The livers and sera were harvested 1 week after the liposome injection. Hepatic mRNA and serum protein levels of IL-1β were measured by qPCR and ELISA, respectively. (D) IL-1β mRNA expression in hepatocytes, Kupffer cells, HSCs, and sinusoidal endothelial cells was measured by qPCR. (E) WT, TLR9−/−, and MyD88−/− Kupffer cells were treated with 5 μg/mL CpG-ODN or non–CpG-ODN, and then IL-1β mRNA levels were measured by qPCR. To convert active IL-1β from proIL-1β, Kupffer cells were treated with 2 mmol/L ATP for 30 minutes after 24 hours of incubation with 5 μg/mL CpG-ODN (n = 4, each group), and then secreted IL-1β levels were measured by ELISA. Data represent mean ± SD; *P < .05, **P < .01; n.s., not significant.
Figure 3
Figure 3
IL-1β promotes lipid metabolism and cell death in hepatocytes. (A–C) WT, TLR9−/−, and IL-1R−/− hepatocytes were treated with 10 ng/mL IL-1β. Veh, vehicle. (A) Lipid accumulation (Oil-Red O staining) and (B) triglyceride content (normalized to protein concentration) were measured in WT, TLR9−/−, and IL-1R−/− hepatocytes treated with IL-1β for 24 hours. (C) Hepatocytes were treated with IL-1β for 8 hours. mRNA expression of DGATs in hepatocytes was determined by quantitative real-time PCR (qPCR). (D–F) Hepatocytes were isolated from 22-week-old mice fed the CSAA (CS) or the CDAA (CD) diet. (D) Hepatocytes were treated with IL-1β for 24 hours. Apoptosis and necrosis were determined by Hoechst33342 and propidium iodide, respectively. (E) ALT and LDH levels in supernatant were measured. (F, left) mRNA levels of Bcl2 and Bax were determined by qPCR. (F, right) NF-κB activity in response to IL-1β was examined by NF-κB–reporter assay. Data represent mean ± SD; *P < .05, **P < .01; n.s., not significant. Original magnification, ×400 (A), ×200 (C).
Figure 4
Figure 4
Kupffer cell–derived IL-1β promotes HSC activation. (A–D) HSCs isolated from WT, TLR9−/−, and IL-1R−/− mice were stimulated with 10 ng/mL IL-1β for 8 hours to measure mRNA expression of TIMP1 (A), PAI-1 (B), collagen (C), and Bambi (D) by quantitative real-time PCR (qPCR), and for 24 hours to determine TIMP1 expression in the supernatant by Western blot (A). (E) Kupffer cell–conditioned medium (Kup-CM) was prepared by treating Kupffer cells with 5 μg/mL CpG-ODN or non–CpG-ODN for 24 hours. Then, WT, TLR9−/−, and IL-1R−/− HSCs were treated with Kup-CM for 8 hours to determine mRNA expression of fibrogenic markers by qPCR (E, left) and for 24 hours to determine TIMP1 protein expression in the supernatant (E, right). Data represent mean ± SD; *P < .05, **P < .01; n.s., not significant.
Figure 5
Figure 5
IL-1R−/− mice show attenuated steatosis and fibrosis. WT and IL-1R−/− mice were fed a CSAA diet (CS; n = 4) or CDAA diet (CD; n = 8) for 22 weeks. Closed bar, WT mice; open bar, IL-1R−/− mice. (A) H&E (arrowheads, inflammatory cells; arrows, ballooning hepatocytes), Oil-Red O, TUNEL (arrows; apoptotic cells), and Sirius red staining were performed. Original magnification, ×200 for H&E and TUNEL, ×100 for Oil-Red O and Sirius red. (B) Number of TUNEL-positive cells and (C) Sirius red–positive area were suppressed in IL-1R−/− mice. (D) NAFLD activity score, steatosis, and fibrosis were suppressed in IL-1R−/− mice. N.D., not detected. (E) Hepatic triglyceride and serum ALT levels were decreased in IL-1R−/− mice. (F) Hepatic mRNA levels of fibrogenic markers were measured by quantitative real-time PCR. Data represent mean ± SD, *P < .05; n.s., not significant.
Figure 6
Figure 6
Gene deletion of MyD88 ameliorates steatohepatitis. WT and MyD88−/− mice were fed a CSAA (CS, n=4) or CDAA (WT-CD, n = 8; MyD88−/− -CD, n = 7) diet for 22 weeks. Closed bar, WT mice; open bar, MyD88−/− mice. (A) H&E (arrowheads, inflammatory cells; arrows, ballooning hepatocytes), Oil-Red O, TUNEL (arrows; apoptotic cells), and Sirius red staining show reduced steatosis, inflammatory cells, apoptosis, and fibrosis in MyD88−/− mice. Original magnification, ×200 for H&E and TUNEL staining, ×100 for Oil-Red O and Sirius red stainings. (B) TUNEL-positive cells, (C) Sirius red–positive area, and (D) NAFLD activity score were suppressed in MyD88−/− mice. N.D., not detected. (E) Hepatic triglyceride and serum ALT levels were suppressed in MyD88−/− mice. (F) Hepatic mRNA levels of fibrogenic markers were determined by quantitative real-time PCR. Data represent mean ± SD; *P < .05, **P < .01; n.s., not significant.
Figure 7
Figure 7
(A) TLR9–MyD88–IL-1R axis promotes insulin resistance. Normal chow (N), CSAA (CS), and CDAA (CD) diets were fed to WT, TLR9−/−, IL-1R−/−, and MyD88−/− mice for 22 weeks (n = 6, each group). Insulin resistance was examined by HOMA-IR. Data represent mean ± SEM; **P < .01; n.s., not significant. (B) The proposed mechanism responsible for the development of NASH through TLR9 and IL-1β. TLR9 ligands, including bacterial DNA or other endogenous mediators, stimulate Kupffer cells to produce IL-1β. Secreted IL-1β acts on hepatocytes to increase lipid accumulation and cell death, causing steatosis and inflammation. IL-1β also stimulates HSCs to produce fibrogenic factors such as collagen and TIMP-1, resulting in fibrogenesis. Simultaneously, the TLR9-MyD88 axis promotes insulin resistance. TLR4 also contributes to this network in NASH as described previously.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Browning JD, Horton JD. Molecular mediators of hepatic steatosis and liver injury. J Clin Invest. 2004;114:147–152. - PMC - PubMed
    1. Marra F, Gastaldelli A, Svegliati Baroni G, et al. Molecular basis and mechanisms of progression of non-alcoholic steatohepatitis. Trends Mol Med. 2008;14:72–81. - PubMed
    1. Maher JJ, Leon P, Ryan JC. Beyond insulin resistance: innate immunity in nonalcoholic steatohepatitis. Hepatology. 2008;48:670–678. - PMC - PubMed
    1. Rivera CA, Adegboyega P, van Rooijen N, et al. Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis. J Hepatol. 2007;47:571–579. - PMC - PubMed
    1. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115:209–218. - PMC - PubMed

Publication types