Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice - PubMed (original) (raw)

Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice

Timea Csak et al. Am J Physiol Gastrointest Liver Physiol. 2011 Mar.

Abstract

Toll-like receptor 4 (TLR4) and its coreceptor, myeloid differentiation factor-2 (MD-2), are key in recognition of lipopolysaccharide (LPS) and activation of proinflammatory pathways. Here we tested the hypothesis that TLR4 and its coreceptor MD-2 play a central role in nonalcoholic steatohepatitis (NASH) and liver fibrosis in nonalcoholic fatty liver disease. Mice of control genotypes and those deficient in MD-2 or TLR4 [knockout (KO)] received methionine choline-deficient (MCD) or methionine choline-supplemented (MCS) diet. In mice of control genotypes, MCD diet resulted in NASH, liver triglycerides accumulation, and increased thiobarbituric acid reactive substances, a marker of lipid peroxidation, compared with MCS diet. These features of NASH were significantly attenuated in MD-2 KO and TLR4 KO mice. Serum alanine aminotransferase, an indicator of liver injury, was increased in MCD diet-fed genotype controls but was attenuated in MD-2 KO and TLR4 KO mice. Inflammatory activation, indicated by serum TNF-α and nictoinamide adenine dinucleotide phosphate oxidase complex mRNA expression and activation, was significantly lower in MCD diet-fed MD-2 KO and TLR4 KO compared with corresponding genotype control mice. Markers of liver fibrosis [collagen by Sirius red and α-smooth muscle actin (SMA) staining, procollagen-I, transforming growth factor-β1, α-SMA, matrix metalloproteinase-2, and tissue inhibitor of matrix metalloproteinase-1 mRNA] were attenuated in MD-2 and TLR4 KO compared with their control genotype counterparts. In conclusion, our results demonstrate a novel, critical role for LPS recognition complex, including MD-2 and TLR4, through NADPH activation in liver steatosis, and fibrosis in a NASH model in mice.

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Figures

Fig. 1.

Deficiency in myeloid differentiation factor-2 (MD-2) and toll-like receptor 4 (TLR4), members of the lipopolysaccharide (LPS) recognition complex, protects from methionine choline-deficient (MCD) diet-induced liver injury. Mice of control genotypes and those deficient [knockout (KO)] in TLR4 (TLR4 KO) and MD-2 (MD-2 KO) were fed MCD or methionine choline-supplemented (MCS) diets for 8 wk. Liver tissue was subjected to hematoxylin and eosin (H&E) (A, left) and OilRed O (A, right) staining and F4/80 immunohistochemistry (D); one representative slide from n = 6–16 mice/group is shown. Liver triglycerides (TG) (B) and serum alanine aminotransferase (ALT) (E) levels were determined as described in

materials and methods

. Serum TNF-α level (F) was determined using the Multiplex assay. Liver macrophages were isolated and stained for TNF-α and the macrophage marker CD68 (ED-1) after cell permeabilization; fluorescence-activated cell sorter analysis of changes in frequency of TNF-α/CD68 double-positive cells compared with the MCS-fed genotype control is shown (C). *P < 0.05 compared with the corresponding MCS group (B, C, E, and F). #P < 0.05, MCD WT compared with MCD TLR4 KO.

Fig. 2.

Deficiency in LPS recognition complex prevents MCD diet-induced upregulation in the expression of NADPH complex and protects from lipid peroxidation. Mice of genotype control, TLR4 KO, and MD-2 KO were fed MCD or MCS diets for 8 wk. Liver thiobarbituric acid reactive substances (TBARS) levels (A) were analyzed as described in

materials and methods

. Expression of liver p47phox (B), p67phox (C), gp91phox (D), and p22phox (E) was quantified by quantitative PCR (qPCR) using specific primers and normalization against the housekeeping gene 18S. NADPH oxidase activity was determined by measuring NADP+-to-NADPH ratios as described in

materials and methods

(F). *P < 0.05 compared with the corresponding MCS group.

Fig. 3.

Deficiency in TLR4 and MD-2 protects from MCD diet-induced liver fibrosis. The livers of MCD and MCS diet-fed genotype controls and MD-2 KO and TLR4 KO mice were stained with Sirius red (A) or α-smooth muscle actin (α-SMA) immunohistochemistry (C); shown here are representative images from n = 6–16/group. Sirius red positive areas were quantified using Image J software (B). Genes associated with fibrosis, including α-SMA (D), procollagen-1 (E), and transforming growth factor (TGF)-β (F), were quantified by qPCR using specific primers and normalization against the housekeeping gene 18S; data (C-F) are shown as means ± SE from n = 6–16/group; *P < 0.05 compared with the corresponding MCS group.

Fig. 4.

Expression of tissue remodeling factors is impaired in mice deficient in MD-2 and TLR4. The livers of MCD and MCS diet-fed genotype controls and MD-2 KO and TLR4 KO mice were analyzed for matrix metalloproteinase (MMP)-2 and tissue inhibitor of metalloproteinase (TIMP)-1 expression by qPCR using specific primers and normalization against the housekeeping gene 18S; data are shown as means ± SE from n = 6–16/group; *P < 0.05 compared with the corresponding MCS group.

References

1. Adams LA, Lindor KD. Nonalcoholic fatty liver disease. Ann Epidemiol 17: 863–869, 2007 - PubMed
1. Akashi S, Saitoh S, Wakabayashi Y, Kikuchi T, Takamura N, Nagai Y, Kusumoto Y, Fukase K, Kusumoto S, Adachi Y, Kosugi A, Miyake K. Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14. J Exp Med 198: 1035–1042, 2003 - PMC - PubMed
1. Asea A, Rehli M, Kabingu E, Boch JA, Bare O, Auron PE, Stevenson MA, Calderwood SK. Novel signal transduction pathway utilized by extracellular hsp70: role of Toll-like receptor (TLR) 2 and TLR4. J Biol Chem 277: 15028–15034, 2002 - PubMed
1. Bataller R, Schwabe RF, Choi YH, Yang L, Paik YH, Lindquist J, Qian T, Schoonhoven R, Hagedorn CH, Lemasters JJ, Brenner DA. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis. J Clin Invest 112: 1383–1394, 2003 - PMC - PubMed
1. Bergheim I, Weber S, Vos M, Krämer S, Volynets V, Kaserouni S, McClain CJ, Bischoff SC. Antibiotics protect against fructose-induced hepatic lipid accumulation in mice: role of endotoxin. J Hepatol 48: 983–992, 2008 - PubMed

Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice - PubMed (original) (raw)