Deficiency in myeloid differentiation factor-2 and Toll-like receptor 4 expression attenuates non-alcoholic steatohepatitis and fibrosis in mice

Timea Csak, University of Massachusetts Medical School
Arumugam Velayudham, University of Massachusetts Medical School
Istvan Hritz, University of Massachusetts Medical School
Jan Petrasek, University of Massachusetts Medical School
Ivan Levin, University of Massachusetts Medical School
Dora Lippai, University of Massachusetts Medical School
Donna Catalano, University of Massachusetts Medical School
Pranoti Mandrekar, University of Massachusetts Medical School
Angela Dolganiuc, University of Massachusetts Medical School
Evelyn A. Kurt-Jones, University of Massachusetts Medical School
Gyongyi Szabo, University of Massachusetts Medical School

Abstract

Toll-like receptor 4 (TLR4), and its co-receptor, Myeloid Differentiation Factor 2 (MD-2), are key in recognition of lipopolysaccharide (LPS) and activation of pro-inflammatory pathways. Here we tested the hypothesis that TLR4 and its co-receptor MD-2 play a central role in non-alcoholic steatohepatitis (NASH) and liver fibrosis in non-alcoholic fatty liver disease. Mice of control genotypes and those deficient in MD-2 or TLR4 (knock-out, KO) received methionine-choline-deficient (MCD) or methionine-choline-supplemented (MCS) diet. In mice of control genotypes MCD diet resulted in non-alcoholic steatohepatitis, liver triglycerides accumulation and increased Thiobarbituric Acid Reactive Substances (TBARS), a marker of lipid peroxidation, compared to MCS diet. These features of NASH were significantly attenuated in MD-2-KO and TLR4-KO mice. Serum alanine aminotransferase (ALT), 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 TNFalpha and nictoinamide adenine dinucleotide phosphate (NADPH) oxidase complex mRNA expression and activation, was significantly lower in MCD-diet-fed MD-2-KO and TLR4-KO compared to corresponding genotype control mice. Markers of liver fibrosis (collagen by Sirius red and alphaSMA staining, procollagen-I, TGFbeta1,alphaSMA, MMP2 and TIMP1 mRNA) were attenuated in MD2- and TLR4-KO compared to 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.