The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88

Abstract

The Toll-like receptor 4 (TLR4) that recognizes endotoxin, a trigger of inflammation in alcoholic liver disease (ALD), activates two signaling pathways utilizing different adapter molecules: the common TLR adapter, myeloid differentiation factor 88 (MyD88), or Toll/interleukin immune-response-domain-containing adaptor inducing interferon (IFN)-beta. The MyD88 pathway induces proinflammatory cytokine activation, a critical mediator of ALD. Here we evaluated the role of MyD88 in alcohol-induced liver injury in wild-type, TLR2-deficient, TLR4-deficient, or MyD88-deficient (knockout [KO]) mice after administration of the Lieber-De-Carli diet (4.5% volume/volume ethanol) or an isocaloric liquid control diet for 5 weeks. Alcohol feeding resulted in a significant increase in serum alanine aminotransferase levels, liver steatosis and triglyceride levels suggesting liver damage in WT, TLR2-KO, and MyD88-KO but not in TLR4-KO mice. Expression of inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) and TLR4 coreceptors (CD14 and MD2) was significantly higher in livers of alcohol-fed WT, TLR2-KO, or MyD88-KO, but not in TLR4-KO mice, compared to controls. Reactive oxygen radicals produced by cytochrome P450 and the nicotinamide adenine dinucleotide phosphate complexes contribute to alcoholic liver damage. Alcohol feeding-induced expression and activation of cytochrome P450 and the nicotinamide adenine dinucleotide phosphate complex were prevented by TLR4-deficiency but not by MyD88-deficiency. Liver expression of interferon regulatory factor 3 (IRF3), a MyD88-independent signaling molecule, was not affected by chronic alcohol treatment in whole livers of WT mice or in any of the KO mice. However, the induction of IRF7, an IRF3-inducible gene, was found in Kupffer cells of alcohol-fed WT mice. Alcohol feeding also induced nuclear factor-kappaB activation in a TLR4-dependent MyD88-independent manner.

Conclusion: While TLR4 deficiency was protective, MyD88 deficiency failed to prevent alcohol-induced liver damage and inflammation. These results suggest that the common TLR adapter, MyD88, is dispensable in TLR4-mediated liver injury in ALD.

Fig. 1.

TLR4-deficiency but not MyD88-deficiency protects against alcohol-induced liver damage. C57BL/6 (WT), TLR4-deficient, or MyD88-deficient mice (five to six per group) received the Lieber-DeCarli diet with 4.5 volume/volume% of ethanol or isocaloric liquid control diet for 5 weeks. After the 5-week feeding period, the mice were sacrificed. Serum was separated from whole blood and analyzed for (A) ALT, (B) alcohol, and (C) endotoxin levels. Mean values ± standard deviation (SD) are shown.

Fig. 2.

Chronic alcohol feeding induces liver steatosis in WT, TLR2-deficient, and MyD88-deficient mice, but not in TLR4-deficient mice. C57BL/6 (WT), TLR2-deficient, TLR4-deficient, or MyD88-deficient mice (five to six per group) received the Lieber-DeCarli diet as described in Materials and Methods. (A) Representative sections of formalin-fixed, paraffin-embedded livers stained with hematoxylin and eosin of each group are shown at ×100 (higher magnification is provided in Supplementary Fig. 1). (B) Liver triglyceride levels and (C) liver/body weight ratios are shown as mean ± standard deviation (SD) values from N = 5 mice/group.

Fig. 3.

Chronic alcohol feeding increases liver mRNA expression of TNFα and IL-6 and TLR4 coreceptors, CD14 and MD2, in WT, TLR2-deficient, and MyD88-deficient mice, but not in TLR4-KO mice. C57BL/6, TLR2-deficient, TLR4-deficient, or MyD88-deficient mice received the Lieber-DeCarli diet. Liver RNA levels of (A) TNFα, (B) IL-6, (C) CD14, and (D) MD2 were analyzed by real-time quantitative polymerase chain reaction (qPCR). The values were normalized to 18S and are shown as the fold increase over the pair-fed WT control group from N = 5–6 per group.

Fig. 4.

TLR4 deficiency protects from alcohol-induced increase of liver CYP2E1 expression. C57BL/6, TLR4-deficient, or MyD88-deficient mice (five to six per group) received the Lieber-DeCarli diet. (A) Liver RNA levels of CYP2E1 were analyzed by real-time quantitative polymerase chain reaction (qPCR). CYP2E1 protein level in liver microsomal fractions of (B) WT, (C) TLR4-deficient, and (D) MyD88-deficient mice was analyzed by western blot; calnexin expression was used as housekeeping control. A representative blot (top) and the densitometric analysis normalized to loading control from N = 3 (bottom) is shown.

Fig. 5.

Chronic alcohol feeding increases liver mRNA expression of the NADPH-complex subunits in WT, TLR2-deficient and MyD88-deficient mice but not in TLR4-KO mice. C57BL/6, TLR2-deficient, TLR4-deficient, or MyD88-deficient mice (five to six per group) received the Lieber-DeCarli diet. Liver RNA levels of (A) p22phox, (B) gp91phox, (C) p47phox, (D) p67phox, and (F) Nox4 were analyzed by real-time quantitative polymerase chain reaction (qPCR). The values were normalized to 18S and shown as fold increase over the pair-fed WT control group. (E) p47phox protein expression in whole liver membrane fractions was analyzed by western blot in alcohol-fed and pair-fed WT mice; pancadherin expression was used as a loading control. The densitometric analysis of p47phox protein expression (normalized to pan-cadherin; N = 3) is shown.

Fig. 6.

Chronic alcohol feeding results in activation of TLR4-mediated MyD88-independent pathways. C57BL/6, TLR4-deficient, or MyD88-deficient mice (five to six per group) received the Lieber-DeCarli or a pair-fed diet for 5 weeks. (A) IRF3 and (C) IRF7 RNA levels in the whole liver, and (B) IRF3 and IRF7 levels in the purified hepatocytes and Kupffer cells were analyzed by real-time quantitative polymerase chain reaction (qPCR). (D) NF-κB activity in the whole livers was analyzed using electrophoretic mobility shift assay; a representative gel (top) and the densitometric analysis from N = 5 mice/group (bottom) are shown. *P < 0.05 compared to the corresponding pair-fed control.