Activation of interferon and NF-kB pathways in early human alcoholic liver disease development

Starkel, Peter [UCL] Lanthier, Nicolas [UCL] De Saeger, Christine [UCL] Sempoux, Christine [UCL] Leclercq, Isabelle [UCL] Horsmans, Yves [UCL]

Background and aims : Animal data suggest a pathogenic role for TLR4 with subsequent activation of NFkB in alcohol liver injury. Here, we assessed cytokines, TLR and TLR-dependent pathways expressions in human ALD. Methods : Liver biopsies from 41 actively drinking ALD patients were compared with normal liver tissue (n=6) and with liver biopsies from ALD patients after 2 weeks of alcohol abstinence (n=27). Quantitative PCR and Western blotting were used for mRNA and protein analysis, respectively, immunohistochemistry for cellular localization of inflammatory processes. The study was approved by our local ethics committee. Results : Compared with normal livers, already early ALD (with minimal fibrosis) showed a significant activation of NFkB with increased total and phosphorylated p65 protein expression and up-regulation of NFkB responsive genes mRNA levels TNFalphaand IL-1beta. P65 immunohistochemistry evidenced positive staining mainly in inflammatory cells in ALD patients. Interestingly, TLR4 mRNA expression remained unchanged whereas CD14 and TNF receptor 1 mRNA were down-regulated in active ALD. Only IL-1beta returned to control levels after abstinence. STAT3 phosphorylation was also significantly induced in early ALD patients. Intriguingly, STAT3 mRNA expression as well as STAT3 responsive genes SOCS3, IL-6 and MCP-1 were significantly downregulated in ALD patients suggesting an overall inhibition of the STAT3 pathway. STAT3 inhibition did not recover following 2 weeks of alcohol abstinence. The interferon pathway was strongly activated in early ALD with significant increase in total and phosphorylated interferon regulatory factor-3, up-regulation of interferon-beta and interferon responsive genes ISG6-16 and OAS-1 mRNA levels. Interferon-beta levels significantly correlated with up-regulation of TLR3 and 7 mRNA. Activation of the interferon pathway was not reversed after abstinence. CD68 mRNA was significantly increased and both CD68 and CD163 immunohistochemistry revealed enlarged, aggregated Kupffer cells and small inflammatory cells in early ALD compared to scattered Kupffer cells in normal liver. Conclusions: Besides NFkB, early activation of the interferon pathway could play an important role in human ALD pathogenesis. Inhibition of the STAT3 pathway could contribute to impaired regenerative capacity in ALD.