Introduction Sepsis is a major cause of mortality in patients with alcohol-induced acute and chronic liver failure (ALF/CLF). Neutrophils are a major innate immune cell subset involved in the first line of defence against infection and circulating neutrophil dysfunction has been reported in patients with ALF/CLF. However, there is a paucity of understanding regarding the mechanisms involved in this dysfunction. In this study we aimed to characterise the precise relationship between neutrophil dysfunction and alcohol-induced liver damage with a novel in vitro model mimicking the in vivo interactions of neutrophils and hepatocytes.
Methods We cultured a well-characterised neutrophil-line HL-60 either directly with ethanol or with supernatants taken from ethanol metabolising-human hepatoma cell lines VL-17A (positive for alcohol-dehydrogenase and CYP2E1) cultured in the presence of safe levels (10 mM) and toxic levels (250 mM) of ethanol reflecting real-life human alcohol consumption for 24 h. Neutrophil function was evaluated by TLR expression, chemotaxis, phagocytosis and respiratory burst assays. Cell supernatants were also collected for cytokine profiling and to quantitate levels of ammonia and ethanol metabolites. The effect of ethanol on the functional activities of neutrophils isolated from both normal and ALF/CLF patients will also be assessed.
Results Supernatants collected from the hepatoma line VL17A cultured with 250 mM ethanol (representative of an alcohol binge) significantly reduced the phagocytic capacity of the HL-60-neutrophil line (p<0.05). This was greater than the effect of the same concentration of ethanol applied directly to the neutrophils (p<0.05). Our preliminary data also suggests that the metabolised ethanol inhibits chemotaxis of the HL60-neutrophil cells towards a gradient of fMLP. Furthermore, we observed a reduction of TLR4 expression.
Conclusion We describe a novel model for investigating the correlates of dysfunctional innate immunity during acute alcohol-induced liver injury. We identify that alcohol does impair neutrophil function directly but this is profoundly increased after hepatocyte alcohol metabolism implicating a causal link between liver injury and impairment of antibacterial neutrophil functions.
Competing interests None declared.
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