Mitochondrial Dysfunction May Explain Innate Immunoparesis And Susceptibility To Infection Of Patients With Alcoholic Hepatitis

Background Alcoholic hepatitis (AH) is the most florid form of alcohol related liver disease. Infection develops in 50% of patients and is strongly associated with mortality. Innate immune paresis is recognised in this condition, but mechanisms have been elusive. Mitochondrial damage within hepatocytes in AH is a strong prognostic finding however mitochondrial defects in immune cells have not been investigated. We therefore sought to explain innate immune defects in patients with AH by probing the mitochondrial function of circulating monocytes and neutrophils in AH. Methods Experiments were performed on whole blood after red cell lysis from 5 AH patients and 4 healthy controls (HC) with and without 10 ng/ml or 100 ng/ml lipopolysaccharide (LPS) stimulation for 4 hours. Flow cytometry was used to quantify mitochondrial mass, polarization and superoxide leak using MitoTracker Green (MT), MitoStatus Red (MS), MitoSOX Red probes. To control for variations in mass Mitochondrial Polarization Ratio (MPR) was calculated from MS:MT fluorescence ratio. IL-1b, IL-6, IL-8 & TNFa production were quantified using intracellular cytokine signalling. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial membrane depolarising agent, was used to recapitulate membrane depolarisation in healthy monocyte mitochondria at doses ranging from 0–100μM. Results Stimulated TNFa production was impaired in AH monocytes (12070 vs 37200 MFI; p=0.02). Mitochondrial mass was greater in AH monocytes vs HC (23802 vs 12926 MFI; p=0.02) and had greater mitochondrial superoxide leak (1791 vs 894 MFI; p=0.03). Unstimulated AH monocyte MPR was lower than HC (0.522 vs 0.815; p=0.032). Unlike HC, AH monocytes failed to hyperpolarize with stimulation (HC 10 ng/mL vs 100 ng/mL LPS 1.28 vs 1.57 MPR, p=0.046 compared to AH 10 ng/mL vs 100 ng/mL LPS 0.58 vs 0.6 MPR, p=ns: figure 1). Similar trends were demonstrated in AH neutrophils. Monocyte MPR correlated strongly with TNFa production (r=0.733; p=0.031) whereas mitochondrial superoxide leak had strong negative correlation (r=-0.8776; p=0.004). Exposure of HC to mitochondrial depolarizer CCCP reduced production of TNFa in a dose dependant manner (0 vs 10 vs 50 vs 100μM CCCP; TNFa 45850 vs 39404 vs 29130 vs 14698 MFI). Conclusion AH monocytes and neutrophils harbour dysfunctional mitochondria. Specific defects in mitochondrial membrane polarization and superoxide leak correlate strongly with impaired TNFa production from AH monocytes, a phenomenon that has been repeatedly associated with increased susceptibility to infection. Further investigation into the precise nature of these mitochondrial defects may identify reversible targets to reduce susceptibility to infection for alcoholic hepatitis patients.