The influence of gut derived antigens and hepatic steatosis-associated fatty acids on chemotaxis, innate immunity and pro-inflammatory cytokine expression in patients with non-alcoholic fatty liver disease.
Background: Emerging data suggests that enhanced chemotaxis, innate immunity and pro-inflammatory mediators, namely cytokines and oxylipins are critical in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Data also suggests that gut-derived antigens and hepatic steatosis-associated fatty acids are potent regulators of these activities. To date, there have been no studies documenting the extent of correlation between these factors and the extent of hepatic inflammatory activity in NAFLD. Objectives: The objectives of this study were: 1) To compare plasma oxylipin levels in 43 different NAFLD patients and healthy controls (HCs). 2) To compare basal and stimulated levels of chemokine receptor (CCR), Toll-like receptor (TLR),cytokines IL-1β, IL-6, TNF-α and IL-10 in peripheral blood mononuclear cells (PBMCs) and monocytes derived from NAFLD patients and HCs, and 3) To determine whether the differences observed in NAFLD patients correlate with hepatic apoptotic activity as reflected by levels of the hepatic apoptotic marker plasma ck18. Methods: 35 adult NAFLD patients and 8 HCs participated in the study. Plasma oxylipin levels were determined by mass spectrometry. PBMCs and monocytes were isolated and analyzed in vitro prior to and following exposure to varying concentrations of the gut-derived antigens, lipopolysaccharide (LPS) and Pam3CSK4 (PAM) or the hepatic steatosis-associated fatty acid, palmitate (PAL) for 12-24 hours. CCR1, CCR2, TLR2, TLR4, IL-1β, IL-6, IL-10 and TNF-α surface or secreted protein levels were measured by flow cytometry and ELISA respectively. Results: Plasma oxylipins, 20-HETE and 8-HETrE were elevated in NAFLD patients with 5-HETE, 8-HETE, 15-HETE, 20-HETE, 11,12-DiHETrE and 14,15-DiHETrE correlating positively with ck18 levels. At baseline, CCR, TLR and cytokine levels were similar in NAFLD patients and HCs. In NAFLD patients, the levels of CCR, TLR and cytokines did not correlate with plasma ck18 levels. Following stimulation with LPS, Pam3CSK or palmitate, CCR1 and CCR2 levels decreased in both NAFLD patients and HCs but to a lesser extent in HCs. The extent of the CCR1 but not CCR2 decrease inversely correlated with plasma ck18 levels. TLR2 levels significantly increased following stimulation while TLR4 expression was found to decrease following LPS, but not Pam3CSK4 or palmitate stimulation. The changes in toll-like receptors were similar in NAFLD patients and HCs and did not correlate with plasma ck18 levels in NAFLD patients. The levels of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α increased following stimulation in NAFLD patients and HCs. Importantly, gut derived antigen-induced IL-1β and IL-6 levels were increased in NAFLD patients compared with HCs and positively correlated with ck18 levels. Conclusions: The results of this study suggest that specific oxylipin levels differ between NAFLD subjects compared to HCs. In addition, NAFLD was associated with an increased PBMC sensitivity to gut antigens reflected by increased stimulated IL-1β and IL-6 production. The increased PBMC sensitivity in simple steatosis is likely prior to the inflammation in the liver which might contribute to NASH development. The increased PBMC sensitivity correlated with hepatic apoptosis levels which indicated that PBMC may play a role in hepatic inflammation and cell death. We also found CCR1 expression was decreased after stimulation, possibly reflecting reduced monocytes trafficking from the site of inflammation which suggests hepatic apoptosis is associated with an increased ability of monocytes to stay in the liver with gut derived stimuli.