Innate Immune Cell Activation in a Human in-vitro Model of Nonalcoholic Steatohepatitis (NASH)

Background:

The immune system likely plays a significant role in the pathogenesis of fatty liver disease. The metabolic changes induce immunological responses resulting in nonalcoholic steatohepatitis (NASH) and further aggravation of the metabolic derangement in a feed-forward loop. However, most of the evidence regarding the immune system role in the context of NASH is based on the use of mouse models, which makes it difficult to rely on these conclusions for humans. Therefore, we aimed in this study to analyze the activation and function of several liver resident innate cell populations in a human in-vitro cell culture model of steatosis.

Methods:

Intrahepatic lymphocytes (IHLs) and peripheral blood mononuclear cells (PBMCs) were isolated from patients undergoing liver resections. As a human in vitro model for steatosis, fully differentiated HepaRG cells were treated with 0.5 mM free fatty acids (FFAs) at a 2:1 ratio of oleate (OA) and palmeate (PA) for 24h. Induction of steatosis was confirmed by oil red O staining. FFA-treated HepaRG cells were co-cultured with or without IHLs and PBMCs. Expression levels of activation markers such as CD69 and HLA-DR on natural killer (NK), mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells were assessed by multicolor flow cytometry.

Results:

While intrahepatic NK cells were not activated in response to co-culture with FFA-treated HepaRG cells, MAIT cells showed an increased frequency and expression level of CD69 (75 vs. 79%; P = 0.03 and MFI 1974 vs. 2207; P = 0.03) as well as HLA-DR (22 vs. 30%; P = 0.04 and MFI 733 vs. 1038; P = 0.03). Remarkably, intrahepatic invariant NKT cells were activated after co-culture with FFA-treated HepaRG cells with an increased frequency and expression level of CD69 (68 vs. 86%; P = 0.03 and MFI 1345 vs. 1931; P = 0.03) as well as HLA-DR (31 vs. 51%; P = 0.04 and MFI 603 vs. 1410; P = 0.03). Peripheral blood iNKT cells were activated to a lesser extent with an increased frequency and expression level of HLA-DR (9 vs. 15%; P = 0.03 and MFI 265 vs. 827; P = 0.03). Activation of iNKT cells was diminished when lymphocytes were separated from FFA-treated HepaRG cells by a transwell system, indicating that cell-to-cell contact was necessary for activation. Next, production of cytokines was assessed by intracellular staining and flow cytometry after an additional 2h of stimulation with αGalCer in the presence of brefeldin A. After co-culture with FFA-treated HepaRG cells, iNKT cells were more frequently positive for the pro-inflammatory Th1 cytokine IFNγ.

Conclusion:

Collectively, these data show that invariant NKT cells are activated in response to FFA-treated HepaRG cells by direct cell-to-cell contact, leading to increased secretion of the Th1 cytokine IFNγ, which could contribute to the pro-inflammatory environment within a NASH liver.

Corresponding author: Werner, Jens M

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