Low TLR9 expression in CD4+ and CD8+ human peripheral blood cells might play a role in NAFLD

BILLORDO LA; PONCINO DANIEL; ALMEIGEIRAS B; ALEGRE NADIA S.; BENAVIDEZ JAVIER; GARCIA DANIEL; ALEJANDRA C CHERÑAVSKY; CECILIA GARCIA; COLOMBATO LUIS; ROMEO JM

Mar del Plata

Congreso; LXIV Reunion Anual de la Soc Argentina de Inmunología; 2016

Sociedad Argentina de Inmunología

Background: Liver exposure to intestinal-derived bacterial products promotes a pro-inflammatory milieu that induces innate immunity and contributes to the pathogenesis of nonalcoholic fatty liver disease (NAFLD) via pattern-recognition receptors such as Toll-like receptors (TLRs). TLRs are expressed and co-stimulate T cell activation. Particularly TLR9 recognize bacterial DNA and is involved in Kupffer cell activation leading to nonalcoholic steatohepatitis (NASH). To study whether TLR9 modulates T cell activation in human NAFLD, we evaluated TLR9 expression in circulating and liver T cells, and TLR9 co-stimulatory role in peripheral blood (PB) T cell activation. Methods: Blood and liver samples came from 16 adult NAFLD patients (4 simple steatosis, 12 NASH) and 8 healthy controls (CO). PB mononuclear cells (PBMC) were obtained by Ficoll-Hypaque density gradient and liver cell suspensions by mechanical disruption. Cells were stained with conjugated anti-CD4, -CD8, -TLR9 and IgG2a isotype and analyzed by flow cytometry (FC). Functional assay were performed using CD3+ cells obtained from PBMC by negative selection. The CD3+ fraction was stimulated with soluble anti-CD3 [saCD3; 250 ng/ml] and/or ODN 2395 [2 mM] for 24 hs. Cells were harvested and stained with conjugated anti-CD4, -CD8, -CD69 and analyzed by FC. Mann-Whitney test was used. All patients provided written informed consent. Results: PB CD4+ and CD8+ cells from NAFLD showed lower expression of TLR9 (p=0,0490 and 0,0034; vs. CO, respectively) . CD8+ but not CD4+ T cells from NAFLD stimulated with saCD3 and co-stimulated with ODN 2395 showed less expression of CD69 than CO (p= 0,0043). T cells from CO and NAFLD were similarly stimulated with saCD3 (p=ns) but no stimulation was found with ODN 2395 alone. Conclusions: a diminished TLR9 expression together with a lower in vitro activation of CD8+ T cells from NAFLD patients could contribute to counterbalance the pro-inflammatory environment.