The potential involvement of LPS in the maintenance of a Th1/citotoxic biased response in NAFLD

INZAUGARAT, ME; ALEGRE NADIA S.; CECILIA GARCPIA; FLORENCIA FERRO; BILLORDO LA; ROMEO JM; ROMERO GA; SAROTTO L; A CHERÑAVSKY

Mar del Plata

Congreso; LIX Reunion anual de la Sociedad Argentina de Inmunología; 2014

Background and aims: Toll-like receptor (TLR)-4 which recognized lipopolysaccharide (LPS) has recently been identified on certain T cell subsets including activated and memory T cells.We have previously demonstrated a characteristic Th1/Tcytotoxic profile of peripheral T cells in nonalcoholic fatty liver diseases (NAFLD). Based on the hypothesis that LPS might modulate T cell responses, we aimed to evaluate TLR4 expression in peripheral central and effector memory T cells and in hepatic lymphocytes in NAFLD patients. Also, to analyze the activating role of LPS on CD4 and CD8 peripheral T cells. Patients and Methods: Blood and liver biopsies were obtained from adult patients with NAFLD (NAFLD, n=15) and metabolically healthy individuals (Co, n=15). PBMC were obtained by Ficoll-Hypaque density gradient. CD3+ or CD45RO cells were further obtained from PBMC by negative selection utilizing inmunobeads. The CD3+ fraction was stimulated with soluble anti-CD3 [500ng/ml] ± LPS[1μg/ml] for 72 hours. CD3+, CD45RO cells or liver cell suspensions were stained with anti-CD4 or -CD8/anti-CCR7/anti-TLR4 mAbs and analyzed by Flow Cytometry. ELISA test was performed according to manufacturer´s protocol. Mann-Whitney test was used. Results: NAFLD patients showed increased %TLR4 CCR7- CD4+ and CD8+ cells (p=0.02 y p=0.02, respectively) while %TLR4 CCR7+ CD4+ and CD8+ cells were similar to Co. No differences were found in Median Fluorescence Intensity for TLR4 expression in CD4+ and CD8+ between NAFLD and Co. NAFLD patients showed an increased hepatic % CD4+TLR4+ and CD8+TLR4+ cells (p=0.02 y p=0.02, vs. control liver). LPS stimulation of CD3+ cells from NAFLD patients showed a trend to up regulate the percentages of CD4+TLR4+ and CD8+TLR4+ cells and IFN- production. Conclusions: The increased plasma levels of LPS usually present in NAFLD patients together with the overall higher expression of TLR4 observed in periphery and liver suggest that LPS can contribute to peripheral Th1/Cytotoxic T cell pool maintenance.