BRUCELLA ABORTUS PROMOTES A FIBROTIC PHENOTYPE IN HEPATIC STELLATE CELLS THROUGH AUTOPHAGY PATHWAY ACTIVATION.

ARRIOLA BENITEZ PAULA C; COMERCI DIEGO J; GIAMBARTOLOMEI GUILLERMO H.; DELPINO M. VICTORIA

Congreso; LIX REUNIÓN CIENTÍFICA ANUAL Sociedad Argentina de Investigación Clínica LXII REUNIÓN ANUAL Sociedad Argentina de Inmunología; 2014

The liver is frequently affected in patients with active brucellosis.Previously, we demonstrated that B. abortus (Ba) inhibits thebasal levels of MMP-9 secretion and induces collagen depositionby hepatic stellate cells (LX-2). These phenomena were dependenton a functional type IV secretion system (virB) and the secretedprotein BPE005. It has been recently demonstrated that upregulationof autophagy drives the fibrogenic response in stellate cells.Our aim is determine if Ba infection induces autophagy pathwayactivation in LX-2 cells. To this end, Ba-infected LX-2 cells werelysed to determine LC3II and Beclin-1 expression by Western blot.Apoptosis was determined by TUNEL and caspase-3 expressionsby immunofluorescence. MMPs production was determined bygelatin zymography and collagen deposition by Sirius red staining.Ba infection of LX-2 cells did not induce apoptosis but increasedthe levels of LC3II (p<0.01) and Beclin-1(p<0.01) expression at24 h post infection (pi), by a mechanism that depends on virB andBPE005 as we demonstrated using isogenic mutants. In addition,when Ba infection experiments were performed in the presenceof bafilomycin A we did not observe inhibition of MMP-9 secretionand induction of collagen deposition. This indicated a correlatebetween autophagy induction and fibrotic phenotype inducted byBa infection. At 48 h pi, our results indicated that Ba infectionbut not virB and BPE005 mutant induced Beclin-1 cleavage. Inconcordance with this finding we found an increase in caspase-3expression. In addition, Ba infection induced apoptosis as wasdetermined by TUNEL. This result indicated that caspase-mediatedcleavage of beclin-1 inactivates beclin-1-induced autophagy andenhances apoptosis. Taking together our results indicated that Bainduced the activation of autophagy pathway in LX-2 cells and thisactivation is involved in the fibrotic phenotype previously observedand conducted ultimately to apoptosis cell dead.