Brucella abortus promotes a fibrotic phenotype in hepatic stellate cells through autophagy pathway activation

ARRIOLA BENITEZ P. C., COMERCI D. J., GIAMBARTOLOMEI G. H., DELPINO M. V

Congreso; LXII Reunión Científica de la Sociedad Argentina de Inmunología. Mar del Plata. Noviembre de 2014; 2014

The liver is frequently affected in patients with active brucellosis. Previously, we demonstrated that B. abortus (Ba) inhibits the basal levels of MMP-9 secretion and induces collagen deposition by hepatic stellate cells (LX-2). These phenomena were dependent on a functional type IV secretion system (virB) and the secreted protein BPE005. It has been recently demonstrated that upregulation of autophagy drives the fibrogenic response in stellate cells. Our aim is determine if Ba infection induces autophagy pathway activation in LX-2 cells. To this end, Ba-infected LX-2 cells were lysed to determine LC3II and Beclin-1 expression by Western blot. Apoptosis was determined by TUNEL and caspase-3 expressions by immunofluorescence. MMPs production was determined by gelatin zymography and collagen deposition by Sirius red staining. Ba infection of LX-2 cells did not induce apoptosis but increased the levels of LC3II (p<0.01) and Beclin-1(p<0.01) expression at 24 h post infection (pi), by a mechanism that depends on virB and BPE005 as we demonstrated using isogenic mutants. In addition, when Ba infection experiments were performed in the presence of bafilomycin A we did not observe inhibition of MMP-9 secretion and induction of collagen deposition. This indicated a correlate between autophagy induction and fibrotic phenotype inducted by Ba infection. At 48 h pi, our results indicated that Ba infection but not virB and BPE005 mutant induced Beclin-1 cleavage. In concordance with this finding we found an increase in caspase-3 expression. In addition, Ba infection induced apoptosis as was determined by TUNEL. This result indicated that caspase-mediated cleavage of beclin-1 inactivates beclin-1-induced autophagy and enhances apoptosis. Taking together our results indicated that Ba induced the activation of autophagy pathway in LX-2 cells and this activation is involved in the fibrotic phenotype previously observed and conducted ultimately to apoptosis cell dead.