Resistance to apoptosis in Leishmania infantum-infected human macrophages: a critical role for anti-apoptotic Bcl-2 protein and cellular IAP1/2.

Abstract

Apoptosis is essential for maintaining tissue homoeostasis in multi-cellular organisms, also occurring as a defence mechanism against a number of infectious agents, such as parasites. Among intracellular protozoan parasites reported to interfere with the apoptotic machinery of the host cell, Leishmania (L.) sp. have been described, although the various species might activate different pathways in their host cells. Since until now it is not yet well clarified the signalling pathway involved in the apoptosis modulation by L. infantum, the aim of this work was to investigate the role of the anti-apoptotic protein, Bcl-2, and the inhibitors of apoptosis IAP1/2 (cIAP1/2) in cell death resistance showed in L. infantum-infected human macrophages. We observed that actinomycin D-induced apoptosis in U-937 cells, evaluated by Annexin V-CY3, DNA fragmentation and caspase-3, caspase-8, caspase-9 activation assays, was inhibited in the presence of L. infantum promastigotes and that, in these conditions, Bcl-2 protein expression resulted significantly upregulated. Interestingly, L. infantum infection in combination with the Bcl-2 inhibitor, ABT-737, significantly increased the apoptotic process in actinomycin D-treated cells, suggesting a role for Bcl-2 in the anti-apoptotic regulation of human macrophages induced by L. infantum infection. Moreover, Western blotting analysis demonstrated not only a significantly upregulation of cIAP1/2 in infected U-937 cells, but also that the inhibition of cIAPs, employing specific siRNAs, restored the apoptotic effect of actinomycin in infected macrophages. These results clearly support the hypothesis that Bcl-2 and cIAPs are strongly involved in the anti-apoptotic action played by L. infantum in human macrophages.