INVESTIGATION OF XIAP AND MITOCHONDRIAL APOPTOSIS SIGNALLING DURING LISTERIA MONOCYTOGENES INFECTION OF MACROPHAGES

Apart from its well-established role as an inhibitor of apoptotic caspase proteases, X-Linked Inhibitor of Apoptosis Protein (XIAP) has recently been suggested to play a role in the regulation of mitochondrial outer membrane permeabilization (MOMP) in response to different stresses. XIAP has been shown to translocate to mitochondria alongside Rab5+ and Rab7+ endolysosomes in response to mitochondrial depolarization and intrinsic apoptosis initiation. Mitochondrial translocation of XIAP and endolysosomes impacts Bax-mediated pore formation in the outer mitochondrial membrane and MOMP-related cell death signalling. In addition, XIAP also plays a pro-survival role by activating the NF-κB pathway using its BIR1 domain. However not much is known about the role of mitochondria and MOMP specifically in Listeria monocytogenes infection. To study the potential role of XIAP in macrophages in regulating mitochondrial apoptosis during infection with L. monocytogenes we investigated the bacterial burden, Bax activation, cytochrome c release, P-p65 translocation into the nucleus and Rab5 colocalization with mitochondria at 2, 3, 6 and 24 hours post-infection, in wildtype and XIAP KO THP-1 macrophages. Our results suggest that XIAP plays an important role in promoting the growth and internalisation of the bacteria leading up to 6 hours. In XIAP KO cells, cytochrome c release was increased and Rab5+ endosomes colocalized with mitochondria at earlier time points. The NF-κB pathway did not appear to be significantly activated at these time points. These results give further insights into the role of XIAP in host innate immune responses to intracellular pathogens. Furthermore, we also investigated the potential anti-apoptotic role of LPS on Valinomycin-treated J774 macrophages by quantifying MOMP markers Bax and cytochrome c. LPS is shown to protect the J774 macrophages from MOMP by activating NF-κB which was confirmed by increased P-p65 translocation in LPS pre-treated, Valinomycin-treated cells. Together, our results support that XIAP plays a central role in the mitochondria-related immune response elicited by L. monocytogenes in macrophages