Autophagy and Inflammation During Rickettsial Infection

Rickettsiae are obligately intracellular bacteria that replicate in the cytoplasm of host cells. In my dissertation, I have studied the induction of autophagy by rickettsiae as well as the role of autophagy in the pathogenesis of rickettsioses in vitro and in vivo. I found that autophagy is induced in murine bone marrow macrophages (BMMs) upon infection with R. australis and bacteria are specifically targeted by autophagosomes. Using BMMs from Atg5flox/flox Lyz-Cre and Atg5flox/flox mice, I showed that rickettsia-induced autophagy is Atg5-dependent. Additionally, deletion of autophagy gene Atg5 in Atg5flox/flox Lyz-Cre BMMs significantly reduced bacterial replication compared to Atg5flox/flox littermates. Furthermore, rickettsial loads in liver, lung and spleens of autophagy-deficient Atg5flox/flox Lyz-Cre animals were significantly less compared to Atg5flox/flox littermates, suggesting that autophagy in macrophages promoted rickettsial replication in vivo. R. australis-infected BMMs of Atg5flox/flox Lyz-Cre mice secreted significantly higher levels of several proinflammatory cytokines including IL-1α, IL-6 and TNF-α and the inflammasome-dependent IL-1β when infected with rickettsiae, suggesting that autophagy suppresses the induction of these inflammatory cytokines. To investigate the regulation of autophagy induced by rickettsiae, I examined the mammalian target of rapamycin (mTOR), a master regulator of autophagy, during infection of BMMs with R. australis. Rickettsiae induced mTOR phosphorylation and activation of the downstream protein P70S6K, a measurement of mTORC1 activation. Furthermore, treatment with mTOR inhibitors, rapamycin and PP242, which induce autophagy, significantly promoted rickettsial replication in BMMs compared to untreated controls. Additionally, I showed for the first time that rickettsiae not only infect, but also replicate in human macrophages in vitro. Interestingly, I demonstrated that rickettsiae did not induce autophagy in human endothelial cells at time points up to 48 hours post infection. Inhibition of autophagy by treatment with 3-MA did not affect rickettsial replication in endothelial cells compared to untreated controls. My studies suggest that rickettsiae induce autophagy at the early stage of infection in mouse macrophages to escape the bactericidal effect possibly mediated by inflammatory cytokines including IL-1β. These data also suggest that rickettsiae interact with the autophagy system in cell-type specific mechanisms.