Abstract
ABSTRACT An important feature of Mycobacterium tuberculosis pathogenesis is the ability to control cell death in infected host cells, including inhibition of apoptosis and stimulation of necrosis. Recently an alternative form of programmed cell death, necroptosis, has been described where necrotic cell death is induced by apoptotic stimuli under conditions where apoptotic execution is inhibited. We show for the first time that M. tuberculosis and TNFα synergise to induce necroptosis in murine fibroblasts via RIPK1-dependent mechanisms and characterized by phosphorylation of Ser345 of the MLKL necroptosis death effector. However, in murine macrophages M. tuberculosis and TNFα induce non-necroptotic cell death that is RIPK1-dependent but independent of MLKL phosphorylation. Instead, M. tuberculosis-infected macrophages undergo RIPK3-dependent cell death which occurs both in the presence and absence of TNFα and involves the production of mitochondrial ROS. Immunocytochemical staining for MLKL phosphorylation further demonstrated the occurrence of necroptosis in vivo in murine M. tuberculosis granulomas. Phosphorylated-MLKL immunoreactivity was observed associated with the cytoplasm and nucleus of fusiform cells in M. tuberculosis lesions but not in proximal macrophages. Thus whereas pMLKL-driven necroptosis does not appear to be a feature of M. tuberculosis-infected macrophage cell death, it may contribute to TNFα-induced cytotoxicity of the lung stroma and therefore contribute to necrotic cavitation and bacterial dissemination.
Highlights
IntroductionAn important component of M. tuberculosis pathogenesis is the complex control over the mode and timing of host cell death
In order to investigate the presence of necroptosis in response to M. tuberculosis infection, we first compared the capability of murine fibroblasts and human and murine macrophages to undergo necroptosis induced by tumour necrosis factor alpha (TNFa) C zVAD treatment, as this capacity is not universal in eukaryotic cells.[27]
L929 fibroblasts,[27] and J774A.1 macrophages underwent cell death in response to TNFaCzVAD treatment, and cell death could be inhibited by Nec-1 (Fig. 1d-e)
Summary
An important component of M. tuberculosis pathogenesis is the complex control over the mode and timing of host cell death. Several studies have demonstrated that apoptosis of infected macrophages results in killing of mycobacteria,[6,7,8,9,10] probably by efferocytosis of mycobacteria-containing apoptotic bodies and subsequent lysosomal digestion or oxidative killing.[11,12] macrophage apoptosis stimulates protective T cell responses through the “detour” pathway of antigen presentation.[13,14,15] In contrast, necrosis has been observed to facilitate release of viable bacteria from infected macrophages[8,16] which may be taken up by phagocytes attracted by damage associated molecular patterns (DAMPs) released by the necrotic macrophage.[17,18] This would allow further intracellular replication producing a cycle of host cell infection, necrosis and reinfection that may represent an important part of the generation of necrotic granuloma
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