Targeting NETosis for the Treatment of Tuberculosis

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Abstract Neutrophils are the most abundant and predominantly-infected cell type in the sputum, bronchoalveolar lavage fluid, and caseum contents from resected lung tissue of active tuberculosis (TB) patients. Studies of TB in mice, non-human primates, and humans have identified a correlation between neutrophil abundance and increased disease severity. Although there is a growing appreciation for the association of increased neutrophil abundance with active TB disease, it was still unknown if the presence of neutrophils in the lungs of active TB patients is consequential, or if the neutrophils are bystanders reacting to an uncontrolled infection. In particular, the details on how specific neutrophil responses and effector functions impact TB disease have remained elusive. In response to Mycobacterium tuberculosis (Mtb) infection, neutrophils deploy a number of defenses including the extrusion of neutrophil extracellular traps (NETs) via a process of cell death termed NETosis. NETosis usually follows the general steps of 1) histone citrullination, 2) chromatin decondensation, and 3) release of web-like chromatin structures decorated with antimicrobial granule proteins with the potential to bind, trap, and kill pathogens. We have recently discovered that NETosis directly promotes Mtb replication, where genetic or chemical inhibition of NETosis mediates better control of Mtb infection in vitro and in vivo, thus validating NETosis as a potential target for host-directed therapies to treat TB. We have used genetic and chemical approaches to mechanistically dissect the process of NETosis during Mtb infection, which has identified a number of regulatory nodes that can be manipulated to lead to better control of Mtb pathogenesis. We find that in response to Mtb infection in neutrophils, protein arginine deiminase 4 (PAD4) citrullinates histones to decondense chromatin that gets packaged into vesicles for release as NETs in a manner that promotes Mtb replication. We discovered that type I interferon (IFN), which has been associated with NETosis in numerous contexts but without a known mechanism, promotes the formation of chromatin-containing vesicles and NET release. In addition, we discovered a new autophagy-independent role for the ATG5 protein in suppressing NETosis by blocking type I IFN-dependent induction of PAD4 activity during Mtb infection, where increased NETosis in the absence of ATG5 expression in neutrophils leads to susceptibility to Mtb. Multiple studies have linked increased levels of type I IFN signaling with TB pathology in mice and humans. Based on our data that NETosis promotes Mtb replication and pathogenesis, NETosis could contribute to the ways that type I IFN signaling impedes control of Mtb infection. In this proposal, we will dissect how NETosis is regulated during Mtb infection (Aim 1), how NETosis contributes to Mtb replication (Aim 2), and how NETosis contributes to loss of control of infection (Aim 3). By pursuing our Aims, we will identify regulatory mechanisms to be targeted by HDTs aimed at blocking NETosis during Mtb infection as well as determine what consequences result from blocking NETosis during Mtb infection.