NETosis associates with human TB lung tissue destruction and disease pathogenesis.

Understanding drivers of tuberculosis (TB) associated lung pathological damage is vital in identifying targets for host directed therapies (HDT). NETosis is a neutrophil specific cell death characterized by release of neutrophil extracellular traps (NETs). The role of NETosis in TB-associated lung damage and disease pathogenesis is still poorly understood. We analysed human lung TB granuloma samples using a proteomics approach, which revealed enrichment of neutrophil-associated proteins in necrotic regions of caseous and cavitary granulomas. Using immunohistochemistry (IHC), we validated the abundance of neutrophil-associated proteins, including myeloperoxidase (MPO), cytochrome b-245 beta chain (CYBB) and neutrophil cytosolic factor 1(NCF1), as well as NETosis markers, neutrophil elastase (NE) and citrullinated H3, in necrotizing caseum of human TB granulomas. MPO protein expression was also more abundant in the plasma of TB patients compared to healthy and latently infected (LTBI) participants. MPO directly correlated with an inflammatory disease marker, IP-10. In addition, MPO and IP-10 colocalized in caseous lesions. In-vitro drug inhibition assays were used to investigate potential drivers of NETosis, with pharmaceutical inhibition of MPO, NE and CYBB resulting in reduction of NETosis induced by Mycobacterium tuberculosis (Mtb). Using RT-qPCR we analysed the expression of 18 neutrophil associated genes in the blood of healthy (n = 20), latent TB infection (LTBI) (n = 20) and TB (n = 30) participants. We found that MPO, NCF1 and NCF2 were upregulated in the TB group. Furthermore, the NETosis-associated genes were induced in a human standardized antigen challenge model. Our data shows evidence of NETosis as an associate of lung pathological damage in TB and identifies key drivers of the neutrophil cell death that can be intercepted as potential HDT targets to reduce neutrophil driven lung pathological damage.