title: Human macrophages infected with a high burden of ESAT-6-expressing M. tuberculosis undergo caspase-1- and cathepsin B-independent necrosis.
abstract: Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1β release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.
authors.affiliation: Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
authors.forename: Amanda
authors.initials: A
authors.lastname: Welin
authors.forename: Daniel
authors.initials: D
authors.lastname: Eklund
authors.forename: Olle
authors.initials: O
authors.lastname: Stendahl
authors.forename: Maria
authors.initials: M
authors.lastname: Lerm
mesh_terms: D000942:Antigens, Bacterial / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D020170:Caspase 1 / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism; D016923:Cell Death; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism; D042541:Intracellular Space / Q000382:microbiology; D008264:Macrophages / Q000201:enzymology* / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D009336:Necrosis / Q000473:pathology*; D010587:Phagocytosis
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D053583:Interleukin-1beta; D002401:Cathepsin B; D020170:Caspase 1