Abstract
Mycobacterium tuberculosis infection generates pulmonary granulomas that consist of a caseous, necrotic core surrounded by an ordered arrangement of macrophages, neutrophils and T cells. This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes. The most abundant phagocyte in active necrotic granulomas is the neutrophil. Here we show that the ESAT-6 protein secreted by the ESX-1 type VII secretion system causes necrosis of the neutrophils. ESAT-6 induced an intracellular Ca2+ overload followed by necrosis of phosphatidylserine externalised neutrophils. This necrosis was dependent upon the Ca2+ activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis. We also observed that the ESAT-6 induced increase in intracellular Ca2+, stimulated the production of neutrophil extracellular traps characterised by extruded DNA and myeloperoxidase. Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission.
Highlights
We know that the bacterium has evolved mechanisms to regulate the mode and timing of macrophage cell death.[3,4,5,6,7] After initial infection into the lungs, it is supposed that the bacterium is phagocytosed by alveolar macrophages which migrate into the interstitium of the lung.[8]
We are beginning to understand the mechanisms of macrophage cell death control,[4,5,6] we know very little about how M. tuberculosis modulates neutrophil death
It is clear that in some circumstances neutrophils have an antimycobacterial capacity,[10,11] which may be mediated by the direct generation of reactive oxygen species (ROS) or by apoptosis of the infected neutrophils and subsequent efferocytosis of the apoptotic body combined with ROS-dependent killing.[12,13]
Summary
We know that the bacterium has evolved mechanisms to regulate the mode and timing of macrophage cell death.[3,4,5,6,7] After initial infection into the lungs, it is supposed that the bacterium is phagocytosed by alveolar macrophages which migrate into the interstitium of the lung.[8]. The most recent to be described is ‘NETosis’,18,19 whereby death of the neutrophil results in formation of a structure made of DNA with a histone backbone which contains neutrophil elastase, myeloperoxidase (MPO) and metalloproteinases. These ‘traps’ are known to be produced in vivo and associate with bacteria in some infections.[18,20,21,22]. This study aimed to determine if ESAT-6 had a leukocidin action, and if so, whether this was dependent on intracellular flux of Ca2+, activation of calpain, and further, if this resulted in the formation of neutrophil extracellular traps (NETs)
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