Sera from Patients with Tuberculosis Increase the Phagocytic-Microbicidal Activity of Human Neutrophils, and ESAT-6 is Implicated in the Phenomenon

Abstract

Background: It has been reported that sera from patients with active pulmonary tuberculosis (APT) induced nuclear changes in normal neutrophils that included pyknosis, swelling, apoptosis, and production of extracellular traps (NETs). Similar changes were observed with some sera from their household contacts but not with sera from healthy, unrelated individuals. It was suggested that those sera from household contacts that induced neutrophil nuclear changes might correspond to people with subclinical tuberculosis. Thus, our experimental approach might serve to identify individuals with early, ongoing disease. Methods: Nuclear changes in neutrophils were fully evident by 3 h of contact and beyond. Circulating mycobacterial antigens were the most likely candidates for this effect. We wanted to know whether the nuclear changes induced on neutrophils by the sera of APT patients would negatively affect the phagocytic/microbicidal ability of neutrophils exposed to APT sera for short periods. Results: We now provide evidence that short-term contact (30 min) with sera from patients with pulmonary tuberculosis increases several phagocytic parameters of normal neutrophils, including endocytosis, myeloperoxidase levels, production of free reactive oxygen species, phagolysosome fusion, and microbicidal activity on Staphylococcus aureus, with these effects not being observed with sera from healthy donors. We also give evidence that suggests that ESAT-6 and CFP-10 are involved in the phenomenon. Conclusion: We conclude that activation is a stage that precedes lethal nuclear changes in neutrophils and suggests that autologous neutrophils must circulate in an altered state in the APT patients, thus contributing to the pathology of the disease.

Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps

“…Our findings revealed no significant induction of NETs by ESAT-6 and CFP-10 in vitro , which aligns with the observations reported by Rojas-Espinosa et al. ( 50 ).…”

“…Previously, Rojas et al. demonstrated that a concentration of 10µg/mL of Mtb soluble recombinant protein ESAT-6 was effective in inducing visible changes in the nuclear morphology of neutrophils ( 50 ). Furthermore, in immunization studies, PE/PPE antigens have been delivered at a concentration of 10µg/mL ( 51 , 52 ).…”

“…Similarly, NET formation has also been characterized upon stimulation with the classical Mtb ESAT-6 protein (an ESX1-derived antigen) resulting in controversial findings by two different studies. On one hand, neutrophils were found to undergo nuclear changes when stimulated with ESAT-6 and CFP-10 in vitro , but no NET structures were identified ( 50 ). On the other hand, a different study showed the formation of NETs by ESAT-6-treated necrotic human-derived neutrophils as referred to by co-localization of ESAT-6 and MPO ( 19 ).…”

Role of phagocyte extracellular traps during Mycobacterium tuberculosis infections and tuberculosis disease processes

“…Interestingly, necrosis by neutrophils depends on ROS and was blocked when inhibiting MPO, thus restoring the mechanisms for mycobacterial growth control by macrophages ( Dallenga et al, 2017 ). Furthermore, Rojas-Espinosa et al (2021) observed morphological and functional changes in the nuclei of neutrophils from blood from TB patients including DNA extrusion, chromatin decondensation and nuclear swelling, suggesting that M.tb infection may induce NETosis. Nevertheless, recombinant proteins ESAT-6 and CFP-10, while inducing nuclear changes in stimulated neutrophils, did not activate formation of NETs ( Rojas-Espinosa et al, 2021 ).…”

“…Furthermore, Rojas-Espinosa et al (2021) observed morphological and functional changes in the nuclei of neutrophils from blood from TB patients including DNA extrusion, chromatin decondensation and nuclear swelling, suggesting that M.tb infection may induce NETosis. Nevertheless, recombinant proteins ESAT-6 and CFP-10, while inducing nuclear changes in stimulated neutrophils, did not activate formation of NETs ( Rojas-Espinosa et al, 2021 ). Conversely, Francis et al (2014) suggested in their study that ESAT-6-treated necrotic neutrophils produced NETs in a Ca 2+ dependent manner with co-localization of ESAT-6 and MPO, supporting a role for ESX-1 in NET formation.…”