Extracellular Vesicles Released from Mycobacterium tuberculosis-Infected Neutrophils Promote Macrophage Autophagy and Decrease Intracellular Mycobacterial Survival

Abstract

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb). In the lungs, macrophages and neutrophils are the first immune cells that have contact with the infecting mycobacteria. Neutrophils are phagocytic cells that kill microorganisms through several mechanisms, which include the lytic enzymes and antimicrobial peptides that are found in their lysosomes, and the production of reactive oxygen species. Neutrophils also release extracellular vesicles (EVs) (100–1,000 nm in diameter) to the extracellular milieu; these EVs consist of a lipid bilayer surrounding a hydrophilic core and participate in intercellular communication. We previously demonstrated that human neutrophils infected in vitro with Mtb H37Rv release EVs (EV-TB), but the effect of these EVs on other cells relevant for the control of Mtb infection, such as macrophages, has not been completely analyzed. In this study, we characterized the EVs produced by non-stimulated human neutrophils (EV-NS), and the EVs produced by neutrophils stimulated with an activator (PMA), a peptide derived from bacterial proteins (fMLF) or Mtb, and observed that the four EVs differed in their size. Ligands for toll-like receptor (TLR) 2/6 were detected in EV-TB, and these EVs favored a modest increase in the expression of the co-stimulatory molecules CD80, a higher expression of CD86, and the production of higher amounts of TNF-α and IL-6, and of lower amounts of TGF-β, in autologous human macrophages, compared with the other EVs. EV-TB reduced the amount of intracellular Mtb in macrophages, and increased superoxide anion production in these cells. TLR2/6 ligation and superoxide anion production are known inducers of autophagy; accordingly, we found that EV-TB induced higher expression of the autophagy-related marker LC3-II in macrophages, and the co-localization of LC3-II with Mtb inside infected macrophages. The intracellular mycobacterial load increased when autophagy was inhibited with wortmannin in these cells. In conclusion, our results demonstrate that neutrophils produce different EVs in response to diverse activators, and that EV-TB activate macrophages and promote the clearance of intracellular Mtb through early superoxide anion production and autophagy induction, which is a novel role for neutrophil-derived EVs in the immune response to Mtb.

Mycobacterium tuberculosis reactivates HIV via exosomes mediated resetting of cellular redox potential and bioenergetics

“…In this context, our data indicate a critical role of oxidative stress in reactivating HIV-1 by Mtb exosomes. Consistent with this, a recent study demonstrated that exosomes from Mtb infected neutrophils trigger superoxide production in macrophages [76]. However, the mechanism of superoxide generation remains uncharacterized.…”

Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles

“…The majority of previous studies focused on exogenous addition of EVs and its effects on host cells (31), but unequivocal demonstration of EV secretion within infected host cells had not been shown. Two reports describe synthesis of EVs by Mycobacterium tuberculosis (55) and L. pneumophila (56), during intracellular residence in macrophages. The infected macrophage cells secreted disparate populations of EVs with one population carrying solely host proteins, whereas the second population carried mostly bacterial products.…”

Extracellular vesicles deliver Mycobacterium RNA to promote host immunity and bacterial killing

“…Both signals are needed as EVs or IFN‐γ alone failed to induce phagosome maturation through a LC3‐associated pathway. Recently, EVs from M.tb ‐infected human neutrophils were found to promote M.tb ‐associated autophagy in human macrophages by triggering superoxide anion production and TLR2/6 signaling pathway . It is presently unclear how the EVs from M.tb ‐infected neutrophils stimulate reactive oxygen species and whether classic or alternative autophagic pathway is activated by EVs.…”

“…These EV-carrying M.tb PAMPs may be detected by PRRs on recipient cells to activate or attenuate cellular responses [5][6][7][8][9][10]. More recently, EVs released by M.tb-infected human neutrophils were also found to regulate proinflammatory response in recipient cells [11]. EVs from M.tb-infected macrophages trigger the TNF-a production in THP-1 human macrophages and naïve mouse bone marrow-derived macrophages (BMMs) [5,12].…”