Mechanism Of IL-32ε-Mediated Apoptosis And Antibacterial Effect During Mycobacterium Tuberculosis Infection

Tuberculosis is a zoonotic infection caused by Mycobacterium tuberculosis that can lead to a large-scale epidemic with malformations and even death.As of 2015,tuberculosis has surpassed HIV infection to become the leading cause of death in the world,killing millions and inflicting huge economic losses every year.Due to the lack of effective treatment options and the emergence of various resistant bacterial strains,the prevention and treatment of tuberculosis remains one of the major global challenges today.Macrophages are the first line of defense against invading pathogens.They can phagocytose the pathogens,initiate inflammatory reactions,direct adaptive immune cells,and eventually clear the pathogens,eliminate inflammation and restore homeostasis.However,as a powerful intracellular parasite,M.tuberculosis has developed various strategies to evade the host’s defense mechanisms during its long-term battle with the host cells.It has been able to establish suitable conditions in the macrophages to facilitate its survival and spread.It utilizes the macrophages as a natural habitat and a tool for transmission within the host.Therefore,a thorough understanding of the immune response elicited by M.tuberculosis in macrophages and its escape mechanism,is of great significance in the development of effective vaccines and targeted immunotherapy against persistent intracellular infections.A growing body of evidence suggests that the proinflammatory cytokine IL-32 plays an important role in the host defense against pathogens such as M.tuberculosis.Due to alternative splicing,many IL-32 isoforms exist,and each of these isoforms can perform different biological functions.Specifically,IL-32γ can inhibit the proliferation of M.tuberculosis in cells via mechanisms such as,induction of apoptosis and activation of the antibacterial vitamin D pathway.However,few studies have evaluated the effects of other isoforms on M.tuberculosis infection.In the present study,we investigated the effects of IL-32 in macrophages,on bacterial proliferation,during M.tuberculosis infection.The mechanisms underlying the IL-32ε–induced antibacterial effects and apoptosis were also elucidated.The key points of the study are as follows:1.We examined the changes in the expression of different IL-32 isoforms in macrophages,following infection with M.tuberculosis.The results showed that in macrophages infected with M.tuberculosis,the expression of many IL-32 isoforms increased,and the difference was statistically significant.Specifically,the expression of IL-32γ was the highest after infection,and the changes in the expression of IL-32ε was the largest infected with M.tuberculosis.2.Since M.tuberculosis induced the expression of many IL-32 isoforms,we further investigated the antituberculosis effects of them.The results showed that,different isoforms of IL-32 exerted different effects on the proliferation of M.tuberculosis in macrophages.In particular,IL-32γ and IL-32ε displayed extremely potent antibacterial effects throughout the experiment.IL-32β and IL-32δ also inhibited the bacterial proliferation,but only in the latter half of the experiment,whereas the antibacterial effects of IL-32α and IL-32ζ were not significant.3.As apoptosis is a key way for IL-32 in resisting tuberculosis,we evaluated the effects of different IL-32 isoforms on apoptosis.The results showed that,different IL-32 isoforms exhibited different capacity for apoptosis induction in macrophages.IL-32β,IL-32γ,and IL-32ε significantly upregulated the rate of apoptosis in macrophages.IL-32δ caused a minor increase in the apoptotic rate,whereas IL-32α and IL-32ζ had no effect on apoptosis.Further investigations revealed that,unlike IL-32β and IL-32γ that induced a high activation rate of Caspase-3,the activation of Caspase-3 was not detected in IL-32ε-overexpressing cells.In addition,quantitative analysis of the apoptosis-related genes showed that only Caspase-6 and c-Myc were upregulated in the IL-32ε–overexpressing cells.Therefore,we hypothesized that IL-32ε induces apoptosis via a Caspase-3–independent mechanism.4.To further investigate the signaling pathway involved in the IL-32ε-mediated apoptosis,we focused on analyzing the correlations of IL-32ε with Nmi and β-catenin.The results showed that IL-32ε induced the expression of Nmi in macrophages.Furthermore,knockdown of Nmi using siRNA abrogated the IL-32ε-mediated antibacterial effects and apoptosis.In addition,the level of β-catenin protein was significantly reduced in the IL-32ε-overexpressing cells,and the addition of β-catenin to the IL-32ε-overexpressing cells also inhibited the IL-32ε-mediated apoptosis.Therefore,this study demonstrated that IL-32ε induces apoptosis via upregulating Nmi expression and reducing the intracellular level of the β-catenin protein.5.To investigate the mechanism underlying the upregulation of Nmi expression induced by IL-32ε,dual-luciferase reporter assay was used to progressively select the core region of the Nmi promoter that responded to the IL-32ε induction.Further investigations revealed that Pax6 and CP2 were the key transcription factors implicated in the IL-32ε-induced Nmi transcription.In addition,we also found that p38 MAPK served as a link in this process,whereby IL-32ε upregulated the transcriptional activities of Pax6 and CP2 via activating the p38 MAPK signaling pathway.Ultimately,this study delineated the mechanism by which IL-32ε induced Nmi expression.In summary,the expression of IL-32 isoforms was up-regulated in macrophages infected with M.tuberculosis,and different isoforms have different antibacterial properties.Specifically,M.tuberculosis induces IL-32ε expression in macrophages,which activates the p38 MAPK signaling pathway and the transcription factors Pax6 and CP2,thereby promoting Nmi expression.Subsequently,the upregulated Nmi reduces the levels of β-catenin protein,ultimately leading to Caspase-6-and c-Myc-dependent apoptosis.This mechanism plays a significant role in the resistance to M.tuberculosis infection by macrophages and may provide novel targets for the prevention and treatment of tuberculosis.