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Autophagy induction targeting mTORC1 enhances Mycobacterium tuberculosis replication in HIV co-infected human macrophages.

ABSTRACT: To survive and replicate in macrophages Mycobacterium tuberculosis (Mtb) has developed strategies to subvert host defence mechanisms, including autophagy. Autophagy induction has the potential to clear Mtb, but little is known about its effect during controlled tuberculosis and HIV co-infection. Mammalian target of rapamycin complex1 (mTORC1) inhibitors were used to induce autophagy in human macrophages pre-infected with HIV-1BaL and infected with a low dose of Mtb (co-infected), or single Mtb infected (single infected). The controlled Mtb infection was disrupted upon mTOR inhibition resulting in increased Mtb replication in a dose-dependent manner which was more pronounced during co-infection. The increased Mtb replication could be explained by the marked reduction in phagosome acidification upon mTOR inhibition. Autophagy stimulation targeting mTORC1 clearly induced a basal autophagy with flux that was unlinked to the subcellular environment of the Mtb vacuoles, which showed a concurrent suppression in acidification and maturation/flux. Overall our findings indicate that mTOR inhibition during Mtb or HIV/Mtb co-infection interferes with phagosomal maturation, thereby supporting mycobacterial growth during low-dose and controlled infection. Therefore pharmacological induction of autophagy through targeting of the canonical mTORC1-pathway should be handled with caution during controlled tuberculosis, since this could have serious consequences for patients with HIV/Mtb co-infection.

SUBMITTER: Andersson AM

PROVIDER: S-EPMC4908603 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Autophagy induction targeting mTORC1 enhances Mycobacterium tuberculosis replication in HIV co-infected human macrophages.

Andersson Anna-Maria AM Andersson Blanka B Lorell Christoffer C Raffetseder Johanna J Larsson Marie M Blomgran Robert R

Scientific reports 20160615

To survive and replicate in macrophages Mycobacterium tuberculosis (Mtb) has developed strategies to subvert host defence mechanisms, including autophagy. Autophagy induction has the potential to clear Mtb, but little is known about its effect during controlled tuberculosis and HIV co-infection. Mammalian target of rapamycin complex1 (mTORC1) inhibitors were used to induce autophagy in human macrophages pre-infected with HIV-1BaL and infected with a low dose of Mtb (co-infected), or single Mtb i ...[more]

PMID: 27302320

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Project description:Macrophages are one of the first innate immune infiltrates in the cornea of mice following ocular infection with herpes simplex virus 1 (HSV-1). Using gamma interferon (IFN-γ) and interleukin-4 (IL-4) injections to polarize macrophages into M1 and M2, respectively, and in M1 and M2 conditional knockout mice, we have shown that M1 macrophages play an important role in suppressing both virus replication in the eye and eye disease in HSV-1-infected mice. Autophagy is also important in controlling HSV infection and integrity of infected cells. To determine if blocking autophagy in M1 and M2 macrophages affects HSV-1 infectivity and eye disease, we generated two transgenic mouse strains expressing the HSV-1 γ34.5 autophagy gene under the M1 promoter (M1-γ34.5) or the M2 promoter (M2-γ34.5). We found that blocking autophagy in M1 macrophages increased both virus replication in the eyes and eye disease in comparison to blocking autophagy in M2 macrophages or wild-type (WT) control mice, but blocked autophagy did not affect latency-reactivation. However, blocking autophagy affected fertility in both M1 and M2 transgenic mice. Analysis of 62 autophagy genes and 32 cytokines/chemokines from infected bone marrow-derived macrophages from M1-γ34.5, M2-γ34.5, and WT mice suggested that upregulation of autophagy-blocking genes (i.e., Hif1a, Mtmr14, mTOR, Mtmr3, Stk11, and ULK2) and the inflammatory tumor necrosis factor alpha (TNF-α) gene in M1-γ34.5 transgenic mice correlated with increased pathogenicity, while upregulation of proautophagy genes (Nrbf2 and Rb1cc1) in M2-γ34.5 macrophages correlated with reduced pathogenicity. The in vivo and in vitro responses of M1-γ34.5 and M2-γ34.5 transgenic mice to HSV-1 infection were independent of the presence of the γ34.5 gene in wild-type HSV-1. Our results suggest that M1 macrophages, but not M2 macrophages, play an important role in autophagy relative to primary virus replication in the eye and eye disease in infected mice. IMPORTANCE Autophagy plays a critical role in clearing, disassembling, and recycling damaged cells, thus limiting inflammation. The HSV-1 γ34.5 gene is involved in neurovirulence and immune evasion by blocking autophagy in infected cells. We found that blocking autophagy in M1 macrophages enhances HSV-1 virus replication in the eye and eye disease in ocularly infected transgenic mice. Our results also show the suppressive effects of γ34.5 on immune responses to infection, suggesting the importance of intact autophagy in M1 but not M2 macrophages in controlling primary infection and eye disease.

Project description:BackgroundTuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis (Mtb) remains a significant health problem worldwide with a third of the world population infected and nearly nine million new cases claiming 1.1 million deaths every year. The outcome following infection by Mtb is determined by a complex and dynamic host-pathogen interaction in which the phenotype of the pathogen and the immune status of the host play a role. However, the molecular mechanism by which Mtb strains induce different responses during intracellular infection of the host macrophage is not fully understood. To explore the early molecular events triggered upon Mtb infection of macrophages, we studied the transcriptional responses of murine bone marrow-derived macrophages (BMM) to infection with two clinical Mtb strains, CDC1551 and HN878. These strains have previously been shown to differ in their virulence/immunogenicity in the mouse and rabbit models of pulmonary TB.ResultsIn spite of similar intracellular growth rates, we observed that compared to HN878, infection by CDC1551 of BMM was associated with an increased global transcriptome, up-regulation of a specific early (6 hours) immune response network and significantly elevated nitric oxide production. In contrast, at 24 hours post-infection of BMM by HN878, more host genes involved in lipid metabolism, including cholesterol metabolism and prostaglandin synthesis were up-regulated, compared to infection with CDC1551.In association with the differences in the macrophage responses to infection with the 2 Mtb strains, intracellular CDC1551 expressed higher levels of stress response genes than did HN878.ConclusionsIn association with the early and more robust macrophage activation, intracellular CDC1551 cells were exposed to a higher level of stress leading to increased up-regulation of the bacterial stress response genes. In contrast, sub-optimal activation of macrophages and induction of a dysregulated host cell lipid metabolism favored a less stressful intracellular environment for HN878. Our findings suggest that the ability of CDC1551 and HN878 to differentially activate macrophages during infection probably determines their ability to either resist host cell immunity and progress to active disease or to succumb to the host protective responses and be driven into a non-replicating latent state in rabbit lungs.

Project description:BAG2 (BCL2 associated athanogene 2) is associated with cell fate determination in response to various pathological conditions. However, the effects of BAG2 on M. tuberculosis-induced endoplasmic reticulum (ER) stress remain elusive. Herein, we report that M. tuberculosis infection of macrophages triggered ER stress and downregulated BAG2 expression. Overexpression of BAG2 enhanced autophagic flux and activated macroautophagy/autophagy targeted to the ER (reticulophagy). In addition, through increasingly localizing SQSTM1 to the ER in BAG2-overexpressing macrophages, we found that the autophagy receptor protein SQSTM1/p62 (sequestosome 1) is associated with the BAG2-induced reticulophagy. Our data also confirmed that BAG2 could render cells resistant to M. tuberculosis-induced cellular damage, and the anti-apoptotic effects of BAG2 in M. tuberculosis-treated macrophages were partially abolished by the autophagic flux inhibitor bafilomycin A1. Furthermore, the dissociation of BECN1 and BCL2 mediated by activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) was responsible for BAG2-activated autophagy. In addition, XBP1 downstream of the ERN1/IRE1 signaling pathway was bound to the Bag2 promoter region and transcriptionally inhibited BAG2 expression. Collectively, these results indicated that BAG2 has anti-apoptotic effects on M. tuberculosis-induced ER stress, which is dependent on the promotion of autophagic flux and the induction of selective autophagy. We revealed a potential host defense mechanism that links BAG2 to ER stress and autophagy during M. tuberculosis infection.AbbreviationsATF6: activating transcription factor 6; BECN1: beclin 1; Baf A1: bafilomycin A1; CASP3: caspase 3; DDIT3/CHOP/GADD153: DNA damage inducible transcript 3; DAPI: 4',6-diamidino-2-phenylindole; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; HSPA5/GRP78/BiP: heat shock protein 5; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK/ERK: mitogen-activated protein kinase; SQSTM1/p62: sequestosome 1; UPR: unfolded protein response; XBP1: x-box binding protein 1.

Project description:A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with naïve macrophages produced an antimicrobial effect, but only if naïve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the naïve macrophages. The antimicrobial effect of naïve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the naïve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of naïve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis.

Dataset Information

Autophagy induction targeting mTORC1 enhances Mycobacterium tuberculosis replication in HIV co-infected human macrophages.

Publications

Autophagy induction targeting mTORC1 enhances Mycobacterium tuberculosis replication in HIV co-infected human macrophages.

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