IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen.     

IL17A augments autophagy in Mycobacterium tuberculosis-infected monocytes from patients with active tuberculosis in association with the severity of the disease

During mycobacterial infection, macroautophagy/autophagy, a process modulated by cytokines, is essential for mounting successful host responses. Autophagy collaborates with human immune responses against Mycobacterium tuberculosis (Mt) in association with specific IFNG secreted against the pathogen. However, IFNG alone is not sufficient to the complete bacterial eradication, and other cytokines might be required. Actually, induction of Th1 and Th17 immune responses are required for protection against Mt. Accordingly, we showed that IL17A and IFNG expression in lymphocytes from tuberculosis patients correlates with disease severity. Here we investigate the role of IFNG and IL17A during autophagy in monocytes infected with Mt H37Rv or the mutant MtΔRD1. Patients with active disease were classified as high responder (HR) or low responder (LR) according to their T cell responses against Mt. IL17A augmented autophagy in infected monocytes from HR patients through a mechanism that activated MAPK1/ERK2-MAPK3/ERK1 but, during infection of monocytes from LR patients, IL17A had no effect on the autophagic response. In contrast, addition of IFNG to infected monocytes, increased autophagy by activating MAPK14/p38 α both in HR and LR patients. Interestingly, proteins codified in the RD1 region did not interfere with IFNG and IL17A autophagy induction. Therefore, in severe tuberculosis patients' monocytes, IL17A was unable to augment autophagy because of a defect in the MAPK1/3 signaling pathway. In contrast, both IFNG and IL17A increased autophagy levels in patients with strong immunity to Mt, promoting mycobacterial killing. Our findings might contribute to recognize new targets for the development of novel therapeutic tools to fight the pathogen.     

Autophagy restricts Chlamydia trachomatis growth in human macrophages via IFNG-inducible guanylate binding proteins

Interferon γ (IFNG) is a key host response regulator of intracellular pathogen replication, including that of Chlamydia spp The antichlamydial functions of IFNG manifest in a strictly host, cell-type and chlamydial strain dependent manner. It has been recently shown that the IFNG-inducible family of immunity-related GTPases (IRG) proteins plays a key role in the defense against nonhost adapted chlamydia strains in murine epithelial cells. In humans, IFN-inducible guanylate binding proteins (hGBPs) have been shown to potentiate the antichlamydial effect of IFNG; however, how hGBPs regulate this property of IFNG is unknown. In this study, we identified hGBP1/2 as important resistance factors against C. trachomatis infection in IFNG-stimulated human macrophages. Exogenous IFNG reduced chlamydial infectivity by 50 percent in wild-type cells, whereas shRNA hGBP1/2 knockdown macrophages fully supported chlamydial growth in the presence of exogenous IFNG. hGBP1/2 were recruited to bacterial inclusions in human macrophages upon stimulation with IFNG, which triggered rerouting of the typically nonfusogenic bacterial inclusions for lysosomal degradation. Inhibition of lysosomal activity and autophagy impaired the IFNG-mediated elimination of inclusions. Thus, hGBP1/2 are critical effectors of antichlamydial IFNG responses in human macrophages. Through their capacity to remodel classically nonfusogenic chlamydial inclusions and stimulate fusion with autophagosomes, hGBP1/2 disable a major chlamydial virulence mechanism and contribute to IFNG-mediated pathogen clearance.     

Inducible costimulator: a modulator of IFN-gamma production in human tuberculosis

Effective host defense against Mycobacterium tuberculosis requires the induction of Th1 cytokine responses. We investigated the regulated expression and functional role of the inducible costimulator (ICOS), a receptor known to regulate Th cytokine production, in the context of human tuberculosis. Patients with active disease, classified as high responder (HR) or low responder (LR) patients according to their in vitro T cell responses against the Ag, were evaluated for T cell expression of ICOS after M. tuberculosis-stimulation. We found that ICOS expression significantly correlated with IFN-gamma production by tuberculosis patients. ICOS expression levels were regulated in HR patients by Th cytokines: Th1 cytokines increased ICOS levels, whereas Th2-polarizing conditions down-regulated ICOS in these individuals. Besides, in human polarized Th cells, engagement of ICOS increased M. tuberculosis IFN-gamma production with a magnitude proportional to ICOS levels on those cells. Moreover, ICOS ligation augmented Ag-specific secretion of the Th1 cytokine IFN-gamma from responsive individuals. In contrast, neither Th1 nor Th2 cytokines dramatically affected ICOS levels on Ag-stimulated T cells from LR patients, and ICOS activation did not enhance IFN-gamma production. However, simultaneous activation of ICOS and CD3 slightly augmented IFN-gamma secretion by LR patients. Together, our data suggest that the regulation of ICOS expression depends primarily on the response of T cells from tuberculosis patients to the specific Ag. IFN-gamma released by M. tuberculosis-specific T cells modulates ICOS levels, and accordingly, ICOS ligation induces IFN-gamma secretion. Thus, ICOS activation may promote the induction of protective Th1 cytokine responses to intracellular bacterial pathogens.     

Selective autophagy gets more selective: Uncoupling of autophagy flux and xenophagy flux in Mycobacterium tuberculosis-infected macrophages

Induction of autophagy has been reported as a potential means to eliminate intracellular pathogens. Corroborating that, many studies report inhibition of autophagy as a survival strategy of bacterial pathogens. Incidentally, autophagy at the basal level is critical for survival of host cells including macrophages. We asked how a bacterial pathogen could inhibit autophagy for its survival if the inhibition resulted in cell death. In a recent study we show distinct regulation of autophagy in Mycobacterium tuberculosis (Mtb)-infected macrophages where Mtb containing- and nonMtb-containing autophagosomes show different fates in terms of maturation. We show that upon Mtb infection, there is no dramatic change in the autophagy flux in macrophages. However, autophagosomes that contain the virulent strains of Mtb show selective resilience to the maturation phase of autophagy. Surprisingly, nonMtb-containing autophagosomes in the infected cells continue to mature into autolysosomes. The block in the xenophagy flux is missing in the case of avirulant infections. We show that this selectivity is achieved through selective exclusion of RAB7 from virulent Mtb-containing autophagosomes, thereby restricting the formation of amphisomes.     

Unexpected Role for IL-17 in Protective Immunity against Hypervirulent Mycobacterium tuberculosis HN878 Infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), infects one third of the world''s population. Among these infections, clinical isolates belonging to the W-Beijing appear to be emerging, representing about 50% of Mtb isolates in East Asia, and about 13% of all Mtb isolates worldwide. In animal models, infection with W-Beijing strain, Mtb HN878, is considered “hypervirulent” as it results in increased mortality and causes exacerbated immunopathology in infected animals. We had previously shown the Interleukin (IL) -17 pathway is dispensable for primary immunity against infection with the lab adapted Mtb H37Rv strain. However, it is not known whether IL-17 has any role to play in protective immunity against infection with clinical Mtb isolates. We report here that lab adapted Mtb strains, such as H37Rv, or less virulent Mtb clinical isolates, such as Mtb CDC1551, do not require IL-17 for protective immunity against infection while infection with Mtb HN878 requires IL-17 for early protective immunity. Unexpectedly, Mtb HN878 induces robust production of IL-1β through a TLR-2-dependent mechanism, which supports potent IL-17 responses. We also show that the role for IL-17 in mediating protective immunity against Mtb HN878 is through IL-17 Receptor signaling in non-hematopoietic cells, mediating the induction of the chemokine, CXCL-13, which is required for localization of T cells within lung lymphoid follicles. Correct T cell localization within lymphoid follicles in the lung is required for maximal macrophage activation and Mtb control. Since IL-17 has a critical role in vaccine-induced immunity against TB, our results have far reaching implications for the design of vaccines and therapies to prevent and treat emerging Mtb strains. In addition, our data changes the existing paradigm that IL-17 is dispensable for primary immunity against Mtb infection, and instead suggests a differential role for IL-17 in early protective immunity against emerging Mtb strains.     

Effect of Helicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells

Host cell responses to Helicobacter pylori infection are complex and incompletely understood. Here, we report that autophagy is induced within human-derived gastric epithelial cells (AGS) cells in response to H. pylori infection. These autophagosomes were distinct and different from the large vacuoles induced during H. pylori infection. Autophagosomes were detected by transmission electron microscopy, conversion of LC3-I to LC3-II, GFP-LC3 recruitment to autophagosomes, and depended on Atg5 and Atg12. The induction of autophagy depended on the vacuolating cytotoxin (VacA) and, moreover, VacA was sufficient to induce autophagosome formation. The channel forming activity of VacA was necessary for inducing autophagy. Intracellular VacA partially co-localized with GFP-LC3, indicating that the toxin associates with autophagosomes. The inhibition of autophagy increased the stability of intracellular VacA, which in turn resulted in enhanced toxin-mediated cellular vacuolation. These findings suggest that the induction of autophagy by VacA may represent a host mechanism to limit toxin-induced cellular damage.     

Induction of autophagy through CLEC4E in combination with TLR4: an innovative strategy to restrict the survival of Mycobacterium tuberculosis

ABSTRACT

Host-directed therapies are gaining considerable impetus because of the emergence of drug-resistant strains of pathogens due to antibiotic therapy. Therefore, there is an urgent need to exploit alternative and novel strategies directed at host molecules to successfully restrict infections. The C-type lectin receptor CLEC4E and Toll-like receptor TLR4 expressed by host cells are among the first line of defense in encountering pathogens. Therefore, we exploited signaling of macrophages through CLEC4E in association with TLR4 agonists (C₄.T₄) to control the growth of Mycobacterium tuberculosis (Mtb). We observed significant improvement in host immunity and reduced bacterial load in the lungs of Mtb-infected mice and guinea pigs treated with C₄.T₄ agonists. Further, intracellular killing of Mtb was achieved with a 10-fold lower dose of isoniazid or rifampicin in conjunction with C₄.T₄ than the drugs alone. C₄.T₄ activated MYD88, PtdIns3K, STAT1 and RELA/NFKB, increased lysosome biogenesis, decreased Il10 and Il4 gene expression and enhanced macroautophagy/autophagy. Macrophages from autophagy-deficient (atg5 knockout or Becn1 knockdown) mice showed elevated survival of Mtb. The present findings also unveiled the novel role of CLEC4E in inducing autophagy through MYD88, which is required for control of Mtb growth. This study suggests a unique immunotherapeutic approach involving CLEC4E in conjunction with TLR4 to restrict the survival of Mtb through autophagy.     

Increase of CD4+CD25highFoxP3+ cells impairs in vitro human microbicidal activity against Mycobacterium tuberculosis during latent and acute pulmonary tuberculosis

BackgroundRegulatory T cells (Tregs) play a critical role during Mycobacterium tuberculosis (Mtb) infection, modulating host responses while neutralizing excessive inflammation. However, their impact on regulating host protective immunity is not completely understood. Here, we demonstrate that Treg cells abrogate the in vitro microbicidal activity against Mtb.MethodsWe evaluated the in vitro microbicidal activity of peripheral blood mononuclear cells (PBMCs) from patients with active tuberculosis (TB), individuals with latent tuberculosis infection (LTBI, TST+/IGRA+) and healthy control (HC, TST-/IGRA-) volunteers. PBMCs, depleted or not of CD4⁺CD25⁺ T-cells, were analyzed to determine frequency and influence on microbicidal activity during in vitro Mtb infection with four clinical isolates (S1, S5, R3, and R6) and one reference strain (H37Rv).ResultsThe frequency of CD4⁺CD25^highFoxP3⁺ cells were significantly higher in Mtb infected whole blood cultures from both TB patients and LTBI individuals when compared to HC. Data from CD4⁺CD25⁺ T-cells depletion demonstrate that increase of CD4⁺CD25^highFoxP3⁺ is associated with an impairment of Th-1 responses and a diminished in vitro microbicidal activity of LTBI and TB groups.ConclusionsTregs restrict host anti-mycobacterial immunity during active disease and latent infection and thereby may contribute to both disease progression and pathogen persistence.     

Ir gene-controlled response to haptenated hen ovomucoid: isotypic specificity and dominant nonresponsiveness

We have examined the isotypic pattern of the response of mice to the Ir gene-controlled antigen, dinitrophenyl-ovomucoid (DNP-OM). H-2 kappa mice are high responders (HR); (H-2b,d mice are low responders (LR). The isotype patterns of HR and LR strains differ both quantitatively and qualitatively. In the primary response to doses of 20-100 micrograms DNP-OM, HR strains produce IgM and IgG antibodies, whereas LR strains produce only IgM. Background genes modify the kinetics of the IgGl primary response in HR strains, but no background was found which allowed an IgG response in a LR strain. In secondary responses, priming with 0.2 microgram DNP-OM increases secondary responses in HR strains, and decreases them in LR strains. Control of this response maps to I-A, and is not altered by the bm 12 I-Ab mutation. The LR phenotype is dominant in (HR X LR)F1 mice.     

Autophagy is induced by the type III secretion system of <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> in several mammalian cell lines

Vibrio alginolyticus is a gram-negative bacterium and has been recognized as an opportunistic pathogen in marine animals as well as humans. Here, we further characterized a cell death mechanism caused by this bacterium in several mammalian cell lines. The T3SS of V. alginolyticus killed HeLa cells by a very similar cell cytolysis mechanism in fish cells, as evidenced by cell rounding and LDH release; however, DNA fragmentation was not observed. Further studies showed that caspase-1 and caspase-3 were not activated during the T3SS-mediated cell death, indicating that the death mechanism is completely independent of pyroptosis and apoptosis in HeLa cells. Conversely, autophagy was detected during the T3SS-mediated cell death by the appearance of MDC-labeled punctate fluorescence and accumulation of autophagic vesicles. Moreover, western blot analysis revealed increase in conversion of LC3-I to LC3-II in infected mammalian cell lines, confirming that autophagy occurs during the process. Together, these data demonstrate that the death process used by V. alginolyticus in mammalian cells is different from that in fish cells, including induction of autophagy, cell rounding and osmotic lysis. This study provides some evidences hinting that differences in death mechanism in responses to V. alginolyticus infection may be attributed to the species of infected cells from which it was derived.     

Clonal Expansions of CD8+ T Cells with IL-10 Secreting Capacity Occur during Chronic Mycobacterium tuberculosis Infection

The exact role of CD8⁺ T cells during Mycobacterium tuberculosis (Mtb) infection has been heavily debated, yet it is generally accepted that CD8⁺ T cells contribute to protection against Mtb. In this study, however, we show that the Mtb-susceptible CBA/J mouse strain accumulates large numbers of CD8⁺ T cells in the lung as infection progresses, and that these cells display a dysfunctional and immunosuppressive phenotype (PD-1⁺, Tim-3⁺, CD122⁺). CD8⁺ T cell expansions from the lungs of Mtb-infected CBA/J mice were also capable of secreting the immunosuppressive cytokine interleukin-10 (IL-10), although in vivo CD8⁺ T cell depletion did not significantly alter Mtb burden. Further analysis revealed that pulmonary CD8⁺ T cells from Mtb-infected CBA/J mice were clonally expanded, preferentially expressing T cell receptor (TcR) Vβ chain 8 (8.2, 8.3) or Vβ 14. Although Vβ8⁺ CD8⁺ T cells were responsible for the majority of IL-10 production, in vivo depletion of Vβ8⁺ did not significantly change the outcome of Mtb infection, which we hypothesize was a consequence of their dual IL-10/IFN-γ secreting profiles. Our data demonstrate that IL-10-secreting CD8⁺ T cells can arise during chronic Mtb infection, although the significance of this T cell population in tuberculosis pathogenesis remains unclear.     

Multiple cytokines are released when blood from patients with tuberculosis is stimulated with Mycobacterium tuberculosis antigens

Background

Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection.

Methods

Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens.

Results

Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines.

Conclusions

Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone.     

DRAM1 promotes the targeting of mycobacteria to selective autophagy

Autophagy provides an important defense mechanism against intracellular bacteria, such as Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease (TB). We recently reported that pathogen recognition and antibacterial autophagy are connected by the induction of the DNA damage-regulated autophagy modulator DRAM1 via the toll-like receptor (TLR)-MYD88-NFKB innate immunity signaling pathway. Having shown that DRAM1 colocalizes with Mtb in human macrophages, we took advantage of a zebrafish model for TB to investigate the function of DRAM1 in autophagic host defense in vivo. We found that DRAM1 protects the zebrafish host from infection with Mycobacterium marinum (Mm), a close relative of Mtb. Overexpression of DRAM1 increases autophagosome formation and promotes autophagic flux by a mechanism dependent on the cytosolic DNA sensor TMEM173/STING and the ubiquitin receptor SQSTM1/p62. Here we summarize and discuss the implications of these findings.     

Immunodiagnosis of pulmonary tuberculosis by concomitant detection of antigen and antibodies of excretory-secretory protein ofMycobacterium tuberculosis H37Ra

With a view to diagnosing tuberculosis in populations in endemic areas, excretory-secretory antigen fraction(Mtb EST-6) of purifiedMycobacterium tuberculosis H₃₇Ra and affinity purified polyclonal antibodies againstMtb EST were used to detect both antibodies and circulating antigen in the sera of patients and disease-free individuals. Indirect stick penicillinase ELISA system usingMtb EST-6 detected antigen-specific IgG antibody in 84% of sputum positive, 77% of sputum negative pulmonary tuberculosis patients and 7% of healthy and 11% of subjects with nontub~rculosis diseases. Similarly, a sandwich penicillinase ELISA system using affinity purified antiMtb EST antibodies detected circulating antigen in 83% and 61% of sputum positive and negative pulmonary tuberculosis subjects. In contrast only 24% of healthy and 18% of disease controls showed seropositivity. Antibody assay showed higher sensitivity and specificity (83% and 91% respectively) compared to antigen detection (sensitivity of 79% and specificity of 79%). However, by concomitant use of both assays it was possible to enhance the specificity of detection to 98%, though sensitivity was reduced marginally to 70%. The present study confirms the presence of both antigen and specific antibodies in the circulation during clinical disease and draws attention to the utility ofMtb EST-6 as a diagnostic marker of pulmonary tuberculosis.     

The Defect in Autophagy Induction by Clinical Isolates of Mycobacterium Tuberculosis Is Correlated with Poor Tuberculosis Outcomes

Background

Tuberculosis (TB) represents a major global health problem. The prognosis of clinically active tuberculosis depends on the complex interactions between Mycobacterium tuberculosis (Mtb) and its host. In recent years, autophagy receives particular attention for its role in host defense against intracellular pathogens, including Mtb. In present study, we aim to investigate the relationship of autophagy induction by clinical isolates of Mtb with the clinical outcomes in patients with TB.

Methodology/Principal Findings

We collected 185 clinical isolates of Mtb, and determined the effect of these Mtb isolates on autophagy induction in macrophages. It was found that most of clinical isolates of Mtb were able to induce autophagosome formation in macrophages, however, the autophagy-inducing ability varied significantly among different isolates. Of importance, our results revealed that patients infected by Mtb with poor autophagy-inducing ability displayed more severe radiographic extent of disease (p<0.001), and were more likely to have unfavorable treatment outcomes (p<0.001). No significant association was observed between the extent of Mtb-induced autophagy with some socio-demographic characteristics (such as gender, age and tobacco consumption), and some laboratory tests (such as hemoglobin, leukocyte count and erythrocyte sedimentation rate). Furthermore, results from logistic regression analysis demonstrated that the defect in autophagy induction by clinical isolates of Mtb was an independent risk factor for far-advanced radiographic disease (aOR 4.710 [1.93–11.50]) and unfavorable treatment outcomes (aOR 8.309 [2.22–28.97]) in TB.

Conclusion/Significance

These data indicated that the defect in autophagy induction by Mtb isolates increased the risk of poor clinical outcomes in TB patients, and detection of clinical isolates-induced autophagosome formation might help evaluate the TB outcomes.     

Serum antibody profiles in individuals with latent Mycobacterium tuberculosis infection

One‐third of the world's humans has latent tuberculosis infection (LTBI), representing a large pool of potentially active TB. Recent LTBI carries a higher risk of disease progression than remote LTBI. Recent studies suggest important roles of antibodies in TB pathology, prompting us to investigate serum antibody profiles in a cohort with LTBI. In this single‐center prospective observational study, we analyzed IgG‐antibody concentrations against five major Mycobacterium tuberculosis (Mtb) antigens (including 6 kDa early secretory antigenic target (ESAT6), CFP10, and antigen 85A, which are expressed mainly in the growth phase; and mycobacterial DNA‐binding protein 1 (MDP1) and alpha‐crystallin like protein (Acr), which are expressed in the dormant phases) in individuals with recent (n=13) or remote (n=12) LTBI, no Mtb infection (n=19), or active TB (n=15). Antibody titers against ESAT6 and MDP1 were significantly higher in individuals with recent LTBI than in those with no Mtb infection or remote LTBI. All pairwise antibody titers against these five major antigens were significantly correlated throughout the stages of Mtb infection. Five individuals with recent LTBI had significantly higher antibody titers against ESAT6 (P = 0.03), Ag85A (P = 0.048), Acr (P = 0.057), and MDP1 (P = 0.0001) than in individuals with remote LTBI; they were also outside the normal range (+2 SDs). One of these individuals was diagnosed with active pulmonary TB at 18‐month follow‐up examination. These findings indicated that concentrations of antibodies against both multiplying and dormant Mtb are higher in recent LTBI and that individuals with markedly higher antibody titers may be appropriate candidates for prophylactic therapy.