Critical and Independent Role for SOCS3 in Either Myeloid or T Cells in Resistance to Mycobacterium tuberculosis

Suppressor of cytokine signalling 3 (SOCS3) negatively regulates STAT3 activation in response to several cytokines such as those in the gp130-containing IL-6 receptor family. Thus, SOCS3 may play a major role in immune responses to pathogens. In the present study, the role of SOCS3 in M. tuberculosis infection was examined. All Socs3^fl/fl LysM cre, Socs3^fl/fl lck cre (with SOCS3-deficient myeloid and lymphoid cells, respectively) and gp130^F/F mice, with a mutation in gp130 that impedes binding to SOCS3, showed increased susceptibility to infection with M. tuberculosis. SOCS3 binding to gp130 in myeloid cells conveyed resistance to M. tuberculosis infection via the regulation of IL-6/STAT3 signalling. SOCS3 was redundant for mycobacterial control by macrophages in vitro. Instead, SOCS3 expression in infected macrophages and DCs prevented the IL-6-mediated inhibition of TNF and IL-12 secretion and contributed to a timely CD4+ cell-dependent IFN-γ expression in vivo. In T cells, SOCS3 expression was essential for a gp130-independent control of infection with M. tuberculosis, but was neither required for the control of infection with attenuated M. bovis BCG nor for M. tuberculosis in BCG-vaccinated mice. Socs3^fl/fl lck cre mice showed an increased frequency of γδ+ T cells in different organs and an enhanced secretion of IL-17 by γδ+ T cells in response to infection. Socs3^fl/fl lck cre γδ+ T cells impaired the control of infection with M. tuberculosis. Thus, SOCS3 expression in either lymphoid or myeloid cells is essential for resistance against M. tuberculosis via discrete mechanisms.     

Mycobacterium tuberculosis Rv3402c Enhances Mycobacterial Survival within Macrophages and Modulates the Host Pro-Inflammatory Cytokines Production via NF-Kappa B/ERK/p38 Signaling

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H₂O₂ exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1β compared with controls. Rv3402c-induced TNF-α and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.     

Combination of Cytokine Responses Indicative of Latent TB and Active TB in Malawian Adults

Background

An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease.

Methods

Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested.

Results

Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses.

Conclusions

CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB.     

Normalization of the levels of inflammatory molecules in Mycobacterium smegmatis-infected U937 cells by fibrate pretreatment

Background

Tuberculosis (TB) is a respiratory tract disease caused by Mycobacterium tuberculosis infection. M. tuberculosis exploits immune privilege to grow and divide in pleural macrophages. Fibrates are associated with the immune response and control lipid metabolism through glycolysis with β-oxidation of fatty acids.

Results

In this study, we investigated the effect of fibrate pretreatment on the immune response during M. smegmatis infection in U937 cells, a human leukemic monocyte lymphoma cell line. The protein expression of tumor necrosis factor α (TNF-α), an inflammatory marker, and myeloid differentiation primary response gene 88 (MyD88), a toll like receptor adaptor molecule, in the infected group increased at 1 and 6 h after M. smegmatis infection of U937 cells. Acetyl coenzyme A acetyl transferase-1 (ACAT-1), peroxisome proliferator-activated receptor-α (PPAR-α), TNF-α, and MyD88 decreased in U937 cells treated with fibrates at 12 and 24 h after treatment. More than a 24 h pretreatment with fibrate resulted in similar expression levels of ACAT-1 and PPAR-α between infected vehicle control and infected groups which were pretreated with fibrate for 24 h. However, upon exposure to M. smegmatis, the cellular expression of the TNF-α and MyD88 in the infected groups pretreated with fibrate for 24 h decreased significantly compared to that in the infected vehicle group.

Conclusion

These results suggest that fibrate pretreatment normalized the levels of inflammatory molecules in Mycobacterium smegmatis-infected U937 cells. Further studies are needed to confirm the findings on pathophysiology and immune defense mechanism of U937 by fibrates during M. tuberculosis infection.     

Involvement of CD244 in Regulating CD4+ T Cell Immunity in Patients with Active Tuberculosis

CD244 (2B4) is a member of the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors and it plays an important role in modulating NK cell and CD8⁺ T cell immunity. In this study, we investigated the expression and function of CD244/2B4 on CD4⁺ T cells from active TB patients and latent infection individuals. Active TB patients had significantly elevated CD244/2B4 expression on M. tuberculosis antigen-specific CD4⁺ T cells compared with latent infection individuals. The frequencies of CD244/2B4-expressing antigen-specific CD4⁺ T cells were significantly higher in retreatment active TB patients than in new active TB patients. Compared with CD244/2B4-dull and -middle CD4⁺ T cells, CD244/2B4-bright CD4⁺ T cell subset had significantly reduced expression of IFN-γ, suggesting that CD244/2B4 expression may modulate IFN-γ production in M. tuberculosis antigen-responsive CD4⁺ T cells. Activation of CD244/2B4 signaling by cross-linking led to significantly decreased production of IFN-γ. Blockage of CD244/2B4 signaling pathway of T cells from patients with active TB resulted in significantly increased production of IFN-γ, compared with isotype antibody control. In conclusion, CD244/2B4 signaling pathway has an inhibitory role on M. tuberculosis antigen-specific CD4⁺ T cell function.     

Mycobacterium tuberculosis Controls MicroRNA-99b (miR-99b) Expression in Infected Murine Dendritic Cells to Modulate Host Immunity

Mycobacterium tuberculosis resides and replicates within host phagocytes by modulating host microbicidal responses. In addition, it suppresses the production of host protective cytokines to prevent activation of and antigen presentation by M. tuberculosis-infected cells, causing dysregulation of host protective adaptive immune responses. Many cytokines are regulated by microRNAs (miRNAs), a newly discovered class of small noncoding RNAs, which have been implicated in modulating host immune responses in many bacterial and viral diseases. Here, we show that miRNA-99b (miR-99b), an orphan miRNA, plays a key role in the pathogenesis of M. tuberculosis infection. We found that miR-99b expression was highly up-regulated in M. tuberculosis strain H37Rv-infected dendritic cells (DCs) and macrophages. Blockade of miR-99b expression by antagomirs resulted in significantly reduced bacterial growth in DCs. Interestingly, knockdown of miR-99b in DCs significantly up-regulated proinflammatory cytokines such as IL-6, IL-12, and IL-1β. Furthermore, mRNA and membrane-bound protein data indicated that inhibition of miR-99b augments TNF-α and TNFRSF-4 production. Thus, miR-99b targets TNF-α and TNFRSF-4 receptor genes. Treatment of anti-miR-99b-transfected DCs with anti-TNF-α antibody resulted in increased bacterial burden. Thus, our findings unveil a novel host evasion mechanism adopted by M. tuberculosis via miR-99b, which may open up new avenues for designing miRNA-based vaccines and therapies.     

Critical Role of Toll-Like Receptor 9 in Morphine and Mycobacterium tuberculosis–Induced Apoptosis in Mice

Background

Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive.

Methodology/Principal Findings

We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-α, IL-1β, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3β in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3β in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration.

Conclusions/Significance

These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection.     

The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90–95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis.     

An Anaerobic-Type α-Ketoglutarate Ferredoxin Oxidoreductase Completes the Oxidative Tricarboxylic Acid Cycle of Mycobacterium tuberculosis

Aerobic organisms have a tricarboxylic acid (TCA) cycle that is functionally distinct from those found in anaerobic organisms. Previous reports indicate that the aerobic pathogen Mycobacterium tuberculosis lacks detectable α-ketoglutarate (KG) dehydrogenase activity and drives a variant TCA cycle in which succinyl-CoA is replaced by succinic semialdehyde. Here, we show that M. tuberculosis expresses a CoA-dependent KG dehydrogenase activity, albeit one that is typically found in anaerobic bacteria. Unlike most enzymes of this family, the M. tuberculosis KG: ferredoxin oxidoreductase (KOR) is extremely stable under aerobic conditions. This activity is absent in a mutant strain deleted for genes encoding a previously uncharacterized oxidoreductase, and this strain is impaired for aerobic growth in the absence of sufficient amounts of CO₂. Interestingly, inhibition of the glyoxylate shunt or exclusion of exogenous fatty acids alleviates this growth defect, indicating the presence of an alternate pathway that operates in the absence of β-oxidation. Simultaneous disruption of KOR and the first enzyme of the succinic semialdehyde pathway (KG decarboxylase; KGD) results in strict dependence upon the glyoxylate shunt for growth, demonstrating that KG decarboxylase is also functional in M. tuberculosis intermediary metabolism. These observations demonstrate that unlike most organisms M. tuberculosis utilizes two distinct TCA pathways from KG, one that functions concurrently with β-oxidation (KOR-dependent), and one that functions in the absence of β-oxidation (KGD-dependent). As these pathways are regulated by metabolic cues, we predict that their differential utilization provides an advantage for growth in different environments within the host.     

Porins Increase Copper Susceptibility of Mycobacterium tuberculosis

Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis.     

Discovery of Mycobacterium tuberculosis α-1,4-Glucan Branching Enzyme (GlgB) Inhibitors by Structure- and Ligand-based Virtual Screening

GlgB (α-1,4-glucan branching enzyme) is the key enzyme involved in the biosynthesis of α-glucan, which plays a significant role in the virulence and pathogenesis of Mycobacterium tuberculosis. Because α-glucans are implicated in the survival of both replicating and non-replicating bacteria, there exists an exigent need for the identification and development of novel inhibitors for targeting enzymes, such as GlgB, involved in this pathway. We have used the existing structural information of M. tuberculosis GlgB for high throughput virtual screening and molecular docking. A diverse database of 330,000 molecules was used for identifying novel and efficacious therapeutic agents for targeting GlgB. We also used three-dimensional shape as well as two-dimensional similarity matrix methods to identify diverse molecular scaffolds that inhibit M. tuberculosis GlgB activity. Virtual hits were generated after structure and ligand-based screening followed by filters based on interaction with human GlgB and in silico pharmacokinetic parameters. These hits were experimentally evaluated and resulted in the discovery of a number of structurally diverse chemical scaffolds that target M. tuberculosis GlgB. Although a number of inhibitors demonstrated in vitro enzyme inhibition, two compounds in particular showed excellent inhibition of in vivo M. tuberculosis survival and its ability to get phagocytosed. This work shows that in silico docking and three-dimensional chemical similarity could be an important therapeutic approach for developing inhibitors to specifically target the M. tuberculosis GlgB enzyme.     

The Effect of Hyperglycaemia on In Vitro Cytokine Production and Macrophage Infection with Mycobacterium tuberculosis

Type 2 diabetes mellitus is an established risk factor for tuberculosis but the underlying mechanisms are largely unknown. We examined the effects of hyperglycaemia, a hallmark of diabetes, on the cytokine response to and macrophage infection with Mycobacterium tuberculosis. Increasing in vitro glucose concentrations from 5 to 25 mmol/L had marginal effects on cytokine production following stimulation of peripheral blood mononuclear cells (PBMCs) with M. tuberculosis lysate, LPS or Candida albicans, while 40 mmol/L glucose increased production of TNF-α, IL-1β, IL-6 and IL-10, but not of IFN-γ, IL-17A and IL-22. Macrophage differentiation under hyperglycaemic conditions of 25 mmol/L glucose was also associated with increased cytokine production upon stimulation with M. tuberculosis lysate and LPS but in infection experiments no differences in M. tuberculosis killing or outgrowth was observed. The phagocytic capacity of these hyperglycaemic macrophages also remained unaltered. The fact that only very high glucose concentrations were able to significantly influence cytokine production by macrophages suggests that hyperglycaemia alone cannot fully explain the increased susceptibility of diabetes mellitus patients to tuberculosis.     

Mycobacterium tuberculosis Promotes Anti-apoptotic Activity of the Macrophage by PtpA Protein-dependent Dephosphorylation of Host GSK3α

Mycobacterium tuberculosis tyrosine phosphatase PtpA inhibits two key cellular events in macrophages required for the elimination of invading organisms, phagosome acidification, and maturation. Kinome analysis revealed multiple PtpA-dependent changes to the phosphorylation status of macrophage proteins upon M. tuberculosis infection. Among those proteins we show that PtpA dephosphorylates GSK3α on amino acid Tyr²⁷⁹, which leads to modulation of GSK3α anti-apoptotic activity, promoting pathogen survival early during infection.     

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis

The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen’s persistence.     

Mycobacterium tuberculosis inhibits the NLRP3 inflammasome activation via its phosphokinase PknF

Mycobacterium tuberculosis (Mtb) has evolved to evade host innate immunity by interfering with macrophage functions. Interleukin-1β (IL-1β) is secreted by macrophages after the activation of the inflammasome complex and is crucial for host defense against Mtb infections. We have previously shown that Mtb is able to inhibit activation of the AIM2 inflammasome and subsequent pyroptosis. Here we show that Mtb is also able to inhibit host cell NLRP3 inflammasome activation and pyroptosis. We identified the serine/threonine kinase PknF as one protein of Mtb involved in the NLRP3 inflammasome inhibition, since the pknF deletion mutant of Mtb induces increased production of IL-1β in bone marrow-derived macrophages (BMDMs). The increased production of IL-1β was dependent on NLRP3, the adaptor protein ASC and the protease caspase-1, as revealed by studies performed in gene-deficient BMDMs. Additionally, infection of BMDMs with the pknF deletion mutant resulted in increased pyroptosis, while the IL-6 production remained unchanged compared to Mtb-infected cells, suggesting that the mutant did not affect the priming step of inflammasome activation. In contrast, the activation step was affected since potassium efflux, chloride efflux and the generation of reactive oxygen species played a significant role in inflammasome activation and subsequent pyroptosis mediated by the Mtb pknF mutant strain. In conclusion, we reveal here that the serine/threonine kinase PknF of Mtb plays an important role in innate immune evasion through inhibition of the NLRP3 inflammasome.     

Variation of Mycobacterium tuberculosis Antigen-Specific IFN-γ and IL-17 Responses in Healthy Tuberculin Skin Test (TST)-Positive Human Subjects

Objective

To determine the variation of IFN-γ and IL-17 responses to M. tuberculosis antigens in healthy TST+ humans.

Methods

We isolated peripheral blood mononuclear cells from 21 TST+ healthy adults, stimulated them with phytohemagglutinin (PHA), PPD, Ag85B, ESAT-6, and live M. bovis BCG, and assayed IFN-γ and IL-17 secretion by ELISA in supernatants after 24 or 72 hours of incubation respectively.

Results

As in other studies, we found a wide range of IFN-γ responses to M. tuberculosis antigens; the variation significantly exceeded that observed in the same donors to the polyclonal T cell stimulus, phytohemagglutinin (PHA). In addition, we assayed IL-17 secretion in response to the same stimuli, and found less subject-to-subject variation. Analysis of the ratio of IFN-γ to IL-17 secretion on a subject-to-subject basis also revealed a wide range, with the majority of results distributed in a narrow range, and a minority with extreme results all of which were greater than that in the majority of subjects. The data suggest that study of exceptional responses to M. tuberculosis antigens may reveal immunologic correlates with specific outcomes of M. tuberculosis infection.

Conclusion

Variation of IFNγ and IFN-γ/IL-17 responses to mycobacterial antigens exceeds that of responses to the polyclonal stimulus, PHA, in TST positive healthy humans. This indicates a quantitative spectrum of human immune responses to infection with M. tuberculosis. Since the outcome of human infection with M. tuberculosis varies greatly, systematic study of multiple immune responses to multiple antigens is likely to reveal correlations between selected immune responses and the outcomes of infection.