Persistent expression of activation markers on Mycobacterium tuberculosis-specific CD4 T cells in smear negative TB patients.

BACKGROUND: T cell activation (HLA-DR, CD-38), proliferation (KI-67), and functional (IFN-γ, TNF-α) markers have recently been shown to be useful in predicting and monitoring anti-TB responses in smear positive TB, but previous research did not characterize the activation and proliferation profiles after therapy of smear negative TB. METHODOLOGY: In this study, we used polychromatic flow cytometry to assess selected PPD-specific T cell markers using fresh PBMC of smear negative and positive pulmonary tuberculosis (PTB) patients, recruited from health facilities in Addis Ababa. RESULT: Levels of activation (HLA-DR, CD38) and proliferation (Ki-67) among total unstimulated CD4 T cells decreased significantly after therapy, particularly at month 6. Similarly, levels of PPD-specific T cell activation markers (HLA-DR, CD-38) were significantly lower in smear positive PTB patients following treatment, whereas a consistent decline in these markers was less apparent among smear negative PTB patients at the sixth month. CONCLUSION: After six months of standard anti-TB therapy, persistent levels of activation of HLA-DR and CD-38 from PPD specific CD4+T cells in this study could indicate that those markers have little value in monitoring and predicting anti-TB treatment response in smear negative pulmonary TB patients in Ethiopian context.

Isoniazid and rifapentine treatment effectively reduces persistent M. tuberculosis infection in macaque lungs.

A once-weekly oral dose of isoniazid and rifapentine for 3 months (3HP) is recommended by the CDC for treatment of latent tuberculosis infection (LTBI). The aim of this study is to assess 3HP-mediated clearance of M. tuberculosis bacteria in macaques with asymptomatic LTBI. Twelve Indian-origin rhesus macaques were infected with a low dose (~10 CFU) of M. tuberculosis CDC1551 via aerosol. Six animals were treated with 3HP and 6 were left untreated. The animals were imaged via PET/CT at frequent intervals. Upon treatment completion, all animals except 1 were coinfected with SIV to assess reactivation of LTBI to active tuberculosis (ATB). Four of 6 treated macaques showed no evidence of persistent bacilli or extrapulmonary spread until the study end point. PET/CT demonstrated the presence of significantly more granulomas in untreated animals relative to the treated group. The untreated animals harbored persistent bacilli and demonstrated tuberculosis (TB) reactivation following SIV coinfection, while none of the treated animals reactivated to ATB. 3HP treatment effectively reduced persistent infection with M. tuberculosis and prevented reactivation of TB in latently infected macaques.

Mycobacterium tuberculosis antigen-specific T-cell responses in smear-negative pulmonary tuberculosis patients.

Despite recent improvements in microbial detection, smear-negative TB remains a diagnostic challenge. In this study, we investigated the potential discriminatory role of polychromatic flow cytometry of M. tuberculosis antigen-specific T cells to discriminate smear-negative TB from health controls with or without latent TB infection, and non-TB respiratory illnesses in an endemic setting. A cross-sectional study was conducted on HIV negative, newly diagnosed smear-positive PTB (n = 34), smear-negative/GeneXpert negative PTB (n = 29) patients, non-TB patients with respiratory illness (n = 33) and apparently healthy latent TB infected (n = 30) or non-infected (n = 23) individuals. The expression of activation (HLA-DR, CD-38), proliferation (Ki-67), and functional (IFN-γ, TNF-α) T-cell markers using polychromatic flow cytometry was defined after stimulation with PPD antigens. Sputum samples were collected and processed from all patients for Mtb detection using a concentrated microscopy, LJ/MGIT culture, and RD9 typing by PCR. Our study showed CD4 T cells specific for PPD co-expressed activation/proliferation markers together with induced cytokines IFN-γ or TNF-α were present at substantially higher levels among patients with smear-positive and smear-negative pulmonary TB than among healthy controls and to a lesser extent among patients with non-TB illness. Our study conclude that smear-negative TB can be distinguished from non-TB respiratory illness and healthy controls with a flow cytometric assay for PPD-specific T cells co-expressing activation/proliferation markers and cytokines.

Characterizing Early T Cell Responses in Nonhuman Primate Model of Tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading infectious disease killer worldwide with 1.4 million TB deaths in 2019. While the majority of infected population maintain an active control of the bacteria, a subset develops active disease leading to mortality. Effective T cell responses are critical to TB immunity with CD4+ and CD8+ T cells being key players of defense. These early cellular responses to TB infection have not yet been studied in-depth in either humans or preclinical animal models. Characterizing early T cell responses in a physiologically relevant preclinical model can provide valuable understanding of the factors that control disease development. We studied Mtb-specific T cell responses in the lung compartment of rhesus macaques infected with either a low- or a high-dose of Mtb CDC1551 via aerosol. Relative to baseline, significantly higher Mtb-specific CD4+IFN-γ+ and TNF-α+ T cell responses were observed in the BAL of low dose infected macaques as early as week 1 post TB infection. The IFN-γ and TNF-a response was delayed to week 3 post infection in Mtb-specific CD4+ and CD8+T cells in the high dose group. The manifestation of earlier T cell responses in the group exposed to the lower Mtb dose suggested a critical role of these cytokines in the antimycobacterial immune cascade, and specifically in the granuloma formation to contain the bacteria. However, a similar increase was not reflected in the CD4+ and CD8+IL-17+ T cells at week 1 post infection in the low dose group. This could be attributed to either a suppression of the IL-17 response or a lack of induction at this early stage of infection. On the contrary, there was a significantly higher IL-17+ response in Mtb-specific CD4+ and CD8+T cells at week 3 in the high dose group. The results clearly demonstrate an early differentiation in the immunity following low dose and high dose infection, largely represented by differences in the IFN-γ and TNF-α response by Mtb-specific T cells in the BAL. This early response to antigen expression by the bacteria could be critical for both bacterial growth control and bacterial containment.

Pulmonary Mycobacterium tuberculosis control associates with CXCR3- and CCR6-expressing antigen-specific Th1 and Th17 cell recruitment.

Mycobacterium tuberculosis-specific (M. tuberculosis-specific) T cell responses associated with immune control during asymptomatic latent tuberculosis infection (LTBI) remain poorly understood. Using a nonhuman primate aerosol model, we studied the kinetics, phenotypes, and functions of M. tuberculosis antigen-specific T cells in peripheral and lung compartments of M. tuberculosis-infected asymptomatic rhesus macaques by longitudinally sampling blood and bronchoalveolar lavage, for up to 24 weeks postinfection. We found substantially higher frequencies of M. tuberculosis-specific effector and memory CD4+ and CD8+ T cells producing IFN-γ in the airways compared with peripheral blood, and these frequencies were maintained throughout the study period. Moreover, M. tuberculosis-specific IL-17+ and IL-17+IFN-γ+ double-positive T cells were present in the airways but were largely absent in the periphery, suggesting that balanced mucosal Th1/Th17 responses are associated with LTBI. The majority of M. tuberculosis-specific CD4+ T cells that homed to the airways expressed the chemokine receptor CXCR3 and coexpressed CCR6. Notably, CXCR3+CD4+ cells were found in granulomatous and nongranulomatous regions of the lung and inversely correlated with M. tuberculosis burden. Our findings provide insights into antigen-specific T cell responses associated with asymptomatic M. tuberculosis infection that are relevant for developing better strategies to control TB.

Immunology of Mycobacterium tuberculosis Infections.

Tuberculosis (TB) is a serious global public health challenge that results in significant morbidity and mortality worldwide. TB is caused by infection with the bacilli Mycobacterium tuberculosis (M. tuberculosis), which has evolved a wide variety of strategies in order to thrive within its host. Understanding the complex interactions between M. tuberculosis and host immunity can inform the rational design of better TB vaccines and therapeutics. This chapter covers innate and adaptive immunity against M. tuberculosis infection, including insights on bacterial immune evasion and subversion garnered from animal models of infection and human studies. In addition, this chapter discusses the immunology of the TB granuloma, TB diagnostics, and TB comorbidities. Finally, this chapter provides a broad overview of the current TB vaccine pipeline.

Mucosal-activated invariant T cells do not exhibit significant lung recruitment and proliferation profiles in macaques in response to infection with Mycobacterium tuberculosis CDC1551.

TB is a catastrophic infectious disease, affecting roughly one third of the world's population. Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize vitamin B metabolites produced by bacteria, possess effector memory phenotype, and express tissue-homing markers driving migration to sites of infection. Previous research in both Mtb and HIV infections has shown that MAIT cells are depleted in the human periphery, possibly migrating to the tissue sites of infection. We investigated this hypothesis using rhesus macaques (RMs) with active TB, latent TB (LTBI), and SIV-coinfection to explore the effects of different disease states on the MAIT cell populations in vivo. Early in infection, we observed that MAIT cells increased in the blood and bronchoalveolar lavage fluid (BAL) of all infected RMs, irrespective of clinical outcome. However, the frequency of MAIT cells rapidly normalized such that they had returned to baseline levels prior to endpoint. Furthermore, following infection, the chemokines expressed on MAIT cells reflected a strong shift towards a Th1 phenotype from a shared Th1/Th17 phenotype. In conclusion, MAIT cells with enhanced Th1 functions migrating to the site of Mtb-infection. The anti-mycobacterial effector functions of MAIT cells, particularly during the early stages of Mtb infection, had been of interest in promoting protective long-term TB immunity. Our research shows, however, that they have relatively short-acting responses in the host.

Deletion of BCG Hip1 protease enhances dendritic cell and CD4 T cell responses.

Dendritic cells (DCs) play a key role in the generation of CD4 T cell responses to pathogens. Mycobacterium tuberculosis (Mtb) harbors immune evasion mechanisms that impair DC responses and prevent optimal CD4 T cell immunity. The vaccine strain Mycobacterium bovis Bacille Calmette-Guérin (BCG) shares many of the immune evasion proteins utilized by Mtb, but the role of these proteins in DC and T cell responses elicited by BCG is poorly understood. We previously reported that the Mtb serine protease, Hip1, promotes sub-optimal DC responses during infection. Here, we tested the hypothesis that BCG Hip1 modulates DC functions and prevents optimal antigen-specific CD4 T cell responses that limit the immunogenicity of BCG. We generated a strain of BCG lacking hip1 (BCGΔhip1) and show that it has superior capacity to induce DC maturation and cytokine production compared with the parental BCG. Furthermore, BCGΔhip1-infected DCs were more effective at driving the production of IFN-γ and IL-17 from antigen-specific CD4 T cells in vitro. Mucosal transfer of BCGΔhip1-infected DCs into mouse lungs induced robust CD4 T cell activation in vivo and generated antigen-specific polyfunctional CD4 T cell responses in the lungs. Importantly, BCGΔhip1-infected DCs enhanced control of pulmonary bacterial burden following Mtb aerosol challenge compared with the transfer of BCG-infected DCs. These results reveal that BCG employs Hip1 to impair DC activation, leading to attenuated lung CD4 T cell responses with limited capacity to control Mtb burden after challenge.

In vivo inhibition of tryptophan catabolism reorganizes the tuberculoma and augments immune-mediated control of Mycobacterium tuberculosis.

Mycobacterium tuberculosis continues to cause devastating levels of mortality due to tuberculosis (TB). The failure to control TB stems from an incomplete understanding of the highly specialized strategies that M. tuberculosis utilizes to modulate host immunity and thereby persist in host lungs. Here, we show that M. tuberculosis induced the expression of indoleamine 2,3-dioxygenase (IDO), an enzyme involved in tryptophan catabolism, in macrophages and in the lungs of animals (mice and macaque) with active disease. In a macaque model of inhalation TB, suppression of IDO activity reduced bacterial burden, pathology, and clinical signs of TB disease, leading to increased host survival. This increased protection was accompanied by increased lung T cell proliferation, induction of inducible bronchus-associated lymphoid tissue and correlates of bacterial killing, reduced checkpoint signaling, and the relocation of effector T cells to the center of the granulomata. The enhanced killing of M. tuberculosis in macrophages in vivo by CD4+ T cells was also replicated in vitro, in cocultures of macaque macrophages and CD4+ T cells. Collectively, these results suggest that there exists a potential for using IDO inhibition as an effective and clinically relevant host-directed therapy for TB.

Engaging the CD40-CD40L pathway augments T-helper cell responses and improves control of Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (Mtb) impairs dendritic cell (DC) functions and induces suboptimal antigen-specific CD4 T cell immune responses that are poorly protective. Mucosal T-helper cells producing IFN-γ (Th1) and IL-17 (Th17) are important for protecting against tuberculosis (TB), but the mechanisms by which DCs generate antigen-specific T-helper responses during Mtb infection are not well defined. We previously reported that Mtb impairs CD40 expression on DCs and restricts Th1 and Th17 responses. We now demonstrate that CD40-dependent costimulation is required to generate IL-17 responses to Mtb. CD40-deficient DCs were unable to induce antigen-specific IL-17 responses after Mtb infection despite the production of Th17-polarizing innate cytokines. Disrupting the interaction between CD40 on DCs and its ligand CD40L on antigen-specific CD4 T cells, genetically or via antibody blockade, significantly reduced antigen-specific IL-17 responses. Importantly, engaging CD40 on DCs with a multimeric CD40 agonist (CD40LT) enhanced antigen-specific IL-17 generation in ex vivo DC-T cell co-culture assays. Further, intratracheal instillation of Mtb-infected DCs treated with CD40LT significantly augmented antigen-specific Th17 responses in vivo in the lungs and lung-draining lymph nodes of mice. Finally, we show that boosting CD40-CD40L interactions promoted balanced Th1/Th17 responses in a setting of mucosal DC transfer, and conferred enhanced control of lung bacterial burdens following aerosol challenge with Mtb. Our results demonstrate that CD40 costimulation by DCs plays an important role in generating antigen-specific Th17 cells and targeting the CD40-CD40L pathway represents a novel strategy to improve adaptive immunity to TB.

Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis.

Tuberculosis (TB) is a global pandaemic, partially due to the failure of vaccination approaches. Novel anti-TB vaccines are therefore urgently required. Here we show that aerosol immunization of macaques with the Mtb mutant in SigH (MtbΔsigH) results in significant recruitment of inducible bronchus-associated lymphoid tissue (iBALT) as well as CD4(+) and CD8(+) T cells expressing activation and proliferation markers to the lungs. Further, the findings indicate that pulmonary vaccination with MtbΔsigH elicited strong central memory CD4(+) and CD8(+) T-cell responses in the lung. Vaccination with MtbΔsigH results in significant protection against a lethal TB challenge, as evidenced by an approximately three log reduction in bacterial burdens, significantly diminished clinical manifestations and granulomatous pathology and characterized by the presence of profound iBALT. This highly protective response is virtually absent in unvaccinated and BCG-vaccinated animals after challenge. These results suggest that future TB vaccine candidates can be developed on the basis of MtbΔsigH.

Biomarkers on patient T cells diagnose active tuberculosis and monitor treatment response.

BACKGROUND: The identification and treatment of individuals with tuberculosis (TB) is a global public health priority. Accurate diagnosis of pulmonary active TB (ATB) disease remains challenging and relies on extensive medical evaluation and detection of Mycobacterium tuberculosis (Mtb) in the patient's sputum. Further, the response to treatment is monitored by sputum culture conversion, which takes several weeks for results. Here, we sought to identify blood-based host biomarkers associated with ATB and hypothesized that immune activation markers on Mtb-specific CD4+ T cells would be associated with Mtb load in vivo and could thus provide a gauge of Mtb infection. METHODS: Using polychromatic flow cytometry, we evaluated the expression of immune activation markers on Mtb-specific CD4+ T cells from individuals with asymptomatic latent Mtb infection (LTBI) and ATB as well as from ATB patients undergoing anti-TB treatment. RESULTS: Frequencies of Mtb-specific IFN-γ+CD4+ T cells that expressed immune activation markers CD38 and HLA-DR as well as intracellular proliferation marker Ki-67 were substantially higher in subjects with ATB compared with those with LTBI. These markers accurately classified ATB and LTBI status, with cutoff values of 18%, 60%, and 5% for CD38+IFN-γ+, HLA-DR+IFN-γ+, and Ki-67+IFN-γ+, respectively, with 100% specificity and greater than 96% sensitivity. These markers also distinguished individuals with untreated ATB from those who had successfully completed anti-TB treatment and correlated with decreasing mycobacterial loads during treatment. CONCLUSION: We have identified host blood-based biomarkers on Mtb-specific CD4+ T cells that discriminate between ATB and LTBI and provide a set of tools for monitoring treatment response and cure. TRIAL REGISTRATION: Registration is not required for observational studies. FUNDING: This study was funded by Emory University, the NIH, and the Yerkes National Primate Center.

DosS Is required for the complete virulence of mycobacterium tuberculosis in mice with classical granulomatous lesions.

Mycobacterium tuberculosis (Mtb) must counter hypoxia within granulomas to persist. DosR, in concert with sensor kinases DosS and DosT, regulates the response to hypoxia. Yet Mtb lacking functional DosR colonize the lungs of C57Bl/6 mice, presumably owing to the lack of organized lesions with sufficient hypoxia in that model. We compared the phenotype of the Δ-dosR, Δ-dosS, and Δ-dosT mutants to Mtb using C3HeB/FeJ mice, an alternate mouse model where lesions develop hypoxia. C3HeB/FeJ mice were infected via aerosol. The progression of infection was analyzed by tissue bacterial burden and histopathology. A measure of the comparative global immune responses was also analyzed. Although Δ-dosR and Δ-dosT grew comparably to wild-type Mtb, Δ-dosS exhibited a significant defect in bacterial burden and pathology in vivo, accompanied by ablated proinflammatory response. Δ-dosS retained the ability to induce DosR. The Δ-dosS mutant was also attenuated in murine macrophages ex vivo, with evidence of reduced expression of the proinflammatory signature. Our results show that DosS, but not DosR and DosT, is required by Mtb to survive in C3HeB/FeJ mice. The attenuation of Δ-dosS is not due to its inability to induce the DosR regulon, nor is it a result of the accumulation of hypoxia. That the in vivo growth restriction of Δ-dosS could be mimicked ex vivo suggested sensitivity to macrophage oxidative burst. Anoxic caseous centers within tuberculosis lesions eventually progress to cavities. Our results provide greater insight into the molecular mechanisms of Mtb persistence within host lungs.

Mycobacterium tuberculosis Hip1 modulates macrophage responses through proteolysis of GroEL2.

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.

The intracellular environment of human macrophages that produce nitric oxide promotes growth of mycobacteria.

Nitric oxide (NO) is a diffusible radical gas produced from the activity of nitric oxide synthase (NOS). NOS activity in murine macrophages has a protective role against mycobacteria through generation of reactive nitrogen intermediates (RNIs). However, the production of NO by human macrophages has remained unclear due to the lack of sensitive reagents to detect NO directly. The purpose of this study was to investigate NO production and the consequence to mycobacteria in primary human macrophages. We found that Mycobacterium bovis BCG or Mycobacterium tuberculosis infection of human macrophages induced expression of NOS2 and NOS3 that resulted in detectable production of NO. Treatment with gamma interferon (IFN-γ), l-arginine, and tetrahydrobiopterin enhanced expression of NOS2 and NOS3 isoforms, as well as NO production. Both of these enzymes were shown to contribute to NO production. The maximal level of NO produced by human macrophages was not bactericidal or bacteriostatic to M. tuberculosis or BCG. The number of viable mycobacteria was increased in macrophages that produced NO, and this requires expression of nitrate reductase. An narG mutant of M. tuberculosis persisted but was unable to grow in human macrophages. Taken together, these data (i) enhance our understanding of primary human macrophage potential to produce NO, (ii) demonstrate that the level of RNIs produced in response to IFN-γ in vitro is not sufficient to limit intracellular mycobacterial growth, and (iii) suggest that mycobacteria may use RNIs to enhance their survival in human macrophages.

Distinct effector memory CD4+ T cell signatures in latent Mycobacterium tuberculosis infection, BCG vaccination and clinically resolved tuberculosis.

Two billion people worldwide are estimated to be latently infected with Mycobacterium tuberculosis (Mtb) and are at risk for developing active tuberculosis since Mtb can reactivate to cause TB disease in immune-compromised hosts. Individuals with latent Mtb infection (LTBI) and BCG-vaccinated individuals who are uninfected with Mtb, harbor antigen-specific memory CD4(+) T cells. However, the differences between long-lived memory CD4(+) T cells induced by latent Mtb infection (LTBI) versus BCG vaccination are unclear. In this study, we characterized the immune phenotype and functionality of antigen-specific memory CD4(+) T cells in healthy BCG-vaccinated individuals who were either infected (LTBI) or uninfected (BCG) with Mtb. Individuals were classified into LTBI and BCG groups based on IFN-γ ELISPOT using cell wall antigens and ESAT-6/CFP-10 peptides. We show that LTBI individuals harbored high frequencies of late-stage differentiated (CD45RA(-)CD27(-)) antigen-specific effector memory CD4(+) T cells that expressed PD-1. In contrast, BCG individuals had primarily early-stage (CD45RA(-)CD27(+)) cells with low PD-1 expression. CD27(+) and CD27(-) as well as PD-1(+) and PD-1(-) antigen-specific subsets were polyfunctional, suggesting that loss of CD27 expression and up-regulation of PD-1 did not compromise their capacity to produce IFN-γ, TNF-α and IL-2. PD-1 was preferentially expressed on CD27(-) antigen-specific CD4(+) T cells, indicating that PD-1 is associated with the stage of differentiation. Using statistical models, we determined that CD27 and PD-1 predicted LTBI versus BCG status in healthy individuals and distinguished LTBI individuals from those who had clinically resolved Mtb infection after anti-tuberculosis treatment. This study shows that CD4(+) memory responses induced by latent Mtb infection, BCG vaccination and clinically resolved Mtb infection are immunologically distinct. Our data suggest that differentiation into CD27(-)PD-1(+) subsets in LTBI is driven by Mtb antigenic stimulation in vivo and that CD27 and PD-1 have the potential to improve our ability to evaluate true LTBI status.

Mycobacterium tuberculosis Hip1 dampens macrophage proinflammatory responses by limiting toll-like receptor 2 activation.

Mycobacterium tuberculosis is a highly successful human pathogen that evades host innate immunity by interfering with macrophage functions. In addition to avoiding macrophage microbicidal activities, M. tuberculosis triggers secretion of proinflammatory cytokines and chemokines in macrophages. The levels of proinflammatory cytokines induced by clinical M. tuberculosis isolates are thought to play an important role in determining tuberculosis disease progression and severity, but the mechanisms by which M. tuberculosis modulates the magnitude of inflammatory responses remain unclear. Here we show that M. tuberculosis restricts robust macrophage activation and dampens proinflammatory responses through the cell envelope-associated serine hydrolase Hip1 (hydrolase important for pathogenesis 1). By transcriptionally profiling macrophages infected with either wild-type or hip1 mutant bacteria, we found that the hip1 mutant induced earlier and significantly higher levels of several proinflammatory cytokines and chemokines. We show that increased activation of Toll-like receptor 2 (TLR2)- and MyD88-dependent signaling pathways mediates the enhanced cytokine secretion induced by the hip1 mutant. Thus, Hip1 restricts the onset and magnitude of proinflammatory cytokines by limiting TLR2-dependent activation. We also show that Hip1 dampens TLR2-independent activation of the inflammasome and limits secretion of interleukin-18 (IL-18). Dampening of TLR2 signaling does not require viable M. tuberculosis or phagocytosis but does require Hip1 catalytic activity. We propose that M. tuberculosis restricts proinflammatory responses by masking cell surface interactions between TLR2 agonists on M. tuberculosis and TLR2 on macrophages. This strategy may allow M. tuberculosis to evade early detection by host immunity, delay the onset of adaptive immune responses, and accelerate disease progression.

Mycobacterium tuberculosis Rv2224c modulates innate immune responses.

Tuberculosis remains a major global health problem that kills up to 2 million people annually. Central to the success of Mycobacterium tuberculosis (Mtb) as a pathogen is its ability to evade host immunity and to establish a chronic infection. Although its primary intracellular niche is within macrophages, the underlying molecular mechanisms are poorly understood. Here we show that Rv2224c, a cell envelope-associated predicted protease, is critical for Mtb virulence. Disruption of Rv2224c led to prolonged survival of infected mice and highly reduced lung pathology. Absence of Rv2224c enhanced host innate immune responses, compromised the intracellular survival of Mtb in macrophages, and increased its susceptibility to lysozyme. We provide insights into the molecular basis for Rv2224c function by showing that Rv2224c activity promotes processing and extracellular release of the Mtb protein, GroEL2. Inhibition of Rv2224c and its targets offers opportunities for therapeutic interventions and immune-modulatory strategies.

Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages.

Macrophages are central to host defense against microbes, but intracellular pathogens have evolved to evade their antimicrobial functions. Mycobacterium tuberculosis (MTB) has successfully exploited macrophages as its primary niche in vivo, but the bacterial genome-wide requirements that promote its intracellular survival remain undefined. Here we comprehensively identify the MTB genes required for survival by screening for transposon mutants that fail to grow within primary macrophages. We identify mutants showing decreased growth in macrophage environments that model stages of the host immune response. By systematically analyzing several biologically relevant data sets, we have been able to identify putative pathways that could not be predicted by genome organization alone. In one example, phosphate transport, requiring physically unlinked genes, was found to be critical for MTB growth in macrophages and important for establishing persistent infection in lungs. Remarkably, the majority of MTB genes found by this analysis to be required for survival are constitutively expressed rather than regulated by macrophages, revealing the host-adapted lifestyle of an evolutionarily selected intracellular pathogen.

Interferon regulatory factor 4 (IRF4) interacts with NFATc2 to modulate interleukin 4 gene expression.

Proteins of the nuclear factor of activated T cells (NFAT) family of transcription factors are critical for lymphocyte activation in the immune system. In particular, NFATs are important regulators of inducible IL-4 gene expression. Interferon regulatory factor 4 (IRF4) is an immune system-restricted interferon regulatory factor that is required for lymphocyte activation, but its molecular functions in the T lineage remain to be elucidated. We demonstrate that IRF4 potently synergizes with NFATc2 to specifically enhance NFATc2-driven transcriptional activation of the IL-4 promoter. This function is dependent on the physical interaction of IRF4 with NFATc2. IRF4 synergizes with NFATc2 and the IL-4-inducing transcription factor, c-maf, to augment IL-4 promoter activity as well as to elicit significant levels of endogenous IL-4 production. Furthermore, naïve T helper cells from mice lacking IRF4 are compromised severely for the production of IL-4 and other Th2 cytokines. The identification of IRF4 as a partner for NFATc2 in IL-4 gene regulation provides an important molecular function for IRF4 in T helper cell differentiation.