Mtb-infected Mice Research Articles

The introduction of mesenchymal stromal cells induces different immunological responses in the lungs of healthy and M. tuberculosis infected mice.

Mesenchymal stromal cells (MSC) have strong immunomodulatory properties and therefore can be used to control inflammation and tissue damage. It was suggested recently that MSC injections can be used to treat multi-drug resistant tuberculosis (TB). However, MSC trafficking and immunomodulatory effects of MSC injections during Mycobacterium tuberculosis (Mtb) infection have not been studied. To address this issue we have analyzed MSC distribution in tissues and local immunological effects of MSC injections in Mtb infected and uninfected mice. After intravenous injection, MSC accumulated preferentially in the lungs where they were located as cell aggregates in the alveolar walls. Immunological analysis of MSC effects included detection of activated, IFN-γ and IL-4 producing CD4+ lymphocytes, the frequency analysis of dendritic cells (CD11c+F4/80) and macrophages (CD11c-F4/80+) located in the lungs, the expression of IA/IE and CD11b molecules by these cells, and evaluation of 23 cytokines/chemokines in lung lysates. In the lungs of uninfected mice, MSC transfer markedly increased the percentage of IFN-γ+ CD4+ lymphocytes and dendritic cells, elevated levels of IA/IE expression by dendritic cells and macrophages, augmented local production of type 2 cytokines (IL-4, IL-5, IL-10) and chemokines (CCL2, CCL3, CCL4, CCL5, CXCL1), and downregulated type 1 and hematopoietic cytokines (IL-12p70, IFN-γ, IL-3, IL-6, GM-CSF). Compared to uninfected mice, Mtb infected mice had statistically higher “background” frequency of activated CD69+ and IFN-γ+ CD4+ lymphocytes and dendritic cells, and higher levels of cytokines in the lungs. The injections of MSC to Mtb infected mice did not show statistically significant effects on CD4+ lymphocytes, dendritic cells and macrophages, only slightly shifted cytokine profile, and did not change pathogen load or slow down TB progression. Lung section analysis showed that in Mtb infected mice, MSC could not be found in the proximity of the inflammatory foci. Thus, in healthy recipients, MSC administration dramatically changed T-cell function and cytokine/chemokine milieu in the lungs, most likely, due to capillary blockade. But, during Mtb infection, i.e., in the highly-inflammatory conditions, MSC did not affect T-cell function and the level of inflammation. The findings emphasize the importance of the evaluation of MSC effects locally at the site of their predominant post-injection localization and question MSC usefulness as anti-TB treatment.

Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis

CD4 Tcells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 Tcells have conferred little or no protection. Here we examined CD4 Tcell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific Tcells to be more differentiated than Ag85B-specific Tcells. The ability of each Tcell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific Tcells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific Tcells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required tooptimize protection mediated by Tcells recognizing antigens expressed at distinct stages of Mtb infection.

Inhibition of heme oxygenase-1 activity suppresses Mycobacterium tuberculosis infection in vivo by a mechanism dependent on T lymphocytes and IFN-γ production

Abstract Infection with M. tuberculosis (Mtb) induces the upregulation of heme oxygenase-1 (HO-1), a powerful antioxidant enzyme that catalyzes the degradation of heme into iron, biliverdin and carbon monoxide. We observed that administration of tin protoporphyrin IX (SnPPIX), a well-characterized HO-1 activity inhibitor, to Mtb-infected mice resulted in pulmonary bacterial load reduction. Moreover, adjunct administration of SnPPIX to Mtb-infected mice receiving antibiotic treatment improved and accelerated pathogen clearance. Interestingly, both the induction of HO-1 expression in lungs of Mtb-infected mice and the efficacy of in vivo SnPPIX-mediated bacterial burden reduction were dependent on the presence of host TCR-α+ lymphocytes as well as IFN-γ suggesting a role for the immune response in the activity of the drug. Further investigation revealed that in the lungs of these immunodeficient mice the numbers of parenchymal CD11b+ myeloid cells staining positive for HO-1 were substantially reduced. SnPPIX treatment of Mtb-infected mouse bone marrow derived macrophages also resulted in diminished bacterial loads in vitro and this effect was reverted by iron supplementation and enhanced by iron chelation. Based on these observations we propose that HO-1 inhibition promotes control of Mtb by limiting the availability of nutrient iron and that the host Th1 response regulates this effect by controlling the recruitment to the lungs of a subset of myeloid cells that is both capable of HO-1 expression and permissive to bacterial infection. This work was supported by the intramural research program of the NIAID.

Human and Mouse Hematopoietic Stem Cells Are a Depot for Dormant Mycobacterium tuberculosis.

An estimated third of the world’s population is latently infected with Mycobacterium tuberculosis (Mtb), with no clinical signs of tuberculosis (TB), but lifelong risk of reactivation to active disease. The niches of persisting bacteria during latent TB infection remain unclear. We detect Mtb DNA in peripheral blood selectively in long-term repopulating pluripotent hematopoietic stem cells (LT-pHSCs) as well as in mesenchymal stem cells from latently infected human donors. In mice infected with low numbers of Mtb, that do not develop active disease we, again, find LT-pHSCs selectively infected with Mtb. In human and mouse LT-pHSCs Mtb are stressed or dormant, non-replicating bacteria. Intratracheal injection of Mtb-infected human and mouse LT-pHSCs into immune-deficient mice resuscitates Mtb to replicating bacteria within the lung, accompanied by signs of active infection. We conclude that LT-pHSCs, together with MSCs of Mtb-infected humans and mice serve as a hitherto unappreciated quiescent cellular depot for Mtb during latent TB infection.

Wnt5a Deficiency Regulates Inflammatory Cytokine Secretion, Polarization, and Apoptosis in Mycobacterium tuberculosis-Infected Macrophages.

Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis (MTB), is one of the global public health catastrophes. Wnt signaling has recently been identified to exert immunoregulatory functions in a variety of inflammatory and infectious diseases, including tuberculosis. The opposite expression of Wnt5a in human and mice during MTB infection drives us to explore the roles and biological significances of reduced Wnt5a for MTB-treated mice. In our study, the reduction of WNT5A in MTB-treated mice lung tissues or MTB-infected mice bone marrow-derived macrophages (BM-Mø) was in a dose- and time-dependent manner. Then, WNT5A-silenced mice, secreted frizzled-related protein 1 (SFRP1)-overexpressed or -silenced mice BM-Mø, were constructed to regulate Wnt5a levels. When Wnt5a is deficient, MTB-induced increases of pro-inflammatory cytokines (TNF-α, IL-1β, IL-12, and IL-6) can be markedly attenuated in mice lung tissues or BM-Mø. Besides, external disturbance triggered that Wnt5a lower expression can induce Mø to be M2 phenotype and enhance cell apoptosis of MTB-infected mice BM-Mø. Hence, the reduction of Wnt5a is a tactful strategy adopted by Mø to resistant MTB-induced immune responses and to enhance MTB-induced Mø apoptosis in mice. Our study revealed a new style for Mø to manipulate themselves against MTB infection. Our research identifies that Wnt5a deficiency can regulate inflammatory cytokine secretion, polarization, and apoptosis in MTB-infected Mø.

NK-CD11c+ Cell Crosstalk in Diabetes Enhances IL-6-Mediated Inflammation during Mycobacterium tuberculosis Infection.

In this study, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and identified factors that increase susceptibility of T2DM mice to infection by Mycobacterium tuberculosis (Mtb). All Mtb-infected T2DM mice and 40% of uninfected T2DM mice died within 10 months, whereas all control mice survived. In Mtb-infected mice, T2DM increased the bacterial burden and pro- and anti-inflammatory cytokine and chemokine production in the lungs relative to those in uninfected T2DM mice and infected control mice. Levels of IL-6 also increased. Anti-IL-6 monoclonal antibody treatment of Mtb-infected acute- and chronic-T2DM mice increased survival (to 100%) and reduced pro- and anti-inflammatory cytokine expression. CD11c+ cells were the major source of IL-6 in Mtb-infected T2DM mice. Pulmonary natural killer (NK) cells in Mtb-infected T2DM mice further increased IL-6 production by autologous CD11c+ cells through their activating receptors. Anti-NK1.1 antibody treatment of Mtb-infected acute-T2DM mice increased survival and reduced pro- and anti-inflammatory cytokine expression. Furthermore, IL-6 increased inflammatory cytokine production by T lymphocytes in pulmonary tuberculosis patients with T2DM. Overall, the results suggest that NK-CD11c+ cell interactions increase IL-6 production, which in turn drives the pathological immune response and mortality associated with Mtb infection in diabetic mice.

Mycobacterium indicus pranii as a booster vaccine enhances BCG induced immunity and confers higher protection in animal models of tuberculosis

BCG, the only approved vaccine protects against severe form of childhood tuberculosis but its protective efficacy wanes in adolescence. BCG has reduced the incidence of infant TB considerably in endemic areas; therefore prime-boost strategy is the most realistic measure for control of tuberculosis in near future. Mycobacterium indicus pranii (MIP) shares significant antigenic repertoire with Mtb and BCG and has been shown to impart significant protection in animal models of tuberculosis. In this study, MIP was given as a booster to BCG vaccine which enhanced the BCG mediated immune response, resulting in higher protection. MIP booster via aerosol route was found to be more effective in protection than subcutaneous route of booster immunization. Pro-inflammatory cytokines like IFN-γ, IL-12 and IL-17 were induced at higher level in infected lungs of ‘BCG-MIP’ group both at mRNA expression level and in secretory form when compared with ‘only BCG’ group. BCG-MIP groups had increased frequency of multifunctional T cells with high MFI for IFN-γ and TNF-α in Mtb infected mice. Our data demonstrate for the first time, potential application of MIP as a booster to BCG vaccine for efficient protection against tuberculosis. This could be very cost effective strategy for efficient control of tuberculosis.

Bis-biguanide dihydrochloride inhibits intracellular replication of M. tuberculosis and controls infection in mice.

While there is an urgent need to develop new and effective drugs for treatment of tuberculosis (TB) and multi-drug resistant TB (MDR-TB), repurposing FDA (U.S. Food and Drug Administration) -approved drugs for development of anti-TB agents may decrease time and effort from bench to bedside. Here, we employed host cell-based high throughput screening (HTS) assay to screen and characterize FDA-approved, off-patent library drugs for anti-Mycobacterium tuberculosis (MTB) activities. The cell-based HTS allowed us to identify an anti-cancer drug of bis-biguanide dihydrochloride (BBD) as potent anti-mycobacteria agent. Further characterization showed that BBD could inhibit intracellular and extracellular growth of M. smegmatis and slow-growing M. bovis BCG. BBD also potently inhibited replication of clinically-isolated MTB and MDR-TB strains. The proof-of-concept study showed that BBD treatment of MTB-infected mice could significantly decrease CFU counts in the lung and spleen. Notably, comparative evaluation showed that MTB CFU counts in BBD-treated mice were lower than those in rifampicin-treated mice. No apparent BBD side effects were found in BBD-treated mice. Thus, our findings support further studies to develop BBD as a new and effective drug against TB and MDR-TB.

HIV-1 Tat protein vaccination in mice infected with Mycobacterium tuberculosis is safe, immunogenic and reduces bacterial lung pathology.

BackgroundThe therapeutic HIV-1 Tat protein vaccine is in advanced clinical development. Tuberculosis, the main AIDS co-infection, is highly endemic in areas where AIDS prevention through vaccination is needed. However, safety and immunogenicity of Tat vaccination in the course of Mycobacterium tuberculosis (Mtb) infection is still unknown and it prevents the possibility to administer the vaccine to Mtb-infected individuals. We addressed the interplay and effects of Tat vaccination on Mtb infection in immunocompetent mice.MethodsC57BL/6 mice were vaccinated or not with Bacillus Calmette-Guerin (BCG), the current tuberculosis vaccine, and after 5 weeks were infected with Mtb by intravenous route. The Tat protein was injected intradermally at 1, 2 and 4 weeks after Mtb challenge. Eight weeks after Mtb infection, all mice were sacrificed, and both the degree of pathology and immune responses to Mtb and Tat were evaluated. As additional control, some mice were either vaccinated or not with BCG, were not challenged with Mtb, but received the Tat protein. Statistical significances were evaluated by one-way or two-way ANOVA and Tukey’s multiple comparisons post-test.ResultsIn the lungs of Mtb-infected mice, Tat-vaccine did not favour Mtb replication and indeed reduced both area of cellular infiltration and protein levels of Interferon-γ, Chemokine (C-C motif) ligand-4 and Interleukin-1β, pathological events triggered by Mtb-infection. Moreover, the protection against Mtb infection conferred by BCG remained good after Tat protein treatment. In spleen cells of Mtb-infected mice, Tat vaccination enhanced Mtb-specific Interferon-γ and Interleukin-17 responses, which may have a protective role. Of note, Mtb infection reduced, but did not suppress, the development of anti-Tat antibodies, required for Tat vaccine efficacy and the titer of anti-Tat IgG was potentiated by BCG vaccination in Mtb-free mice. In general, Tat treatment was well tolerated in both Mtb-infected and Mtb-free mice.ConclusionsTat protein vaccine, administered in Mtb-infected mice with a protocol resembling that used in the clinical trials, was safe, immunogenic, limited the lung Mtb-associated immunopathology and did not abrogate the protective efficacy of BCG. These data provide preliminary evidence for a safe use of Tat vaccine in people vaccinated with BCG and/or suffering from tuberculosis.

Microfold Cells Actively Translocate Mycobacterium tuberculosis to Initiate Infection

The prevailing paradigm is that tuberculosis infection is initiated when patrolling alveolar macrophages and dendritic cells within the terminal alveolus ingest inhaled Mycobacterium tuberculosis (Mtb). However, definitive data for this model are lacking. Among the epithelial cells of the upper airway, a specialized epithelial cell known as a microfold cell (M cell) overlies various components of mucosa-associated lymphatic tissue. Here, using multiple mouse models, we show that Mtb invades via M cells to initiate infection. Intranasal Mtb infection in mice lacking M cells either genetically or by antibody depletion resulted in reduced invasion and dissemination to draining lymph nodes. M cell-depleted mice infected via aerosol also had delayed dissemination to lymph nodes and reduced mortality. Translocation of Mtb across two M cell transwell models was rapid and transcellular. Thus, M cell translocation is a vital entry mechanism that contributes to the pathogenesis of Mtb.

Quantifying Limits on Replication, Death, and Quiescence of Mycobacterium tuberculosis in Mice.

When an individual is exposed to Mycobacterium tuberculosis (Mtb) three outcomes are possible: bacterial clearance, active disease, or latent infection. It is generally believed that most individuals exposed to Mtb become latently infected and carry the mycobacteria for life. How Mtb is maintained during this latent infection remains largely unknown. During an Mtb infection in mice, there is a phase of rapid increase in bacterial numbers in the murine lungs within the first 3 weeks, and then bacterial numbers either stabilize or increase slowly over the period of many months. It has been debated whether the relatively constant numbers of bacteria in the chronic infection result from latent (dormant, quiescent), non-replicating bacteria, or whether the observed Mtb cell numbers are due to balance between rapid replication and death. A recent study of mice, infected with a Mtb strain carrying an unstable plasmid, showed that during the chronic phase, Mtb was replicating at significant rates. Using experimental data from this study and mathematical modeling we investigated the limits of the rates of bacterial replication, death, and quiescence during Mtb infection of mice. First, we found that to explain the data the rates of bacterial replication and death could not be constant and had to decrease with time since infection unless there were large changes in plasmid segregation probability over time. While a decrease in the rate of Mtb replication with time since infection was expected due to depletion of host's resources, a decrease in the Mtb death rate was counterintuitive since Mtb-specific immune response, appearing in the lungs 3–4 weeks after infection, should increase removal of bacteria. Interestingly, we found no significant correlation between estimated rates of Mtb replication and death suggesting the decline in these rates was driven by independent mechanisms. Second, we found that the data could not be explained by assuming that bacteria do not die, suggesting that some removal of bacteria from lungs of these mice had to occur even though the total bacterial counts in these mice always increased over time. Third and finally, we showed that to explain the data the majority of bacterial cells (at least ~60%) must be replicating in the chronic phase of infection further challenging widespread belief of nonreplicating Mtb in latency. Our predictions were robust to some changes in the structure of the model, for example, when the loss of plasmid-bearing cells was mainly due to high fitness cost of the plasmid. Further studies should determine if more mechanistic models for Mtb dynamics are also able to accurately explain these data.

TGF-β-mediated inhibition of IFN-γ production by Mycobacterium tuberculosis-specific T cells in the infected lung

Abstract Tuberculosis (TB) is a major global health problem killing over one million people every year. The emergence of multi and extremely-drug resistant (MDR and XDR) strains of Mycobacterium tuberculosis (Mtb) has made treatment difficult and has spurred interest in developing host-directed immunotherapeutics. A critical roadblock is our failure to understand how Mtb escapes eradication despite an apparently robust adaptive immune response. Interferon-γ (IFN-γ) producing CD4 T cells are critical for immunity, yet their abundance in the blood does not correlate with protection to TB, suggesting that their activity may be blocked locally at the primary site of infection in the lung. A leading candidate for IFN-γ suppression in the lung is transforming growth factor-β (TGF-β), a multifunctional cytokine that is capable of inhibiting IFN-γ. TGF-β is produced in excess at sites of active Mtb infection in both murine and human TB. Neutralization of TGF-β improves T-cell responses of active TB patients in vitro, but the extent of its role during in vivo TB is unknown. Here, we generated mixed bone marrow chimeras using WT mice and mice with a T cell-specific conditional deletion of TGF-β receptor II (dLck-Cre:TGFbRIIfl/fl). Mtb-specific TGF-βRII knockout cells outcompeted their WT counterparts and exhibited increased IFN-γ production during Mtb infection. Furthermore, Mtb-infected dLck-Cre:TGFbRIIfl/fl mice had decreased bacterial loads early during infection. Finally, two pharmacological inhibitors of TGF-β signaling lowered the bacterial loads of lungs from Mtb-infected mice both early and late during infection. Targeting TGF-β may pave the way for new host-directed immunotherapeutics for TB.

Role of secreted IgM in regulating the host response to Mycobacterium tuberculosis

Abstract B cell aggregates, which display characteristics of germinal center reactions, are a prominent feature in the lungs of Mycobacterium tuberculosis (Mtb)-infected hosts. Using the B cell-deficient mouse strain μMT, we have previously shown that B cells are required for optimal protection against aerosol Mtb infection. μMT mice display exacerbated lung granulomatous inflammation during acute tuberculosis (TB), and this can be ameliorated by adoptive transfer of B cells or treatment with immune serum, suggesting a role for immunoglobulin in modulating the local lung response to Mtb. To investigate the role of antibody in a protective host response to Mtb, we utilized mice deficient in various immunoglobulins. Mice unable to secrete IgM (μS−/−) were more susceptible to aerosol Mtb infection than those lacking all other immunoglobulin isotypes, and displayed increased mortality during chronic TB. Relative to wild-type (WT) mice, Mtb-infected μS−/− mice had fewer lymphoid aggregates and a delayed granulomatous response in the lungs, despite having greater numbers of activated pulmonary CD4+ T cells. Treatment of μS−/− mice with immune serum (derived from Mtb-infected WT mice) during acute infection, led to increased lymphoid aggregates and plasma cells in the lungs, more pulmonary infiltrate, a concomitant decrease in the lung bacterial burden, and ultimately delayed the onset of death of μS−/− mice during chronic TB. These data suggest IgM plays an important role in controlling Mtb infection by orchestrating local granulomatous responses in the lungs, which leads to improved host survival during the chronic phase of infection.

Intracerebral Mycobacterium tuberculosis infection-induced immune response in the central nervous system

Abstract The World Health Organization declared a tuberculosis (TB) global emergency. Central nervous system (CNS) TB is a rare, highly devastating manifestation of extra-pulmonary TB. To date, research has focused on pulmonary TB, with no significant advancements being made in understanding the mechanisms associated with CNS TB. This study uses a small animal model of CNS TB that permits us to investigate the immune responses following CNS Mycobacterium tuberculosis (Mtb) infection. We use intracranial infection of mice with the H37Rv strain of Mtb which has been fluorescently tagged with a TdTomato plasmid, allowing us to track it in vivo, and analyzed the cellular composition of the subsequent immunological response in the brain. At 7 days post infection (PI), we show a robust infiltration of immune cells into the CNS including the infiltration of CD11c+ dendritic cells. We can also see Mtb-containing granuloma-like lesions. We adoptively transferred Mtb-specific P25 CD4+ T and found that these cells also accumulate in the brain and were found activated in the peripheral lymphoid tissue, including the spleen and cervical draining lymph nodes. The P25 T cells present in the brain display extensive proliferation. Moreover, P25 T cells present in the peripheral lymphoid organs of Mtb infected mice show increased proliferation when compared to non-infected controls suggesting a systemic sampling of CNS Mtb antigen. We also show that at 7 days PI there is a significantly higher percentage of activated, INFγ producing, CD4+ T in the brain. Both INFγ and IL-17 producing T cell populations increase in size by day 21. This study will provide knowledge into the pathogenesis of CNS TB, and provide possible treatment strategies to lessen its destructive outcome.

Identifying key regulators of CD4 T cell function in the Mycobacterium tuberculosis-infected lung using systems analysis

Abstract Mycobacterium tuberculosis (Mtb) infection is the leading infectious killer worldwide and an effective vaccine remains elusive. Although CD4 T cells are known to be important for protection, the specific requirements for a protective CD4 response are unclear. Our lab has shown that differences in antigen exposure drive significant phenotypic variation within Mtb-specific CD4 T cells. CD4 T cells specific for the Mtb protein Ag85B, which is only expressed early during infection, remain capable of robust cytokine production. Conversely, T cells specific for ESAT-6, which is expressed for the duration of infection, exhibit blunted cytokine production. To identify regulators that govern this T cell exhaustion, we employed a systems biology approach. We performed RNA-seq on unstimulated and stimulated Ag85B and ESAT-6-specific CD4 T cells isolated from the lungs of Mtb-infected mice and used cMonkey, a biclustering algorithm, to create gene clusters based on expression patterns and identify transcription factors predicted to regulate the clusters. This analysis revealed eight transcription factors that were themselves differentially expressed between Ag85B and ESAT-6-specific CD4 T cells and also predicted to regulate clusters of differentially expressed genes. We are currently focusing our analysis on two of these transcription factors, VDR and Lyl-1. Mtb infection of WT:VDR−/− mixed bone marrow chimeras have validated the systems-based predictions by showing that VDR regulates distinct processes in Ag85B-specific compared to ESAT-6-specific CD4 T cells. Understanding the disparate regulatory pathways operant in functional vs. exhausted T cells may inform new avenues for host-directed therapy of tuberculosis.

Mycobacterium tuberculosis Rv3628 drives Th1-type T cell immunity via TLR2-mediated activation of dendritic cells and displays vaccine potential against the hyper-virulent Beijing K strain.

Identification of vaccine target antigens (Ags) that induce Ag-specific Th1 immunity is the first step toward the development of a tuberculosis vaccine. Here, we evaluated the Mycobacterium tuberculosis (Mtb) protein Rv3628, a soluble inorganic pyrophosphatase, as a vaccine target and characterized the molecular details of its interaction with dendritic cells (DCs). Rv3628 activated DCs, increasing their expression of cell surface molecules and augmenting their production of TNF-α, IL-1β, IL-6, and IL-12p70. Rv3628 mediated these effects by binding to TLR2 and activating downstream MyD88-, MAPK- and NF-κB-dependent signaling pathways. Rv3628-stimulated DCs induced the expansion of OVA-specific CD4+ and CD8+ T cells, which secreted IFN-γ and IL-2. Rv3628-specific effector/memory T cells expanded to a similar extent as those stimulated with ESAT-6 Ag in samples of lung and spleen cells collected from Mtb-infected mice. Finally, an Rv3628 subunit vaccine adjuvanted with dimethyldioctadecylammonium liposomes containing monophosphoryl lipid-A caused significant reductions in bacterial counts and lung inflammation after challenge with the hyper-virulent Mtb K strain. Importantly, protective efficacy was correlated with the generation of Rv3628-specific CD4+ T cells co-producing IFN-γ, TNF-α and IL-2 and exhibiting an elevated IFN-γ recall response. Thus, Rv3628 polarizes DCs toward a Th1 phenotype and promotes protective immunity against Mtb infection.

Mycobacterium tuberculosis PE27 activates dendritic cells and contributes to Th1-polarized memory immune responses during in vivo infection

A gradual understanding of the proline-glutamate (PE) and proline-proline-glutamate (PPE) families, which compromise 10% of the coding regions in the Mycobacterium tuberculosis (Mtb) genome, has uncovered unique roles in host–pathogen interactions. However, the immunological function of PE27 (Rv2769c), the largest PE member, remains unclear. Here, we explored the functional roles and related signaling mechanisms of PE27 in the interaction with dendritic cells (DCs) to shape the T cell response. PE27 phenotypically and functionally induces DC maturation by up-regulating CD80, CD86, MHC class I and MHC class II expression on the DC surface to promote the production of TNF-α, IL-1β, IL-6, and IL-12p70 but not IL-10. Additionally, we found that PE27-mediated DC activation requires the participation of mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) signaling pathways. Interestingly, PE27-treated DCs directed naïve CD4+ T cells to secrete IFN-γ and activate T-bet but not GATA-3. PE27 also induced IFN-γ-producing memory T cell responses in Mtb-infected mice, indicating that PE27 contributes to Th1-polarization. Taken together, these findings suggest that PE27 possesses Th1-polarizing potential through DC maturation and could be useful in the design of TB vaccines.

Helminth-induced arginase-1 exacerbates lung inflammation and disease severity in tuberculosis.

Parasitic helminth worms, such as Schistosoma mansoni, are endemic in regions with a high prevalence of tuberculosis (TB) among the population. Human studies suggest that helminth coinfections contribute to increased TB susceptibility and increased rates of TB reactivation. Prevailing models suggest that T helper type 2 (Th2) responses induced by helminth infection impair Th1 immune responses and thereby limit Mycobacterium tuberculosis (Mtb) control. Using a pulmonary mouse model of Mtb infection, we demonstrated that S. mansoni coinfection or immunization with S. mansoni egg antigens can reversibly impair Mtb-specific T cell responses without affecting macrophage-mediated Mtb control. Instead, S. mansoni infection resulted in accumulation of high arginase-1-expressing macrophages in the lung, which formed type 2 granulomas and exacerbated inflammation in Mtb-infected mice. Treatment of coinfected animals with an antihelminthic improved Mtb-specific Th1 responses and reduced disease severity. In a genetically diverse mouse population infected with Mtb, enhanced arginase-1 activity was associated with increased lung inflammation. Moreover, in patients with pulmonary TB, lung damage correlated with increased serum activity of arginase-1, which was elevated in TB patients coinfected with helminths. Together, our data indicate that helminth coinfection induces arginase-1-expressing type 2 granulomas, thereby increasing inflammation and TB disease severity. These results also provide insight into the mechanisms by which helminth coinfections drive increased susceptibility, disease progression, and severity in TB.

Mycobacterium tuberculosis RpfE promotes simultaneous Th1- and Th17-type T-cell immunity via TLR4-dependent maturation of dendritic cells.

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation-promoting factor (Rpf) E, a latency-associated member of the Rpf family, in promoting naïve CD4(+) T-cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL-6, IL-1β, IL-23p19, IL-12p70, and TNF-α but not IL-10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF-κB signaling. RpfE-treated DCs effectively caused naïve CD4(+) T cells to secrete IFN-γ, IL-2, and IL-17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T-bet and RORγt but not GATA-3. Furthermore, lung and spleen cells from Mtb-infected WT mice but not from TLR4(-/-) mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1- and Th17-polarized T-cell expansion.

STIM1 controls T cell-mediated immune regulation and inflammation in chronic infection.

Chronic infections induce a complex immune response that controls pathogen replication, but also causes pathology due to sustained inflammation. Ca2+ influx mediates T cell function and immunity to infection, and patients with inherited mutations in the gene encoding the Ca2+ channel ORAI1 or its activator stromal interaction molecule 1 (STIM1) are immunodeficient and prone to chronic infection by various pathogens, including Mycobacterium tuberculosis (Mtb). Here, we demonstrate that STIM1 is required for T cell-mediated immune regulation during chronic Mtb infection. Compared with WT animals, mice with T cell-specific Stim1 deletion died prematurely during the chronic phase of infection and had increased bacterial burdens and severe pulmonary inflammation, with increased myeloid and lymphoid cell infiltration. Although STIM1-deficient T cells exhibited markedly reduced IFN-γ production during the early phase of Mtb infection, bacterial growth was not immediately exacerbated. During the chronic phase, however, STIM1-deficient T cells displayed enhanced IFN-γ production in response to elevated levels of IL-12 and IL-18. The lack of STIM1 in T cells was associated with impaired activation-induced cell death upon repeated TCR engagement and pulmonary lymphocytosis and hyperinflammation in Mtb-infected mice. Chronically Mtb-infected, STIM1-deficient mice had reduced levels of inducible regulatory T cells (iTregs) due to a T cell-intrinsic requirement for STIM1 in iTreg differentiation and excessive production of IFN-γ and IL-12, which suppress iTreg differentiation and maintenance. Thus, STIM1 controls multiple aspects of T cell-mediated immune regulation to limit injurious inflammation during chronic infection.