Mtb-infected Lung Research Articles

Subunit vaccine H56/CAF01 induces a population of circulating CD4 T cells that traffic into the Mycobacterium tuberculosis-infected lung

The capacity of CD4 T cells to protect against Mycobacterium tuberculosis (Mtb) is governed by their ability to localize to the lung site of infection. Subunit vaccine H56/CAF01, a liposome-adjuvanted fusion protein of Mtb antigens Ag85B, ESAT-6, and Rv2660, conferred durable protection and elicited polyfunctional CD4 T cells that preferentially localized to the lung parenchyma. These lung-resident T cells had reduced KLRG1 and increased CXCR3 expression, an intermediate state of Th1 differentiation that has been associated with Mtb protection. Importantly, KLGR1− CXCR3+ cells were also enriched in the lung vasculature and peripheral circulation of vaccinated animals, but not controls. Moreover, S1P1R blockade rapidly cleared this population from the blood and adoptive transfer of T cells recovered from the vasculature of vaccinated, but not control, mice efficiently trafficked into the Mtb-infected lung parenchyma. Thus, durable immunity elicited by H56/CAF01 vaccination is associated with the maintenance of circulating CD4 T cells that selectively home to the lung parenchyma.

IL-6 regulates pro- and anti-inflammatory cytokine production and mortality of Mycobacterium tuberculosis infected type 2 diabetic mice (INC2P.418)

Abstract In this study, to determine whether diabetic (DM) mice are susceptible to Mycobacterium tuberculosis (Mtb) infection, we developed experimentally induced diabetes in wild type C57BL/6 mice with streptozotocin & nicotinamide. We then infected non-DM and DM mice with Mtb H37Rv by aerosol. Around 50% of Mtb infected DM (p=0.05) and 25% of uninfected DM (p=0.07) mice died in 5 to 6 months compared to no deaths in infected control mice. Six months after Mtb infection, there was a statistically significant but marginal increase in bacterial burden in the lungs of Mtb infected DM mice compared to non-DM infected mice (3.1 ± 0.4 x106 vs 0.9 ± 0.1 x 106, p=0.001) suggesting increased mortality was not due to increased bacterial burden. Real time PCR analysis of Mtb infected DM lungs indicated significantly increased pro- and anti-inflammatory cytokine gene expression compared to uninfected DM and infected control mice, including increased levels of IL-6 mRNA. Anti-IL-6 antibody treatment of Mtb infected DM mice enhanced the survival (100% vs 60% survival in isotype control antibody treated, p=0.02) and reduced pro- and anti-inflammatory cytokine production. Dendritic cells (DC), but not other immune cells were the major source for IL-6 in Mtb infected DM mice. NK cells of Mtb infected DM mice further enhanced IL-6 production by autologous DCs. Our results suggest NK-DC interaction enhance IL-6 production which drives the immune pathology and mortality of Mtb infected diabetic mice

Etanercept exacerbates inflammation and pathology in a rabbit model of active pulmonary tuberculosis.

Treatment of chronic inflammatory diseases with tumor necrosis factor alpha (TNF-α) antagonists has been associated with increased risk of tuberculosis (TB). We examined the usefulness of the rabbit model of active pulmonary TB for studying the impact of the human immune modulatory reagent etanercept on the host immune response. Control of Mycobacterium tuberculosis (Mtb) infection, disease pathology, and the global transcriptional response in Mtb-infected lungs of rabbits were studied. Etanercept treatment exacerbated disease pathology and reduced bacillary control in the lungs, compared with infected untreated animals. Reduced collagen and fibrin deposition in the granulomas was associated with significant downregulation of the collagen metabolism and fibrosis network genes and upregulation of genes in the inflammatory response and cell recruitment networks in the lungs of etanercept treated, compared with untreated rabbits. Our results suggest that targeting the TNF-α signaling pathway disrupts the tissue remodeling process, which is required for the formation and maintenance of well-differentiated granulomas and for control of Mtb growth in the lungs. These results validate the use of the rabbit model for investigating the impact of selected human immune modulatory drugs, such as a TNF-α antagonist, on the host immune response and pathogenesis in TB.

Human lung epithelial cells contain Mycobacterium tuberculosis in a late endosomal vacuole and are efficiently recognized by CD8⁺ T cells.

Mycobacterium tuberculosis (Mtb) is transmitted via inhalation of aerosolized particles. While alveolar macrophages are thought to play a central role in the acquisition and control of this infection, Mtb also has ample opportunity to interact with the airway epithelium. In this regard, we have recently shown that the upper airways are enriched with a population of non-classical, MR1-restricted, Mtb-reactive CD8+ T cells (MAIT cells). Additionally, we have demonstrated that Mtb-infected epithelial cells lining the upper airways are capable of stimulating IFNγ production by MAIT cells. In this study, we demonstrate that airway epithelial cells efficiently stimulate IFNγ release by MAIT cells as well as HLA-B45 and HLA-E restricted T cell clones. Characterization of the intracellular localization of Mtb in epithelial cells indicates that the vacuole occupied by Mtb in epithelial cells is distinct from DC in that it acquires Rab7 molecules and does not retain markers of early endosomes such as Rab5. The Mtb vacuole is also heterogeneous as there is a varying degree of association with Lamp1 and HLA-I. Although the Mtb vacuole shares markers associated with the late endosome, it does not acidify, and the bacteria are able to replicate within the cell. This work demonstrates that Mtb infected lung epithelial cells are surprisingly efficient at stimulating IFNγ release by CD8+ T cells.

CXCR5-CXCL-13-pathway in protective immunity to tuberculosis (P3190)

Abstract One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Although most infected people remain asymptomatic, they have a 10% lifetime risk of developing active tuberculosis (TB). Thus, the current challenge is to identify immune parameters that distinguish individuals with latent TB from those with active TB. Using human and experimental models of Mtb infection, we show that organized ectopic lymphoid structures containing CXCR5+ T cells are found in Mtb-infected lungs. In addition, we show that in experimental Mtb infection models, the presence of CXCR5+ T cells inside ectopic lymphoid structures are associated with immune control. Furthermore, in a mouse model of Mtb infection, we show that activated CD4+ CXCR5+ T cells accumulate in Mtb-infected lungs, and produce proinflammatory cytokines. Absence of CXCR5 results in increased susceptibility to TB due to defective T cell localization within the lung parenchyma. We show that CXCR5 expression on T cells mediates correct T cell localization within TB granulomas, efficient macrophage activation, promoting protection against Mtb infection and facilitating lymphoid follicle formation. We also show that inducing T helper 17 (Th17) memory responses using mucosal immunization strategies improves vaccine-induced immunity against TB highlighting the potential use of IL-17-CXCR5-CXCL-13 axis in future TB vaccine design and therapy.

CXCR5+ T helper cells mediate protective immunity against tuberculosis

One third of the world's population is infected with Mycobacterium tuberculosis (Mtb). Although most infected people remain asymptomatic, they have a 10% lifetime risk of developing active tuberculosis (TB). Thus, the current challenge is to identify immune parameters that distinguish individuals with latent TB from those with active TB. Using human and experimental models of Mtb infection, we demonstrated that organized ectopic lymphoid structures containing CXCR5+ T cells were present in Mtb-infected lungs. In addition, we found that in experimental Mtb infection models, the presence of CXCR5+ T cells within ectopic lymphoid structures was associated with immune control. Furthermore, in a mouse model of Mtb infection, we showed that activated CD4+CXCR5+ T cells accumulated in Mtb-infected lungs and produced proinflammatory cytokines. Mice deficient in Cxcr5 had increased susceptibility to TB due to defective T cell localization within the lung parenchyma. We demonstrated that CXCR5 expression in T cells mediated correct T cell localization within TB granulomas, promoted efficient macrophage activation, protected against Mtb infection, and facilitated lymphoid follicle formation. These data demonstrate that CD4+CXCR5+ T cells play a protective role in the immune response against TB and highlight their potential use for future TB vaccine design and therapy.

Increased (6 exon) interleukin-7 production after M. tuberculosis infection and soluble interleukin-7 receptor expression in lung tissue

Interleukin-7 (IL-7) and the IL-7 receptor (IL-7R) have been shown to be alternatively spliced in infectious diseases. We tested IL-7 and IL-7R splicing in a tuberculosis (TB)-vaccine/Mycobacterium tuberculosis (Mtb)-challenge model in non-human primates (NHPs). Differential IL-7 splicing was detected in peripheral blood mononuclear cells (PBMCs) from 15/15 NHPs showing 6 different IL-7 spliced isoforms. This pattern did not change after infection with virulent Mtb. We demonstrated increased IL-7 (6 exon) and IL-17 protein production in lung tissue along with concomitant decreased transforming growth factor-β (TGF-β) from NHPs (vaccinated with a recombinant BCG (rBCG)) who showed increased survival after Mtb challenge. IL-7 increased IL-17 and interferon-γ (IFN-γ) gene and protein expression in PBMCs. Mtb-infected NHPs showed differential IL-7R splicing associated with the anatomical location and tissue origin, that is, in lung tissue, hilus, axillary lymph nodes (LNs) and spleen. Differential splicing of the IL-7R was typical for healthy (non-Mtb infected) and for Mtb-infected lung tissue with a dominant expression of soluble IL-7R (sIL-7R) receptor lacking exon 6 (9:1 ratio of sIL-7R/cell-bound IL-7R). Differential ratios of cell-bound vs sIL-7R could be observed in hilus and axillary LNs from Mtb-infected NHPs with an inversed ratio of 1:9 (sIL-7R/cell-bound IL-7R) in spleen and PBMCs. Soluble IL-7R is exclusively present in lung tissue.

IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung. (150.1)

Abstract IL-23 is required for effective IL-17 responses to aerosol infection with Mycobacterium tuberculosis (Mtb), however it does not appear to impact early control of bacterial growth in the lung. By extending the time course of infection using mice deficient for the p19 component of the IL-23 cytokine, we report that there is increased bacterial growth in the lungs of these mice compared to wild type infected mice. This increased bacterial growth is associated with decreased maintenance of B cell follicles within lungs of infected mice. While mRNA expression for molecules associated with limiting bacterial growth such as Interferon gamma (IFNγ) were equivalent between wild type and IL-23p19 deficient mice, reduced expression of both IL-17 and IL-22 mRNA was observed in IL-23p19 deficient Mtb-infected lungs. Importantly, we show that in mice deficient for IL-23p19, B cell follicle size was reduced during chronic Mtb infection and this was temporally associated with the increased bacterial burden. In mice deficient in the IL-17 receptor (IL-17R), B cell follicle generation was affected early in infection whereas the absence of IL-22 influenced the size of B cell follicles in the mid-term. Interestingly, the absence of IL-22 or IL-17R alone did not impact bacterial control. Thus, we show novel data that while IL-23 is required for long term control of Mtb growth in the lung, there is a compensatory effect of IL-22 and IL-17 during tuberculosis.

Human Mucosal Associated Invariant T Cells Detect Bacterially Infected Cells

Author SummaryAbout one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb), yet thanks to a robust immune response most infected people remain healthy. CD8 T cells are unique in detecting intracellular infections. Surprisingly, Mtb-reactive CD8 T cells are found in humans with no prior exposure to Mtb. We show that mucosal associated invariant T (MAIT) cells, which have no previously known in vivo function, make up a proportion of these Mtb-reactive CD8 T cells and detect Mtb-infected cells via a specific major histocompatibility molecule called MHC-related molecule 1, which is evolutionarily conserved among mammals. Mtb-reactive MAIT cells are enriched in lung and detect primary Mtb-infected lung epithelial cells from the airway where initial exposure to Mtb occurs. We go on to show that MAIT cells are not specific for Mtb since they can detect cells infected with a variety of other bacteria. Curiously, Mtb-reactive MAIT cells are absent in the blood of individuals with active tuberculosis. We postulate that MAIT cells are innate detectors of bacterial infection poised to play a role in control of intracellular infection.

Suppressed induction of mycobacterial antigen-specific Th1-type CD4+ T cells in the lung after pulmonary mycobacterial infection

Although the importance of T(h)1-type immune response in protection against mycobacterial infection is well recognized, its regulatory mechanism in the Mycobacterium tuberculosis (Mtb)-infected lung is not well characterized. To address this issue, we analyzed kinetics of induction of mycobacterial antigen-specific CD4(+) T(h)1 T cells after mycobacterial infection in P25 TCR-transgenic (Tg) mice which express TCR alpha and beta chains from a mycobacterial Ag85B-specific MHC class II A(b)-restricted CD4(+) T-cell clone. To supply normal regulatory T-cell repertoire, we transferred normal spleen T cells into the P25 TCR-Tg mice before infection. High dose subcutaneous infection with Mtb or Mycobacterium bovis bacillus Calmette-Guérin (BCG) induced P25 TCR-Tg CD4(+) T(h)1 cells within a week. In contrast, high-dose Mtb or BCG infection into the lung failed to induce P25 TCR-Tg CD4(+) T(h)1 cells at the early stage of the infection. Furthermore, low-dose Mtb infection into the lung induced P25 TCR-Tg CD4(+) T(h)1 cells on day 21 in the mediastinal lymph node but not in the lung. IL-10 was partially involved in the suppression of T(h)1 induction in the lung because pretreatment of mice with anti-IL-10 antibody resulted in increase of P25 TCR-Tg CD4(+) T(h)1 cells in the Mtb-infected lung on day 21 of the infection, whereas neutralization of transforming growth factor-beta, another important suppressive cytokine in the lung, showed no effects on the T(h)1 induction. Our data suggest that induction of anti-mycobacterial CD4(+) T(h)1 cells is suppressed in the mycobacteria-infected lung partially by IL-10.

Mycobacterium tuberculosis-Specific CD8+ T Cells Require Perforin to Kill Target Cells and Provide Protection In Vivo

Optimal immunity to Mycobacterium tuberculosis (Mtb) infection requires CD8(+) T cells, and several current Mtb vaccine candidates are being engineered to elicit enhanced CD8(+) T cell responses. However, the function of these T cells and the mechanism by which they provide protection is still unknown. We have previously shown that CD8(+) T cells specific for the mycobacterial Ags CFP10 and TB10.4 accumulate in the lungs of mice following Mtb infection and have cytolytic activity in vivo. In this study, we determine which cytolytic pathways are used by these CD8(+) T cells during Mtb infection. We find that Mtb-specific CD8(+) T cells lacking perforin have reduced cytolytic capacity in vivo. In the absence of perforin, the residual cytolytic activity is CD95 and TNFR dependent. This is particularly true in Mtb-infected lung tissue where disruption of both perforin and CD95 eliminates target cell lysis. Moreover, adoptive transfer of immune CD8(+) T cells isolated from wild-type, but not perforin-deficient mice, protect recipient mice from Mtb infection. We conclude that CD8(+) T cells elicited following Mtb infection use several cytolytic pathways in a hierarchical and compensatory manner dominated by perforin-mediated cytolysis. Finally, although several cytolytic pathways are available, adoptively transferred Mtb-specific CD8(+) T cells require perforin-mediated cytolysis to protect animals from infection. These data show that CD8(+) T cell-mediated protection during Mtb infection requires more than the secretion of IFN-gamma and specifically defines the CD8(+) cytolytic mechanisms utilized and required in vivo.

Mycobacterium tuberculosis-induced apoptotic neutrophils trigger a pro-inflammatory response in macrophages through release of heat shock protein 72, acting in synergy with the bacteria

Mycobacterium tuberculosis (Mtb) survive inside macrophages by manipulating microbicidal functions such as phago-lysosome fusion, production of reactive oxygen species and nitric oxide, and by rendering macrophages non-responsive to IFN-γ. Mtb-infected lung tissue does however not only contain macrophages, but also significant numbers of infiltrating polymorphonuclear neutrophils (PMN). These are able to phagocytose and kill ingested Mtb, but are short-lived cells that constantly need to be removed from tissues to avoid tissue damage. Phagocytosis of aged or UV-induced apoptotic PMN by macrophages induce an anti-inflammatory response in macrophages. However, in the present study, we show that engulfment of Mtb-induced apoptotic PMN by macrophages initiates secretion of TNF-α from the macrophages, reflecting a pro-inflammatory response. Moreover, Mtb-induced apoptotic PMN up-regulate heat shock proteins 60 and 72 (Hsp60, Hsp72) intracellularly and also release Hsp72 extracellularly. We found that both recombinant Hsp72 and released Hsp72 enhanced the pro-inflammatory response to both Mtb-induced apoptotic PMN and Mtb. This stimulatory effect of the supernatant was abrogated by depleting the Hsp72 with immunoprecipitation. These findings indicate that released Hsp72 from Mtb-infected PMN can trigger macrophage activation during the early stage of Mtb infections, thereby creating a link between innate and adaptive immunity.

Expansion and function of Foxp3-expressing T regulatory cells during tuberculosis

Mycobacterium tuberculosis (Mtb) frequently establishes persistent infections that may be facilitated by mechanisms that dampen immunity. T regulatory (T reg) cells, a subset of CD4+ T cells that are essential for preventing autoimmunity, can also suppress antimicrobial immune responses. We use Foxp3-GFP mice to track the activity of T reg cells after aerosol infection with Mtb. We report that during tuberculosis, T reg cells proliferate in the pulmonary lymph nodes (pLNs), change their cell surface phenotype, and accumulate in the pLNs and lung at a rate parallel to the accumulation of effector T cells. In the Mtb-infected lung, T reg cells accumulate in high numbers in all sites where CD4+ T cells are found, including perivascular/peribronchiolar regions and within lymphoid aggregates of granulomas. To determine the role of T reg cells in the immune response to tuberculosis, we generated mixed bone marrow chimeric mice in which all cells capable of expressing Foxp3 expressed Thy1.1. When T reg cells were depleted by administration of anti-Thy1.1 before aerosol infection with Mtb, we observed ∼1 log less of colony-forming units of Mtb in the lungs. Thus, after aerosol infection, T reg cells proliferate and accumulate at sites of infection, and have the capacity to suppress immune responses that contribute to the control of Mtb.

Mycobacterium tuberculosis Up-Regulates Matrix Metalloproteinase-1 Secretion from Human Airway Epithelial Cells via a p38 MAPK Switch

Pulmonary cavitation is vital to the persistence and spread of Mycobacterium tuberculosis (MTb), but mechanisms underlying this lung destruction are poorly understood. Fibrillar type I collagen provides the lung's tensile strength, and only matrix metalloproteinases (MMPs) can degrade it at neutral pH. We investigated MTb-infected lung tissue and found that airway epithelial cells adjacent to tuberculosis (Tb) granulomas expressed a high level of MMP-1 (interstitial collagenase). Conditioned media from MTb-infected monocytes (CoMTb) up-regulated epithelial cell MMP-1 promoter activity, gene expression, and secretion, whereas direct MTb infection did not. CoMTb concurrently suppressed tissue inhibitor of metalloprotease-1 (TIMP-1) secretion, further promoting matrix degradation, and in Tb patients very low TIMP-1 expression was detected. MMP-1 up-regulation required synergy between TNF-alpha and G protein-coupled receptor signaling pathways. CoMTb stimulated p38 MAPK phosphorylation, and this is the point of TNF-alpha synergy with G protein-coupled receptor activation. Furthermore, p38 phosphorylation was the switch up-regulating MMP-1 activity and decreasing TIMP-1 secretion. Activated p38 localized to MMP-1-secreting airway epithelial cells in Tb patients. These data reveal a monocyte-epithelial cell network whereby MTb may drive tissue destruction, and they demonstrate that p38 phosphorylation is a key regulatory point in the generation of a matrix-degrading phenotype.

Induction of M3-restricted cytotoxic T lymphocyte responses by N-formylated peptides derived from Mycobacterium tuberculosis.

Major histocompatibility complex (MHC) class I-restricted CD8(+) T cells play a critical role in the protective immunity against Mycobacterium tuberculosis (Mtb). However, only a few Mtb peptides recognized by MHC class Ia-restricted CD8(+) T cells have been identified. Information on epitopes recognized by class Ib-restricted T cells is even more limited. M3 is an MHC class Ib molecule that preferentially presents N-formylated peptides to CD8(+) T cells. Because bacteria initiate protein synthesis with N-formyl methionine, the unique binding specificity of M3 makes it especially suitable for presenting these particular bacterial epitopes. We have scanned the full sequence of the Mtb genome for NH2-terminal peptides that share features with other M3-binding peptides. Synthetic peptides corresponding to these sequences were tested for their ability to bind to M3 in an immunofluorescence-based peptide-binding assay. Four of the N-formylated Mtb peptides were able to elicit cytotoxic T lymphocytes (CTLs) from mice immunized with peptide-coated splenocytes. The Mtb peptide-specific, M3-restricted CTLs lysed the Mtb-infected macrophages effectively, suggesting that these N-formylated Mtb peptides are presented as the naturally processed epitopes by Mtb-infected cells. Furthermore, T cells from Mtb-infected lungs, spleen, and lymph nodes responded to N-formylated Mtb peptides in an M3-restricted manner. Taken together, our data suggest that M3-restricted T cells may participate in the immune response to Mtb.