Elevated Cyclic AMP Inhibits Mycobacterium tuberculosis-Stimulated T-cell IFN-γ Secretion Through Type I Protein Kinase A.

Cyclic adenosine monophosphate (cAMP) is critical in immune regulation, and its role in tuberculosis infection remains unclear. We determined the levels of cAMP in peripheral blood mononuclear cells (PBMC) from tuberculosis patients and the mechanisms for cAMP suppression of IFN-γ production. PBMC from tuberculosis patients contained significantly elevated cAMP than latent tuberculosis infected subjects (LTBI), with an inverse correlation with IFN-γ production. Consistent with this, the expression of cAMP response element binding protein (CREB), activating transcription factor (ATF)-2 and c-Jun were reduced in tuberculosis patients compared with LTBI. PKA type I specific cAMP analogs inhibited Mtb-stimulated IFN-g production by PBMC through suppression of Mtb-induced IFN-γ promoter binding activities of CREB, ATF-2, and c-Jun and also miR155, the target miRNA of these transcription factors. Neutralizing both IL-10 and TGF-ß1 or supplementation of IL-12 restored cAMP-suppressed IFN-g production. We conclude that increased cAMP inhibits IFN-g production through PKA type I pathway in tuberculosis infection.

A rho GDP dissociation inhibitor produced by apoptotic T-cells inhibits growth of Mycobacterium tuberculosis.

In this study, we found that a subpopulation of CD4(+)CD25(+) (85% Foxp3(+)) cells from persons with latent tuberculosis infection (LTBI) inhibits growth of M. tuberculosis (M. tb) in human monocyte-derived macrophages (MDMs). A soluble factor, Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic CD4(+)CD25(+) (85% Foxp3(+)) cells is responsible for this inhibition of M. tb growth in human macrophages and in mice. M. tb-expanded CD4(+C)D25(+)Foxp3(+)D4GDI(+) cells do not produce IL-10, TGF-ß and IFN-γ. D4GDI inhibited growth of M. tb in MDMs by enhancing production of IL-1ß, TNF-α and ROS, and by increasing apoptosis of M. tb-infected MDMs. D4GDI was concentrated at the site of disease in tuberculosis patients, with higher levels detected in pleural fluid than in serum. However, in response to M. tb, PBMC from tuberculosis patients produced less D4GDI than PBMC from persons with LTBI. M. tb-expanded CD4+CD25+ (85% Foxp3(+)) cells and D4GDI induced intracellular M. tb to express the dormancy survival regulator DosR and DosR-dependent genes, suggesting that D4GDI induces a non-replicating state in the pathogen. Our study provides the first evidence that a subpopulation of CD4(+)CD25(+) (85% Foxp3+) cells enhances immunity to M. tb, and that production of D4GDI by this subpopulation inhibits M. tb growth.

Phosphorylated STAT3 and PD-1 regulate IL-17 production and IL-23 receptor expression in Mycobacterium tuberculosis infection.

We studied the factors that regulate IL-23 receptor expression and IL-17 production in human tuberculosis infection. Mycobacterium tuberculosis (M. tb)-stimulated CD4(+) T cells from tuberculosis patients secreted less IL-17 than did CD4(+) T cells from healthy tuberculin reactors (PPD(+) ). M. tb-cultured monocytes from tuberculosis patients and PPD(+) donors expressed equal amounts of IL-23p19 mRNA and protein, suggesting that reduced IL-23 production is not responsible for decreased IL-17 production by tuberculosis patients. Freshly isolated and M. tb-stimulated CD4(+) T cells from tuberculosis patients had reduced IL-23 receptor and phosphorylated STAT3 (pSTAT3) expression, compared with cells from PPD(+) donors. STAT3 siRNA reduced IL-23 receptor expression and IL-17 production by CD4(+) T cells from PPD(+) donors. Tuberculosis patients had increased numbers of PD-1(+) T cells compared with healthy PPD(+) individuals. Anti-PD-1 antibody enhanced pSTAT3 and IL-23R expression and IL-17 production by M. tb-cultured CD4(+) T cells of tuberculosis patients. Anti-tuberculosis therapy decreased PD-1 expression, increased IL-17 and IFN-γ production and pSTAT3 and IL-23R expression. These findings demonstrate that increased PD-1 expression and decreased pSTAT3 expression reduce IL-23 receptor expression and IL-17 production by CD4(+) T cells of tuberculosis patients.

Protecting against post-influenza bacterial pneumonia by increasing phagocyte recruitment and ROS production.

Seasonal and especially pandemic influenza predispose patients to secondary bacterial pneumonias, which are a major cause of deaths and morbidity. Staphylococcus aureus is a particularly common and deadly form of post-influenza pneumonia, and increasing staphylococcal drug resistance makes the development of new therapies urgent. We explored an innate immune-mediated model of the lung to define novel mechanisms by which the host can be protected against secondary staphylococcal pneumonia after sub-lethal influenza infection. We found that stimulating the innate immunity in the lung by overexpression of GM-CSF will result in resistance to S. aureus pneumonia after sublethal influenza infection. Resistance was mediated by alveolar macrophages and neutrophils, and was associated with increased production of reactive oxygen species (ROS) by alveolar macrophages. Resistance was abrogated by treatment with agents that scavenged ROS. We conclude that stimulating innate immunity in the lung markedly reduces susceptibility to post-influenza staphylococcal pneumonia and that this may represent a novel immunomodulatory strategy for prevention and treatment of secondary bacterial pneumonia after influenza.

Interleukin 22 inhibits intracellular growth of Mycobacterium tuberculosis by enhancing calgranulin A expression.

Previously, we found that interleukin 22 (IL-22) inhibits intracellular growth of Mycobacterium tuberculosis in human monocyte-derived macrophages (MDMs). In the current study, we determined the mechanisms underlying these effects. We found that W7, a phagolysosomal fusion inhibitor, abrogates IL-22-dependent M. tuberculosis growth inhibition in MDMs, suggesting that IL-22 acts through enhanced phagolysosomal fusion. Our microarray analysis indicated that recombinant IL-22 (rIL-22) enhances the expression of an intracellular signaling molecule, calgranulin A. This was confirmed by real-time polymerase chain reaction, Western blot, and confocal microscopy. Calgranulin A small interfering RNA (siRNA) abrogated rIL-22-dependent growth inhibition of M. tuberculosis in MDMs. IL-22 enhanced Rab7 expression and downregulated Rab14 expression of M. tuberculosis-infected MDMs, and these effects were reversed by calgranulin A siRNA. These results suggest that M. tuberculosis growth inhibition by IL-22 depends on calgranulin A and enhanced phagolysosomal fusion, which is associated with increased Rab7 and reduced Rab14 expression.

Phosphorylation of mitogen-activated protein kinases contributes to interferon γ production in response to Mycobacterium tuberculosis.

Immune control of Mycobacterium tuberculosis depends on interferon γ (IFN-γ)-producing CD4(+) lymphocytes. Previous studies have shown that T cells from patients with tuberculosis produce less IFN-γ, compared with healthy donors, in response to mycobacterial antigens, although IFN-γ responses to mitogens are preserved. In this work, we found that M. tuberculosis-induced IFN-γ production by human T cells correlated with phosphorylation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38. Moreover, the majority of IFN-γ-producing T cells expressed signaling lymphocyte activation molecule (SLAM), and SLAM activation further increased ERK phosphorylation. Interestingly, patients with tuberculosis had delayed activation of ERK and p38, and this was most marked in patients with the poorest IFN-γ responses (ie, low responders). Besides, SLAM signaling failed to phosphorylate ERK in low responders. Our findings suggest that activation of p38 and ERK, in part through SLAM, mediates T-cell IFN-γ production in response to M. tuberculosis, a pathway that is defective in patients with tuberculosis.

Early secreted antigenic target of 6-kDa protein of Mycobacterium tuberculosis primes dendritic cells to stimulate Th17 and inhibit Th1 immune responses.

Early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis is a T cell Ag that is a potential vaccine candidate, but it is also a virulence factor that mediates pathogenicity. To better understand the effects of ESAT-6 on the immune response, we studied the effect of ESAT-6 on human dendritic cells (DCs). Peripheral blood monocytes were treated with GM-CSF and IL-4 to yield immature DCs, which were matured by addition of LPS and CD40 ligand (CD40L), with or without ESAT-6. ESAT-6 inhibited LPS/CD40L-induced DC expression of costimulatory molecules, reduced DC-stimulated allogeneic T cell proliferation and IL-2 and IFN-γ production, and enhanced IL-17 production. ESAT-6-treated DCs also increased IL-17 and reduced IFN-γ production by M. tuberculosis-specific autologous T cells. ESAT-6 inhibited LPS/CD40L-induced DC production of IL-12 and enhanced that of IL-23 and IL-1ß, without affecting secretion of TNF-α, IL-6, or IL-8 through specific interaction with immature DCs. The effects of ESAT-6 were not mediated through cAMP or p38 MAPK. Medium from ESAT-6-conditioned DCs increased IL-17 and reduced IFN-γ production by T cells stimulated with anti-CD3 plus anti-CD28, and ESAT-6-induced IL-17 production was blocked by neutralizing both IL-23 and IL-1ß. ESAT-6 reduced LPS/CD40L-stimulated transcription of IL-12p35 and enhanced that of IL-23p19 through inhibition of IFN regulatory factor-1 and upregulation of activating transcription factor-2 and c-Jun, transcriptional regulators of IL-12p35 and IL-23p19, respectively. We conclude that ESAT-6 increases DC production of IL-23 and IL-1ß while inhibiting that of IL-12, thus enhancing Th17 at the expense of protective Th1 responses.

NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis.

We previously found that human NK cells lyse Mycobacterium tuberculosis-infected monocytes and alveolar macrophages and upregulate CD8(+) T cell responses. We also found that human NK cells produce IL-22, which inhibits intracellular growth of M. tuberculosis, and that NK cells lyse M. tuberculosis-expanded CD4(+)CD25(+)FOXP3(+) T regulatory cells (Tregs). To determine the role of NK cells during the protective immune response to vaccination in vivo, we studied the NK cell and T cell responses in a mouse model of vaccination with bacillus Calmette-Guérin (BCG), followed by challenge with virulent M. tuberculosis H37Rv. BCG vaccination enhanced the number of IFN-γ-producing and IL-22-producing NK cells. Depletion of NK1.1(+) cells at the time of BCG vaccination increased the number of immunosuppressive Tregs (CD4(+)CD25(hi), 95% Foxp3(+)) after challenge with M. tuberculosis H37Rv, and NK1.1(+) cells lysed expanded but not natural Tregs in BCG-vaccinated mice. Depletion of NK1.1(+) cells at the time of BCG vaccination also increased the bacillary burden and reduced T cell responses after challenge with M. tuberculosis H37Rv. IL-22 at the time of vaccination reversed these effects and enhanced Ag-specific CD4(+) cell responses in BCG-vaccinated mice after challenge with M. tuberculosis H37Rv. Our study provides evidence that NK1.1(+) cells and IL-22 contribute to the efficacy of vaccination against microbial challenge.

Immune regulatory activities of early secreted antigenic target of 6-kD protein of Mycobacterium tuberculosis and implications for tuberculosis vaccine design.

Although ESAT-6 was originally identified as a strong T cell immunogen in short-term culture filtrate of Mtb, and has therefore been a candidate vaccine antigen for many years, recent work has demonstrated that ESAT-6 is also a virulence factor that mediates pathogenicity of Mtb. The studies described in this review suggest that ESAT-6 secreted by Mtb subverts host immunity by manipulating intracellular signaling pathways in macrophages and T cells, which are critical in protection against Mtb. Furthermore, ESAT-6 elicits pro-inflammatory responses that can be detrimental to the host. Understanding the molecular mechanisms through which ESAT-6 inhibits immunity will permit design of ESAT-6-based vaccine constructs that elicit protective immune responses with minimal negative effects.

The Mycobacterium tuberculosis early secreted antigenic target of 6 kDa inhibits T cell interferon-γ production through the p38 mitogen-activated protein kinase pathway.

We reported previously that the early secreted antigenic target of 6 kDa (ESAT-6) from Mycobacterium tuberculosis directly inhibits human T cell IFN-γ production and proliferation in response to stimulation with anti-CD3 and anti-CD28. To determine the mechanism of this effect, we treated T cells with kinase inhibitors before stimulation with ESAT-6. Only the p38 MAPK inhibitor, SB203580, abrogated ESAT-6-mediated inhibition of IFN-γ production in a dose-dependent manner. SB203580 did not reverse ESAT-6-mediated inhibition of IL-17 and IL-10 production, suggesting a specific effect of SB203580 on IFN-γ production. SB203580 did not act through inhibition of AKT (PKB) as an AKT inhibitor did not affect ESAT-6 inhibition of T cell IFN-γ production and proliferation. ESAT-6 did not reduce IFN-γ production by expanding FoxP3(+) T regulatory cells. Incubation of T cells with ESAT-6 induced phosphorylation and increased functional p38 MAPK activity, but not activation of ERK or JNK. Incubation of peripheral blood mononuclear cells with ESAT-6 induced activation of p38 MAPK, and inhibition of p38 MAPK with SB203580 reversed ESAT-6 inhibition of M. tuberculosis-stimulated IFN-γ production by peripheral blood mononuclear cells from subjects with latent tuberculosis infection. Silencing of p38α MAPK with siRNA rendered T cells resistant to ESAT-6 inhibition of IFN-γ production. Taken together, our results demonstrate that ESAT-6 inhibits T cell IFN-γ production in a p38 MAPK-dependent manner.

GM-CSF in the lung protects against lethal influenza infection.

RATIONALE: Alveolar macrophages contribute to host defenses against influenza in animal models. Enhancing alveolar macrophage function may contribute to protection against influenza. OBJECTIVES: To determine if increased expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the lung increases resistance to influenza. METHODS: Wild-type mice and transgenic mice that expressed GM-CSF in the lung were infected with influenza virus, and lung pathology, weight loss, and mortality were measured. We also administered GM-CSF to the lungs of wild-type mice that were infected with influenza virus. MEASUREMENTS AND MAIN RESULTS: Wild-type mice all died after infection with different strains of influenza virus, but all transgenic mice expressing GM-CSF in the lungs survived. The latter also had greatly reduced weight loss and lung injury, and showed histologic evidence of a rapid host inflammatory response that controlled infection. The resistance of transgenic mice to influenza was abrogated by elimination of alveolar phagocytes, but not by depletion of T cells, B cells, or neutrophils. Transgenic mice had far more alveolar macrophages than did wild-type mice, and they were more resistant to influenza-induced apoptosis. Delivery of intranasal GM-CSF to wild-type mice also conferred resistance to influenza. CONCLUSIONS: GM-CSF confers resistance to influenza by enhancing innate immune mechanisms that depend on alveolar macrophages. Pulmonary delivery of this cytokine has the potential to reduce the morbidity and mortality due to influenza virus.

Programmed death 1 and cytokine inducible SH2-containing protein dependent expansion of regulatory T cells upon stimulation With Mycobacterium tuberculosis.

We previously found that CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) expand in response to Mycobacterium tuberculosis infection in individuals who are healthy tuberculin reactors, but not in tuberculin-negative individuals. We also found that the M. tuberculosis mannose-capped lipoarabinomannan and prostaglandin E2 produced by monocytes are involved in Treg expansion. In this study, we found that Tregs expanded from CD4(+)CCR4(+) cells but not from CCR4(-) cells. However, introduction of CCR4 small interfering RNA (siRNA) into CD4(+) cells only marginally reduced expansion of Tregs. Using siRNA and neutralizing antibodies, we found that expansion of Tregs by M. tuberculosis required expression of programmed death1 (PD-1) and expression of the signaling molecule, cytokine inducible SH2-containing protein (CISH). Anti-PD-1 siRNA inhibited expression of CISH by expanded Tregs. M. tuberculosis-expanded Tregs produced transforming growth factor ß and interleukin 10 and reduced the frequency of interferon γ-producing autologous CD8(+) cells. We conclude that M. tuberculosis infection induces development of Tregs from CCR4(+) cells through a process that depends on PD-1and CISH.

c-Maf-dependent growth of Mycobacterium tuberculosis in a CD14(hi) subpopulation of monocyte-derived macrophages.

Macrophages are a major component of the innate immune response, comprising the first line of defense against various intracellular pathogens, including Mycobacterium tuberculosis. In this report, we studied the factors that regulate growth of M. tuberculosis H37Rv in subpopulations of human monocyte-derived macrophages (MDMs). In healthy donors, M. tuberculosis H37Rv grew 5.6-fold more rapidly in CD14(hi) MDMs compared with that in CD14(lo)CD16(+) MDMs. Compared with CD14(lo)CD16(+) cells, M. tuberculosis H37Rv-stimulated CD14(hi) monocytes produced more IL-10 and had increased mRNA expression for c-Maf, a transcription factor that upregulates IL-10 gene expression. c-Maf small interfering RNA (siRNA) inhibited IL-10 production and growth of M. tuberculosis in CD14(hi) cells. Compared with CD14(lo)CD16(+) monocytes, M. tuberculosis H37Rv-stimulated CD14(hi) cells had increased expression of 22 genes whose promoters contained a c-Maf binding site, including hyaluronan synthase 1 (HAS1). c-Maf siRNA inhibited HAS1 expression in M. tuberculosis-stimulated CD14(hi) monocytes, and HAS1 siRNA inhibited growth of M. tuberculosis in CD14(hi) MDMs. M. tuberculosis H37Rv upregulated expression of HAS1 protein and its product, hyaluronan, in CD14(hi) MDMs. We conclude that M. tuberculosis grows more rapidly in CD14(hi) than in CD14(lo)CD16(+) MDMs because CD14(hi) cells have increased expression of c-Maf, which increases production of two key factors (hyaluronan and IL-10) that promote growth of M. tuberculosis.

Exposure to cigarette smoke inhibits the pulmonary T-cell response to influenza virus and Mycobacterium tuberculosis.

Smoking is associated with increased susceptibility to tuberculosis and influenza. However, little information is available on the mechanisms underlying this increased susceptibility. Mice were left unexposed or were exposed to cigarette smoke and then infected with Mycobacterium tuberculosis by aerosol or influenza A by intranasal infection. Some mice were given a DNA vaccine encoding an immunogenic M. tuberculosis protein. Gamma interferon (IFN-γ) production by T cells from the lungs and spleens was measured. Cigarette smoke exposure inhibited the lung T-cell production of IFN-γ during stimulation in vitro with anti-CD3, after vaccination with a construct expressing an immunogenic mycobacterial protein, and during infection with M. tuberculosis and influenza A virus in vivo. Reduced IFN-γ production was mediated through the decreased phosphorylation of transcription factors that positively regulate IFN-γ expression. Cigarette smoke exposure increased the bacterial burden in mice infected with M. tuberculosis and increased weight loss and mortality in mice infected with influenza virus. This study provides the first demonstration that cigarette smoke exposure directly inhibits the pulmonary T-cell response to M. tuberculosis and influenza virus in a physiologically relevant animal model, increasing susceptibility to both pathogens.

Postnatal tissue-specific disruption of transcription factor FoxN1 triggers acute thymic atrophy.

The transcription factor FoxN1 is essential for differentiation of thymic epithelial cell (TEC) progenitors during thymic organogenesis. However, limited information is available on the postnatal contribution of FoxN1 to thymic maintenance. To address this question, we generated a loxP-floxed FoxN1 (fx) mouse with three different promoter-driven inducible CreER(T) transgenes. Postnatal ubiquitous deletion of FoxN1 caused dramatic thymic atrophy in 5 days and more severe deterioration in medullary TECs (mTECs) than in cortical TECs (cTECs). Induction of FoxN1 deletion selectively in K5 promoter-driven somatic epithelial cells (mostly mTECs and possibly some adult epithelial stem cells) was sufficient to cause significant thymic atrophy, whereas FoxN1 deletion in K18 promoter-driven somatic epithelial cells (mostly cTECs) was not. Thymic atrophy resulted from increased apoptosis and was associated with activation of the p53 gene in mature mTECs. Although FoxN1 is required for the development of both mTECs and cTECs in thymic organogenesis, it is most important for the maintenance of mTECs in the postnatal thymus, which are in turn necessary to prevent thymic atrophy.

Progress in understanding the human immune responses to Mycobacterium tuberculosis.

Development of an effective vaccine against tuberculosis hinges on an improved understanding of the human immune response to Mycobacterium tuberculosis. Work in this area at the University of Texas Health Science Center at Tyler has led to advances in four areas: (1) natural killer cells contribute to innate immunity by lysing M. tuberculosis-infected mononuclear phagocytes, and to adaptive immunity by enhancing the CD8+ T-cell effector function and inhibiting expansion of T regulatory cells; (2) Interferon-gamma plays a central role in resistance to many intracellular pathogens, including M. tuberculosis, and we have identified three transcription factors that bind to the Interferon-gamma proximal promoter and increase Interferon-gamma transcription in live T-cells that are activated by M. tuberculosis antigens; (3) A DNA vaccine that encodes the M. tuberculosis 10fts;kDa culture filtrate protein and the lysosomal integral membrane protein-2 was produced to direct vaccine antigens to the MHC class II processing and presentation pathway. When this vaccine was coated with polyethylenimine and administered to mice, it yielded a remarkably potent pulmonary immune response that reduced the bacillary burden by 90% after M. tuberculosis challenge; (4) The early secreted antigenic target of 6fts;kDa (ESAT-6) is a putative vaccine antigen. We found that high concentrations of this antigen markedly inhibit Interferon-gamma production by T-cells and are working to understand the molecular mechanisms underlying this effect. Developing methods to enhance NK cell functions that favor protective immunity, increase interferon-gamma transcription, elicit protective pulmonary immune responses and prevent ESAT-6 from inhibiting T-cell function will contribute significantly to development of antituberculosis vaccines.

Mycobacterium tuberculosis ESX-1 system-secreted protein ESAT-6 but not CFP10 inhibits human T-cell immune responses.

The secreted proteins of M. tuberculosis, early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP10), have been identified as antigenic proteins with potent T-cell stimulatory effects, and therefore have been the focus of tuberculosis vaccine studies. However, recent work showed that secretion of these proteins by the specialized ESAT-6 secretion system (ESX)-1 of M. tuberculosis is associated with virulence and pathogenesis. The studies demonstrated that ESAT-6 inhibits antigen-presenting cell function by reducing IL-12 production by macrophages through interrupting TLR2 signaling pathways and inducing macrophage apoptosis. However, the effect of ESAT-6 on T cells remains unexplored. To address this question, we studied the effect of recombinant ESAT-6 and CFP10 on human primary T-cell IFN-gamma secretion and proliferation. ESAT-6, but not CFP10, inhibited IFN-gamma production by T cells stimulated with M. tuberculosis or with anti-CD3 plus anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T-cell production of IL-17 and TNF-a, but not IL-2. Presence of CFP10 as part of the ESAT-6/CFP10 heterodimer did not affect ESAT-6 inhibition of T-cell IFN-gamma production. ESAT-6 inhibited the proliferation of CD3+ cells in response to TCR stimulation. ESAT-6 decreased T-cell IFN-gamma secretion by mechanisms independent of cytotoxicity or apoptosis. ESAT-6 reduced IFN-gamma mRNA levels by inhibiting the expression of the transcription factors, ATF-2, c-Jun and CREB, which upregulate IFN-gamma gene expression in T cells through binding to the IFN-gamma proximal promoter. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing phosphorylation of ZAP70, a proximal TCR signaling molecule. We conclude that ESAT-6 directly inhibits human T-cell responses by affecting TCR signaling pathways downstream of ZAP70.

Innate and adaptive immune responses to human Mycobacterium tuberculosis infection.

Tuberculosis is a leading cause of death from infectious diseases world-wide, and multidrug-resistant (MDR) tuberculosis continues to spread in many parts of the world. MDR tuberculosis is a potential bioterrorist threat, as therapy is prolonged with potentially toxic agents, and the cure rate is much lower than that for treatment of drug-susceptible tuberculosis. Development of methods to enhance innate and adaptive defenses against M. tuberculosis are an attractive means to provide protection against both MDR and drug-susceptible tuberculosis. Before such strategies can be developed, an improved understanding must be gained of the immune response to M. tuberculosis. Our laboratory is mainly focused on understanding the mechanisms by which natural killer (NK) cells lyse M. tuberculosis-infected cells, determining the molecular mechanisms involved in the induction of regulatory T cells (Tregs), and characterizing the mechanisms by which NK cells affect expansion of Tregs in M. tuberculosis infection. As several studies demonstrated defective immune responses in tuberculosis patients, our studies will pinpoint the nature of this defective immune response and permit development of methods to reverse this defect. In the long run, these findings will permit development of novel methods to stimulate immunity against tuberculosis, a strategy that will contribute to development of an effective vaccine to prevent tuberculosis and novel immunotherapy to treat the disease.

IL-22 produced by human NK cells inhibits growth of Mycobacterium tuberculosis by enhancing phagolysosomal fusion.

We determined whether human NK cells could contribute to immune defenses against Mycobacterium tuberculosis through production of IL-22. CD3(-)CD56(+) NK cells produced IL-22 when exposed to autologous monocytes and gamma-irradiated M. tuberculosis, and this depended on the presence of IL-15 and IL-23, but not IL-12 or IL-18. IL-15-stimulated NK cells expressed 10.6 times more DAP10 mRNA compared with control NK cells, and DAP10 siRNA inhibited IL-15-mediated IL-22 production by NK cells. Soluble factors produced by IL-15-activated NK cells inhibited growth of M. tuberculosis in macrophages, and this effect was reversed by anti-IL-22. Addition of rIL-22 to infected macrophages enhanced phagolysosomal fusion and reduced growth of M. tuberculosis. We conclude that NK cells can contribute to immune defenses against M. tuberculosis through production of IL-22, which inhibits intracellular mycobacterial growth by enhancing phagolysosomal fusion. IL-15 and DAP-10 elicit IL-22 production by NK cells in response to M. tuberculosis.

NKG2D-dependent IL-17 production by human T cells in response to an intracellular pathogen.

We studied the factors that control IL-17 production in human Mycobacterium tuberculosis infection. CD4(+) cells from healthy tuberculin reactors produced IL-17 in response to autologous M. tuberculosis-stimulated monocytes, and most IL-17(+) cells were Ag experienced, CD4(+)CD62L(-). IL-17 production by CD4(+) cells was inhibited by anti-IL-23, but not by Abs to IL-1, IL-6, or TGF-beta. Anti-NKG2D reduced IL-17 production and the frequency of CD4(+)CD62(-) IL-17(+) cells, suggesting that NKG2D stimulates IL-17 production. CD4(+)NKG2D(+) cells did not produce IL-17. Monocytes and alveolar macrophages from healthy donors produced IL-23 in response to M. tuberculosis. Addition of CD4(+) cells markedly enhanced IL-23 production by M. tuberculosis-stimulated monocytes, and this was inhibited by anti-NKG2D and by Abs to UL-16 binding protein (ULB)1, a ligand for NKG2D on APCs. We conclude that binding of NKG2D to UL-16 binding protein (ULB)1 contributes to IL-23-dependent IL-17 production by CD4(+) cells in human M. tuberculosis infection.