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In this study, we determined the role of IL-21R signaling in Mycobacterium tuberculosis infection, using IL-21R knockout (KO) mice. A total of 50% of M. tuberculosis H37Rv–infected IL-21R KO mice died in 6 mo compared with no deaths in infected wild type (WT) mice. M. tuberculosis–infected IL-21R KO mice had enhanced bacterial burden and reduced infiltration of Ag-specific T cells in lungs compared with M. tuberculosis–infected WT mice. Ag-specific T cells from the lungs of M. tuberculosis–infected IL-21R KO mice had increased expression of T cell inhibitory receptors, reduced expression of chemokine receptors, proliferated less, and produced less IFN- γ, compared with Ag-specific T cells from the lungs of M. tuberculosis–infected WT mice. T cells from M. tuberculosis–infected IL-21R KO mice were unable to induce optimal macrophage responses to M. tuberculosis. This may be due to a decrease in the Ag-specific T cell population. We also found that IL-21R signaling is associated with reduced expression of a transcriptional factor Eomesodermin and enhanced functional capacity of Ag-specific T cells of M. tuberculosis–infected mice. The sum of our findings suggests that IL-21R signaling is essential for the optimal control of M. tuberculosis infection.

Mycobacterium tuberculosis (Mtb) remains to be a leading cause of morbidity and mortality, causing an estimated 1.3 million deaths annually. It is known that intranasal Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccination in mice has proven to provide superior protection against pulmonary TB, as compared to parenterally administered BCG. In the current study, we determined the phenotype and function of lung-resident lymphocytes in intranasally BCG vaccinated mice. C57BL/6 mice were vaccinated intranasally with live-attenuated BCG and lung lymphocytes were isolated and stained for immunophenotyping via flow cytometry at 24, 48, and 72 hours post-vaccination. At 48 hours post-vaccination, we have observed an expansion of CD69+, CD103+, and CD69+CD103+ lymphocytes within the lung of vaccinated mice. Studies are underway to determine the phenotype and function of lung-resident lymphocyte subsets and their expansion capacity at three months after BCG vaccination and Mtb infection.

Alveolar macrophages (AMs) are the first cells encountered by TB pathogen and serve as the primary defense against Mycobacterium tuberculosis (Mtb) in the lungs. Studies have shown that liver macrophages (Kupffer cells; KCs) can control Mtb growth but, it is not clear how KCs completely eliminate mycobacterial infections. Thus, identification of these mechanisms will facilitate the development of immunomodulatory strategies to boost AM-mediated immunity to Mtb. In the current study, we compared Mtb growth in murine AMs, peritoneal (PMs), liver macrophages (Kupffer cells; KCs) and bone marrow-derived monocytes (BDMs). KCs restricted Mtb growth more efficiently than all other macrophages and monocytes despite equivalent infections. Differences in Mtb growth restriction were not due to differences in cytokine production, expression of Toll-like receptors 2 and 4, M1/M2 paradigm or apoptosis. We provide evidence that the enhanced autophagy efficiently restricts Mtb growth in KCs using flow cytometry, western blot, RT-PCR and confocal microscopy. A metabolomic comparison of Mtb-infected macrophages by liquid chromatography mass spectrometry indicated that ornithine (VIP=1.8) and imidazole (VIP=1.6) were two top-scoring metabolites found in Mtb-infected KCs and that acetylcholine was top-scoring in Mtb-infected AMs. Ornithine and imidazole inhibited Mtb growth in AMs by enhancing AMPK mediated autophagy whereas imidazole directly killed Mtb by reducing cytochrome P450 activity. Intranasal delivery of ornithine or imidazole or together restricted Mtb growth in Mtb-infected mouse lungs. Our study demonstrates that the metabolic differences in Mtb-infected AMs and KCs leads to differences in the restriction of Mtb growth.

Previously, we found that a Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic T regulatory cells inhibit Mycobacterium tuberculosis (Mtb) growth in human macrophages through reactive oxygen species (ROS) mediated IL-1β production. Mitochondria is an important source of ROS and critical for antibacterial properties. In the current study, we evaluated the effects of D4GDI on mitochondrial homeostasis and anti-microbial scheme during Mtb infection. Recombinant D4GDI (rD4GDI) treatment of Mtb infected human macrophages enhanced the accumulation of LC3B protein on the mitochondria and triggered mitophagy. rD4GDI treatment significantly enhanced the expression of a mitophagy receptor BCL2 and adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L). BNIP3L siRNA inhibited rD4GDI dependent accumulation of LC3B protein on the mitochondria and enhanced Mtb growth in human macrophages. Further we found that rD4GDI treatment of Mtb infected macrophages induces the expression of antimicrobial peptide phospholipase family protein phospholipase A2 group VII (PLA2G7). Further studies are underway to determine the interactions between BNIP3L, PLA2G7 and mitophagy in rD4GDI treated macrophages to inhibit Mtb growth in human macrophages.

Diabetes is a significant risk factor for the development of active tuberculosis. In this study, we used mouse model of Streptozotocin/Nicotinamide (STZ/NA) induced non-obese type 2 diabetes mellitus (T2DM) to determine the effect of prior BCG vaccination on survival and immune responses to Mycobacterium tuberculosis (Mtb) infection. We found that at 6–7 months post-Mtb infection, 90% of the Mtb-infected T2DM mice died, whereas only 50% of BCG-vaccinated T2DM-Mtb-infected mice died. Moreover, 40% of the PBS-treated uninfected T2DM mice and 30% of the uninfected BCG-vaccinated T2DM mice died, whereas all uninfected and infected nondiabetic mice survived. BCG vaccination was less effective in reducing the lung bacterial burden of Mtb infected T2DM mice compared to Mtb-infected non-diabetic mice, however it reduced immunopathology of lung tissues. Further, we found increased survival of BCG vaccinated Mtb infected T2DM mice is associated with 2-fold expansion of IL-13 producing CXCR3+ T-regulatory cells as measured by flow cytometry, qRT-PCR and confocal microscopy. We also found that prior BCG vaccination restored the immunosuppressive function of T-regulatory cells of Mtb-infected T2DM mice and reduced inflammation. IL-13 producing T-regulatory cells of BCG vaccinated Mtb-infected T2DM mice converted proinflammatory M1 macrophages (iNOS) to anti-inflammatory M2 macrophage (Arg1) phenotype to suppress the inflammation. In contrast, anti-IL-13R antibody inhibited the conversion of macrophages from M1 to the M2 phenotype and enhanced the inflammatory cytokines (IL-6 and TNF-α) production. Our findings suggest a novel role for BCG in preventing excessive inflammation and mortality in T2DM mice infected with Mtb.

Previously, we found experimentally induced type 2 diabetes mellitus (T2DM) mice are highly susceptible to Mycobacterium tuberculosis (Mtb) infection. In the present study, we determined whether prior BCG vaccination has any effect on the susceptibility of T2DM mice infected with Mtb. C57BL/6 mice were vaccinated with BCG or treated with PBS, three months later some mice were induced with T2DM and after another month fifty percent of all groups of mice were challenged with Mtb. All Mtb infected T2DM mice and 40% of uninfected T2DM mice died within 10 months. In contrast, only 40% of Mtb infected BCG vaccinated T2DM mice and 20% of uninfected BCG vaccinated T2DM mice died. Lung bacterial burden was significantly less in Mtb infected BCG vaccinated T2DM mice compared to PBS treated Mtb infected mice. Pro and anti-inflammatory cytokine levels were significantly high in the lungs of Mtb infected BCG vaccinated T2DM mice compared to all other groups of vaccinated and PBS treated mice infected with Mtb. Our findings suggest that prior BCG vaccination protects Mtb infected mice from T2DM induced pathogenesis. Studies are underway to determine the BCG induced mechanism/s those protect T2DM mice challenged with Mtb.

IL-22 play an important role in protective immune responses against bacterial infections including Mtb and maintains homeostasis by regulating excess inflammation. IL-22 can also regulate glucose homeostasis, suggesting IL-22 pathway as a novel target for therapeutic intervention. Previously, we developed a mouse model of type 2 diabetes mellitus (T2DM) and found that pathological immune response enhances mortality of Mycobacterium tuberculosis (Mtb) infected T2DM mice. In the current study, we determined the role of IL-22 during Mtb infection in T2DM mice. We found innate lymphoid 3 cells (ILC3) are the major source for IL-22 and IL- 22 production was significantly reduced in the lungs and serum of T2DM mice infected with Mtb. Recombinant IL- 22 or adoptive transfer of ILC3 cells prolonged the survival of Mtb infected T2DM mice. Recombinant IL-22 prevented neutrophil accumulation near alveoli, reduced the serum insulin level and improved the lipid metabolism. Recombinant IL-22 also prevented neutrophil-mediated epithelial cell damage by inhibiting elastase production by neutrophils. Further, we found serum IL-22 levels were significantly less in tuberculosis (TB) patients with T2DM compared to TB patients without T2DM. Our findings suggest that IL-22 produced by ILC3 cells is essential to inhibit excess inflammation, epithelial cell damage in T2DM mice infected with Mtb.

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+CD25hi, 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.

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 CD14hi MDMs compared with that in CD14loCD16+ MDMs. Compared with CD14loCD16+ cells, M. tuberculosis H37Rv-stimulated CD14hi 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 CD14hi cells. Compared with CD14loCD16+ monocytes, M. tuberculosis H37Rv-stimulated CD14hi 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 CD14hi monocytes, and HAS1 siRNA inhibited growth of M. tuberculosis in CD14hi MDMs. M. tuberculosis H37Rv upregulated expression of HAS1 protein and its product, hyaluronan, in CD14hi MDMs. We conclude that M. tuberculosis grows more rapidly in CD14hi than in CD14loCD16+ MDMs because CD14hi cells have increased expression of c-Maf, which increases production of two key factors (hyaluronan and IL-10) that promote growth of M. tuberculosis.

In the current study, we determined the effects of chronic alcohol consumption on the mortality of young and old mice and immune responses during Mycobacterium tuberculosis (Mtb) infection. Eighty percent of Mtb H37Rv infected alcohol-fed young mice died in five months compared to twenty-five percent in Mtb infected alcohol-fed old mice. There is no significant difference in lung bacterial burden of control and alcohol diet fed young and old mice. IFN-α levels were significantly higher in the lungs of Mtb infected alcohol-fed young mice and treatment with anti-IFNAR-1 antibody enhanced their survival. There are significantly higher numbers of CD11b+Ly6G+ neutrophils in the lungs of Mtb infected alcoholic young mice compared to Mtb infected alcohol-fed old mice and Mtb infected control diet-fed young mice. CD11b+Ly6G+ neutrophils are the major source of IFN-α in Mtb-infected alcohol-fed young mice and IFN-α enhanced the expression of RIP-1 and RIP-3 molecules, which are known to be involved in necroptosis. Alcohol-fed old Mtb infected mice and Mtb infected control diet-fed old and young mice expressed low level of IFN-α and RIP-1 and RIP-3 in their lungs. In response to Mtb stimulation, peripheral blood mononuclear cells (PBMC) from young healthy alcoholic individuals with latent tuberculosis infection (LTBI) produced significantly higher amounts of IFN-α compared to non-alcoholic young, alcoholic and non-alcoholic old healthy LTBI+ individuals. Our findings demonstrate that in Mtb infected young mice, alcohol enhances CD11b+Ly6G+ neutrophil infiltration in lungs and excess IFN-α production by neutrophils causes lung macrophage necroptosis and enhanced mortality.

Kupffer cells protect liver from bacterial infections. In the current study, we compared Mycobacterium tuberculosis (M.tb) growth and cytokine production by mice kupffer cells and alveolar macrophages. M.tb H37Rv infected kupffer cells and alveolar macrophages produced equal amounts of TNF-α, IL-6, IL-10 & IL-1β. In contrast, kupffer cells restricted M.tb growth better than alveolar macrophages (1 ± 0.346×106 CFU vs. 4 ± 0.916×106 CFU, p

Previously, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and found that T2DM mice are susceptible to Mycobacterium tuberculosis (Mtb) infection. We also found that alveolar macrophages from T2DM mice were more permissive to Mtb growth ex vivo compared to non-diabetic controls. In the current study, we determined the defective mechanisms that make T2DM mice alveolar macrophages more susceptible to Mtb infection. Mtb infected alveolar macrophages from T2DM mice produced more TNF-α (973.8 ± 13.3 pg/ml vs. 614.6 ± 27.2 pg/ml, p

CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-β binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-β caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-β in regulating CD1d transcription.

CD4+CD25+FoxP3+ cells (Tregs) inhibit inflammatory immune responses to allografts. Here we found that co-transplantation of allogeneic pancreatic islets with Tregs that are defective in JNK1 signaling prolong the islet allograft survival in the liver parenchyma of the T1D mice. Deletion of the JNK1 in Tregs increased the expression of anti-apoptotic molecules Mcl-1, Bcl-Xl and anti-inflammatory cytokine IL-10 production. JNK1−/− Tregs persist for long period in the transplanted liver and prolong the islet allograft survival compared to WT Tregs. JNK1−/− Tregs demonstrated improved control of allo-inflammation compared to WT Tregs in the T1D liver upon co-transplantation. JNK1−/− Tregs specifically inhibit IL-17 and IL-21 medited alloimmune response. Inhibition of alloimmune response by JNK1−/− Tregs independently mediated through the LAG3 on its cell surface and the increased production of IL-10. Our study identifies a novel role of JNK1 signaling in the Tregs function and co-transplantation of JNK1 deficient Tregs enhances the islet allograft survival in the liver parenchyma of T1D mice.

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

Dietary fibre can mediate effects on mucosal immunity and the gut microbial community. The aim of this study was to examine the impact of dietary fibre intake on immunization efficacy, and to assess potential sex-based differences. Following a 6 week feeding trial, male and female rats fed purified diets containing single sources of fermentable material (cellulose; C, wheat bran; WB, oat bran; OB, resistant starch; RS or oligofructan; OF) were parenterally immunized with 0.3 mg/kg Keyhole Limpet Hemocyanin (KLH). Anti-KLH and total antibody concentrations were evaluated at intervals from tail vein blood, and splenic lymphocyte percentages and cytokine levels were measured at the end of the trial. Circulating anti-KLH IgG, IgG2a and IgG2b levels decreased significantly in females fed OB, while anti-KLH IgG2a levels decreased in males fed either RS or OF-supplemented diets compared to control-fed rats. In contrast to females, males fed OB had significantly lower splenic TGF-b1/IFN-g ratios and higher total IgG antibody levels. Additionally, females fed RS or OF had significantly reduced splenic CD3+ and CD3+4+ percentages while only CD3+ percentages were significantly reduced in males fed OF, relative to their control-fed counterparts. Collectively these results suggest dietary fibre can impact systemic immune responses to immunization, and that these effects can differ between sexes.

In the current study we asked whether modified CD4+CD25+FoxP3+ regulatory T-cells (T-regs) can induce long term allograft tolerance. Pancreatic islets from BALB/c mice survived in a type 1 diabetic C57BL/6 mice (recipient mice) liver up to 18-21 days (n=5). Preloading islets with T-regs from WT (wild type) C57BL/6 mice, enhanced survival up to 37 days (N=5 and p = 0.003). Further preloading islets with T-regs from JNK1-/- mice (C57BL/6 background) enhanced survival up to 107 days (N=5 and p = 0.002). In contrast preloading islets with T-regs from FAS-/- (C57BL/6 background) did not enhance islet survival beyond 45 days. T-cell receptor stimulation of T-regs from JNK1-/- mice produced significant high amount of IL-10 (14.67 ± 2.784 vs 3.803 ± 0.8325, p = 0.009) and TGF-β (27.93 ± 7.563 vs 9.920 ± 1.212, p = 0.05) compared to T-regs from WT mice. Preloading islets with T-regs from JNK1-/- mice reduced IL-1β (30.91 ± 2.41 vs 16.13 ± 2.76; p = 0.003), TNF-α (3.802 ± 0.69 vs 1.888 ± 0.26; p = 0.03) IL-17 (24.82 ± 4.07 vs 4.708 ± 1.57; p = 0.0017). Studies are underway to determine the JNK1-/-T-regs mediated mechanisms involved in prolongation of allograft survival.

IL-21 is a Th17 cytokine and is known for its anti-tumor and anti-viral effects. In the current study using IL-21receptor (IL-21r-/-) and IL-21 knockout (IL-21-/-) mice, we determined the role of IL-21 and IL-21r in Mycobacterium tuberculosis (M. tb) infection. Around 50% of M. tb H37Rv infected IL-21r-/- mice died in six months compared to no deaths in infected control mice. M. tb infected IL-21r-/- mice have enhanced bacterial burden (7.3 ± 1.0 x 106 vs. 0.7 ± 0.08 x 106,p

The intestinal epithelium interacts with varied microbial components and metabolites, integrating multiple signals that influence immunity. We examined the time and dose-dependent effects of a Toll-Like Receptor (TLR) 2 agonist lipoprotein Pam3Cys (P3C) and the bacterial fermentation metabolite butyric acid (BA) (10mM and 25mM) on TLR3 agonist (poly(I:C)) (pIC)-induced chemokine production by HT-29 intestinal epithelial cells (IEC). Pre-treatment of HT-29 IEC with P3C enhanced (p(IC))-induced IL-8 production. However, pre-treatment of HT-29 IEC with P3C and 25mM BA together inhibited p(I:C)-induced IL-8 production, suggesting BA blocked the enhancing effect of this TLR2 agonist on TLR3-induced chemokine production. This inhibitory effect was not observed when IEC were pre-treated with a lower concentration of BA (10mM). Co-incubation of IEC with P3C and p(I:C) did not enhance TLR3 agonist-induced IL-8 production, while co-incubation with 10mM or 25mM BA acid significantly reduced p(I:C)-induced IL-8 and CXCL-10 production by HT-29 IEC. In addition, a p38 blocker (SB203580) significantly inhibited p(I:C)-induced IL-8 production by HT-29 IEC, while co-incubation with SB203580 and 10mM BA showed no additive inhibition. These findings suggest that while TLR2 agonists can upregulate TLR3-induced chemokine production, butyric acid inhibits this process, and the effects of TLR2 agonists and BA are dependent on timing of contact with IEC relative to TLR3 agonist stimulation.

We determined the role of liver NK1.1 cells in allograft survival. Pancreatic islets from BALB/c mice survived in a type 1 diabetic C57BL/6 mice liver up to 15-21 days (n=5, p=0.001). Anti NK1.1 but not isotype control antibody treatment of type 1 diabetic C57BL/6 mice before and after pancreatic islet transfer rejected graft within 3 days (n=5, p=0.001). Anti-NK1.1 antibody treatment enhanced IL-1β (45.13 ± 5.9 vs 94.25 ± 5.5; p = 0.0003), TNF-α (4.746 ± 2.4 vs 11.92 ± 1.8; p = 0.047) and IFN-γ (2.714 ± 0.45 vs 4.344 ± 0.4; p = 0.036) and inhibited IL-22 (24.82 ± 4.07 vs 11.01 ± 2.186; p = 0.01) gene expression by liver lymphocytes of recipient mice. NK1.1 cells are major source of IL-22 in islets recipient mice liver and anti-IL-22 antibody treatment of recipient mice reduced allograft survival to 6-9 days compared to 18-21 days in isotype control antibody treated mice. Recombinant IL-22 but not IFN-γ enhanced insulin production by pancreatic islets of BALB/c mice (102.0 ± 22.65 vs 33.41 ± 12.07; p = 0.03) and enhanced expression of genes Reg2, Reg3a and Reg3g involved in islet survival. Further studies indicated that immunization and challenge of type 1 diabetic C57BL/6 mice with TLR9 agonist can enhance IL-22 production by NK1.1 cells and prolongs pancreatic allograft survival from 15 days to 45 days. The sum of our studies suggests that IL-22 produced by liver NK1.1 cells enhance allograft survival and TLR9 agonist can be used to prolong pancreatic allograft survival.

Tissue Factor (TF) is a trans membrane glycoprotein important for initiation of the coagulation cascade. Recent observations indicate that TF is expressed in cancer, metabolic diseases and infections. In the current study, we determined the role of TF in M. tuberculosis (M. tb) infection using mice lacking the TF gene in myeloid cells (Cre+). One month after infection with the M. tb virulent laboratory strain, H37Rv, the lung bacterial burden was higher in Cre+ than Cre- mice (3.7 ± 0.2 X 106 vs 1.9 ± 0.1 X 106 CFU, p=0.01). M.tb infected Cre+ mice had more CD4+ and CD8+ lymphocytes and expressed 33 and 38 fold less of IFN-γ and IL-17 mRNA in the lungs, compared to Cre- mice, as measured by real time PCR. In contrast, lung IL-10 mRNA levels were higher in M. tb-infected Cre+ mice than Cre- mice (16.04 ± 3.3 vs 4.4 ± 1 fold, p=0.03). Immunohistochemical analysis indicated that M. tb-infected Cre+ mice had more foamy macrophages in the lungs than Cre- mice. M. tbH37Rv grew more rapidly in peritoneal exudate macrophages of Cre+ mice, compared to Cre- macrophages (6.1 ± 1 X 106 vs 1.5 ± 0.6 X 106 CFU, p=0.02). Further studies indicated that M. tb-infected Cre+ macrophages undergo less mitochondrial apoptosis than Cre- macrophages (16.2 ± 2.1vs 27.9 ± 1.4 %, p=0.01). Studies are underway to determine the mechanism/s involved in increased IL-10 production, reduced mitochondrial apoptosis and increased bacterial burden of M tb infected Cre+ mice.

Previously, we found that IL-22 produced by NK cells inhibits intracellular mycobacterial growth in human macrophages (Mϕ) by enhancing phagolysosomal fusion. In the current study, we determined the mechanism/s by which IL-22 inhibits virulent M. tuberculosis H37Rv (M. tb) growth in Mϕ. First we asked whether IL-22 restricts M. tb growth in phagosomes that leads to enhanced phagolysosomal fusion. W7, a phagolysosomal fusion inhibitor abrogated IL-22 dependent M. tb growth in Mϕ (11.38 ± 3.3 x 106 vs 2.95 ± 1.7 x 106 CFU per well, p=0.05) suggesting IL-22 inhibits M. tb growth after phagolysosomal fusion. In microarray analysis we found that mRNA expression for 41 genes was >1-fold higher in rIL-22 cultured infected Mϕ compared to infected Mϕ. Of these genes, we selected Indolamine 2,3-dioxegenase (IDO1) and Calgranulin A for further study. rIL-22 enhanced Calgranulin A expression in M. tb infected Mϕ by three fold (3.3 ± 0.83 vs 1.0 arbitrary units, p=0.04) compared to infected Mϕ as measured by real time PCR and confirmed by confocal microscopy. In contrast, IDO1 expression of infected Mϕ was not effected by rIL-22. Calgranulin A siRNA abrogated rIL-22 dependent growth inhibition of M. tb in macrophages (18.4 ± 1.8 x 106 vs 7.97 ± 0.8 x 106 CFU per well, p=0.0005). Studies are underway to determine the effect of Calgranulin A siRNA on phagolysosomal fusion using early endosomal, late endosomal and lysosomal markers.

We studied the mechanisms of IL-22 production by NK cells in human Mycobacterium tuberculosis (M. tb) infection. CD3-CD56+ NK cells from 17 healthy donors produced IL-22 upon culturing with autologous monocytes and irradiated M. tb (417 ± 99 vs 52 ± 22 pg/ml, p < 0.001). In 14 healthy donors anti-IL-15 and anti-IL-23 reduced IL-22 production by NK cells from 494 ± 110 pg/ml to 193 ± 50 pg/ml (p = 0.01) and 256.1 ± 64.7 pg/ml (p = 0.03) respectively. In contrast, anti-IL-12 and anti-IL-18 had no effect. Recombinant IL-15 induced IL-23 receptor expression (18.7 ± 5.1% vs 6.7 ± 1.9%, p =0.03). Recombinant IL-15 induced IL-22 production by NK cells (4294 ± 1627 vs 43.50 ± 29 pg/ml, p=0.04), but recombinant IL-12 and 1L-18 had no effect. To identify signaling pathways involved in IL-15 mediated IL-22 production, we cultured NK cells from healthy donors with recombinant IL-15 for 48 hrs. In 5 healthy donors IL-15-stimulated NK cells expressed 12 times more DAP10 mRNA compared to control NK cells. In 8 healthy donors DAP10 siRNA inhibited IL-15-mediated IL-22 production by NK cells, compared to control siRNA-transfected NK cells (346 ± 121 vs 1248 ± 465 pg/ml, p =0.05). Studies are underway to determine the effect of IL-22 on intracellular survival of M. tuberculosis in macrophages. We conclude that IL-15 and DAP-10 are involved in IL-22 production by NK cells in human M. tb infection.