Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.

Cigarette smoke has been associated with susceptibility to different pulmonary and airway diseases. Impaired alveolar macrophages (AMs) that are major phagocytes in the lung have been associated with patients with airway diseases and active smokers. In the current report, we show that exposure to second-hand cigarette smoke (SHS) significantly reduced efferocytosis in vivo. More importantly, delivery of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) to the alveolar space restored and refurbished the efferocytosis capability of AMs. Exposure to SHS significantly reduced expression of CD16/32 on AMs, and treatment with GM-CSF not only restored but also significantly increased the expression of CD16/32 on AMs. GM-CSF treatment increased uptake and digestion/removal of apoptotic cells by AMs. The latter was attributed to increased expression of Rab5 and Rab7. Increased efferocytosis of AMs was also tested in a disease condition. AMs from GM-CSF-treated, influenza-infected, SHS-exposed mice showed significantly better efferocytosis activity, and mice had significantly less morbidity compared with phosphate-buffered saline-treated group. GM-CSF-treated mice had increased amphiregulin levels in the lungs, which in addition to efferocytosis of AMs may have attributed to their protection against influenza. These results will have great implications for developing therapeutic approaches by harnessing mucosal innate immunity to treat lung and airway diseases and protect against pneumonia.

Background Since adaptive immunity is thought to be central to immunity against influenza A virus (IAV) pneumonias, preventive strategies have focused primarily on vaccines. However, vaccine efficacy has been variable, in part because of antigenic shift and drift in circulating influenza viruses. Recent studies have highlighted the importance of innate immunity in protecting against influenza. Methods Granulocyte-macrophage colony stimulating factor (GM-CSF) contributes to maturation of mononuclear phagocytes, enhancing their capacity for phagocytosis and cytokine production. Results Overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) in the lung of transgenic mice provides remarkable protection against IAV, which depends on alveolar macrophages (AM). In this study, we report that pulmonary delivery of GM-CSF to wild type young and aged mice abrogated mortality from IAV. Conclusion We also demonstrate that protection is species specific and human GM-CSF do not protect the mice nor stimulates mouse immunity. We also show that IAV-induced lung injury is the culprit for side-effects of GM-CSF in treating mice after IAV infection, and introduce a novel strategy to deliver the GM-CSF to and retain it in the alveolar space even after IAV infection.

In response to Roux and Ricard [1], we wish to highlight a few facts regarding the use of granulocytemacrophage colony-stimulating factor (GM-CSF) for preventing microbial pneumonia.Recent studies by us and others show that overexpression of GM-CSF in the lungs protects mice against secondary bacterial pneumonia following sublethal influenza A virus infection [2,3].We demonstrated that GM-CSF mediates protection against secondary bacterial pneumonia through enhanced function of alveolar macrophages and neutrophils.However, our previous study indicated that overexpression of pulmonary GM-CSF provides 100% protection against lethal influenza virus infection and that alveolar macrophages, not neutrophils, play a vital role in protection [4].Bacterial pneumonia significantly contributes to complications and mortality during influenza virus infection [5-8] and ventilator associated pneumonia (VAP).VAP is a common infectious disease in intensive care units, and Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, and other bacterial species have been isolated from patients with VAP.Studies have shown an association between Candida albicans and P. aeruginosa in VAP [9], indicating that prior infections or insults to the lung increase the predisposition to secondary bacterial pneumonia.GM-CSF has been shown to reverse the immunosuppressive effect and restore the fungicidal activity against Aspergillus conidia in mice [10], and prophylactic use of GM-CSF in patients

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.

Aging increases susceptibility to infection, in part because thymic involution culminates in reduced naïve T-lymphocyte output. Thymic epithelial cells (TECs) are critical to ensure normal maturation of thymocytes and production of peripheral T cells. The forkhead-class transcription factor, encoded by FoxN1, regulates development, differentiation, and function of TECs, both in the prenatal and postnatal thymus. We recently showed that expression of FoxN1, by keratin 14 (K14)-expressing epithelial cells is essential for maintenance of thymic medullary architecture, and deletion of FoxN1 in K14 promoter-driven TECs inhibited development of mature TECs and reduced the number of total thymocytes. These findings are reminiscent of changes observed during normal thymic aging. In the current report, we compared the effects of K14-driven FoxN1 deletion on peripheral T cell function in response to influenza virus infection with those associated with normal aging in a mouse model. FoxN1-deleted mice had reduced numbers of peripheral CD62L+CD44- naïve T-cells. In addition, during influenza infection, these animals had reduced antigen-specific CD8+ T-cell and IgG responses to influenza virus, combined with increased lung injury, weight loss and mortality. These findings paralleled those observed in aged wild type mice, providing the first evidence that K14-mediated FoxN1 deletion causes changes in T-cell function that mimic those in aging during an immune response to challenge with an infectious agent.

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/macrophagecolony-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.

ABSTRACTSmoking 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 withMycobacterium tuberculosisby aerosol or influenza A by intranasal infection. Some mice were given a DNA vaccine encoding an immunogenicM. tuberculosisprotein. 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 stimulationin vitrowith anti-CD3, after vaccination with a construct expressing an immunogenic mycobacterial protein, and during infection withM. tuberculosisand influenza A virusin 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 withM. tuberculosisand 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 toM. tuberculosisand influenza virus in a physiologically relevant animal model, increasing susceptibility to both pathogens.

Early secreted antigenic target of 6 kD (ESAT-6) of Mycobacterium tuberculosis (M. tb) is a potent antigen with vaccine potential, but is also a virulence factor. We studied the effect of recombinant His-tagged ESAT-6 on IFN-γ secretion by human primary T cells, using the 10 kD culture filtrate protein (CFP10) as a control. ESAT-6 but not CFP10 started to inhibit IFN-γ secretion by M-tb stimulated PBMC at 1.6 μM and this reached 90% inhibition, compared to that with CFP10 (12.5 + 5.4 vs. 1.3 + 1.2 ng/ml) at 3.3 μM (20 μg/ml). Dimerization of ESAT-6 with CFP10 did not affect inhibition. ESAT-6 inhibited IFN-γ production of α-CD3/CD28 mAb-stimulated T cells by 89% (14.5 + 1.8 vs. 1.6 + 0.4 ng/ml), and proliferation, as measured by CFSE assay. Depleting ESAT-6 with a nickel resin abrogated the inhibition. The inhibitory effect of ESAT-6 was not due to T cell toxicity or apoptosis, as measured by MTT assay and Annexin V staining. ESAT-6 inhibited expression of M. tb-induced IFN-γ mRNA (> 90%) and the transcription factors, ATF-2 and c-Jun, which regulate IFN-γ transcription. ESAT-6 bound to T cells and inhibited expression of CD69 and CD25, without affecting proximal TCR signaling, ZAP70 activation. We conclude that high concentrations of ESAT-6 inhibit T cell responses to M. tb by affecting signaling pathways downstream of ZAP70, and may be a mechanism by which M. tb inhibits effective immunity. (NIH AI063514)

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-γ 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-γ proximal promoter and increase Interferon-γ 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-γ 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-γ transcription, elicit protective pulmonary immune responses and prevent ESAT-6 from inhibiting T-cell function will contribute significantly to development of antituberculosis vaccines.

The Mycobacterium tuberculosis early secreted Ag of 6 kDa (ESAT-6) is a potent Ag for human T cells and is a putative vaccine candidate. However, ESAT-6 also contributes to virulence in animal models, mediates cellular cytolysis, and inhibits IL-12 production by mononuclear phagocytes. We evaluated the effects of ESAT-6 and its molecular chaperone, culture filtrate protein of 10 kDa (CFP10), on the capacity of human T cells to produce IFN-gamma and proliferate in response to TCR activation. Recombinant ESAT-6, but not CFP10, markedly inhibited IFN-gamma production by T cells stimulated with M. tuberculosis or with the combination of anti-CD3 and anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T cell production of IL-17 and TNF-alpha but not IL-2. Preincubation of ESAT-6 with CFP10 under conditions that favor dimer formation did not affect inhibition of IFN-gamma. ESAT-6 decreased IFN-gamma transcription and reduced expression of the transcription factors, ATF-2 and c-Jun, which normally bind to the IFN-gamma proximal promoter and stimulate mRNA expression. ESAT-6 inhibited T cell IFN-gamma secretion through mechanisms that did not involve cellular cytotoxicity or apoptosis. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing activation of ZAP70. We conclude that ESAT-6 directly inhibits human T cell responses to mycobacterial Ags by affecting TCR signaling pathways downstream of ZAP70. The Journal of Immunology,2009,182: 3668-3677.

The Mycobacterium tuberculosis early secreted Ag of 6 kDa (ESAT-6) is a potent Ag for human T cells and is a putative vaccine candidate. However, ESAT-6 also contributes to virulence in animal models, mediates cellular cytolysis, and inhibits IL-12 production by mononuclear phagocytes. We evaluated the effects of ESAT-6 and its molecular chaperone, culture filtrate protein of 10 kDa (CFP10), on the capacity of human T cells to produce IFN-γ and proliferate in response to TCR activation. Recombinant ESAT-6, but not CFP10, markedly inhibited IFN-γ production by T cells stimulated with M. tuberculosis or with the combination of anti-CD3 and anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T cell production of IL-17 and TNF-α but not IL-2. Preincubation of ESAT-6 with CFP10 under conditions that favor dimer formation did not affect inhibition of IFN-γ. ESAT-6 decreased IFN-γ transcription and reduced expression of the transcription factors, ATF-2 and c-Jun, which normally bind to the IFN-γ proximal promoter and stimulate mRNA expression. ESAT-6 inhibited T cell IFN-γ secretion through mechanisms that did not involve cellular cytotoxicity or apoptosis. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing activation of ZAP70. We conclude that ESAT-6 directly inhibits human T cell responses to mycobacterial Ags by affecting TCR signaling pathways downstream of ZAP70.

To determine the immunogenicity and protective efficacy of the Mycobacterium tuberculosis 10 kD culture filtrate protein (CFP10), and to evaluate strategies that enhance local immunity, we used C57Bl/6 DR4 mice that were transgenic for human HLA DRB1 0401, because CFP10 contains epitopes for DRB1 0401 but not for C57Bl/6 mice. Intramuscular immunization with a DNA vaccine encoding CFP10 elicited production of IFN-gamma by systemic CD4+ T cells, and one intravenous dose of the CFP10-based DNA vaccine coated with polyethylenimine (PEI) stimulated IFN-gamma production by lung CD4+ cells and reduced the pulmonary bacillary burden. We conclude that CFP10 is a potential vaccine candidate and that coating vaccines with PEI enhances local protective immunity to tuberculosis

IFN-γ production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-γ, −73 to −48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-γ in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-γ upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-γ proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-γ and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-γ proximal promoter and that they up-regulate IFN-γ transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation.

CD4+ T cells are believed to play a dominant role in human defenses against Mycobacterium tuberculosis through production of interferon (IFN)-gamma, cytolytic T-cell (CTL) activity, and inhibition of intracellular mycobacterial growth. Most functional studies of CD4+ cells have used bulk T-cells that recognize crude mycobacterial antigens, and the functional capacity of individual human T cells is not well defined. We studied the functional capacity of human CD4+ T-cell clones that recognize a specific mycobacterial peptide. Clone B9 produced high concentrations of IFN-gamma and exhibited potent CTL activity, whereas clone D3 produced IFN-gamma but showed poor CTL activity. The CTL activity of clone B9 was inhibited by SrCl(2) and concanamycin A but not by anti-Fas antibodies. Clone B9 also reduced the mycobacterial burden in dendritic cells by more than 90%, and this antimycobacterial activity was inhibited by SrCl(2) and concanamycin A. We conclude that: (1) individual human peptide-specific CD4+ T-cell clones have differential capacity to produce Th1 cytokines and to lyse M tuberculosis-infected target cells; and (2) both granulysin and perforin contribute to the capacity of human CD4+ T-cells to lyse infected targets and to inhibit intracellular mycobacterial growth.

IFN-gamma production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-gamma, -73 to -48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-gamma in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-gamma upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-gamma proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-gamma and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-gamma proximal promoter and that they up-regulate IFN-gamma transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation.

Dendritic cells (DC) present lipid and peptide antigens to T cells on CD1 and MHC Class II (MHCII), respectively. The relative contribution of these systems during the initiation of adaptive immunity after microbial infection is not characterized. MHCII molecules normally acquire antigen and rapidly traffic from phagolysosomes to the plasma membrane as part of DC maturation, whereas CD1 molecules instead continually recycle between these sites before, during, and after DC maturation. We find that in Mycobacterium tuberculosis (Mtb)-infected DCs, CD1 presents antigens quickly. Surprisingly, rapid DC maturation results in early failure and delay in MHCII presentation. Whereas both CD1b and MHCII localize to bacterial phagosomes early after phagocytosis, MHCII traffics from the phagosome to the plasma membrane with a rapid kinetic that can precede antigen availability and loading. Thus, rather than facilitating antigen presentation, a lack of coordination in timing may allow organisms to use DC maturation as a mechanism of immune evasion. In contrast, CD1 antigen presentation occurs in the face of Mtb infection and rapid DC maturation because a pool of CD1 molecules remains available on the phagolysosome membrane that is able to acquire lipid antigens and deliver them to the plasma membrane.

IFN-gamma is essential for resistance to many intracellular pathogens, including Mycobacterium tuberculosis. Transcription of the IFN-gamma gene in activated T cells is controlled by the proximal promoter element (-73 to -48 bp). CREB binds to the IFN-gamma proximal promoter, and binding is enhanced by phosphorylation of CREB. Studies in human T cell lines and in transgenic mice have yielded conflicting results about whether CREB is a positive or a negative regulator of IFN-gamma transcription. To determine the role of CREB in mediating IFN-gamma production in response to a microbial pathogen, we evaluated the peripheral blood T cell response to M. tuberculosis in healthy tuberculin reactors. EMSAs, chromatin immunoprecipitation, and Western blotting demonstrated that stimulation of PBMC with M. tuberculosis induced phosphorylation and enhanced binding of CREB to the IFN-gamma proximal promoter. Neutralization of CREB with intracellular Abs or down-regulation of CREB levels with small interfering RNA decreased M. tuberculosis-induced production of IFN-gamma and IFN-gamma mRNA expression. In addition, M. tuberculosis-stimulated T cells from tuberculosis patients, who have ineffective immunity, showed diminished IFN-gamma production, reduced amounts of CREB binding to the IFN-gamma proximal promoter, and absence of phosphorylated CREB. These findings demonstrate that CREB positively regulates IFN-gamma production by human T cells that respond to M. tuberculosis.