Scott M. Reba

Inhibition of Major Histocompatibility Complex II Expression and Antigen Processing in Murine Alveolar Macrophages by Mycobacterium bovis BCG and the 19-Kilodalton Mycobacterial Lipoprotein

ABSTRACT Alveolar macrophages constitute a primary defense against Mycobacterium tuberculosis, but they are unable to control M. tuberculosis without acquired T-cell immunity. This study determined the antigen-presenting cell function of murine alveolar macrophages and the ability of the model mycobacterium, Mycobacterium bovis BCG, to modulate it. The majority (80 to 85%) of alveolar macrophages expressed both CD80 (B7.1) and CD11c, and 20 to 30% coexpressed major histocompatibility complex II (MHC-II). Gamma interferon (IFN-γ) enhanced MHC-II but not B7.1 expression. Naive or IFN-γ-treated alveolar macrophages did not express CD86 (B7.2), CD11b, Mac-3, CD40, or F4/80. M. bovis BCG and the 19-kDa mycobacterial lipoprotein inhibited IFN-γ-regulated MHC-II expression on alveolar macrophages, and inhibition was dependent on Toll-like receptor 2. The inhibition of MHC-II expression by the 19-kDa lipoprotein was associated with decreased presentation of soluble antigen to T cells. Thus, susceptibility to tuberculosis may result from the ability of mycobacteria to interfere with MHC-II expression and antigen presentation by alveolar macrophages.

Neutrophil-Mediated Mycobacteriocidal Immunity in the Lung during Mycobacterium bovis BCG Infection in C57BL/6 Mice

ABSTRACT Although neutrophils have been identified as sources of inflammatory cytokines and chemokines, little is known about their immunologic function during mycobacterial infection in the lungs. In this study, we examined the growth of Mycobacterium bovis BCG in the lungs under experimental conditions that altered neutrophil recruitment to the lungs. Depletion and recruitment of neutrophils was associated with respective increases and decreases in M. bovis BCG growth. Thus, neutrophils may enhance mycobacteriocidal immunity in the lungs.

Mycobacterium bovis BCG decreases MHC-II expression in vivo on murine lung macrophages and dendritic cells during aerosol infection

Mycobacterium tuberculosis and M. bovis BCG infect APCs. In vitro, mycobacteria inhibit IFN-gamma-induced MHC-II expression by macrophages, but the effects of mycobacteria on lung APCs in vivo remain unclear. To assess MHC-II expression on APCs infected in vivo, mice were aerosol-infected with GFP-expressing BCG. At 28 d, approximately 1% of lung APCs were GFP+ by flow cytometry and CFU data. Most GFP+ cells were CD11b(high)/CD11c(neg-mid) lung macrophages (58-68%) or CD11b(high)/CD11c(high) DCs (28-31%). Lung APC MHC-II expression was higher in infected mice than naïve mice. Within infected lungs, however, MHC-II expression was lower in GFP+ cells than GFP- cells for both macrophages and DCs. MHC-II expression was also inhibited on purified lung macrophages and DCs that were infected with BCG in vitro. Thus, lung APCs that harbor mycobacteria in vivo have decreased MHC-II expression relative to uninfected APCs from the same lung, possibly contributing to evasion of T cell responses.

Mycobacteria lacking the RD1 region do not induce necrosis in the lungs of mice lacking interferon‐γ

The genetic region of difference 1 (RD1) in Mycobacterium tuberculosis has recently been hypothesized to encode for proteins that are cytotoxic to the host cell in nature. We demonstrate here that while M. tuberculosis grew progressively in the lungs of gene disrupted mice (GKO) unable to produce interferon‐γ (IFN‐γ), similar mice infected instead with M. bovis bacillus Calmette–Guérin (BCG) reproducibly exhibited an obvious slowing of the disease after about 20 days. Closer examination of BCG‐infected GKO mice showed a florid granulomatous inflammation in the lungs, whereas similar mice infected with M. tuberculosis exhibited wholesale progressive necrosis. In the BCG‐infected GKO mice large numbers of activated effector T cells, some strongly positive for the cytokine tumour necrosis factor, as well as activated natural killer cells accumulated in the lungs. To further test the hypothesis that the differences observed were directly associated with the loss of the RD1 region, it was then shown that a mutant of M. tuberculosis lacking RD1 grew progressively in both normal and GKO mice but failed to induce any degree of necrosis in either animal despite reaching similar levels in the lungs. However, when mice were infected with this mutant, in which the RD1 region had been restored by complementation, wholesale necrosis of the lungs again occurred. These data support the hypothesis that proteins encoded in the RD1 region are a major cause of necrosis and contribute significantly to the pathogenesis of the disease.

Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection

Bacteria can utilize a mammalian host siderophore to usurp host iron; however, the host can respond by down-regulating siderophore expression and up-regulating expression of an inhibitory siderophore-binding protein.

Interferon‐αβ mediates partial control of early pulmonary Mycobacterium bovis bacillus Calmette–Guérin infection

The role of type I interferon (IFN‐αβ) in modulating innate or adaptive immune responses against mycobacterial infection in the lung is unclear. In this study we investigated the susceptibility of IFN‐αβ‐receptor‐deficient (IFN‐αβR–/–) mice to pulmonary infection with aerosolized Mycobacterium bovis bacillus Calmette–Guérin (BCG). During early infection (2–3 weeks), enhanced growth of BCG was measured in the lungs of IFN‐αβR–/– mice compared to wild‐type mice. However, during late infection the burden of BCG was similar in the lungs of IFN‐αβR–/– and wild‐type mice. Although control of BCG growth was delayed, recruitment and activation of T and natural killer cells, production of IFN‐γ, and cytokine expression were all similar in wild‐type and IFN‐αβR–/– mice. However, decreased expression of nitric oxide in bronchoalveolar lavage fluids from IFN‐αβR–/– mice correlated with enhanced growth of BCG. Bone marrow‐derived macrophages from IFN‐αβR–/– mice also produced less nitric oxide following infection with BCG in vitro. These findings suggest that IFN‐αβ contributes to innate immunity to pulmonary mycobacterial infection by augmenting production of nitric oxide.

Modulation of Pulmonary Dendritic Cell Function during Mycobacterial Infection

ABSTRACT We have previously reported that during mycobacterial infection, naïve CD4+ T-cell activation is enhanced in the lungs. We investigated the role of chemokine receptor CCR7 and its ligands in the ability of CD11c+ lung dendritic cells (DCs) to activate naïve CD4+ T cells during pulmonary infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection resulted in the accumulation and maturation in the lungs of DCs that persisted as the mycobacterial burden declined. Lung DCs from infected mice expressed more major histocompatibility complex class II (MHC-II) than those from uninfected mice. CCR7 expression levels on lung DCs were comparable among uninfected and infected mice. The gene expression of the CCR7 ligand CCL19 progressively increased throughout BCG infection, and its expression was MyD88 dependent. CD11c+ lung cells from BCG-infected mice activated ovalbumin (OVA)-specific naïve CD4+ T cells more than CD11c+ lung cells from uninfected mice. Interestingly, during peak mycobacterial infection, CD11chi MHChi lung DCs had slightly decreased chemotaxis toward the CCR7 ligand CCL21 and less efficiency in activating naive CD4+ T cells than DCs from mice during late-stage infection, when few bacilli are found in the lung. These findings suggest that during BCG infection, the inflammation and sustained expression of CCL19 result in the recruitment, activation, and retention in the lung of DCs that can activate naïve CD4+ T cells in situ.

TLR2 engagement on CD4+ T cells enhances effector functions and protective responses to Mycobacterium tuberculosis

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4+ T cells and upregulate TCR‐triggered IFN‐γ secretion and cell proliferation in vitro. Here we examined the role of CD4+ T‐cell‐expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag‐specific T‐cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4+ T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1‐like response was observed in the context of both polyclonal and Ag‐specific TCR stimulation. To evaluate the role of T‐cell TLR2 in priming of CD4+ T cells in vivo, naive MTB Ag85B‐specific TCR transgenic CD4+ T cells (P25 TCR‐Tg) were adoptively transferred into Tlr2−/− recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam3Cys‐SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN‐γ‐secreting P25 TCR‐Tg T cells 1 week after immunization. P25 TCR‐Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4+ T cells increases MTB Ag‐specific responses and may contribute to protection against MTB infection.

Modulation of Naive CD4+ T-Cell Responses to an Airway Antigen during Pulmonary Mycobacterial Infection

ABSTRACT During pulmonary mycobacterial infection, there is increased trafficking of dendritic cells from the lungs to the draining lymph nodes. We hypothesized that ongoing mycobacterial infection would modulate recruitment and activation of antigen-specific naive CD4+ T cells after airway antigen challenge. BALB/c mice were infected by aerosol with Mycobacterium bovis BCG. At peak bacterial burden in the lungs (4 to 6 weeks postinfection), carboxy-fluorescein diacetate succinimidyl ester-labeled naive ovalbumin-specific DO11.10 T cells were adoptively transferred into infected and uninfected mice. Recipient mice were challenged intranasally with soluble ovalbumin (OVA), and OVA-specific T-cell responses were measured in the lungs, draining mediastinal lymph nodes (MLN), and spleens. OVA challenge resulted in increased activation and proliferation of OVA-specific T cells in the draining MLN of both infected and uninfected mice. However, only BCG-infected mice had prominent OVA-specific T-cell activation, proliferation, and Th1 differentiation in the lungs. BCG infection caused greater distribution of airway OVA to pulmonary dendritic cells and enhanced presentation of OVA peptide by lung CD11c+ cells. Together, these data suggest that an existing pulmonary mycobacterial infection alters the phenotype of lung dendritic cells so that they can activate antigen-specific naive CD4+ T cells in the lungs in response to airway antigen challenge.

Latency-associated peptide of transforming growth factor beta enhances mycobacteriocidal immunity in the lung during Mycobacterium bovis BCG infection in C57BL/6 mice.

ABSTRACT Latency-associated peptide of transforming growth factor β (TGF-β) (LAP) was used to determine whether in vivo modulation of TGF-β bioactivity enhanced pulmonary immunity to Mycobacterium bovis BCG infection in C57BL/6 mice. LAP decreased BCG growth in the lung and enhanced antigen-specific T-cell proliferation and gamma interferon mRNA expression. Thus, susceptibility of the lung to primary BCG infection may be partially mediated by the immunosuppressive effects of TGF-β.