Bovis BCG-infected Macrophages Research Articles

Rab6b localizes to the Golgi complex in murine macrophages and promotes tumor necrosis factor release in response to mycobacterial infection.

The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in the host control of mycobacterial infections. Expression and release of TNF are tightly regulated, yet the molecular mechanisms that control the release of TNF by mycobacteria-infected host cells, in particular macrophages, are incompletely understood. Rab GTPases direct the transport of intracellular membrane-enclosed vesicles and are important regulators of macrophage cytokine secretion. Rab6b is known to be predominantly expressed in the brain where it functions in retrograde transport and anterograde vesicle transport for exocytosis. Whether it executes similar functions in the context of immune responses is unknown. Here we show that Rab6b is expressed by primary mouse macrophages, where it localized to the Golgi complex. Infection with Mycobacterium bovis bacille Calmette-Guérin (BCG) resulted in dynamic changes in Rab6b expression in primary mouse macrophages in vitro as well as in organs from infected mice in vivo. We further show that Rab6b facilitated TNF release by M. bovis BCG-infected macrophages, in the absence of discernible impact on Tnf messenger RNA and intracellular TNF protein expression. Our observations identify Rab6b as a positive regulator of M. bovis BCG-induced TNF trafficking and secretion by macrophages and positions Rab6b among the molecular machinery that orchestrates inflammatory cytokine responses by macrophages.

GM-CSF targeted immunomodulation affects host response to M. tuberculosis infection

Host directed immunomodulation represents potential new adjuvant therapies in infectious diseases such as tuberculosis. Major cytokines like TNFα exert a multifold role in host control of mycobacterial infections. GM-CSF and its receptor are over-expressed during acute M. tuberculosis infection and we asked how GM-CSF neutralization might affect host response, both in immunocompetent and in immunocompromised TNFα-deficient mice. GM-CSF neutralizing antibodies, at a dose effectively preventing acute lung inflammation, did not affect M. tuberculosis bacterial burden, but increased the number of granuloma in wild-type mice. We next assessed whether GM-CSF neutralization might affect the control of M. tuberculosis by isoniazid/rifampicin chemotherapy. GM-CSF neutralization compromised the bacterial control under sub-optimal isoniazid/rifampicin treatment in TNFα-deficient mice, leading to exacerbated lung inflammation with necrotic granulomatous structures and high numbers of intracellular M. tuberculosis bacilli. In vitro, GM-CSF neutralization promoted M2 anti-inflammatory phenotype in M. bovis BCG infected macrophages, with reduced mycobactericidal NO production and higher intracellular M. bovis BCG burden. Thus, GM-CSF pathway overexpression during acute M. tuberculosis infection contributes to an efficient M1 response, and interfering with GM-CSF pathway in the course of infection may impair the host inflammatory response against M. tuberculosis.

Exosomes Derived from M. Bovis BCG Infected Macrophages Activate Antigen-Specific CD4+ and CD8+ T Cells In Vitro and In Vivo

Activation of both CD4+ and CD8+ T cells is required for an effective immune response to an M. tuberculosis infection. However, infected macrophages are poor antigen presenting cells and may be spatially separated from recruited T cells, thus limiting antigen presentation within a granuloma. Our previous studies showed that infected macrophages release from cells small membrane-bound vesicles called exosomes which contain mycobacterial lipid components and showed that these exosomes could stimulate a pro-inflammatory response in naïve macrophages. In the present study we demonstrate that exosomes stimulate both CD4+ and CD8+ splenic T cells isolated from mycobacteria-sensitized mice. Although the exosomes contain MHC I and II as well as costimulatory molecules, maximum stimulation of T cells required prior incubation of exosomes with antigen presenting cells. Exosomes isolated from M. bovis and M. tuberculosis infected macrophages also stimulated activation and maturation of mouse bone marrow-derived dendritic cells. Interestingly, intranasal administration of mice with exosomes isolated from M. bovis BCG infected macrophages induce the generation of memory CD4+ and CD8+ T cells. The isolated T cells also produced IFN-γ upon restimulation with BCG antigens. The release of exosomes from infected macrophages may overcome some of the defects in antigen presentation associated with mycobacterial infections and we suggest that exosomes may be a promising M. tuberculosis vaccine candidate.

Characterization of human Mycobacterium bovis bacille Calmette-Guérin-reactive CD8+ T cells.

Gamma interferon (IFN-gamma)-secreting CD4+ T cells have long been established as an essential component of the protective immune response against Mycobacterium tuberculosis. It is now becoming evident from studies with the murine model of tuberculosis that an important role also exists for major histocompatibility complex (MHC) class I-restricted CD8+ T cells. These cells are capable of acting as both IFN-gamma secretors and cytotoxic T lymphocyte (CTL) effectors; however, their exact role in immunity against tuberculosis remains unclear. This study demonstrates the presence of Mycobacterium bovis BCG-reactive CD8+ T cells in healthy BCG-vaccinated donors and that these CD8+ T cells are potent cytokine producers as well as cytotoxic effector cells. Using FACScan analysis, we have shown that restimulation with live M. bovis BCG induced more CD8+-T-cell activation than the soluble antigen purified protein derivative and that these cells are actively producing the type 1 cytokines IFN-gamma and tumor necrosis factor alpha (TNF-alpha). These CD8+ T cells also contain the cytolytic granule perforin and are capable of acting as potent CTLs against M. bovis BCG-infected macrophages. The mycobacterial antigens 85A and B (Ag85A and Ag85B, respectively), and to a lesser extent the 19- and 38-kDa proteins, are major antigenic targets for these mycobacterium-specific CD8+ T cells, while whole-M. bovis BCG activated effector cells from these BCG-vaccinated donors, as expected, failed to recognize the 6-kDa ESAT-6 protein. The use of metabolic inhibitors and blocking antibodies revealed that the CD8+ T cells recognize antigen processed and presented via the classical MHC class I pathway. These data suggest that CD8+ T cells may play a critical role in the human immune response to tuberculosis infection.

Attenuation of MHC Class II Expression in Macrophages Infected withMycobacterium bovisBacillus Calmette-Guerin Involves Class II Transactivator and Depends on theNramp1Gene

AbstractThe natural resistance associated macrophage protein 1 (Nramp1) gene determines the ability of murine macrophages to control infection with a group of intracellular pathogens, including Salmonella typhimurium, Leishmania donovani, and Mycobacterium bovis bacillus Calmette-Guérin (BCG). The expression of the resistant allele of the Nramp1 gene in murine macrophages is associated with a more efficient expression of several macrophage activation-associated genes, including class II MHC loci. In this study, we investigated the molecular mechanisms involved in IFN-γ-induced MHC class II expression in three types of macrophages: those expressing a wild-type allele of the Nramp1 gene (B10R and 129/Mφ), those carrying a susceptible form of the Nramp1 gene (B10S), and those derived from 129-Nramp1-knockout mice (129/Nramp1-KO). Previously, we published results showing that Ia protein expression is significantly higher in the IFN-γ-induced B10R macrophages, compared with its susceptible counterpart. In this paper, we also show that the higher expression of Ia protein in B10R cells is associated with higher I-Aβ mRNA expression, which correlates with a higher level of IFN-γ-induced phosphorylation of the STAT1-α protein and subsequently with elevated expression of class II transactivator (CIITA) mRNA, compared with B10S. Furthermore, we demonstrate that the infection of macrophages with M. bovis BCG results in a down-regulation of CIITA mRNA expression and, consequently, in the inhibition of Ia induction. Therefore, our data explain, at least in part, the molecular mechanism involved in the inhibition of I-Aβ gene expression in M. bovis BCG-infected macrophages activated with IFN-γ.

Antigen-specific lymphocytes enhance nitric oxide production in Mycobacterium bovis BCG-infected bovine macrophages.

Nitric oxide (NO) production was evaluated in macrophages isolated from Mycobacterium bovis bacille Calmette-Guérin (BCG)-immunized, and control non-immunized, cattle. Incubation of M. bovis BCG-infected macrophages with recombinant bovine IFN-gamma led to increased nitrite levels in culture supernatants. It was also demonstrated that NO production by autologous M. bovis BCG-infected macrophages increased in a linear relationship with the number of antigen-specific lymphocytes added to cultures. The elevated NO levels were also associated with increased IFN-gamma secretion. Treatment of cultures with the NO inhibitor, N-monomethyl L-arginine (L-NMMA), reduced the levels of NO without affecting the metabolic activity of internalized M. bovis BCG. Our results suggest that synthesis of NO may constitute an integral part of the cell-mediated antigen-specific response against M. bovis BCG. However, although the presence of lymphocytes does partially inhibit multiplication of M. bovis BCG in macrophages, it appears that the activity of NO, or the levels produced in monocyte-derived macrophages, may be insufficient to influence the growth of the intracellular mycobacteria.

Activation of CD8 T cells with specificity for mycobacterial heat shock protein 60 in Mycobacterium bovis bacillus Calmette-Guérin-vaccinated mice

Heat shock protein 60 (hsp60)-specific CD8 T cells lysed Mycobacterium bovis BCG-infected macrophages in vitro and adoptively transferred protection against mycobacterial infection. Moreover, CD8 T cells with this hsp60 specificity were activated in vivo by BCG vaccination. Our studies suggest there is participation of hsp60-specific CD8 T cells in BCG-induced immunity.

Bacterial metabolism, cytokine mRNA transcription and viability of bovine alveolar macrophages infected with Mycobacterium bovis BCG or virulent M bovis

Mycobacterium bovis causes tuberculosis in cattle and many other animals including humans while BCG, an attenuated form of M. bovis, has been used widely as a safe vaccine. Both strains infect host macrophages and their fate is determined by their ability to survive within these phagocytic cells. We compared interactions of these two strains with bovine alveolar macrophages in order to gain an understanding of virulence mechanisms involved in the early pathogenesis of M. bovis infection. Macrophages were infected with bacilli at varying multiplicities of infection and cultured for 1-4 days. Bacterial metabolism within macrophages was assessed by [3H]-uracil uptake and bacterial growth was assessed by culture and acid-fast staining. Induction of TNF-alpha, IL-1 beta and IL-6 cytokine mRNA transcription in macrophages was determined by reverse transcriptase-polymerase chain reaction. Infection of macrophages by virulent M. bovis resulted in enhanced bacterial metabolism, enhanced induction of macrophage cytokines and reduced viability of macrophages when compared to M. bovis BCG-infected macrophages. These differences may reflect virulence mechanisms contributing to the early pathogenesis of bovine tuberculosis.

H2O2 induces monocyte apoptosis and reduces viability of Mycobacterium avium-M. intracellulare within cultured human monocytes.

Mycobacterium avium-M. intracellulare, an intracellular parasite of mononuclear phagocytes, rarely causes disease in immunocompetent individuals. In contrast, in human immunodeficiency virus type 1-infected patients, M. avium-M. intracellulare can infect almost every tissue and organ. This suggests that immunocompetent individuals have a protective mechanism to control or prevent the infection. How mycobacterial may be killed by the host immune response is unclear. We have recently reported that induction of apoptosis of Mycobacterium bovis BCG-infected macrophages with ATP4- was associated with killing of the intracellular mycobacteria. In the present study, a long-term culture of M. avium-M. intracellulare-infected monocytes was used to further evaluate the interaction between M. avium-M. intracellulare and primary human monocytes. In our system, M. avium-M. intracellulare parasitized the human monocytes and appeared to replicate slowly over 14 days within the host cells. To examine the role of apoptotic mechanisms in survival or death of intracellular mycobacteria, M. avium-M. intracellulare-infected human monocytes were treated with a monoclonal antibody to Fas receptor (APO-1/CD95) or with various concentrations of H2O2. Although both of these exogenous agents induced monocyte apoptosis, optimal killing (65% reduction in CFU) of intracellular M. avium-M. intracellulare was observed only when M. avium-M. intracellulare-infected cells were treated with 10 mM H2O2. Fas-induced apoptosis did not affect M. avium-M. intracellulare viability. Our results suggest that not all stimuli of monocyte apoptosis induce killing of intracellular M. avium-M. intracellulare. Since release of H2O2 following phagocytosis of mycobacteria has been documented, H2O2-induced apoptotic death of M. avium-M. intracellulare-infected monocytes and its association with killing of the intracellular bacilli may be a physiological mechanism of host defense against M. avium-M. intracellulare.