Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited. Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types. Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M. tuberculosis for survival in the host.     

Mice lacking SIGNR1 have stronger T helper 1 responses to Mycobacterium tuberculosis

Mycobacterium tuberculosis and the associated disease tuberculosis are health risks causing many deaths worldwide each year in humans. M. tuberculosis targets dendritic cell (DC)-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) to induce immunosuppression, since interaction of DC-SIGN with mycobacterial mannose-capped lipoarabinomannan (ManLAM) induces interleukin (IL)-10 and prevents DC maturation. We investigated the role of a murine homolog of DC-SIGN, SIGN Related 1 (SIGNR1), in a model of M. tuberculosis infection using SIGNR1 deficient (KO) mice. Although SIGNR1 is expressed by macrophages and not by DCs, it also interacts with M. tuberculosis similar to DC-SIGN. Peritoneal macrophages from SIGNR1 KO mice produce less IL-10 upon stimulation with ManLAM than those from wild-type mice, suggesting that the interaction of ManLAM with SIGNR1 can result in immunosuppression similar to its human homolog. Indeed, early in infection, we observed increased T cell activity in SIGNR1 KO mice and increased IFNgamma production by splenocytes in SIGNR1 KO mice. However, we did not detect any differences between WT and KO mice in mycobacterial loads in the lungs or distant organs after M. tuberculosis infection resulting in similar survival rates. Moreover, we found that SIGNR1 is not present on alveolar macrophages of uninfected mice nor is it induced on lung macrophages throughout infection. Therefore, our data suggest that although SIGNR1 has a similar binding specificity as DC-SIGN, its role is limited during murine M. tuberculosis infection.     

Induction of TNF in human alveolar macrophages as a potential evasion mechanism of virulent Mycobacterium tuberculosis.

The ability of macrophages to release cytokines is crucial to the host response to intracellular infection. In particular, macrophage-derived TNF plays an important role in the host response to infection with the intracellular pathogen Mycobacterium tuberculosis. In mice, TNF is indispensable for the formation of tuberculous granulomas, which serve to demarcate the virulent bacterium. TNF is also implicated in many of the immunopathological features of tuberculosis. To investigate the role of TNF in the local immune response, we infected human alveolar macrophages with virulent and attenuated mycobacteria. Infection with virulent strains induced the secretion of significantly higher levels of bioactive TNF than attenuated strains correlating with their ability to multiply intracellularly. Treatment of infected macrophages with neutralizing anti-TNF Abs reduced the growth rate of intracellular bacteria, whereas bacterial replication was augmented by addition of exogenous TNF. Infected and uninfected macrophages contributed to cytokine production as determined by double-staining of M. tuberculosis and intracellular TNF. The induction of TNF by human alveolar macrophages at the site of infection permits the multiplication of intracellular bacteria and may therefore present an evasion mechanism of human pathogens.     

Intracellular replication of attenuated Mycobacterium tuberculosis phoP mutant in the absence of host cell cytotoxicity

Intracellular pathogen Mycobacterium tuberculosis survives and replicates in macrophages but limited information is available on its replication into non-phagocytic cells. Here we study the role of the M. tuberculosis virulence gene phoP in the intracellular growth with rat and human lung fibroblasts. In contrast to macrophages, attenuated M. tuberculosis phoP mutant was able to multiply intracellularly in fibroblasts at the same level as the virulent M. tuberculosis. However, when M. tuberculosis virulence was studied using human foetal lung fibroblasts, MRC-5 cell line, the virulent strain caused a significant damage in cells compared with attenuated strains BCG and M. tuberculosis phoP mutant. We analysed the effect of cytoskeleton inhibitors in NRK-49F fibroblasts. M. tuberculosis invasion was not inhibited, suggesting that mycobacterial uptake was microtubule and microfilament independent. Our results suggest that PhoP in M. tuberculosis does not regulate intracellular replication in fibroblasts, contrary to what happens in macrophages. The ability of M. tuberculosis phoP mutant to replicate within non-phagocytic cells, such as fibroblasts, without causing damage, could be a potential advantage for a live attenuated vaccine against tuberculosis.     

CpG oligonucleotides partially inhibit growth of Mycobacterium tuberculosis, but not Salmonella or Listeria, in human monocyte-derived macrophages

Immunostimulatory DNA sequences and their synthetic oligonucleotide analogs (CpG-ODN) activate innate immunity and can stimulate antibacterial effects against numerous intracellular pathogens. While it has been shown previously that CpG-ODN inhibit growth of Mycobacterium avium in murine and human macrophages, we now report that Mycobacterium tuberculosis growth can be inhibited by CpG-ODN treatment of human monocyte-derived macrophages (hMDM). This inhibitory effect was reversed by IFN-gamma, which has been shown repeatedly to enhance the growth of virulent M. tuberculosis in cultured hMDM. The antibacterial effect of CpG-ODN in human macrophages was specific for M. tuberculosis when compared to other intracellular pathogens including Listeria monocytogenes and Salmonella enterica serovar Dublin. These data indicate that CpG-ODN can improve the ability of hMDM to contain growth of virulent M. tuberculosis.     

ATP stimulates human macrophages to kill intracellular virulent Mycobacterium tuberculosis via calcium-dependent phagosome-lysosome fusion.

Advances in therapy for tuberculosis will require greater understanding of the molecular mechanisms of pathogenesis and the human immune response in this disease. Exposure of Mycobacterium tuberculosis-infected human macrophages to extracellular ATP (ATP(e)) results in bacterial killing, but the molecular mechanisms remain incompletely characterized. In this study, we demonstrate that ATP(e)-induced bactericidal activity toward virulent M. tuberculosis requires an increase in cytosolic Ca(2+) in infected macrophages. Based on our previous work with primary infection of human macrophages, we hypothesized that the Ca(2+) dependence of ATP-induced killing of intracellular M. tuberculosis was linked to promotion of phagosome-lysosome fusion. Using confocal laser-scanning microscopy, we demonstrate that ATP(e) induces fusion of the M. tuberculosis-containing phagosome with lysosomes, defined by accumulation of three lysosomal proteins and an acidophilic dye. Stimulation of phagosome-lysosome fusion by ATP(e) exhibited distinct requirements for both Ca(2+) and phospholipase D and was highly correlated with killing of intracellular bacilli. Thus, key signal transduction pathways are conserved between two distinct models of human macrophage antituberculous activity: primary infection of naive macrophages and physiologic stimulation of macrophages stably infected with M. tuberculosis.     

Production of phthiocerol dimycocerosates protects Mycobacterium tuberculosis from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection

The growth of Mycobacterium tuberculosis mutants unable to synthesize phthiocerol dimycocerosates (DIMs) was recently shown to be impaired in mouse lungs. However, the precise role of these molecules in the course of infection remained to be determined. Here, we provide evidence that the attenuation of a DIM-deficient strain takes place during the acute phase of infection in both lungs and spleen of mice, and that this attenuation results in part from the increased sensitivity of the mutant to the cidal activity of reactive nitrogen intermediates released by activated macrophages. We also show that the DIM-deficient mutant, the growth and survival of which were not impaired within resting macrophages and dendritic cells, induced these cells to secrete more tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 than the wild-type strain. Although purified DIM molecules by themselves had no effect on the activation of macrophages and dendritic cells in vitro, we found that the proper localization of DIMs in the cell envelope of M. tuberculosis is critical to their biological effects. Thus, our findings suggest that DIM production contributes to the initial growth of M. tuberculosis by protecting it from the nitric oxide-dependent killing of macrophages and modulating the early immune response to infection.     

The LFA-1 adhesion molecule is required for protective immunity during pulmonary Mycobacterium tuberculosis infection

Host immunity to Mycobacterium tuberculosis is mediated by T cells that recognize and activate infected macrophages to control intracellular bacterial replication. The early appearance of T cells in the lungs of infected mice correlates with greater resistance to infection. However, it is unknown whether the trafficking of T cells to the lung following infection is dependent upon the expression of certain adhesion molecules. To address this question, we infected knockout (KO) mice that have defective expression of CD11a, CD11b, CD18, CD62, CD103, or beta7. We found that the integrins CD11a and CD18 are absolutely required for host resistance following infection with aerosolized M. tuberculosis. Although Ag-specific T cells are generated following infection of CD11a KO mice, T cell priming is delayed, T cell trafficking to the lung is impaired, and fewer ESAT6-specific CD4+ T cells are found in the lungs of CD11a KO mice compared with control mice. Thus, LFA-1 (CD11a/CD18) plays an essential role in immunity to M. tuberculosis infection.     

Dynamic roles of type I and type II IFNs in early infection with Mycobacterium tuberculosis

Although the protective role of type II IFN, or IFN-γ, against Mycobacterium tuberculosis has been established, the effects of type I IFNs are still unclear. One potential confounding factor is the overlap of function between the two signaling pathways. We used mice carrying null mutations in the type I IFNR, type II IFNR, or both and compared their immune responses to those of wild-type mice following aerosol infection with M. tuberculosis. We discovered that, in the absence of a response to IFN-γ, type I IFNs play a nonredundant protective role against tuberculosis. Mice unable to respond to both types of IFNs had more severe lung histopathology for similar bacterial loads and died significantly earlier than did mice with impaired IFN-γ signaling alone. We excluded a role for type I IFN in T cell recruitment, which was IFN-γ dependent, whereas both types of IFNs were required for optimal NK cell recruitment to the lungs. Type I IFN had a time-dependent influence on the composition of lung myeloid cell populations, in particular by limiting the abundance of M. tuberculosis-infected recruited macrophages after the onset of adaptive immunity. We confirmed that response to IFN-γ was essential to control intracellular mycobacterial growth, without any additional effect of type I IFN. Together, our results imply a model in which type I IFN limit the number of target cells that M. tuberculosis can infect in the lungs, whereas IFN-γ enhances their ability to restrict bacterial growth.     

Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis ( M.tb ) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show that siderocalin expression is upregulated following M.tb infection of mouse macrophage cell lines and primary murine alveolar macrophages. Furthermore, siderocalin added exogenously as a recombinant protein or overexpressed in the RAW264.7 macrophage cell line inhibited the intracellular growth of the pathogen. A variant form of siderocalin, which is expressed only in the macrophage cytosol, inhibited intracellular M.tb growth as effectively as the normal, secreted form, an observation that provides mechanistic insight into how siderocalin might influence iron acquisition by the bacteria in the phagosome. Our findings are consistent with an important role for siderocalin in protection against M.tb infection and suggest that exogenously administered siderocalin may have therapeutic applications in tuberculosis.     

Coronin-1a inhibits autophagosome formation around Mycobacterium tuberculosis-containing phagosomes and assists mycobacterial survival in macrophages

Mycobacterium tuberculosis is an intracellular bacterium that can survive within macrophages. Such survival is potentially associated with Coronin-1a (Coro1a). We investigated the mechanism by which Coro1a promotes the survival of M. tuberculosis in macrophages and found that autophagy was involved in the inhibition of mycobacterial survival in Coro1a knock-down (KD) macrophages. Fluorescence microscopy and immunoblot analyses revealed that LC3, a representative autophagic protein, was recruited to M. tuberculosis-containing phagosomes in Coro1a KD macrophages. Thin-section electron microscopy demonstrated that bacilli were surrounded by the multiple membrane structures in Coro1a KD macrophages. The proportion of LC3-positive mycobacterial phagosomes colocalized with p62/SQSTM1, ubiquitin or LAMP1 increased in Coro1a KD macrophages during infection. These results demonstrate the formation of autophagosomes around M. tuberculosis in Coro1a KD macrophages. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was induced in response to M. tuberculosis infection in Coro1a KD macrophages, suggesting that Coro1a blocks the activation of the p38 MAPK pathway involved in autophagosome formation. LC3 recruitment to M. tuberculosis-containing phagosomes was also observed in Coro1a KD alveolar or bone marrow-derived macrophages. These results suggest that Coro1a inhibits autophagosome formation in alveolar macrophages, thereby facilitating M. tuberculosis survival within the lung.     

Mycobacterium tuberculosis subverts innate immunity to evade specific effectors

The macrophage is the niche of the intracellular pathogen Mycobacterium tuberculosis. Induction of macrophage apoptosis by CD4(+) or CD8(+) T cells is accompanied by reduced bacterial counts, potentially defining a host defense mechanism. We have already established that M. tuberculosis-infected primary human macrophages have a reduced susceptibility to Fas ligand (FasL)-induced apoptosis. To study the mechanisms by which M. tuberculosis prevents apoptotic signaling, we have generated a cell culture system based on PMA- and IFN-gamma-differentiated THP-1 cells recapitulating the properties of primary macrophages. In these cells, nucleotide-binding oligomerization domain 2 or TLR2 agonists and mycobacterial infection protected macrophages from apoptosis and resulted in NF-kappaB nuclear translocation associated with up-regulation of the antiapoptotic cellular FLIP. Transduction of a receptor-interacting protein-2 dominant-negative construct showed that nucleotide-binding oligomerization domain 2 is not involved in protection in the mycobacterial infection system. In contrast, both a dominant-negative construct of the MyD88 adaptor and an NF-kappaB inhibitor abrogated the protection against FasL-mediated apoptosis, showing the implication of TLR2-mediated activation of NF-kappaB in apoptosis protection in infected macrophages. The apoptosis resistance of infected macrophages might be considered as an immune escape mechanism, whereby M. tuberculosis subverts innate immunity signaling to protect its host cell against FasL(+)-specific cytotoxic lymphocytes.     

CD4+ T cells mediate IFN-gamma-independent control of Mycobacterium tuberculosis infection both in vitro and in vivo

Although IFN-gamma is necessary for survival of Mycobacterium tuberculosis infection in people and animal models, it may not be sufficient to clear the infection, and IFN-gamma is not a reliable correlate of protection. To determine whether IFN-gamma-independent mechanisms of immunity exist, we developed a murine ex vivo culture system that directly evaluates the ability of splenic or lung lymphocytes to control the growth of M. tuberculosis within infected macrophages, and that models in vivo immunity to tuberculosis. Surprisingly, CD4(+) T cells controlled >90% of intracellular M. tuberculosis growth in the complete absence of IFN-gamma stimulation of macrophages, via a NO-dependent mechanism. Furthermore, bacillus Calmette-Guerin-vaccinated IFN-gamma-deficient mice exhibited significant protection against M. tuberculosis challenge that was lost upon depletion of CD4(+) T cells. These findings demonstrate that CD4(+) T cells possess IFN-gamma-independent mechanisms that can limit the growth of an intracellular pathogen and are dominant in secondary responses to M. tuberculosis.     

Studies on neutral exopolysaccharides produced by the ectomycorrhiza Thelephora terrestris

We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process.     

Comparative ability of human monocytes and macrophages to control the intracellular growth of Mycobacterium avium and Mycobacterium tuberculosis: effect of interferon-gamma and indomethacin

Abstract Intracellular growth of Mycobacterium avium and M. tuberculosis H37Rv was compared both in human peripheral blood monocytes and in cultured macrophages. The cells were treated with 300 U of human recombinant interferon-gamma (IFNγ) either 48 h prior to phagocytosis or after infection. In some cases, indomethacin (IND, a potent inhibitor of prostaglandin-E₂ synthesis), was added immediately after infection of macrophages. IFNγ pretreatment of monocytes resulted in about 50% lesser uptake of both pathogens, but had no effect in macrophages. Macrophages, as compared to monocytes, were more permissive to M. avium growth suggesting that monocytes may be innately more efficient in controlling the intracellular growth of this pathogen. About ten-fold higher growth of M. avium as compared to M. tuberculosis was observed in both culture systems. IFNγ-treatment alone did not confer any anti- M. avium activity to monocytes and macrophages alike and addition of IND did not change this unresponsiveness. In the case of M. tuberculosis , the IFNγ treatment alone endowed both monocytes and macrophages with significant bacteriostatic activity which was further potentiated by the addition of IND. These observations show innate differences in the ability of human monocytes and macrophages to control the growth of two major mycobacterial pathogens and the immunoregulatory mechanisms involved.     

Macrophage apoptosis in response to high intracellular burden of Mycobacterium tuberculosis is mediated by a novel caspase-independent pathway

We previously reported that macrophage exposure to attenuated strains of pathogenic mycobacteria at multiplicities of infection (MOI) < or = 10 triggers TNF-alpha-mediated apoptosis which reduces the viability of intracellular bacilli. Virulent strains were found to suppress macrophage apoptosis, and it was proposed that apoptosis is an innate defense against intracellular Mycobacterium tuberculosis analogous to apoptosis of virus-infected cells. The potential similarity of host cell responses to intracellular infection with mycobacteria and viruses suggests that M. tuberculosis might lyse infected macrophage when that niche is no longer needed. To investigate this question, we challenged murine macrophages with high intracellular bacillary loads. A sharp increase in cytolysis within 24 h was observed at MOI > or = 25. The primary death mode was apoptosis, based on nuclear morphology and phosphatidyl serine exposure, although the apoptotic cells progressed rapidly to necrosis. Apoptosis at high MOI differs markedly from low MOI apoptosis: it is potently induced by virulent M. tuberculosis, it is TNF-alpha-independent, and it does not reduce mycobacterial viability. Caspase inhibitors failed to prevent high MOI apoptosis, and macrophages deficient in caspase-3, MyD88, or TLR4 were equally susceptible as wild type. Apoptosis was reduced in the presence of cathepsin inhibitors, suggesting the involvement of lysosomal proteases in this novel death response. We conclude that the presence of high numbers of intracellular M. tuberculosis bacilli triggers a macrophage cell death pathway that could promote extracellular spread of infection and contribute to the formation of necrotic lesions in tuberculosis.