Expression of cathelicidin LL-37 during Mycobacterium tuberculosis infection in human alveolar macrophages, monocytes, neutrophils, and epithelial cells

The innate immune response in human tuberculosis is not completely understood. To improve our knowledge regarding the role of cathelicidin hCAP-18/LL37 in the innate immune response to tuberculosis infection, we used immunohistochemistry, immunoelectron microscopy, and gene expression to study the induction and production of the antimicrobial peptide in A549 epithelial cells, alveolar macrophages (AM), neutrophils, and monocyte-derived macrophages (MDM) after infection with Mycobacterium tuberculosis. We demonstrated that mycobacterial infection induced the expression and production of LL-37 in all cells studied, with AM being the most efficient. We did not detect peptide expression in tuberculous granulomas, suggesting that LL-37 participates only during early infection. Through the study of Toll-like receptors (TLR) in MDM, we showed that LL-37 can be induced by stimulation through TLR-2, TLR-4, and TLR-9. This last TLR was strongly stimulated by M. tuberculosis DNA. We concluded that LL-37 may have an important role in the innate immune response against M. tuberculosis.     

Early Secreted Antigenic Target of 6 kDa of Mycobacterium tuberculosis Stimulates Macrophage Chemoattractant Protein‐1 Production by Macrophages and Its Regulation by p38 Mitogen‐Activated Protein Kinases and Interleukin‐4

Early secreted antigenic target of 6 kDa (ESAT‐6), the major virulence factor of Mycobacterium tuberculosis, affects host immunity and the formation of granulomas likely through inflammatory cytokines. To understand its role in this regard further, we investigated the effect of ESAT‐6 on macrophages by determining the production of macrophage chemoattractant protein (MCP)‐1, a major chemokine associated with tuberculosis pathogenesis, by murine bone marrow‐derived macrophages (BMDMs) and its regulation by protein kinases and cytokines. The results revealed that ESAT‐6, but not Ag85A and culture filtrate protein 10 kDa (CFP10), induced MCP‐1 production by BMDMs dose and time dependently. Inhibition of p38 but not other mitogen‐activated protein kinases (MAPK) and PI3K further enhanced ESAT‐6‐induced MCP‐1 production by BMDMs. Inhibition of p38 MAPK enhanced ESAT‐6‐induced MCP‐1 mRNA accumulation without affecting mRNA stability. ESAT‐6 also induced TNF‐α from BMDMs and MCP‐1 from mouse lung epithelial cells, and these were suppressed by p38 MAPK inhibition, implying cytokine‐ and cell‐specific effect of p38 MAPK inhibition on ESAT‐6‐induced MCP‐1 by macrophages. Pretreatment of BMDMs with IL‐4, but not other cytokines (IL‐2, IL‐10, TNF‐α, IFN‐γ and IL‐1α) further elevated ESAT‐6‐stimulated MCP‐1 production although IL‐4 did not induce MCP‐1 without ESAT‐6. Both p38 MAPK inhibitor and IL‐4 did not show additive effect on ESAT‐6‐induced MCP‐1 protein level despite such effect on MCP‐1 mRNA level was evident. In conclusion, these results indicate a specific role for both p38 MAPK and IL‐4 in ESAT‐6‐induced MCP‐1 production by macrophages and suggest a pathway with significance in tuberculosis pathogenesis.     

Characterization of Novel Multiantigenic Vaccine Candidates with Pan-HLA Coverage against Mycobacterium tuberculosis

The low protection by the bacillus Calmette-Guérin (BCG) vaccine and existence of drug-resistant strains require better anti-Mycobacterium tuberculosis vaccines with a broad, long-lasting, antigen-specific response. Using bioinformatics tools, we identified five 19- to 40-mer signal peptide (SP) domain vaccine candidates (VCs) derived from M. tuberculosis antigens. All VCs were predicted to have promiscuous binding to major histocompatibility complex (MHC) class I and II alleles in large geographic territories worldwide. Peripheral mononuclear cells (PBMC) from healthy naïve donors and tuberculosis patients exhibited strong proliferation that correlated positively with Th1 cytokine secretion only in healthy naïve donors. Proliferation to SP VCs was superior to that to antigen-matched control peptides with similar length and various MHC class I and II binding properties. T-cell lines induced to SP VCs from healthy naïve donors had increased CD44^high/CD62L⁺ activation/effector memory markers and gamma interferon (IFN-γ), but not interleukin-4 (IL-4), production in both CD4⁺ and CD8⁺ T-cell subpopulations. T-cell lines from healthy naïve donors and tuberculosis patients also manifested strong, dose-dependent, antigen-specific cytotoxicity against autologous VC-loaded or M. tuberculosis-infected macrophages. Lysis of M. tuberculosis-infected targets was accompanied by high IFN-γ secretion. Various combinations of these five VCs manifested synergic proliferation of PBMC from selected healthy naïve donors. Immunogenicity of the best three combinations, termed Mix1, Mix2, and Mix3 and consisting of 2 to 5 of the VCs, was then evaluated in mice. Each mixture manifested strong cytotoxicity against M. tuberculosis-infected macrophages, while Mix3 also manifested a VC-specific humoral immune response. Based on these results, we plan to evaluate the protection properties of these combinations as an improved tuberculosis subunit vaccine.     

Innate response of human endothelial cells infected with mycobacteria

Endothelial cells are susceptible to infection by several pathogens, but little is known about mycobacterial infection. We analyzed some features of mycobacteria-endothelial cell interactions and the innate response to the infection. Intracellular growth in human umbilical vein endothelial cells (HUVECs) of three Mycobacterium species: M. tuberculosis (MTB), M. abscessus (MAB) and M. smegmatis (MSM) was analyzed. M. smegmatis was eliminated; M. abscessus had an accelerate intracellular replication and M. tuberculosis did not replicate or was eliminated. M. abscessus infection induced profound cytoskeleton rearrangements, with M. tuberculosis infection changes were less marked, and with MSM were slight. Nitric oxide (NO) production was induced differentially: M. abscessus induced the highest levels followed by M. tuberculosis and M. smegmatis; the contrary was true for reactive oxygen species (ROS) production. Only M. tuberculosis infection caused beta-1 defensin over-expression. As a whole, our results describe some aspects of the innate response of HUVEC infected by mycobacteria with different virulence and suggest that a strong cytoskeleton mobilization triggers a high NO production in these cells.     

Mycobacterium massiliense Induces Inflammatory Responses in Macrophages Through Toll-Like Receptor 2 and c-Jun N-Terminal Kinase

Mycobacterium massiliense (Mmass) is an emerging, rapidly growing mycobacterium (RGM) that belongs to the M. abscessus (Mabc) group, albeit clearly differentiated from Mabc. Compared with M. tuberculosis, a well-characterized human pathogen, the host innate immune response against Mmass infection is largely unknown. In this study, we show that Mmass robustly activates mRNA and protein expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in murine bone marrow-derived macrophages (BMDMs). Toll-like receptor (TLR)-2 and myeloid differentiation primary response gene 88 (MyD88), but neither TLR4 nor Dectin-1, are involved in Mmass-induced TNF-α or IL-6 production in BMDMs. Mmass infection also activates the mitogen-activated protein kinase (MAPKs; c-Jun N-terminal kinase (JNK), ERK1/2 and p38 MAPK) pathway. Mmass-induced TNF-α and IL-6 production was dependent on JNK activation, while they were unaffected by either the ERK1/2 or p38 pathway in BMDMs. Additionally, intracellular reactive oxygen species (ROS), NADPH oxidase-2, and nuclear factor-κB are required for Mmass-induced proinflammatory cytokine generation in macrophages. Furthermore, the S morphotype of Mmass showed lower overall induction of pro-inflammatory (TNF-α, IL-6, and IL-1β) and anti-inflammatory (IL-10) cytokines than the R morphotype, suggesting fewer immunogenic characteristics for this clinical strain. Together, these results suggest that Mmass-induced activation of host proinflammatory cytokines is mediated through TLR2-dependent JNK and ROS signaling pathways.     

LL-37 Immunomodulatory Activity during Mycobacterium tuberculosis Infection in Macrophages

Tuberculosis is one of the most important infectious diseases worldwide. The susceptibility to this disease depends to a great extent on the innate immune response against mycobacteria. Host defense peptides (HDP) are one of the first barriers to counteract infection. Cathelicidin (LL-37) is an HDP that has many immunomodulatory effects besides its weak antimicrobial activity. Despite advances in the study of the innate immune response in tuberculosis, the immunological role of LL-37 during M. tuberculosis infection has not been clarified. Monocyte-derived macrophages were infected with M. tuberculosis strain H37Rv and then treated with 1, 5, or 15 μg/ml of exogenous LL-37 for 4, 8, and 24 h. Exogenous LL-37 decreased tumor necrosis factor alpha (TNF-α) and interleukin-17 (IL-17) while inducing anti-inflammatory IL-10 and transforming growth factor β (TGF-β) production. Interestingly, the decreased production of anti-inflammatory cytokines did not reduce antimycobacterial activity. These results are consistent with the concept that LL-37 can modulate the expression of cytokines during mycobacterial infection and this activity was independent of the P2X7 receptor. Thus, LL-37 modulates the response of macrophages during infection, controlling the expression of proinflammatory and anti-inflammatory cytokines.     

MicroRNA-31 mediated by interferon regulatory factor 7 signaling facilitates control of Mycobacterium tuberculosis infection

Tuberculosis (TB) induced by Mycobacterium tuberculosis (M. tuberculosis) infection remains a global most deadly infectious disease. While development of more effective TB vaccines and therapeutics relies on identifications of true biomarkers designating an immune protection against M. tuberculosis infection, exact protective immune components against M. tuberculosis infection remain largely unidentified. We previously found that severe TB induced remarkable up-regulation of interferon regulatory factor 7 (IRF7) and IRF7-related gene signatures, implicating that some unknown downstream molecules in IRF7 signaling cascades may determine the M. tuberculosis infection outcomes and serve as a protective immune component against M. tuberculosis infection. Indeed, here, we observe that genetic ablation of IRF7 leads to more severe lung pathology, increased M. tuberculosis burdens, impaired differentiation of effector/memory T subsets, and extensively elevated expression of pro-inflammatory cytokines in lungs. Importantly, IRF7 is vital for sustaining expression of PD-1/PD-L1 and PD-1/PD-L1-modulated miRNA-31. Moreover, interventions of miRNA-31 expressions via administration of miRNA-31 agomir reduces lung pathology and bacilli burdens via inducing up-regulation of gene sets involved in biological processes of defense response or cellular and chemical homeostasis in lungs. Thus, this study uncovers previously unrecognized importance and mechanisms of IRF7-mediated miRNA-31 as a protective immune component against M. tuberculosis infection.     

Bystander macrophage apoptosis after Mycobacterium tuberculosis H37Ra infection

Human macrophages infected with Mycobacterium tuberculosis may undergo apoptosis. Macrophage apoptosis contributes to the innate immune response against M. tuberculosis by containing and limiting the growth of mycobacteria and also by depriving the bacillus of its niche cell. Apoptosis of infected macrophages is well documented; however, bystander apoptosis of uninfected macrophages has not been described in the setting of M. tuberculosis. We observed that uninfected human macrophages underwent significant bystander apoptosis 48 and 96 h after they came into contact with macrophages infected with avirulent M. tuberculosis. The bystander apoptosis was significantly greater than the background apoptosis observed in uninfected control cells cultured for the same length of time. There was no evidence of the involvement of tumor necrosis factor alpha, Fas, tumor necrosis factor-related apoptosis-inducing ligand, transforming growth factor β, Toll-like receptor 2, or MyD88 in contact-mediated bystander apoptosis. This newly described phenomenon may further limit the spread of M. tuberculosis by eliminating the niche cells on which the bacillus relies.     

β-Glucans inhibit intracellular growth of Mycobacterium bovis BCG but not virulent Mycobacterium tuberculosis in human macrophages

The yeast polysaccharide, β-glucan, has been shown to promote both anti-microbial and anti-tumor activities through its interaction with macrophages. Here we analyzed the effects of an insoluble whole glucan particle (WGP), a 1,3/1,6-β-glucan from Saccharomyces cerevisiae, and a soluble poly-1-6-β-d-glucopyranosyl-1-3-β-d-glucopyranose (PGG), a hydrolytic product of WGP, on the anti-microbial response of human macrophages against mycobacterial infection. Treatment of macrophages with WGP and PGG significantly decreased cell association and intracellular growth of Mycobacterium bovis BCG, but not Mycobacterium tuberculosis (M.tb) when compared to untreated controls. We characterized the influence of β-glucans on the generation of macrophage oxidative products and pro-inflammatory cytokines, two important anti-microbial defense mechanisms. WGP but not PGG treatment enhanced the oxidative response of macrophages as determined by the 2′,7′-dichlorofluorescin (DCF) assay. WGP treatment also induced macrophages to produce pro-inflammatory cytokines. The β-glucan receptor, Dectin-1, was found to be involved in the WGP-induced macrophage oxidative burst and intracellular growth inhibition of M. bovis BCG. This report indicates that although some forms of β-glucan are able to stimulate the respiratory burst and cytokine production in human macrophages, and exhibit anti-microbial properties against M. bovis BCG, the β-glucans tested here did not inhibit growth of M.tb within human macrophages.     

Toll-Like Receptor 2-Dependent Extracellular Signal-Regulated Kinase Signaling in Mycobacterium tuberculosis-Infected Macrophages Drives Anti-Inflammatory Responses and Inhibits Th1 Polarization of Responding T Cells

Mycobacterium tuberculosis survives within macrophages and employs immune evasion mechanisms to persist in the host. Protective T helper type 1 (Th1) responses are induced, and the immune response in most individuals is sufficient to restrict M. tuberculosis to latent infection, but most infections are not completely resolved. As T cells and macrophages respond, a balance is established between protective Th1-associated and other proinflammatory cytokines, such as interleukin-12 (IL-12), interferon gamma (IFN-γ), and tumor necrosis factor alpha, and anti-inflammatory cytokines, such as IL-10. The mechanisms by which M. tuberculosis modulates host responses to promote its survival remain unclear. In these studies, we demonstrate that M. tuberculosis induction of IL-10, suppression of IL-12, and inhibition of class II major histocompatibility complex (MHC-II) molecules in infected macrophages are all driven by Toll-like receptor 2 (TLR2)-dependent activation of the extracellular signal-regulated kinases (ERK). Elimination of ERK signaling downstream of TLR2 by pharmacologic inhibition with U0126 or genetic deletion of Tpl2 blocks IL-10 secretion and enhances IL-12 p70 secretion. We demonstrate that M. tuberculosis regulation of these pathways in macrophages affects T cell responses to infected macrophages. Thus, genetic blockade of the ERK pathway in Tpl2^−/− macrophages enhances Th1 polarization and IFN-γ production by antigen-specific CD4⁺ T cells responding to M. tuberculosis infection. These data indicate that M. tuberculosis and its potent TLR2 ligands activate ERK signaling in macrophages to promote anti-inflammatory macrophage responses and blunt Th1 responses against the pathogen.     

Mycobacterium tuberculosis PPE25 and PPE26 proteins expressed in Mycobacterium smegmatis modulate cytokine secretion in mouse macrophages and enhance mycobacterial survival

PPE25 and PPE26, the Mycobacterium tuberculosis proline-proline-glutamic acid (PPE) family proteins, are members of the M. tuberculosis ESX-5 system associated with virulence of M. tuberculosis. To investigate the roles of PPE25 and PPE26 during M. tuberculosis infection, we expressed them in non-pathogenic fast-growing Mycobacterium smegmatis, respectively, and used these recombinant strains to infect ANA-1 macrophages and BALB/c mice. We observed that both PPE25 and PPE26 enhanced survival of M. smegmatis in ANA-1 macrophages, and prolonged the persistence of M. smegmatis in mouse tissues. M. smegmatis-expressed PPE25 and PPE26 induced a significantly higher level of TNF-α and a slightly higher amount of IL-1β, which was found to be mediated by the NF-κB, ERK and p38 pathways in ANA-1 macrophages. In addition, M. smegmatis-expressed PPE26 inhibited synthase of inducible nitric oxide and induced stronger cell necrosis. In summary, our data suggest that PPE25 and PPE26 enhance non-pathogenic M. smegmatis to survive in ANA-1 macrophages and persistence in mice, modify expression of multiple cytokines and affect host cell necrosis. Our results could help to understand the complex interactions between the host and M. tuberculosis.     

Macrophage defense mechanisms against intracellular bacteria

Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics.     

Pattern recognition receptors and coordinated cellular pathways involved in tuberculosis immunopathogenesis: Emerging concepts and perspectives

Pattern Recognition Receptors (PRRs) play a central role in the recognition of numerous pathogens, including Mycobacterium tuberculosis, resulting in activation of innate and adaptive immune responses. Besides Toll Like Receptors, C-type Lectin Receptors and Nod Like Receptors are now being recognized for their involvement in inducing immune response against M. tuberculosis infection. Although, a functional redundancy of the PRRs has also been reported in many studies, emerging evidences support the notion that a cooperative and coordinated response generated by these receptors is critical to sustain the full immune control of M. tuberculosis infection. Many of the PRRs are now found to be involved in various cellular host defenses, such as inflammasome activation, phagosome biogenesis, endosomal trafficking, and antigen processing pathways that are all very critical for an effective immune response against M. tuberculosis. In support, polymorphism in several of these receptors has also been found associated with increased susceptibility to tuberculosis in humans. Nonetheless, increasing evidences also show that in order to enhance its intracellular survival, M. tuberculosis has also evolved multiple strategies to subvert and reprogram PPR-mediated immune responses. In light of these findings, this review analyzes the interaction of bacterial and host factors at the intersections of PRR signaling pathways that could provide integrative insights for the development of better vaccines and therapeutics for tuberculosis.     

A role for interleukin‐17A in modulating intracellular survival of Mycobacterium bovis bacillus Calmette–Guérin in murine macrophages

Interleukin 17A IL‐17A is a crucial immunomodulator in various chronic immunological diseases including rheumatoid arthritis and inflammatory bowel disease. The cytokine has also been demonstrated to control the pathogenesis of the Mycobacterium tuberculosis by dysregulating production of cytokines and chemokines and promoting granuloma formation. Whether IL‐17A regulates innate defence mechanisms of macrophages in response to mycobacterial infection remains to be elucidated. In the current report, we investigated the effects of IL‐17A on modulating the intracellular survival of Mycobacterium bovis bacillus Calmette–Guérin (BCG) in RAW264.7 murine macrophages. We observed that IL‐17A pre‐treatment for 24 hr was able to synergistically enhance BCG‐induced nitric oxide (NO) production and inducible nitric oxide synthase expression in dose‐ and time‐dependent manners. We further delineated the mechanisms involved in this synergistic reaction. IL‐17A was found to specifically enhanced BCG‐induced phosphorylation of Jun N‐terminal kinase (JNK), but not of extracellular signal‐regulated kinase 1/2 and p38 mitogen‐activated protein kinase. By using a specific JNK inhibitor (SP600125), we found that the production of NO in BCG‐infected macrophages was significantly suppressed. Taken together, we confirmed the involvement of the JNK pathway in IL‐17A‐enhanced NO production in BCG‐infected macrophages. We further demonstrated that IL‐17A significantly enhanced the clearance of intracellular BCG by macrophages through an NO‐dependent killing mechanism. In conclusion, our study revealed an anti‐mycobacterial role of IL‐17A through priming the macrophages to produce NO in response to mycobacterial infection.     

Chemokine Production by a Human Alveolar Epithelial Cell Line in Response to Mycobacterium tuberculosis

To investigate the role of chemokines during the initial local response to Mycobacterium tuberculosis in the human lung, we studied chemokine production by the human alveolar epithelial cell line A549 after infection with M. tuberculosis. M. tuberculosis-infected A549 cells produced mRNAs and protein for monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) but not mRNAs for macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and RANTES. Chemokine production in response to M. tuberculosis was not dependent on production of tumor necrosis factor alpha, IL-1β, or IL-6. Two virulent clinical M. tuberculosis isolates, the virulent laboratory strain H37Rv, and the avirulent strain H37Ra elicited production of comparable concentrations of MCP-1 and IL-8, whereas killed M. tuberculosis and three Mycobacterium avium strains did not. The three virulent M. tuberculosis strains grew more rapidly than the avirulent M. tuberculosis strain in the alveolar epithelial cell line, and the three M. avium strains did not grow intracellularly. These findings suggest that intracellular growth is necessary for mycobacteria to elicit production of MCP-1 and IL-8 by alveolar epithelial cells but that virulence and the rate of intracellular growth do not correlate with chemokine production. Alveolar epithelial cells may contribute to the local inflammatory response in human tuberculosis by producing chemokines which attract monocytes, lymphocytes, and polymorphonuclear cells.     

The role of interferon-gamma in the increased tuberculosis risk in type 2 diabetes mellitus

As patients with diabetes mellitus are at increased risk of developing tuberculosis, we hypothesized that this susceptibility to mycobacterial infection is due to a defective Th1-cytokine response. To explore this hypothesis, we examined four groups of subjects in Indonesia: 23 patients with tuberculosis, 34 patients with tuberculosis and diabetes, 32 patients with diabetes only and 36 healthy controls. Ex-vivo production of interferon (IFN)γ, tumour necrosis factor-α and interleukin (IL)-1β, 6, 10, -12 and -4 was measured following stimulation with Mycobacterium tuberculosis, Escherichia coli lipopolysaccharide and phytohaemagglutinin. Patients with active tuberculosis were found to have lower IFNγ levels and a higher production of other pro-inflammatory cytokines and IL-4, both in the presence and absence of diabetes. Diabetes patients without tuberculosis, however, showed strongly reduced non-specific IFNγ production, which is essential for inhibition of the initial growth of M. tuberculosis. Our data suggest that a defective non-specific immune response in diabetes may contribute to an increased susceptibility to develop tuberculosis.     

Nicotine modulates molecules of the innate immune response in epithelial cells and macrophages during infection with M. tuberculosis

Smoking increases susceptibility to becoming infected with and developing tuberculosis. Among the components of cigarette smoke, nicotine has been identified as the main immunomodulatory molecule; however, its effect on the innate immune system is unknown. In the present study, the effect of nicotine on molecules of the innate immune system was evaluated. Lung epithelial cells and macrophages were infected with Mycobacterium tuberculosis (Mtb) and/or treated with nicotine. The results show that nicotine alone decreases the expression of the Toll-like receptors (TLR)-2, TLR-4 and NOD-2 in all three cell types, as well as the production of the SP-D surfactant protein in type II pneumocytes. Moreover, it was observed that nicotine decreases the production of interleukin (IL)-6 and C-C chemokine ligand (CCL)5 during Mtb infection in epithelial cells (EpCs), whereas in macrophages derived from human monocytes (MDMs) there is a decrease in IL-8, IL-6, tumor necrosis factor (TNF)-α, IL-10, CCL2, C-X-C chemokine ligand (CXCL)9 and CXCL10 only during infection with Mtb. Although modulation of the expression of cytokines and chemokines appears to be partially mediated by the nicotinic acetylcholine receptor α7, blocking this receptor found no effect on the expression of receptors and SP-D. In summary, it was found that nicotine modulates the expression of innate immunity molecules necessary for the defense against tuberculosis.     

Efficient Activation of Human T Cells of Both CD4 and CD8 Subsets by Urease-Deficient Recombinant Mycobacterium bovis BCG That Produced a Heat Shock Protein 70-M. tuberculosis-Derived Major Membrane Protein II Fusion Protein

For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8⁺ T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8⁺ T cells and perforin-producing effector CD8⁺ T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.