Role of Mincle in Alveolar Macrophage-Dependent Innate Immunity against Mycobacterial Infections in Mice

The role of macrophage-inducible C-type lectin Mincle in lung innate immunity against mycobacterial infection is incompletely defined. In this study, we show that wild-type (WT) mice responded with a delayed Mincle induction on resident alveolar macrophages and newly immigrating exudate macrophages to infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), peaking by days 14-21 posttreatment. As compared with WT mice, Mincle knockout (KO) mice exhibited decreased proinflammatory mediator responses and leukocyte recruitment upon M. bovis BCG challenge, and they demonstrated increased mycobacterial loads in pulmonary and extrapulmonary organ systems. Secondary mycobacterial infection on day 14 after primary BCG challenge led to increased cytokine gene expression in sorted alveolar macrophages of WT mice, but not Mincle KO mice, resulting in substantially reduced alveolar neutrophil recruitment and increased mycobacterial loads in the lungs of Mincle KO mice. Collectively, these data show that WT mice respond with a relatively late Mincle expression on lung sentinel cells to M. bovis BCG infection. Moreover, M. bovis BCG-induced upregulation of C-type lectin Mincle on professional phagocytes critically shapes antimycobacterial responses in both pulmonary and extrapulmonary organ systems of mice, which may be important for elucidating the role of Mincle in the control of mycobacterial dissemination in mice.     

A protective and agonistic function of IL-12p40 in mycobacterial infection.

IL-12p35(-/-)p40(-/-) mice are highly susceptible to Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis infection. In this study IL-12p35(-/-) mice, which are able to produce endogenous IL-12p40, cleared M. bovis BCG and showed reduced susceptibility to pulmonary M. tuberculosis infection, which was in striking contrast to the outcome of mycobacterial infection in IL-12p35(-/-)p40(-/-) mice. Resistance in wild-type and IL-12p35(-/-) mice was accompanied by protective granuloma formation and Ag-specific delayed-type hypersensitivity responses, which were impaired in susceptible IL-12p35(-/- )p40(-/-) mice. Furthermore, IL-12p35(-/-) mice, but not IL-12p35(-/-)p40(-/-) mice, mounted Ag-specific Th1 and cytotoxic T cell responses. In vivo therapy with rIL-12p40 homodimer restored the impaired delayed-type hypersensitivity responses in M. bovis BCG-infected IL-12p35(-/-)p40(-/-) mice and reverted them to a more resistant phenotype. Together, these results show evidence for a protective and agonistic role of endogenous and exogenous IL-12p40 in mycobacterial infection, which is independent of IL-12p70.     

IL-17-mediated regulation of innate and acquired immune response against pulmonary Mycobacterium bovis bacille Calmette-Guerin infection

IL-17 is a cytokine that induces neutrophil-mediated inflammation, but its role in protective immunity against intracellular bacterial infection remains unclear. In the present study, we demonstrate that IL-17 is an important cytokine not only in the early neutrophil-mediated inflammatory response, but also in T cell-mediated IFN-gamma production and granuloma formation in response to pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin (BCG). IL-17 expression in the BCG-infected lung was detected from the first day after infection and the expression depended on IL-23. Our observations indicated that gammadelta T cells are a primary source of IL-17. Lung-infiltrating T cells of IL-17-deficient mice produced less IFN-gamma in comparison to those from wild-type mice 4 wk after BCG infection. Impaired granuloma formation was also observed in the infected lungs of IL-17-deficient mice, which is consistent with the decreased delayed-type hypersensitivity response of the infected mice against mycobacterial Ag. These data suggest that IL-17 is an important cytokine in the induction of optimal Th1 response and protective immunity against mycobacterial infection.     

Minimal contribution of Valpha14 natural killer T cells to Th1 response and host resistance against mycobacterial infection in mice

We elucidated the contribution of Valpha14 NKT cells to Th1 response and host resistance against mycobacterial infection. In Valpha14 NKT cell-deficient mice, host defense and DTH response to Mycobacterium bovis BCG were not different from wild-type mice after pulmonary infection. There was no significant difference in the lung concentrations of IFN-gamma between the two strains of mice. In addition, host defense to systemic infection with M. tuberculosis was similar to that of M. bovis. Our results indicate that Valpha14 NKT cells play only a marginal role, if any, in the Th1 response and host resistance to mycobacterial infection.     

Transmembrane TNF induces an efficient cell-mediated immunity and resistance to Mycobacterium bovis bacillus Calmette-Guérin infection in the absence of secreted TNF and lymphotoxin-alpha

The contribution of a transmembrane (Tm) form of TNF to protective immunity against Mycobacterium bovis bacillus Calmette-Guérin (BCG) was studied in transgenic (tg) mice expressing a noncleavable Tm TNF but lacking the TNF/lymphotoxin-alpha (LT-alpha) locus (Tm TNF tg mice). These mice were as resistant to BCG infection as wild-type mice, whereas TNF/LT-alpha(-/-), TNF(-/-), and LT-alpha(-/-) mice succumbed. Tm TNF tg mice developed granulomas of smaller size but at 2- to 4-fold increased frequencies compared with wild-type mice. Granulomas were mainly formed by monocytes and activated macrophages expressing Tm TNF mRNA and accumulating acid phosphatase. NO synthase 2 activation as a key macrophage bactericidal mechanism was low during the acute phase of infection in Tm TNF tg mice but was still sufficient to limit bacterial growth and increased in late infection. While infection with virulent Mycobacterium tuberculosis resulted in very rapid death of TNF/LT-alpha(-/-) mice, it also resulted in survival of Tm TNF tg mice which presented an increase in the number of CFU in spleen (5-fold) and lungs (10-fold) as compared with bacterial load of wild-type mice. In conclusion, the Tm form of TNF induces an efficient cell-mediated immunity and total resistance against BCG even in the absence of LT-alpha and secreted TNF. However, Tm TNF-mediated protection against virulent M. tuberculosis infection can also be efficient but not as strong as in BCG infection, in which cognate cellular interactions may play a more predominant role in providing long-term surveillance and containment of BCG-infected macrophages.     

Mycobacterium bovis bacille Calmette-Guérin infection in the CNS suppresses experimental autoimmune encephalomyelitis and Th17 responses in an IFN-gamma-independent manner

Multiple sclerosis and an animal model resembling multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases of the CNS that are suppressed by systemic mycobacterial infection in mice and BCG vaccination in humans. Host defense responses against Mycobacterium in mice are influenced by T lymphocytes and their cytokine products, particularly IFN-gamma, which plays a protective regulatory role in EAE. To analyze the counter-regulatory role of mycobacterial infection-induced IFN-gamma in the CNS on the function of the pathological Th17 cells and the clinical outcome of EAE, we induced EAE in mice that were intracerebrally infected with Mycobacterium bovis bacille Calmette-Guerin (BCG). In this study, we demonstrate that intracerebral (i.c.) BCG infection prevented inflammatory cell recruitment to the spinal cord and suppressed the development of EAE. Concomitantly, there was a significant decrease in the frequency of myelin oligodendrocyte glycoprotein-specific IFN-gamma-producing CD4(+) T cells in the CNS. IL-17(+)CD4(+) T cell responses were significantly suppressed in i.c. BCG-infected mice following EAE induction regardless of T cell specificity. The frequency of Foxp3(+)CD4(+) T cells in these mice was equivalent to that of control mice. Intracerebral BCG infection-induced protection of EAE and suppression of myelin oligodendrocyte glycoprotein-specific IL-17(+)CD4(+) T cell responses were similar in both wild-type and IFN-gamma-deficient mice. These data show that live BCG infection in the brain suppresses CNS autoimmunity. These findings also reveal that the regulation of Th17-mediated autoimmunity in the CNS can be independent of IFN-gamma-mediated mechanisms.     

Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG

A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed. In contrast to r-S. typhimurium control, oral vaccination of mice with the r-S. typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M. bovis BCG strain. The immune response induced by r-S. typhimurium p30 was accompanied by augmented interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels produced by restimulated splenocytes. These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r-S. typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens.     

TLR3 regulates mycobacterial RNA-induced IL-10 production through the PI3K/AKT signaling pathway

Cytokine induction in response to Mycobacterium tuberculosis (Mtb) infection is critical for pathogen control, by (i) mediating innate immune effector functions and (ii) instructing specific adaptive immunity. IL-10 is an important anti-inflammatory cytokine involved in pathogenesis of tuberculosis (TB). Here, we show that TLR3, a sensor of extracellular viral or host RNA with stable stem structures derived from infected or damaged cells, is essential for Mtb-induced IL-10 production. Upon Mycobacterium bovis Bacillus Calmette–Guérin (BCG) infection, TLR3^−/− macrophages expressed lower IL-10 but higher IL-12p40 production, accompanied by reduced phosphorylation of AKT at Ser473. BCG-infected TLR3^−/− mice exhibited reduced IL-10 but elevated IL-12 expression compared to controls. Moreover, higher numbers of splenic Th1 cells and reduced pulmonary bacterial burden and tissue damage were observed in BCG-infected TLR3^−/− mice. Finally, BCG RNA induced IL-10 in macrophages via TLR3-mediated activation of PI3K/AKT. Our findings demonstrate a critical role of TLR3-mediated regulation in the pathogenesis of mycobacterial infection involving mycobacterial RNA, which induces IL-10 through the PI3K/AKT signaling pathway.     

Characterization of lung gamma delta T cells following intranasal infection with Mycobacterium bovis bacillus Calmette-Guérin

The lungs are considered to have an impaired capacity to contain infection by pathogenic mycobacteria, even in the presence of effective systemic immunity. In an attempt to understand the underlying cellular mechanisms, we characterized the gammadelta T cell population following intranasal infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). The peak of gammadelta T cell expansion at 7 days postinfection preceded the 30 day peak of alphabeta T cell expansion and bacterial count. The expanded population of gammadelta T cells in the lungs of BCG-infected mice represents an expansion of the resident Vgamma2 T cell subset as well as an influx of Vgamma1 and of four different Vdelta gene-bearing T cell subsets. The gammadelta T cells in the lungs of BCG-infected mice secreted IFN-gamma following in vitro stimulation with ionomycin and PMA and were cytotoxic against BCG-infected peritoneal macrophages as well as against the uninfected J774 macrophage cell line. The cytotoxicity was selectively blocked by anti-gammadelta TCR mAb and strontium ions, suggesting a granule-exocytosis killing pathway. Depletion of gammadelta T cells by injection of specific mAb had no effect on the subsequent developing CD4 T cell response in the lungs of BCG-infected mice, but significantly reduced cytotoxic activity and IFN-gamma production by lung CD8 T cells. Thus, gammadelta T cells in the lungs might help to control mycobacterial infection in the period between innate and classical adaptive immunity and may also play an important regulatory role in the subsequent onset of alphabeta T lymphocytes.     

In vivo persistence and protective efficacy of the bacille Calmette Guerin vaccine overexpressing the HspX latency antigen

New strategies to control infection with Mycobacterium tuberculosis, the causative agent of tuberculosis, are urgently required, particularly in areas where acquired immunodeficiencies are prevalent. In this report we have determined if modification of the current tuberculosis vaccine, Mycobacterium bovis BCG, to constitutively express the mycobacterial HspX latency antigen altered its protective effect against challenge with virulent M. tuberculosis. Overexpression of M. tuberculosis HspX in BCG caused reduced growth in aerated cultures compared to control BCG, but growth under limited oxygen availability was not markedly altered. Upon infection of mice, BCG:HspX displayed tissue-specific attenuation compared to control BCG, with reduced growth within the lung and liver but not the spleen. Both BCG:HspX and control BCG protected mice against aerosol M. tuberculosis challenge to a similar extent, however, immunodeficient mice infected with BCG:HspX survived significantly longer than mice infected with the control BCG strain. Therefore, altering the in vivo persistence of BCG by overexpression of HspX may be one important step towards developing a new tuberculosis vaccine with an improved safety profile and suitable protective efficacy against M. tuberculosis infection.     

Mycobacterium tuberculosis triggers host type I IFN signaling to regulate IL-1β production in human macrophages

Mycobacterium tuberculosis is a virulent intracellular pathogen that survives in macrophages even in the presence of an intact adaptive immune response. Type I IFNs have been shown to exacerbate tuberculosis in mice and to be associated with disease progression in infected humans. Nevertheless, the mechanisms by which type I IFNs regulate the host response to M. tuberculosis infection are poorly understood. In this study, we show that M. tuberculosis induces an IFN-related gene expression signature in infected primary human macrophages, which is dependent on host type I IFN signaling as well as the mycobacterial virulence factor, region of difference-1. We further demonstrate that type I IFNs selectively limit the production of IL-1β, a critical mediator of immunity to M. tuberculosis. This regulation occurs at the level of IL1B mRNA expression, rather than caspase-1 activation or autocrine IL-1 amplification and appears to be preferentially used by virulent mycobacteria since avirulent M. bovis bacillus Calmette-Guérin (BCG) fails to trigger significant expression of type I IFNs or release of mature IL-1β protein. The latter property is associated with decreased caspase-1-dependent IL-1β maturation in the BCG-infected macrophages. Interestingly, human monocytes in contrast to macrophages produce comparable levels of IL-1β in response to either M. tuberculosis or BCG. Taken together, these findings demonstrate that virulent and avirulent mycobacteria employ distinct pathways for regulating IL-1β production in human macrophages and reveal that in the case of M. tuberculosis infection the induction of type I IFNs is a major mechanism used for this purpose.     

Murine cytokine responses following multiple oral immunizations using lipid-formulated mycobacterial antigens

Oral vaccination of mice with live Mycobacterium bovis BCG in lipid-formulation induces a gamma-interferon response that can be measured systemically, and confers protection against aerosolized mycobacterial challenge. Here, we have investigated cytokine responses following the vaccination, drawing comparisons between mice that received single or multiple oral immunizations and between mice receiving formulations containing live BCG or non-replicating mycobacterial antigens. Single oral immunization with lipid-formulated live BCG invoked secreted and cellular IFN-gamma responses in mice 8 weeks post-vaccination, the magnitudes of which were significantly elevated in mice receiving multiple immunizations over the 8-week period. Single oral immunization with live BCG also invoked an interleukin-2 response (but not TNF-alpha or IL-4), although the magnitude was not elevated by multiple immunizations. Multiple immunizations with lipid-formulated soluble or particulate non-replicating mycobacterial antigens failed to invoke cytokine responses, except for a low-level IFN-gamma response in mice multiple immunized with lipid-formulated heat-killed BCG. These results are discussed in contrast to the known patterns of cytokine induction following parenteral-route immunization with live or non-replicating mycobacterial antigens and with practical reference to the development of oral-delivery vaccines against tuberculosis.     

Neutrophil elastase cleaves laminin-332 (laminin-5) generating peptides that are chemotactic for neutrophils

Proteolytic processing of laminin-332 by matrix metalloproteinase (MMP)-2 and MMP-14 has been shown to yield fragments that are promigratory for epithelial cells. During acute and chronic inflammation, proteases are elaborated by neutrophils and macrophages that can degrade basement membranes. We investigated the susceptibility of laminin-332 to degradation by the following neutrophil and macrophage proteases: neutrophil elastase (NE), cathepsin G, proteinase-3, and MMPs-2, -8, -9, and -12. Protease-specific differences were seen in the capacity to cleave the individual chains of laminin-332. NE and MMP-12 showed the greatest activity toward the gamma2 chain, generating a fragment similar in size to the gamma2x fragment generated by MMP-2. The digestion pattern of laminin-332 by degranulated neutrophils was nearly identical to that generated with NE alone. Digestion by supernatants of degranulated neutrophils was blocked by an inhibitor of NE, and NE-deficient neutrophils were essentially unable to digest laminin-332, suggesting that NE is the major neutrophil-derived protease that degrades laminin-332. In vivo, laminin gamma2 fragments were found in the bronchoalveolar lavage fluid of wild-type mice treated with lipopolysaccharide, whereas that obtained from NE-deficient mice showed a different cleavage pattern. In addition, NE cleaved a synthetic peptide derived from the region of human laminin gamma2 containing the MMP-2 cleavage site, suggesting that NE may generate laminin-332 fragments that are also promigratory. Both laminin-332 fragments generated by NE digestion and NE-digested laminin gamma2 peptide were found to be chemotactic for neutrophils. Collectively, these data suggest that degradation of laminin-332 by NE generates fragments with important biological activities.     

Inactivation of CD4+ CD25+ regulatory T cells during early mycobacterial infection increases cytokine production but does not affect pathogen load

Mycobacterium tuberculosis uses numerous mechanisms to avoid elimination by the infected host. In this study, we investigated the possibility whether, similar to other pathogens, M. tuberculosis exploits natural CD4+ CD25+ T-regulatory cells (Treg) to suppress the effector function of responding host lymphocytes, thus enhancing its survival. During a Mycobacterium bovis bacille calmette guerin (BCG) pulmonary infection, we observed a 2.8-fold increase in forkhead box P3 (Foxp3+) CD25+ Treg in the lung. To inactivate the Treg in vivo, an mAb was given against CD25 (PC61) 3 days before a pulmonary infection with BCG or M. tuberculosis. Following PC61 treatment, we observed significantly decreased CD25 expression on CD4+ T lymphocytes for at least 23 days in the blood, spleen and lung when compared with the control mice. To determine whether Treg inactivation affected the protective antimycobacterial immune response, we measured cytokine production by flow cytometry. We observed small, but significant increases in the percentages of both IFN-gamma-producing and IL-2-producing CD4+ cells from the spleen and the IL-2-producing CD4+ cells from the lungs of PC61-treated BCG-infected mice compared with the infected control mice. Despite this, there was neither a difference between the lung bacterial burdens of PC61-treated mice and control mice, measured until day 44 postinfection, nor was there an effect on infection-induced lung pathology. Together, these data imply that the absence of natural Treg early after infection results in a small increase in cytokine production, but this does not alter the course of either M. tuberculosis or BCG infections. This contrasts with the important role that natural Treg play in the pathogenesis of many other intracellular infectious organisms.     

Mycobacterium bovis BCG attenuates surface expression of mature class II molecules through IL-10-dependent inhibition of cathepsin S

We have previously shown that macrophage infection with Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) partially inhibits MHC class II surface expression in response to IFN-gamma. The present study examined the nature of class II molecules that do in fact reach the surface of infected cells. Immunostaining with specific Abs that discriminate between mature and immature class II populations showed a predominance of invariant chain (Ii)-associated class II molecules at the surface of BCG-infected cells suggesting that mycobacteria specifically block the surface export of peptide-loaded class II molecules. This phenotype was due to inhibition of IFN-gamma-induced cathepsin S (Cat S) expression in infected cells and the subsequent intracellular accumulation of alphabeta class II dimers associated with the Cat S substrate Ii p10 fragment. In contrast, infection with BCG was shown to induce secretion of IL-10, and addition of blocking anti-IL-10 Abs to cell cultures restored both expression of active Cat S and export of mature class II molecules to the surface of infected cells. Consistent with these findings, expression of mature class II molecules was also restored in cells infected with BCG and transfected with active recombinant Cat S. Thus, M. bovis BCG exploits IL-10 induction to inhibit Cat S-dependent processing of Ii in human macrophages. This effect results in inhibition of peptide loading of class II molecules and in reduced presentation of mycobacterial peptides to CD4(+) T cells. This ability may represent an effective mycobacterial strategy for eluding immune surveillance and persisting in the host.     

The role of histamine in the intracellular survival of Mycobacterium bovis BCG

The course and outcome of infection with mycobacteria are determined by a complex interplay between the immune system of the host and the survival mechanisms developed by the bacilli. Histamine plays an important role in various processes, including cell division, metabolism, and apoptosis, and it modulates innate and adaptive immune responses. In the present study we investigated the intracellular survival of Mycobacterium bovis BCG in murine bone-marrow macrophages isolated from wild-type (WT) and histidine-decarboxylase knock-out [HDC (-/-)] mice. Mycobacterial titers were significantly higher in the HDC (-/-) macrophages as compared with the WT cells. M. bovis BCG growth in WT macrophages could be enhanced by pyrilamine and cimetidine. Exogenously added histamine decreased the intracellular counts of M. bovis BCG in HDC (-/-) macrophages. Infection of activated macrophages with M. bovis BCG elicited apoptosis, but there was no significant difference between the WT and the HDC (-/-) cells. These bacilli induced comparable levels of tumor necrosis factor-alpha production in the WT and the HDC (-/-) macrophages. M. bovis BCG stimulated interleukin-18 (IL-18) production in the macrophages from WT mice, but not in the HDC (-/-) cells. Exogenously added IL-18 decreased the titers of intracellular mycobacteria in HDC (-/-) cells. In conclusion, these data implicate histamine in the intracellular survival of M. bovis BCG. The cellular control mechanisms restricting the growth of M. bovis BCG are complex and involve H1 and H2 receptor-mediated events. Histamine might be an important mediator of M. bovis BCG-induced IL-18 production, which in turn contributes to immune protection.     

Influence of bovine lactoferrin on expression of presentation molecules on BCG-infected bone marrow derived macrophages

The current vaccine for tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is an attenuated strain of Mycobacterium bovis bacillus Calmette-Guerin (BCG). BCG has proven to be effective in children, however, efficacy wanes in adulthood. Lactoferrin, a natural protein with immunomodulatory properties, is a potential adjuvant candidate to enhance efficacy of BCG. These studies define bovine lactoferrin as an enhancer of the BCG vaccine, functioning in part by modulating macrophage ability to present antigen and stimulate T-cells. BCG-infected bone marrow derived macrophages (BMMs) cultured with bovine lactoferrin increased the number of MHC II(+) expressing cells. Addition of IFN-gamma and lactoferrin to BCG-infected BMMs enhanced MHC II expressiona dna increased the ratio of CD86/CD80. Lactoferrin treated BCG-infected BMMs were able to stimulate an increase in IFN-gamma production from presensitized CD3(+) splenocytes. Together, these results demonstrate that bovine lactoferrin is capable of modulating BCG-infected macrophages to enhance T-cell stimulation through increased surface expression of antigen presentation and co-stimulatory molecules, which potentially explains the observed in vivo bovine lactoferrin enhancement of BCG vaccine efficacy to protect against virulent MTB infection.     

Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase.

A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope. In this report, the effect of the overexpression of the M. bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M. bovis BCG is described. The purification of the enzyme was performed to apparent homogeneity from overexpressing M. bovis BCG cells and its kinetic parameters were determined. The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100-1000-fold less efficiently. The best kcat/Km values were found with benzaldehyde > 3-methoxybenzaldehyde > octanal > coniferaldehyde. A phylogenetic analysis clearly revealed that the M. bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium-chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs). Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification. A possible role for the M. bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.