CD43 controls the intracellular growth of Mycobacterium tuberculosis through the induction of TNF-alpha-mediated apoptosis

Establishment of Tuberculosis infection begins with the successful entry and survival of the pathogen within macrophages. We previously showed that macrophage CD43 is required for optimal uptake and growth inhibition of Mycobacterium tuberculosis both in vitro and in vivo. Here, we explore the mechanisms by which CD43 restricts mycobacterial growth in murine macrophages. We found that although M. tuberculosis grows more readily in resting CD43-/- macrophages, priming of cells with IFN-gamma returns the bacterial growth rate to that seen in CD43+/+ cells. To discern the mechanisms by which M. tuberculosis exhibits enhanced growth within resting CD43-/- macrophages, we assessed the induction of inflammatory mediators in response to infection. We found that absence of CD43 resulted in reduced production of TNF-alpha, IL-12 and IL-6 by M. tuberculosis-infected macrophages. We also found that infected resting, but not activated CD43-/- macrophages, showed decreased apoptosis and increased necrosis. Exogenous addition of the pro-inflammatory cytokine TNF-alpha restored control of M. tuberculosis growth and induction of apoptosis to CD43+/+ levels. We propose that CD43 is involved in the inflammatory response to M. tuberculosis and, through the induction of pro-inflammatory mediators, can regulate apoptosis to control intracellular growth of the bacterium.     

Utilization of CD11b knockout mice to characterize the role of complement receptor 3 (CR3, CD11b/CD18) in the growth of Mycobacterium tuberculosis in macrophages

Using CD11b knockout mice as a source of macrophages (Mphi;), we show that complement receptor 3 (CR3) mediates approximately 40-50% of nonopsonic binding and 50-60% of serum-mediated binding of Mycobacterium tuberculosis to resident Mphi;. We demonstrate that opsonic binding of M. tuberculosis to Mphi; is mediated by an immunoglobulin-independent, heat-labile component of serum, in both the presence and the absence of CD11b. The survival and replication of M. tuberculosis in an in vitro Mphi; model and an in vivo mouse model of infection were not significantly affected by the absence of CD11b, indicating that CR3-mediated uptake of M. tuberculosis is not a major factor in controlling the subsequent intracellular survival of the mycobacteria. However, whether a mycobacterium will gain access to the intracellular environment, and the type of Mφ that the bacterium enters, is significantly affected by the presence or absence of CR3.     

Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice

Endotoxin from Gram-negative bacteria bound to CD14 signals through Toll-like receptor (TLR) 4, while components of Gram-positive bacteria, fungi, and Mycobacterium tuberculosis (M.tb.) preferentially use TLR2 signaling. We asked whether TLR4 plays any role in host resistance to M.tb. infection in vivo. Therefore, we infected the TLR4 mutant C3H/HeJ mice and their controls, C3H/HeN mice, with M.tb. by aerosol. TLR4 mutant mice had a reduced capacity to eliminate mycobacteria from the lungs, spread the infection to spleen and liver, with 10-100 times higher CFU organ levels than the wild-type mice and succumbed within 5-7 mo, whereas most of the wild-type mice controlled infection and survived the duration of the experiment. The lungs of TLR4 mutant mice showed chronic pneumonia with increased neutrophil infiltration, reduced macrophages recruitment, and abundant acid-fast bacilli. Furthermore, the pulmonary expression of TNF-alpha, IL-12p40, and monocyte chemoattractant protein 1 was significantly lower in C3H/HeJ mice when compared with the wild-type controls. C3H/HeJ-derived macrophages infected in vitro with M.tb. produced lower levels of TNF-alpha. Finally, the purified mycobacterial glycolipid, phosphatidylinositol mannosides, induced signaling in both a TLR2- and TLR4-dependent manner, thus suggesting that recognition of phosphatidylinositol mannosides in vivo may influence the development of protective immunity. In summary, macrophage recruitment and the proinflammatory response to M.tb. are impaired in TLR4 mutant mice, resulting in chronic infection with impaired elimination of mycobacteria. Therefore, TLR4 signaling is required to mount a protective response during chronic M.tb. infection.     

Abnormally differentiated subsets of intestinal macrophage play a key role in Th1-dominant chronic colitis through excess production of IL-12 and IL-23 in response to bacteria

Disorders in enteric bacteria recognition by intestinal macrophages (Mphi) are strongly correlated with the pathogenesis of chronic colitis; however the precise mechanisms remain unclear. The aim of the current study was to elucidate the roles of Mphi in intestinal inflammation by using an IL-10-deficient (IL-10-/-) mouse colitis model. GM-CSF-induced bone marrow-derived Mphi (GM-Mphi) and M-CSF-induced bone marrow-derived Mphi (M-Mphi) were generated from bone marrow CD11b+ cells. M-Mphi from IL-10-/- mice produced abnormally large amounts of IL-12 and IL-23 upon stimulation with heat-killed whole bacteria Ags, whereas M-Mphi from wild-type (WT) mice produced large amounts of IL-10 but not IL-12 or IL-23. In contrast, IL-12 production by GM-Mphi was not significantly different between WT and IL-10-/- mice. In ex vivo experiments, cytokine production ability of colonic lamina propria Mphi (CLPMphi) but not splenic Mphi from WT mice was similar to that of M-Mphi, and CLPMphi but not splenic Mphi from IL-10-/- mice also showed abnormal IL-12p70 hyperproduction upon stimulation with bacteria. Surprisingly, the abnormal IL-12p70 hyperproduction from M-Mphi from IL-10-/- mice was improved by IL-10 supplementation during the differentiation process. These results suggest that CLPMphi and M-Mphi act as anti-inflammatory Mphi and suppress excess inflammation induced by bacteria in WT mice. In IL-10-/- mice, however, such Mphi subsets differentiated into an abnormal phenotype under an IL-10-deficient environment, and bacteria recognition by abnormally differentiated subsets of intestinal Mphi may lead to Th1-dominant colitis via IL-12 and IL-23 hyperproduction. Our data provide new insights into the intestinal Mphi to gut flora relationship in the development of colitis in IL-10-/- mice.     

NOD2-deficient mice have impaired resistance to Mycobacterium tuberculosis infection through defective innate and adaptive immunity

NOD2/CARD15 mediates innate immune responses to mycobacterial infection. However, its role in the regulation of adaptive immunity has remained unknown. In this study, we examined host defense, T cell responses, and tissue pathology in two models of pulmonary mycobacterial infection, using wild-type and Nod2-deficient mice. During the early phase of aerosol infection with Mycobacterium tuberculosis, Nod2(-/-) mice had similar bacterial counts but reduced inflammatory response on histopathology at 4 and 8 wk postchallenge compared with wild-type animals. These findings were confirmed upon intratracheal infection of mice with attenuated Mycobacterium bovis bacillus Calmette-Guérin. Analysis of the lungs 4 wk after bacillus Calmette-Guérin infection demonstrated that Nod2(-/-) mice had decreased production of type 1 cytokines and reduced recruitment of CD8(+) and CD4(+) T cells. Ag-specific T cell responses in both the spleens and thoracic lymph nodes were diminished in Nod2(-/-) mice, indicating impaired adaptive antimycobacterial immunity. The immune regulatory role of NOD2 was not restricted to the lung since Nod2 disruption also led to reduced type 1 T cell activation following i.m. bacillus Calmette-Guérin infection. To determine the importance of diminished innate and adaptive immunity, we measured bacterial burden 6 mo after aerosol infection with M. tuberculosis and followed a second infected group for assessment of survival. Nod2(-/-) mice had a higher bacterial burden in the lungs 6 mo after infection and succumbed sooner than did wild-type controls. Taken together, these data indicate that NOD2 mediates resistance to mycobacterial infection via both innate and adaptive immunity.     

Infection of B cell-deficient mice with CDC 1551, a clinical isolate of Mycobacterium tuberculosis: delay in dissemination and development of lung pathology

Long-term survival of mice infected with Mycobacterium tuberculosis is dependent upon IFN-gamma and T cells, but events in early phases of the immune response are not well understood. In this study, we describe a role for B cells during early immune responses to infection with a clinical isolate of M. tuberculosis (CDC 1551). Following a low-dose infection with M. tuberculosis CDC 1551, similar numbers of bacteria were detected in the lungs of both B cell knockout (IgH 6-, BKO) and C57BL/6J (wild-type) mice. However, despite comparable bacterial loads in the lungs, less severe pulmonary granuloma formation and delayed dissemination of bacteria from lungs to peripheral organs were observed in BKO mice. BKO mice reconstituted with naive B cells, but not those given M. tuberculosis-specific Abs, before infection developed pulmonary granulomas and dissemination patterns similar to wild-type animals. Further analysis of lung cell populations revealed greater numbers of lymphocytes, especially CD8+ T cells, macrophages, and neutrophils in wild-type and reconstituted mice than in BKO mice. Thus, less severe lesion formation and delayed dissemination of bacteria found in BKO mice were dependent on B cells, not Abs, and were associated with altered cellular infiltrate to the lungs. These observations demonstrate an important, previously unappreciated, role for B cells during early immune responses to M. tuberculosis infections.     

Decreased pathology and prolonged survival of human DC-SIGN transgenic mice during mycobacterial infection

Dendritic cell (DC)-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN: CD209) is a C-type lectin that binds ICAM-2,3 and various pathogens such as HIV, helicobacter, and mycobacteria. It has been suggested that Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, interacts with DC-SIGN to evade the immune system. To directly analyze the role of human DC-SIGN during mycobacterial infection, we generated conventional transgenic (tg) mice (termed "hSIGN") using CD209 cDNA under the control of the murine CD11c promoter. Upon mycobacterial infection, DCs from hSIGN mice produced significantly less IL-12p40 and no significant differences were be observed in the secretion levels of IL-10 relative to control DCs. After high dose aerosol infection with the strain M. tuberculosis H37Rv, hSIGN mice showed massive accumulation of DC-SIGN(+) cells in infected lungs, reduced tissue damage and prolonged survival. Based on our in vivo data, we propose that instead of favoring the immune evasion of mycobacteria, human DC-SIGN may have evolved as a pathogen receptor promoting protection by limiting tuberculosis-induced pathology.     

Maintenance of pulmonary Th1 effector function in chronic tuberculosis requires persistent IL-12 production

The mechanisms that prevent reactivation of latent Mycobacterium tuberculosis infection in asymptomatic individuals are poorly understood. Although IL-12 is critical for the induction of IFN-gamma-dependent host control of M. tuberculosis, the requirement for the cytokine in the maintenance of host resistance and pulmonary Th1 effector function has not yet been formally examined. In this study, we reconstituted IL-12p40-deficient mice with IL-12 during the first 4 wk of infection and then assessed the effects of cytokine withdrawal. Although IL-12 administration initially resulted in restricted mycobacterial growth and prolonged survival, the reconstituted animals eventually succumbed to infection. This breakdown in bacterial control was accompanied by a marked reduction in the numbers of IFN-gamma-producing CD4(+) T cells in lungs. Moreover, whereas CD4(+) T cells isolated from chronically infected wild-type mice expanded and transferred long-term protection to M. tuberculosis-challenged RAG(-/-) mice, they failed to do so in IL-12p40-deficient RAG(-/-) recipients and were clearly reduced in frequency within pulmonary granulomas in the latter animals. These studies establish that continuous IL-12 production is necessary for maintenance of the pulmonary Th1 cells required for host control of persistent M. tuberculosis infection and suggest that breakdown of this mechanism could be a contributing factor in reactivated disease.     

Exploiting type 3 complement receptor for TNF-alpha suppression, immune evasion, and progressive pulmonary fungal infection

TNF-alpha is crucial in defense against intracellular microbes. Host immune cells use type 3 complement receptors (CR3) to regulate excess TNF-alpha production during physiological clearance of apoptotic cells. BAD1, a virulence factor of Blastomyces dermatitidis, is displayed on yeast and released during infection. BAD1 binds yeast to macrophages (Mphi) via CR3 and CD14 and suppresses TNF-alpha, which is required for resistance. We investigated whether blastomyces adhesin 1 (BAD1) exploits host receptors for immune deviation and pathogen survival. Soluble BAD1 rapidly entered Mphi, accumulated intracellularly by 10 min after introduction to cells, and trafficked to early and late endosomes. Inhibition of receptor recycling by monodansyl cadaverine blocked association of BAD1 with Mphi and reversed TNF-alpha suppression in vitro. Inhibition of BAD1 uptake with cytochalasin D and FcR-redirected delivery of soluble BAD1 as Ag-Ab complexes or of wild-type yeast opsonized with IgG similarly reversed TNF-alpha suppression. Hence, receptor-mediated entry of BAD1 is requisite in TNF-alpha suppression, and the route of entry is critical. Binding of soluble BAD1 to Mphi of CR3(-/-) and CD14(-/-) mice was reduced to 50 and 33%, respectively, of that in wild-type mice. Mphi of CR3(-/-) and CD14(-/-) mice resisted soluble BAD1 TNF-alpha suppression in vitro, but, in contrast to CR3(-/-) cells, CD14(-/-) cells were still subject to suppression mediated by surface BAD1 on wild-type yeast. CR3(-/-) mice resisted both infection and TNF-alpha suppression in vivo, in contrast to wild-type and CD14(-/-) mice. BAD1 of B. dermatitidis thus co-opts normal host cell physiology by exploiting CR3 to subdue TNF-alpha production and foster pathogen survival.     

CD8+ T cells accumulate in the lungs of Mycobacterium tuberculosis-infected Kb-/-Db-/- mice,but provide minimal protection

Recent studies have shown that MHC class I molecules play an important role in the protective immune response to Mycobacterium tuberculosis infection. Here we showed that mice deficient in MHC class Ia, but possessing MHC class Ib (K(b-/-)D(b-/-) mice), were more susceptible to aerosol infection with M. tuberculosis than control mice, but less susceptible than mice that lack both MHC class Ia and Ib (beta(2)m(-/-) mice). The susceptibility of K(b-/-)D(b-/-) mice cannot be explained by the failure of CD8(+) T cells (presumably MHC class Ib-restricted) to respond to the infection. Although CD8(+) T cells were a relatively small population in uninfected K(b-/-)D(b-/-) mice, most already expressed an activated phenotype. During infection, a large percentage of these cells further changed their cell surface phenotype, accumulated in the lungs at the site of infection, and were capable of rapidly producing IFN-gamma following TCR stimulation. Histopathologic analysis showed widespread inflammation in the lungs of K(b-/-)D(b-/-) mice, with a paucity of lymphocytic aggregates within poorly organized areas of granulomatous inflammation. A similar pattern of granuloma formation has previously been observed in other types of MHC class I-deficient mice, but not CD8alpha(-/-) mice. Thus, neither the presence of MHC class Ib molecules themselves, nor the activity of a population of nonclassical CD8(+) effector cells, fully restored the deficit caused by the absence of MHC class Ia molecules, suggesting a unique role for MHC class Ia molecules in protective immunity against M. tuberculosis.     

Human toll-like receptors mediate cellular activation by Mycobacterium tuberculosis.

Recent studies have implicated a family of mammalian Toll-like receptors (TLR) in the activation of macrophages by Gram-negative and Gram-positive bacterial products. We have previously shown that different TLR proteins mediate cellular activation by the distinct CD14 ligands Gram-negative bacterial LPS and mycobacterial glycolipid lipoarabinomannan (LAM). Here we show that viable Mycobacterium tuberculosis bacilli activated both Chinese hamster ovary cells and murine macrophages that overexpressed either TLR2 or TLR4. This contrasted with Gram-positive bacteria and Mycobacterium avium, which activated cells via TLR2 but not TLR4. Both virulent and attenuated strains of M. tuberculosis could activate the cells in a TLR-dependent manner. Neither membrane-bound nor soluble CD14 was required for bacilli to activate cells in a TLR-dependent manner. We also assessed whether LAM was the mycobacterial cell wall component responsible for TLR-dependent cellular activation by M. tuberculosis. We found that TLR2, but not TLR4, could confer responsiveness to LAM isolated from rapidly growing mycobacteria. In contrast, LAM isolated from M. tuberculosis or Mycobacterium bovis bacillus Calmette-Guérin failed to induce TLR-dependent activation. Lastly, both soluble and cell wall-associated mycobacterial factors were capable of mediating activation via distinct TLR proteins. A soluble heat-stable and protease-resistant factor was found to mediate TLR2-dependent activation, whereas a heat-sensitive cell-associated mycobacterial factor mediated TLR4-dependent activation. Together, our data demonstrate that Toll-like receptors can mediate cellular activation by M. tuberculosis via CD14-independent ligands that are distinct from the mycobacterial cell wall glycolipid LAM.     

Mice deficient in CD4 T cells have only transiently diminished levels of IFN-gamma, yet succumb to tuberculosis

CD4 T cells are important in the protective immune response against tuberculosis. Two mouse models deficient in CD4 T cells were used to examine the mechanism by which these cells participate in protection against Mycobacterium tuberculosis challenge. Transgenic mice deficient in either MHC class II or CD4 molecules demonstrated increased susceptibility to M. tuberculosis, compared with wild-type mice. MHC class II-/- mice were more susceptible than CD4-/- mice, as measured by survival following M. tuberculosis challenge, but the relative resistance of CD4-/- mice did not appear to be due to increased numbers of CD4-8- (double-negative) T cells. Analysis of in vivo IFN-gamma production in the lungs of infected mice revealed that both mutant mouse strains were only transiently impaired in their ability to produce IFN-gamma following infection. At 2 wk postinfection, IFN-gamma production, assessed by RT-PCR and intracellular cytokine staining, in the mutant mice was reduced by >50% compared with that in wild-type mice. However, by 4 wk postinfection, both mutant and wild-type mice had similar levels of IFN-gamma mRNA and protein production. In CD4 T cell-deficient mice, IFN-gamma production was due to CD8 T cells. Thus, the importance of IFN-gamma production by CD4 T cells appears to be early in infection, lending support to the hypothesis that early events in M. tuberculosis infection are crucial determinants of the course of infection.     

Mycobacterium tuberculosis infection in complement receptor 3-deficient mice

Complement receptor type 3 (CR3) present on macrophages is used by Mycobacterium tuberculosis as one of its major phagocytic receptors. In this study, we examined the in vivo significance of CR3-mediated phagocytosis on the pathogenesis of disease caused by M. tuberculosis. The outcome of tuberculous infection in mice deficient in the CD11b subunit of CR3 (CR3-/-) on a mixed 129SV and C57BL background and control wild-type counterparts was comparable with respect to survival, bacterial burden, granulomatous lesion development, and cytokine expression in the spleen and lungs. M. tuberculosis infection was also examined in CR3-/- mice on C57BL/6 and BALB/c backgrounds and was found to be similar. In conclusion, our results suggest that in the absence of CR3, M. tuberculosis is able to gain entry into host cells via alternative phagocytic receptors and establish infection. The data also indicate that absence of CR3 does not alter disease course in either the relatively resistant C57BL/6 or the relatively susceptible BALB/c strains of mice.     

Codominance of TLR2-dependent and TLR2-independent modulation of MHC class II in Mycobacterium tuberculosis infection in vivo

Mycobacterium tuberculosis is an exceptionally successful human pathogen. A major component of this success is the ability of the bacteria to infect immunocompetent individuals and to evade eradication by an adaptive immune response that includes production of the macrophage-activating cytokine, IFN-gamma. Although IFN-gamma is essential for arrest of progressive tuberculosis, it is insufficient for efficacious macrophage killing of the bacteria, which may be due to the ability of M. tuberculosis to inhibit selected macrophage responses to IFN-gamma. In vitro studies have determined that mycobacterial lipoproteins and other components of the M. tuberculosis cell envelope, acting as agonists for TLR2, inhibit IFN-gamma induction of MHC class II. In addition, M. tuberculosis peptidoglycan and IL-6 secreted by infected macrophages inhibit IFN-gamma induction of MHC class II in a TLR2-independent manner. To determine whether TLR2-dependent inhibition of macrophage responses to IFN-gamma is quantitatively dominant over the TLR2-independent mechanisms in vivo, we prepared mixed bone marrow chimeric mice in which the hemopoietic compartment was reconstituted with a mixture of TLR(+/+) and TLR2(-/-) cells. When the chimeric mice were infected with M. tuberculosis, the expression of MHC class II on TLR2(+/+) and TLR2(-/-) macrophages from the lungs of individual infected chimeric mice was indistinguishable. These results indicate that TLR2-dependent and -independent mechanisms of inhibition of responses to IFN-gamma are equivalent in vivo, and that M. tuberculosis uses multiple pathways to abrogate the action of an important effector of adaptive immunity. This work was supported by National Institutes of Health Grants AI 065357-AI 020010.     

Selectin ligand-independent priming and maintenance of T cell immunity during airborne tuberculosis

Immunity to Mycobacterium tuberculosis infection is critically dependent on the timely priming of T effector lymphocytes and their efficient recruitment to the site of mycobacterial implantation in the lung. E-, P-, and L-selectin counterreceptors control lymphocyte homing to lymph nodes and leukocyte trafficking to peripheral sites of acute inflammation, their adhesive function depending on fucosylation by fucosyltransferases (FucT) IV and VII. To address the relative importance of differentially glycosylated selectin counterreceptors for priming of T cell effector functions in a model of mycobacteria-induced granulomatous pulmonary inflammation, we used aerosol-borne M. tuberculosis to infect FucT-IV-/-, FucT-VII-/-, FucT-IV-/-/FucT-VII-/-, or wild-type control mice. In lymph nodes, infected FucT-IV-/-/FucT-VII-/- and, to a lesser extent, FucT-VII-/- mice had severely reduced numbers of T cells and reduced Ag-specific effector responses. By contrast, recruitment of activated T cells into the lungs was similar in all four groups of mice during infection and expression of T cell, and macrophage effector functions were only delayed in lungs of FucT-IV-/-/FucT-VII-/- mice. Importantly, lungs from all groups expressed CXCL13, CCL21, and CCL19 and displayed organized follicular neolymphoid structures after infection with M. tuberculosis, which suggests that the lung served as a selectin ligand-independent priming site for immune responses to mycobacterial infection. All FucT-deficient strains were fully capable of restricting M. tuberculosis growth in infected organs until at least 150 days postinfection. Our observations indicate that leukocyte recruitment functions dictated by FucT-IV and FucT-VII-dependent selectin ligand activities are not critical for inducing or maintaining T cell effector responses at levels necessary to control pulmonary tuberculosis.     

CD4(+) T cells are required for the development of cytotoxic CD8(+) T cells during Mycobacterium tuberculosis infection.

The control of acute and chronic Mycobacterium tuberculosis infection is dependent on CD4(+) T cells. In a variety of systems CD8(+) T cell effector responses are dependent on CD4(+) T cell help. The development of CD8(+) T cell-mediated immune responses in the absence of CD4(+) T cells was investigated in a murine model of acute tuberculosis. In vitro and in vivo, priming of mycobacteria-specific CD8(+) T cells was unaffected by the absence of CD4(+) T cells. Infiltration of CD8(+) T cells into infected lungs of CD4(-/-) or wild-type mice was similar. IFN-gamma production by lung CD8(+) T cells in CD4(-/-) and wild-type mice was also comparable, suggesting that emergence of IFN-gamma-producing mycobacteria-specific CD8(+) T cells in the lungs was independent of CD4(+) T cell help. In contrast, cytotoxic activity of CD8(+) T cells from lungs of M. tuberculosis-infected mice was impaired in CD4(-/-) mice. Expression of mRNA for IL-2 and IL-15, cytokines critical for the development of cytotoxic effector cells, was diminished in the lungs of M. tuberculosis-infected CD4(-/-) mice. As tuberculosis is frequently associated with HIV infection and a subsequent loss of CD4(+) T cells, understanding the interaction between CD4(+) and CD8(+) T cell subsets during the immune response to M. tuberculosis is imperative for the design of successful vaccination strategies.     

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.     

Multifunctional, high-level cytokine-producing Th1 cells in the lung, but not spleen, correlate with protection against Mycobacterium tuberculosis aerosol challenge in mice

Boosting bacillus Calmette-Guérin (BCG)-primed mice with a recombinant adenovirus expressing Mycobacterium tuberculosis Ag 85A by different administration routes has very different effects on protection against aerosol challenge with M. tuberculosis. Mice boosted intradermally make very strong splenic CD4 and CD8 Th1 cytokine responses to Ag 85A, but show no change in lung mycobacterial burden over BCG primed animals. In contrast, intranasally boosted mice show greatly reduced mycobacterial burden and make a much weaker splenic response but a very strong lung CD4 and CD8 response to Ag 85A and an increased response to purified protein derivative. This effect is associated with the presence in the lung of multifunctional T cells, with high median fluorescence intensity and integrated median fluorescence intensity for IFN-gamma, IL-2, and TNF. In contrast, mice immunized with BCG alone have few Ag-specific cells in the lung and a low proportion of multifunctional cells, although individual cells have high median fluorescence intensity. Successful immunization regimes appear to induce Ag-specific cells with abundant intracellular cytokine staining.     

NKT cells are critical to initiate an inflammatory response after Pseudomonas aeruginosa ocular infection in susceptible mice

CD4(+) T cells produce IFN-gamma contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-gamma and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (-/-; no T/NKT cells), B6.TCR Jalpha281(-/-) (NKT cell deficient), alpha-galactosylceramide (alphaGalCer) (anergized NKT cells) injected and IL-12p40(-/-) vs B6 controls were tested. IFN-gamma mRNA was undetectable in RAG-1(-/-)- and alphaGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jalpha281(-/-), alphaGalCer-treated, and IL-12p40(-/-) (activated CD4(+) T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mphi; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-kappaB, and CD1d, and addition of rIFN-gamma potentiated Mphi CD1d levels. Together, these data suggest that Langerhans cell/Mphi recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-gamma production by NKT cells, that IFN-gamma is required to optimally activate NK cells to produce IFN-gamma, and that depletion of both NKT/NK cells results in earlier corneal perforation.