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CD4+ T cells mediate protection against Mycobacterium tuberculosis (Mtb); however, the phenotype of protective T cells is undefined, thereby confounding vaccination efforts. IL-27 is highly expressed during human tuberculosis (TB), and absence of IL-27R (Il27ra) specifically on T cells results in increased protection. IL-27R deficiency during chronic Mtb infection does not impact antigen-specific CD4+ T cell number but maintains programmed death-1 (PD-1), CD69, and CD127 expression while reducing T-bet and killer cell lectin-like receptor G1 (KLRG1) expression. Furthermore, T-bet haploinsufficiency results in failure to generate KLRG1+, antigen-specific CD4+ T cells, and in improved protection. T cells in Il27ra−/− mice accumulate preferentially in the lung parenchyma within close proximity to Mtb, and antigen-specific CD4+ T cells lacking IL-27R are intrinsically more fit than intact T cells and maintain IL-2 production. Improved fitness of IL-27R–deficient T cells is not associated with increased proliferation but with decreased expression of cell death–associated markers. Therefore, during Mtb infection, IL-27R acts intrinsically on T cells to limit protection and reduce fitness, whereas the IL-27R–deficient environment alters the phenotype and location of T cells. The significant expression of IL-27 in TB and the negative influence of IL-27R on T cell function demonstrate the pathway by which this cytokine/receptor pair is detrimental in TB.

ABSTRACTWe identify an N-ethyl-N-nitrosourea (ENU)-induced I23N mutation in the THEMIS protein that causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium bergheiANKA. ThemisI23Nhomozygous mice show reduced CD4+and CD8+T lymphocyte numbers. ECM resistance in P. bergheiANKA-infected ThemisI23Nmice is associated with decreased cerebral cellular infiltration, retention of blood-brain barrier integrity, and reduced proinflammatory cytokine production. THEMISI23Nprotein expression is absent from mutant mice, concurrent with the decreased THEMISI23Nstability observed in vitro. Biochemical studies in vitroand functional complementation in vivoin ThemisI23N/+:Lck−/+doubly heterozygous mice demonstrate that functional coupling of THEMIS to LCK tyrosine kinase is required for ECM pathogenesis. Damping of proinflammatory responses in ThemisI23Nmice causes susceptibility to pulmonary tuberculosis. Thus, THEMIS is required for the development and ultimately the function of proinflammatory T cells. ThemisI23Nmice can be used to study the newly discovered association of THEMIS(6p22.33) with inflammatory bowel disease and multiple sclerosis.

ABSTRACTIL12RB1is a human gene that is important for resistance to Mycobacterium tuberculosisinfection. IL12RB1is expressed by multiple leukocyte lineages, and encodes a type I transmembrane protein (IL12Rβ1) that associates with IL12p40 and promotes the development of host-protective TH1cells. Recently, we observed that il12rb1—the mouse homolog of IL12RB1—is alternatively spliced by leukocytes to produce a second isoform (IL12Rβ1ΔTM) that has biological properties distinct from IL12Rβ1. Although the expression of IL12Rβ1ΔTM is elicited by M. tuberculosisin vivo, and its overexpression enhances IL12p40 responsiveness in vitro, the contribution of IL12Rβ1ΔTM to controlling M. tuberculosisinfection has not been tested. Here, we demonstrate that IL12Rβ1ΔTM represents a secreted product of il12rb1that, when absent from mice, compromises their ability to control M. tuberculosisinfection in extrapulmonary organs. Furthermore, elevated M. tuberculosisburdens in IL12Rβ1ΔTM-deficient animals are associated with decreased lymph node cellularity and a decline in TH1 development. Collectively, these data support a model wherein IL12Rβ1ΔTM is a secreted product of il12rb1that promotes resistance to M. tuberculosisinfection by potentiating THcells response to IL-12.

During inflammatory responses and wound healing, the conversion of soluble fibrinogen to fibrin, an insoluble extracellular matrix, long has been assumed to create a scaffold for the migration of leukocytes and fibroblasts. Previous studies concluded that fibrinogen is a necessary cofactor for mycobacterial trehalose 6,6'-dimycolate-induced responses, because trehalose dimycolate-coated beads, to which fibrinogen was adsorbed, were more inflammatory than those to which other plasma proteins were adsorbed. Herein, we investigate roles for fibrin(ogen) in an in vivo model of mycobacterial granuloma formation and in infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. In wild-type mice, the subcutaneous injection of trehalose dimycolate-coated polystyrene microspheres, suspended within Matrigel, elicited a pyogranulomatous response during the course of 12 days. In fibrinogen-deficient mice, neutrophils were recruited but a more suppurative lesion developed, with the marked degradation and disintegration of the matrix. Compared to that in wild-type mice, the early formation of granulation tissue in fibrinogen-deficient mice was edematous, hypocellular, and disorganized. These deficiencies were complemented by the addition of exogenous fibrinogen. The absence of fibrinogen had no effect on cell recruitment or cytokine production in response to trehalose dimycolate, nor was there a difference in lung histopathology or overall bacterial burden in mice infected with Mycobacterium tuberculosis. In this model, fibrin(ogen) was not required for cell recruitment, cytokine response, or response to infection, but it promoted granulation tissue formation and suppressed leukocyte necrosis.

Mice lacking expression of granulocyte macrophage‐colony stimulating factor (GM‐CSF KO) are unable to contain Mycobacterium tuberculosis(M. tuberculosis) growth and succumb to infection by 35 days following pulmonary challenge. GM‐CSF KO mice do not express normal levels of the inflammatory cytokine tumor necrosis factor α (TNF‐α) nor the chemokines, regulated on activation, normal T expressed and secreted (RANTES), macrophage‐inflammatory protein‐1β (MIP‐1β), MIP‐1α, and lymphotactin, which are required for recruitment of lymphocytes and expression of a T helper cell type 1 (TH1) response within the lungs. In contrast, transgenic mice overexpressing GM‐CSF in the lungs but with a lack of GM‐CSF in other organs (GM+) are able to recruit lymphocytes and to express a TH1 response with production of TNF‐α and interferon‐γ in the lungs. However, GM+ mice succumb to infection between 60 and 90 days post‐challenge, as they are unable to develop a normal granulomatous response. Although GM+ mice are able to express the chemokine RANTES, they lack the ability to express other inflammatory chemokines such as lymphotactin and MIP‐1β. We conclude that GM‐CSF is essential to the recruitment of lymphocytes and expression of a TH1 response in the lung, to the generation of a normal mononuclear granuloma, and most importantly, to the containment of M. tuberculosisbacterial growth.

Infection by virulent Mycobacterium avium caused progressive severe lymphopenia in C57BL/6 mice due to increased apoptosis rates. T-cell depletion did not occur in gamma interferon (IFN-γ)-deficient mice which showed increased T-cell numbers and proliferation; in contrast, deficiency in nitric oxide synthase 2 did not prevent T-cell loss. Although T-cell loss was IFN-γ dependent, expression of the IFN-γ receptor on T cells was not required for depletion. Similarly, while T-cell loss was optimal if the T cells expressed IFN-γ, CD8+ T-cell depletion could occur in the absence of T-cell-derived IFN-γ. Depletion did not require that the T cells be specific for mycobacterial antigen and was not affected by deficiencies in the tumor necrosis factor receptors p55 or p75, the Fas receptor (CD95), or the respiratory burst enzymes or by forced expression of bcl-2 in hematopoietic cells.

ABSTRACTMice that were transgenic for a T-cell receptor (TCR) specific for ovalbumin peptide323-339(DO11.10) were able to survive an infection with Mycobacterium tuberculosisfor approximately 80 days. This limited early control of infection was associated with gamma interferon production, inducible nitric oxide synthase expression within the lung, and an influx of clonotypic lymphocytes. The control of M. tuberculosiswas lost in DO11.10 mice bred in a ragmutant background, demonstrating that the immune responsiveness was recombinase dependent and likely to be associated with the expression of an alternative α TCR by DO11.10 mice. A characterization of the antigen specificity in DO11.10 TCR transgenic mice demonstrated that the specificity was limited and dominated by the 26-kDa (Rv1411c) lipoprotein of M. tuberculosis. This study identifies this lipoprotein as an important and potent inducer of protective T cells within the lungs of mice infected with M. tuberculosisand therefore as a possible target for vaccination.

Severe combined immunodeficiency (SCID) mice were used to analyze the role of NK cells in resistance to Mycobacterium avium. The neutralization of IFN-gamma in these animals led to an exacerbation of the infection associated with a reduction in macrophage activation, suggesting a role for NK cells in innate immunity to mycobacteria. In contrast, administration of anti-asialo-GM(1) polyclonal serum or mAb specific for Thy1.2 did not affect mycobacterial growth or macrophage activation despite causing the almost complete abrogation of the natural cytolysis of a tumor cell target. Treatment with anti-asialo-GM(1)-specific serum depleted only two-thirds of the Thy1.2+ spleen cells, and anti-Thy1.2 treatment allowed for the persistence of a small number of cells still exhibiting an NK cell marker recognized by mAb DX5 and able to express IFN-gamma as analyzed by flow cytometry. In vivo treatment of B6.SCID mice with anti-NK1.1 mAb again failed to affect resistance to infection and allowed for the persistence of 2-8% of IFN-gamma-producing cells, many of them still expressing the DX5 marker. In vitro depletion studies showed that removal of IFN-gamma-expressing cells required the combined action of anti-Thy1.2, anti-Ly49C and DX5 antibodies in the presence of complement. Our data show that resistance to M. avium mediated by NK cells is independent of their cytolytic activity, and that there is a marked phenotypic and functional heterogeneity of the NK cell lineage in vivo during infection.

ABSTRACTMacrophages produce reactive oxygen species and reactive nitrogen species that have potent antimicrobial activity. Resistance to killing by macrophages is critical to the virulence ofMycobacterium tuberculosis. M. tuberculosishas two genes encoding superoxide dismutase proteins, sodAandsodC. SodC is a Cu,Zn superoxide dismutase responsible for only a minor portion of the superoxide dismutase activity ofM. tuberculosis. However, SodC has a lipoprotein binding motif, which suggests that it may be anchored in the membrane to protect M. tuberculosisfrom reactive oxygen intermediates at the bacterial surface. To examine the role of the Cu,Zn superoxide dismutase in protecting M. tuberculosisfrom the toxic effects of exogenously generated reactive oxygen species, we constructed a null mutation in thesodCgene. In this report, we show that theM. tuberculosis sodCmutant is readily killed by superoxide generated externally, while the isogenic parentalM. tuberculosisis unaffected under these conditions. Furthermore, the sodCmutant has enhanced susceptibility to killing by gamma interferon (IFN-γ)-activated murine peritoneal macrophages producing oxidative burst products but is unaffected by macrophages not activated by IFN-γ or by macrophages from respiratory burst-deficient mice. These observations establish that the Cu,Zn superoxide dismutase contributes to the resistance of M. tuberculosisagainst oxidative burst products generated by activated macrophages.

Interferon-γ is a pivotal cytokine in the protective response to tuberculosis. In its absence rampant bacterial growth results in tissue destruction and death. While macrophage activation is key, this pleiotropic cytokine has other secondary but significant roles. To investigate these roles, both intravenous and aerosol infection of the IFN-γ gene disrupted (GKO) mouse was performed. For the first time we describe the very similar growth of bacteria, during the initial phase of infection, between control and GKO mice. During this initial phase, however, very different histopathologic consequences between control and GKO mice were observed. Key observations included an early increased accumulation of granulocytes and a much more rapid and pronounced interstitial pneumonia in the GKO mice. As infection developed, GKO mice mounted an antigen-specific response; however, lymphocyte activation was much more rapid in these mice. Of interest is the fact that this increased rapidity occurred prior to significant differences in bacterial number. Taken together these data support a role for IFN-γ in limiting both initial cellular recruitment and acquired lymphocytic responses to mycobacterial infection. This role may be key in surviving the kind of chronic stimulatory disease caused by Mycobacterium tuberculosis.

ABSTRACTThe interleukin-12 and gamma interferon (IFN-γ) pathway of macrophage activation plays a pivotal role in controlling tuberculosis. In the murine model, the generation of supplementary nitric oxide by the induction of the nitric oxide synthase 2 (NOS2) gene product is considered the principal antimicrobial mechanism of IFN-γ-activated macrophages. Using a low-dose aerosol-mediated infection model in the mouse, we have investigated the role of nitric oxide in controllingMycobacterium tuberculosisin the lung. In contrast to the consequences of a systemic infection, a low dose of bacteria introduced directly into the lungs of mice lacking the NOS2 gene is controlled almost as well as in intact animals. This is in contrast to the rapid progression of disease in mice lacking IFN-γ or a key member of the IFN signaling pathway, interferon regulatory factor 1. Thus while IFN-γ is pivotal in early control of bacterial growth in the lung, this control does not completely depend upon the expression of the NOS2 gene. The absence of inducible nitric oxide in the lung does, however, result in increased polymorphonuclear cell involvement and eventual necrosis in the pulmonary granulomas of the infected mice lacking the NOS2 gene.

ABSTRACTImmunity to Mycobacterium tuberculosisis dependent upon the generation of a protective gamma interferon (IFN-γ)-producing T-cell response. Recent studies have suggested that interleukin-6 (IL-6) is required for the induction of a protective T-cell response and that IL-4 may suppress the induction of IFN-γ. To evaluate the role of the cytokines IL-6 and IL-4 in the generation of pulmonary immunity to M. tuberculosis, IL-6 and IL-4 knockout mice were infected with M. tuberculosisvia aerosol. The absence of IL-6 led to an early increase in bacterial load with a concurrent delay in the induction of IFN-γ. However, mice were able to contain and control bacterial growth and developed a protective memory response to secondary infection. This demonstrates that while IL-6 is involved in stimulating early IFN-γ production, it is not essential for the development of protective immunity against M. tuberculosis. In contrast, while the absence of IL-4 resulted in increased IFN-γ production, this had no significant effect upon bacterial growth.

ABSTRACTMycobacterium tuberculosisis an important respiratory pathogen the growth of which is controlled primarily by cytokine-activated macrophages. One of the principal mediators of this control is nitric oxide; however, superoxide has recently been shown to be protective in systemic mycobacterial infections. To determine whether superoxide is important in controlling M. tuberculosisduring primary pulmonary infection, mice lacking the cytosolic p47phoxgene (which is essential for effective superoxide production by the NADPH oxidase) were infected aerogenically. The lack of superoxide during an aerosol infection withM. tuberculosisresulted in a significant increase in bacterial growth over the early period of infection. Once antigen-specific gamma interferon-producing lymphocytes were detected in the draining lymph nodes, however, bacterial growth in the lung stopped. One interesting consequence of the lack of superoxide was an increase in neutrophilic infiltrates within the granuloma. This may be a consequence of increased tissue damage due to more rapid bacterial growth or may reflect a role for superoxide in controlling inflammation.

ABSTRACTEvidence showing that neutrophils play a protective role in the host response to infection by different intracellular parasites has been published in the past few years. We assessed this issue with regard to the infection of mice with Mycobacterium tuberculosis. We found a chronic recruitment of neutrophils to the infection foci, namely, to the peritoneal cavity after intraperitoneal infection and to the spleen and liver after intravenous inoculation of the mycobacteria. However, bacilli were never found associated with the recruited neutrophils but rather were found inside macrophages. The intravenous administration of the antineutrophil monoclonal antibody RB6-8C5 during the first week of infection led to selective and severe neutropenia associated with an enhancement of bacillary growth in the target organs of the mice infected by the intravenous route. The neutropenia-associated exacerbation of infection was most important in the liver, where a bacterial load 10-fold higher than that in nonneutropenic mice was found; the exacerbation in the liver occurred both during and after the neutropenic period. Early in infection by M. tuberculosis, neutropenic mice expressed lower levels of mRNAs for gamma interferon and inducible nitric oxide synthase in the liver compared to nondepleted mice. These results point to a protective role of neutrophils in the host defense mechanisms against M. tuberculosis, which occurs early in the infection and is not associated with the phagocytic activity of neutrophils but may be of an immunomodulatory nature.

ABSTRACTIsoxyl (ISO), a thiourea (thiocarlide; 4,4′-diisoamyloxythiocarbanilide), demonstrated potent activity againstMycobacterium tuberculosisH37Rv (MIC, 2.5 μg/ml),Mycobacterium bovisBCG (MIC, 0.5 μg/ml),Mycobacterium avium(MIC, 2.0 μg/ml), andMycobacterium aurumA+ (MIC, 2.0 μg/ml), resulting in complete inhibition of mycobacteria grown on solid media. Importantly, a panel of clinical isolates of M. tuberculosisfrom different geographical areas with various drug resistance patterns were all sensitive to ISO in the range of 1 to 10 μg/ml. In a murine macrophage model, ISO exhibited bactericidal killing of viable intracellular M. tuberculosisin a dose-dependent manner (0.05 to 2.50 μg/ml). The selective action of ISO on mycolic acid synthesis was studied through the use of [1,2-14C]acetate labeling of M. tuberculosisH37Rv, M. bovisBCG, and M. aurumA+. At its MIC for M. tuberculosis, ISO inhibited the synthesis of both fatty acids and mycolic acids (α-mycolates by 91.6%, methoxymycolates by 94.3%, and ketomycolates by 91.1%); at its MIC in M. bovisBCG, ISO inhibited the synthesis of α-mycolates by 87.2% and that of ketomycolates by 88.5%; and the corresponding inhibitions for M. aurumA+ were 87.1% for α-mycolates, 87.2% for ketomycolates, and 86.5% for the wax-ester mycolates. A comparison with isoniazid (INH) and ethionamide (ETH) demonstrated marked similarity in action, i.e., inhibition of the synthesis of all kinds of mycolic acids. However, unlike INH and ETH, ISO also inhibited the synthesis of shorter-chain fatty acids. ISO showed no acute toxicity against primary macrophage cell cultures as demonstrated by diminution of redox activity. A homologous series of ISO derivatives were synthesized. Most derivatives were as effective or more effective than the parent compound in the agar proportion assay. Thus, these thioureas, like INH and ETH, specifically inhibit mycolic acid synthesis and show promise in counteracting a wide variety of drug-sensitive and -resistant strains of M. tuberculosis.

ABSTRACTThe cytokine gamma interferon (IFN-γ) plays a major role in the control of Mycobacterium aviuminfections. We assessed whether the progressive growth of virulent strains of M. aviumwas associated with alterations in the production of this cytokine as evaluated by reverse transcription-PCR and detection of immunoreactive cytokine in the serum and in spleen homogenates. We found that IFN-γ was induced during infection by a virulent strain ofM. aviumto similar or even higher extents than the levels found during infections by a less virulent strain whose growth was controlled. IFN-γ produced during infection by both mycobacterial strains was partly derived from T cells and led to activation of macrophages, namely, those that were infected. Concomitant with the development of the infection with the virulent strain of M. aviumthere was an extensive depletion of lymphocytes in the spleen. Thymectomy alone promoted the proliferation of the virulent, but not of the less virulent, strain of M. avium. Our data indicate that virulent strains of M. aviumresist the antimicrobial mechanisms of IFN-γ-activated macrophages and raise the possibility that a second, T-cell-dependent signal is required for the effective control of mycobacterial replication inside macrophages.

ABSTRACTSeveral studies have shown that γδ T cells influence granuloma development after infection with intracellular pathogens. The role of γδ T cells in controlling the influx of inflammatory cells into the lung after Mycobacterium aviuminfection was therefore examined with gene-disrupted mice (K/O). The mice were infected with either M. avium724, a progressively replicating highly virulent strain of M. avium, or with M. avium2-151 SmT, a virulent strain that induces a chronic infection. γδ-K/O mice infected with M. avium2-151 SmT showed early enhanced bacterial growth within the lung compared to the wild-type mice, although granuloma formation was similar in both strains. γδ-K/O mice infected with M. avium724 showed identical bacterial growth within the lung compared to the wild-type mice, but they developed more-compact lymphocytic granulomas and did not show the extensive neutrophil influx and widespread tissue necrosis seen in wild-type mice. These data support the hypothesis that isolates of M. aviumthat induce protective T-cell-specific immunity are largely unaffected by the absence of γδ T cells. Whereas with bacterial strains that induce poor protective immunity, the absence of γδ T cells led to significant reductions in both the influx of neutrophils and tissue damage within the lungs of infected mice.

ABSTRACTIt remains unknown whether the expression of cell-mediated protective immunity and the capacity to mount a delayed-type hypersensitivity (DTH) reaction in tuberculosis infection represent two manifestations of a basic response or are dissociable events. In this study, we present data in favor of the latter hypothesis, by showing that tuberculosis infection in the lungs of mice possessing only a truncated form of intracellular adhesion molecule 1 due to gene disruption was still adequately controlled by the expression of protective immunity in the absence of any sustained influx of macrophages and the lack of formation of appreciable granulomas. These animals also had no detectable DTH response to mycobacterial proteins in the footpad assay, indicating that the accumulation of blood-borne macrophages at sites of mycobacterial infection or antigen deposition is not essential to control of the infection. These data support the hypothesis that the DTH component of the cellular response is not protective but contributes by walling off the sites of infection to prevent dissemination and reactivation disease.

Immunity to Mycobacterium tuberculosis infection is associated with the emergence of protective CD4 T cells that secrete cytokines, resulting in activation of macrophages and the recruitment of monocytes to initiate granuloma formation. The cytokine-mediating macrophage activation is interferon-γ (IFN-γ), which is largely dependent on interleukin-12 (IL-12) for its induction. To address the role of IL-12 in immunity to tuberculosis, IL-12 p40−/− mice were infected with M. tuberculosis and their capacity to control bacterial growth and other characteristics of their immune response were determined. The IL-12 p40−/− mice were unable to control bacterial growth and this appeared to be linked to the absence of both innate and acquired sources of IFN-γ. T cell activation as measured by delayed type hypersensitivity and lymphocyte accumulation at the site of infection were both markedly reduced in the IL-12 p40−/− mice. Therefore, IL-12 is essential to the generation of a protective immune response to M. tuberculosis, with its main functions being the induction of the expression of IFN-γ and the activation of antigen-specific lymphocytes capable of creating a protective granuloma.

Discussion on how discovery of the pathway by which Mycobacterium tuberculosis(Mtb) induces IL‐17 in naïve human peripheral blood cells aids our understanding of how Mtb interacts with the host immune response.