Immune control of Mycobacterium tuberculosis is dependent on both soluble TNFRp55 and soluble TNFRp75.

Tuberculosis presents a global health challenge, and tumour necrosis factor (TNF) signalling is required for host immunity against Mycobacterium tuberculosis (Mtb). TNF receptor shedding, however, compromises effective immunity by reducing bioactive TNF through the formation of inactive complexes. In this study, we first compared the effect of total soluble TNF receptors using a transgenic p55ΔNS /p75-/- murine strain on host protection during a low-dose aerosol Mtb H37Rv challenge. We report that the presence of membrane-bound TNFRp55 alone in the absence of TNFRp75 results in superior control of a primary Mtb infection where p55ΔNS /p75-/- hyperactive dendritic cells displayed an increased capacity to induce a hyperactive Mtb-specific CD4+ T-cell response. p55ΔNS /p75-/- dendritic cells expressed a higher frequency of MHCII and increased MFIs for both CD86 and MHCII, while CD4+ T cells had higher expression of CD44 and IFN-γ. Next, the relative contributions of soluble TNFRp55 and soluble TNFRp75 to host protection against either primary Mtb infection or during reactivation of latent tuberculosis were delineated by comparing the experimental outcomes of control C57BL/6 mice to transgenic p55ΔNS /p75-/- , p55ΔNS and p75-/- mouse strains. We found that soluble TNFRp55 is redundant for immune regulation during the chronic stages of a primary Mtb infection. However, TNFRp55 together with soluble TNFRp75 has a crucial role in immune regulation of reactivation of latent tuberculosis.

TNFRp75-dependent immune regulation of alveolar macrophages and neutrophils during early Mycobacterium tuberculosis and Mycobacterium bovis BCG infection.

TNF signalling through TNFRp55 and TNFRp75, and receptor shedding is important for immune activation and regulation. TNFRp75 deficiency leads to improved control of Mycobacterium tuberculosis (M. tuberculosis) infection, but the effects of early innate immune events in this process are unclear. We investigated the role of TNFRp75 on cell activation and apoptosis of alveolar macrophages and neutrophils during M. tuberculosis and M. bovis BCG infection. We found increased microbicidal activity against M. tuberculosis occurred independently of IFNy and NO generation, and displayed an inverse correlation with alveolar macrophages (AMs) apoptosis. Both M. tuberculosis and M. bovis BCG induced higher expression of MHC-II in TNFRp75-/- AMs; however, M bovis BCG infection did not alter AM apoptosis in the absence of TNFRp75. Pulmonary concentrations of CCL2, CCL3 and IL-1ß were increased in TNFRp75-/- mice during M, bovis BCG infection, but had no effect on neutrophil responses. Thus, TNFRp75-dependent regulation of mycobacterial replication is virulence dependent and occurs independently of early alveolar macrophage apoptosis and neutrophil responses.

The Use of Murine Infection Models to Investigate the Protective Role of TNF in Central Nervous System Tuberculosis.

Tuberculosis of the central nervous system (CNS-TB) is the most severe form of extra-pulmonary tuberculosis that is often associated with high mortality. Secretion of tumor necrosis factor (TNF) has important protective and immune modulatory functions for immune responses during CNS-TB. Therefore, by combining the approaches of aerosol and intracerebral infection in mice, this chapter describes the methods to investigate the contribution of TNF in protective immunity against CNS-TB infection.

Complete ablation of tumor necrosis factor decreases the production of IgA, IgG, and IgM in experimental central nervous system tuberculosis.

OBJECTIVES: This study aimed to explore the contribution of tumor necrosis factor (TNF) in the recruitment of B-cell and secretion of immunoglobulins (Igs) during cerebral tuberculosis (TB). MATERIALS AND METHODS: In this work, the contributing role of TNF in regulating Ig secretions was investigated by comparing wild type TNF (TNFf/f), B-cell-derived TNF (BTNF-/-), and complete TNF ablation (TNF-/-) in a mouse cerebral Mycobacterium tuberculosis infection. Using flow cytometry and ELISA, we were able to examine the recruitment of B-cell subsets, and the production of Igs; also assessed the expression of surface markers on B cell subsets. RESULTS: Here, we found that TNF-/- mice showed defective expression of IgA, IgG, and IgM antibodies compared with TNFf/f and BTNF-/- mice, which was significantly decreased in the expression of surface markers and co-stimulatory molecules. Moreover, mice that produced low antibody levels were not able to control infection, therefore progressed to disease; providing direct evidence for the TNF gene-regulating humoral immunity during central nervous system (CNS) M. tuberculosis infection. In contrast, BTNF-/- mice controlled the infection and had levels of IgA, IgG, and IgM comparable to TNFf/f mice. CONCLUSION: Together, our results demonstrate that TNF may serve as an essential regulator of antibody-mediated immune responses in CNS TB. However, the protective level exhibited by TNF-producing B cells could be defined as baseline protection that could be used in the development of new therapeutic targets or designing new vaccines.

In vitro and in vivo toxicity evaluation of non-neuroleptic phenothiazines, antitubercular drug candidates.

The phenothiazine-derived antipsychotic drugs, such as chlorpromazine and thioridazine, are bactericidal against drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis, but produce undesirable side effects at clinically relevant doses. We have previously modified four novel phenothiazines and maintained their antimycobacterial activity. This study evaluated the pharmacological and toxicity profiles of these novel non-neuroleptic phenothiazines, PTZ3, PTZ4, PTZ31 and PTZ32, for their metabolic stability, kinetic solubility and potential cytotoxic effects in vitro. To further support the safet use of these drug candidates, the in vivo pharmacological and toxicity profiles were assessed in C57BL/6 mice via single or repeated oral gavage. In acute toxicity studies, all four modified phenothiazines showed favourable safety in mice. When treated daily with 100 mg/kg of PTZ3 and PTZ4 for 2 weeks, mice displayed no signs of toxicity. Alternatively, treatment with PTZ31 resulted in 20% mortality with no toxicity evident in biochemical or histological analysis, while exposure to PTZ32 resulted in a 45% survival with increased serum concentrations of uric acid and alkaline phosphatase. The combined non-neuroleptic and antimycobacterial effects of the novel phenothiazines PTZ3, PTZ4, PTZ31 and PTZ32 demonstrated favourable pharmacological and toxicity profiles in this study, highlight the potential of these compounds as suitable anti-tuberculosis drug candidates.

GM-CSF targeted immunomodulation affects host response to M. tuberculosis infection.

Host directed immunomodulation represents potential new adjuvant therapies in infectious diseases such as tuberculosis. Major cytokines like TNFα exert a multifold role in host control of mycobacterial infections. GM-CSF and its receptor are over-expressed during acute M. tuberculosis infection and we asked how GM-CSF neutralization might affect host response, both in immunocompetent and in immunocompromised TNFα-deficient mice. GM-CSF neutralizing antibodies, at a dose effectively preventing acute lung inflammation, did not affect M. tuberculosis bacterial burden, but increased the number of granuloma in wild-type mice. We next assessed whether GM-CSF neutralization might affect the control of M. tuberculosis by isoniazid/rifampicin chemotherapy. GM-CSF neutralization compromised the bacterial control under sub-optimal isoniazid/rifampicin treatment in TNFα-deficient mice, leading to exacerbated lung inflammation with necrotic granulomatous structures and high numbers of intracellular M. tuberculosis bacilli. In vitro, GM-CSF neutralization promoted M2 anti-inflammatory phenotype in M. bovis BCG infected macrophages, with reduced mycobactericidal NO production and higher intracellular M. bovis BCG burden. Thus, GM-CSF pathway overexpression during acute M. tuberculosis infection contributes to an efficient M1 response, and interfering with GM-CSF pathway in the course of infection may impair the host inflammatory response against M. tuberculosis.

Immunity to the Dual Threat of Silica Exposure and Mycobacterium tuberculosis.

Exposure to silica and the consequent development of silicosis are well-known health problems in countries with mining and other dust producing industries. Apart from its direct fibrotic effect on lung tissue, chronic and immunomodulatory character of silica causes susceptibility to tuberculosis (TB) leading to a significantly higher TB incidence in silica-exposed populations. The presence of silica particles in the lung and silicosis may facilitate initiation of tuberculous infection and progression to active TB, and exacerbate the course and outcome of TB, including prognosis and survival. However, the exact mechanisms of the involvement of silica in the pathological processes during mycobacterial infection are not yet fully understood. In this review, we focus on the host's immunological response to both silica and Mycobacterium tuberculosis, on agents of innate and adaptive immunity, and particularly on silica-induced immunological modifications in co-exposure that influence disease pathogenesis. We review what is known about the impact of silica and Mycobacterium tuberculosis or their co-exposure on the host's immune system, especially an impact that goes beyond an exclusive focus on macrophages as the first line of the defense. In both silicosis and TB, acquired immunity plays a major role in the restriction and/or elimination of pathogenic agents. Further research is needed to determine the effects of silica in adaptive immunity and in the pathogenesis of TB.

Myeloid and T Cell-Derived TNF Protects against Central Nervous System Tuberculosis.

Tuberculosis of the central nervous system (CNS-TB) is a devastating complication of tuberculosis, and tumor necrosis factor (TNF) is crucial for innate immunity and controlling the infection. TNF is produced by many cell types upon activation, in particularly the myeloid and T cells during neuroinflammation. Here we used mice with TNF ablation targeted to myeloid and T cell (MT-TNF-/-) to assess the contribution of myeloid and T cell-derived TNF in immune responses during CNS-TB. These mice exhibited impaired innate immunity and high susceptibility to cerebral Mycobacterium tuberculosis infection, a similar phenotype to complete TNF-deficient mice. Further, MT-TNF-/- mice were not able to control T cell responses and cytokine/chemokine production. Thus, our data suggested that collective TNF production by both myeloid and T cells are required to provide overall protective immunity against CNS-TB infection.

Persistent p55TNFR expression impairs T cell responses during chronic tuberculosis and promotes reactivation.

The pleiotropic activities of TNF are mediated by two structurally related but functionally distinct type I transmembrane receptors, p55TNFR and p75TNFR expressed in most cell types, that can be cleaved and act as TNF scavengers. Here, we investigated the effect of persistent p55TNFR cell surface expression during aerosol inhalation challenge with virulent M. tuberculosis H37Rv. We demonstrated that persistency of p55TNFR in macrophage cultures increased the synthesis of soluble TNF, p75TNFR and NO, however, had no effects on bacteria killing ability. Furthermore, it did not facilitate enhanced protection to primary acute M. tuberculosis infection in p55∆NS mice. Without exacerbated lung inflammation, we found a compensatory increase in p75TNFR shedding and decrease in bioactive TNF in BAL of p55∆NS mice after M. tuberculosis challenge. Defective expressions of CD44 and INFγ attributed to an impaired T cell response during persistent p55TNFR expression that caused marginal transient susceptibility during chronic infection. Moreover, persistent p55TNFR expression induced early reactivation during latent tuberculosis infection. These data indicate a prominent role of p55TNFR shedding in Th1 mediated protection against chronic and latent tuberculosis infection.

Controlled Mycobacterium tuberculosis infection in mice under treatment with anti-IL-17A or IL-17F antibodies, in contrast to TNFα neutralization.

Antibodies targeting IL-17A or its receptor IL-17RA show unprecedented efficacy in the treatment of autoimmune diseases such as psoriasis. These therapies, by neutralizing critical mediators of immunity, may increase susceptibility to infections. Here, we compared the effect of antibodies neutralizing IL-17A, IL-17F or TNFα on murine host responses to Mycobacterium tuberculosis infection by evaluating lung transcriptomic, microbiological and histological analyses. Coinciding with a significant increase of mycobacterial burden and pathological changes following TNFα blockade, gene array analyses of infected lungs revealed major changes of inflammatory and immune gene expression signatures 4 weeks post-infection. Specifically, gene expression associated with host-pathogen interactions, macrophage recruitment, activation and polarization, host-antimycobacterial activities, immunomodulatory responses, as well as extracellular matrix metallopeptidases, were markedly modulated by TNFα blockade. IL-17A or IL-17F neutralization elicited only mild changes of few genes without impaired host resistance four weeks after M. tuberculosis infection. Further, the absence of both IL-17RA and IL-22 pathways in genetically deficient mice did not profoundly compromise host control of M. tuberculosis over a 6-months period, ruling out potential compensation between these two pathways, while TNFα-deficient mice succumbed rapidly. These data provide experimental confirmation of the low clinical risk of mycobacterial infection under anti-IL-17A therapy, in contrast to anti-TNFα treatment.

Innate myeloid cell TNFR1 mediates first line defence against primary Mycobacterium tuberculosis infection.

TNF is crucial for controlling Mycobacterium tuberculosis infection and understanding how will help immunomodulating the host response. Here we assessed the contribution of TNFR1 pathway from innate myeloid versus T cells. We first established the prominent role of TNFR1 in haematopoietic cells for controlling M. tuberculosis in TNFR1 KO chimera mice. Further, absence of TNFR1 specifically on myeloid cells (M-TNFR1 KO) recapitulated the uncontrolled M. tuberculosis infection seen in fully TNFR1 deficient mice, with increased bacterial burden, exacerbated lung inflammation, and rapid death. Pulmonary IL-12p40 over-expression was attributed to a prominent CD11b(+) Gr1(high) cell population in infected M-TNFR1 KO mice. By contrast, absence of TNFR1 on T-cells did not compromise the control of M. tuberculosis infection over 6-months. Thus, the protective TNF/TNFR1 pathway essential for controlling primary M. tuberculosis infection depends on innate macrophage and neutrophil myeloid cells, while TNFR1 pathway in T cells is dispensable.

TNF-dependent regulation and activation of innate immune cells are essential for host protection against cerebral tuberculosis.

BACKGROUND: Tuberculosis (TB) affects one third of the global population, and TB of the central nervous system (CNS-TB) is the most severe form of tuberculosis which often associates with high mortality. The pro-inflammatory cytokine tumour necrosis factor (TNF) plays a critical role in the initial and long-term host immune protection against Mycobacterium tuberculosis (M. tuberculosis) which involves the activation of innate immune cells and structure maintenance of granulomas. However, the contribution of TNF, in particular neuron-derived TNF, in the control of cerebral M. tuberculosis infection and its protective immune responses in the CNS were not clear. METHODS: We generated neuron-specific TNF-deficient (NsTNF(-/-)) mice and compared outcomes of disease against TNF(f/f) control and global TNF(-/-) mice. Mycobacterial burden in brains, lungs and spleens were compared, and cerebral pathology and cellular contributions analysed by microscopy and flow cytometry after M. tuberculosis infection. Activation of innate immune cells was measured by flow cytometry and cell function assessed by cytokine and chemokine quantification using enzyme-linked immunosorbent assay (ELISA). RESULTS: Intracerebral M. tuberculosis infection of TNF(-/-) mice rendered animals highly susceptible, accompanied by uncontrolled bacilli replication and eventual mortality. In contrast, NsTNF(-/-) mice were resistant to infection and presented with a phenotype similar to that in TNF(f/f) control mice. Impaired immunity in TNF(-/-) mice was associated with altered cytokine and chemokine synthesis in the brain and characterised by a reduced number of activated innate immune cells. Brain pathology reflected enhanced inflammation dominated by neutrophil influx. CONCLUSION: Our data show that neuron-derived TNF has a limited role in immune responses, but overall TNF production is necessary for protective immunity against CNS-TB.

Mycobacterium tuberculosis infection of the 'non-classical immune cell'.

Mycobacterium tuberculosis can infect 'non-classical immune cells', which comprise a significant constituency of cells that reside outside of those defined as 'classical immune cells' from myeloid or lymphoid origin. Here we address the influence of specific 'non-classical immune cells' in host responses and their effects in controlling mycobacterial growth or enabling an environment conducive for bacilli persistence. The interaction of M. tuberculosis with epithelial cells, endothelial cells, fibroblasts, adipocytes, glia and neurons and downstream cellular responses that often dictate immune regulation and disease outcome are discussed. Functional integration and synergy between 'classical' and 'non-classical immune cells' are highlighted as critical for determining optimal immune outcomes that favour the host.

Soluble TNFRp75 regulates host protective immunity against Mycobacterium tuberculosis.

Development of host protective immunity against Mycobacterium tuberculosis infection is critically dependent on the inflammatory cytokine TNF. TNF signals through 2 receptors, TNFRp55 and TNFRp75; however, the role of TNFRp75-dependent signaling in immune regulation is poorly defined. Here we found that mice lacking TNFRp75 exhibit greater control of M. tuberculosis infection compared with WT mice. TNFRp75-/- mice developed effective bactericidal granulomas and demonstrated increased pulmonary recruitment of activated DCs. Moreover, IL-12p40-dependent migration of DCs to lung draining LNs of infected TNFRp75-/- mice was substantially higher than that observed in WT M. tuberculosis-infected animals and was associated with enhanced frequencies of activated M. tuberculosis-specific IFN-γ-expressing CD4+ T cells. In WT mice, TNFRp75 shedding correlated with markedly reduced bioactive TNF levels and IL-12p40 expression. Neutralization of TNFRp75 in M. tuberculosis-infected WT BM-derived DCs (BMDCs) increased production of bioactive TNF and IL-12p40 to a level equivalent to that produced by TNFRp75-/- BMDCs. Addition of exogenous TNFRp75 to TNFRp75-/- BMDCs infected with M. tuberculosis decreased IL-12p40 synthesis, demonstrating that TNFRp75 shedding regulates DC activation. These data indicate that TNFRp75 shedding downmodulates protective immune function and reduces host resistance and survival; therefore, targeting TNFRp75 may be beneficial for improving disease outcome.

Novel non-neuroleptic phenothiazines inhibit Mycobacterium tuberculosis replication.

OBJECTIVES: Phenothiazines are a commercially available class of psychotropic drugs known to show antituberculosis activity. At clinically relevant bactericidal doses, however, the psychotropic drugs produce undesirable side effects in addition to their neuroleptic properties. This study aimed to evaluate rationally designed novel phenothiazines as antimycobacterial drug candidates. METHODS: Remodelling of psychotropic drugs by substitution of characteristic N-alkylamine side chains, important for CNS activity, with N-alkylsulphonates gave novel drug candidates, which were then tested for post-synaptic receptor binding affinity in a radioligand displacement assay. The bactericidal activities were screened using green fluorescent protein (GFP) microplate assays, and the efficacy of intracellular bacillus killing was evaluated by cfu enumeration. RESULTS: Of the four selected phenothiazine derivatives (PTZ3, PTZ4, PTZ31 and PTZ32) tested, PTZ31 displayed marginal serotonergic activity. The remaining three derivatives did not exhibit dopamine or serotonin receptor binding activity. In vitro results showed significant growth inhibition of virulent Mycobacterium tuberculosis with MICs of 12.5-25 mg/L. None of the phenothiazine derivatives displayed cytotoxicity in infected primary bone marrow-derived macrophages. Moreover, the phenothiazines showed significant antimycobacterial activity of between 40% and 60% against intracellular (ex vivo) M. tuberculosis. CONCLUSIONS: We demonstrate that structural modification of the phenothiazine core is possible in a manner that does not affect the ability of the phenothiazine derivatives to inhibit M. tuberculosis, but that abolishes undesirable dopamine and serotonin receptor binding.

Type I interferons contribute to experimental cerebral malaria development in response to sporozoite or blood-stage Plasmodium berghei ANKA.

Cerebral malaria is a severe complication of Plasmodium falciparum infection. Although T-cell activation and type II IFN-γ are required for Plasmodium berghei ANKA (PbA)-induced murine experimental cerebral malaria (ECM), the role of type I IFN-α/ß in ECM development remains unclear. Here, we address the role of the IFN-α/ß pathway in ECM devel-opment in response to hepatic or blood-stage PbA infection, using mice deficient for types I or II IFN receptors. While IFN-γR1⁻/⁻ mice were fully resistant, IFNAR1⁻/⁻ mice showed delayed and partial protection to ECM after PbA infection. ECM resistance in IFN-γR1⁻/⁻ mice correlated with unaltered cerebral microcirculation and absence of ischemia, while WT and IFNAR1⁻/⁻ mice developed distinct microvascular pathologies. ECM resistance appeared to be independent of parasitemia. Instead, key mediators of ECM were attenuated in the absence of IFNAR1, including PbA-induced brain sequestration of CXCR3⁺-activated CD8⁺ T cells. This was associated with reduced expression of Granzyme B, IFN-γ, IL-12Rß2, and T-cell-attracting chemokines CXCL9 and CXCL10 in IFNAR1⁻/⁻ mice, more so in the absence of IFN-γR1. Therefore, the type I IFN-α/ß receptor pathway contributes to brain T-cell responses and microvascular pathology, although it is not as essential as IFN-γ for the development of cerebral malaria upon hepatic or blood-stage PbA infection.

Prominent role for T cell-derived tumour necrosis factor for sustained control of Mycobacterium tuberculosis infection.

Tumour Necrosis Factor (TNF) is critical for host control of M. tuberculosis, but the relative contribution of TNF from innate and adaptive immune responses during tuberculosis infection is unclear. Myeloid versus T-cell-derived TNF function in tuberculosis was investigated using cell type-specific TNF deletion. Mice deficient for TNF expression in macrophages/neutrophils displayed early, transient susceptibility to M. tuberculosis but recruited activated, TNF-producing CD4(+) and CD8(+) T-cells and controlled chronic infection. Strikingly, deficient TNF expression in T-cells resulted in early control but susceptibility and eventual mortality during chronic infection with increased pulmonary pathology. TNF inactivation in both myeloid and T-cells rendered mice critically susceptible to infection with a phenotype resembling complete TNF deficient mice, indicating that myeloid and T-cells are the primary TNF sources collaborating for host control of tuberculosis. Thus, while TNF from myeloid cells mediates early immune function, T-cell derived TNF is essential to sustain protection during chronic tuberculosis infection.

The contraceptive depot medroxyprogesterone acetate impairs mycobacterial control and inhibits cytokine secretion in mice infected with Mycobacterium tuberculosis.

The contraceptive depot medroxyprogesterone acetate (DMPA), with progestin as the single active compound, possesses selective glucocorticoid activity and can alter the expression of glucocorticoid receptor-regulated genes. We therefore propose that pharmacological doses of DMPA used for endocrine therapy could have significant immune modulatory effects and impact on susceptibility to, as well as clinical manifestation and outcome of, infectious diseases. We investigated the effect of contraceptive doses of DMPA in two different murine Mycobacterium tuberculosis models. Multiplex bead array analysis revealed that DMPA altered serum cytokine levels of tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), and interleukin 10 (IL-10) in C57BL/6 mice and gamma interferon (IFN-γ) in BALB/c mice. DMPA also suppressed antigen-specific production of TNF-α, G-CSF, IL-10, and IL-6 and induced the production of IP-10 in C57BL/6 mice. In BALB/c mice, DMPA altered the antigen-specific secretion of IFN-γ, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and monocyte chemotactic protein 1 (MCP-1). Furthermore, we show that C57BL/6 mice treated with doses of DMPA, which result in serum concentrations similar to those observed in contraceptive users, have a significantly higher bacterial load in their lungs. Our data show for the first time that DMPA impacts tuberculosis (TB) disease severity in a mouse model and that the effects of this contraceptive are not confined to infections of the genital tract. This could have major implications for the contraceptive policies not only in developing countries like South Africa but also worldwide.

Reactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice.

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.