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The interleukin-1 (IL-1) superfamily of cytokines comprises a set of pivotal mediators of inflammation. Among them, the action of IL-36 cytokines in immune responses has remained elusive. In a recent study, we demonstrated a direct effect of IL-36 on immune cells. Here we show that, among T cells, the IL-36 receptor is predominantly expressed on naive CD4+ T cells and that IL-36 cytokines act directly on naive T cells by enhancing both cell proliferation and IL-2 secretion. IL-36β acts in synergy with IL-12 to promote Th1 polarization and IL-36 signaling is also involved in mediating Th1 immune responses to Bacillus Calmette-Guerin infection in vivo. Our findings point toward a critical function of IL-36 in the priming of Th1 cell responses in vitro, and in adaptive immunity in a model of mycobacterial infection in vivo.

The effects of total and partial inhibition of tumor necrosis factor (TNF) on sensitivity to Mycobacterium bovis BCG infection were investigated by using transgenic mice in which hepatocytes produced different amounts of human soluble TNF receptor 1 (sTNFR1) fused to the Fc fragment of human immunoglobulin G3 that could be detected in the serum. Transgenic mice expressing high serum levels of sTNFR1, neutralizing all circulating TNF, failed to develop differentiated granulomas and bactericidal mechanisms, and they succumbed to BCG infection. sTNFR1 transgenic mice did not activate BCG-induced Th1-type cytokines early in infection, but uncontrolled cytokine release was found late in infection. In this work we also evaluated the effect of partial inhibition of TNF on resistance to BCG infection. Transgenic mice expressing low levels of sTNFR1 were protected against BCG infection, and they developed increased bactericidal mechanisms, such as enhanced inducible nitric oxide synthase activity, increased macrophage activation, and showed higher numbers of liver granulomas early in infection compared to their negative littermates. Our data suggest that while total inhibition of TNF prevented BCG-induced cell-mediated immune responses, partial inhibition of TNF could contribute to macrophage activation, induction of bactericidal mechanisms, and granuloma formation in the early phase of BCG infection.

Tumor necrosis factor (TNF) is an important cytokine for host defense against pathogens but is also associated with the development of human immunopathologies. TNF blockade effectively ameliorates many chronic inflammatory conditions but compromises host immunity to tuberculosis. The search for novel, more specific human TNF blockers requires the development of a reliable animal model. We used a novel mouse model with complete replacement of the mouse TNF gene by its human ortholog (human TNF [huTNF] knock-in [KI] mice) to determine resistance to Mycobacterium bovis BCG and M. tuberculosis infections and to investigate whether TNF inhibitors in clinical use reduce host immunity. Our results show that macrophages from huTNF KI mice responded to BCG and lipopolysaccharide similarly to wild-type macrophages by NF-κB activation and cytokine production. While TNF-deficient mice rapidly succumbed to mycobacterial infection, huTNF KI mice survived, controlling the bacterial burden and activating bactericidal mechanisms. Administration of TNF-neutralizing biologics disrupted the control of mycobacterial infection in huTNF KI mice, leading to an increased bacterial burden and hyperinflammation. Thus, our findings demonstrate that human TNF can functionally replace murine TNF in vivo , providing mycobacterial resistance that could be compromised by TNF neutralization. This new animal model will be helpful for the testing of specific biologics neutralizing human TNF.

IL-36 cytokines are members of the IL-1 family of cytokines that stimulate dendritic cells and T cells leading to enhanced T helper 1 responses in vitro and in vivo ; however, their role in host defense has not been fully addressed thus far. The objective of this study was to examine the role of IL-36R signaling in the control of mycobacterial infection, using models of systemic attenuated M . bovis BCG infection and virulent aerogenic M . tuberculosis infection. IL-36γ expression was increased in the lung of M . bovis BCG infected mice. However, IL-36R deficient mice infected with M . bovis BCG showed similar survival and control of the infection as compared to wild-type mice, although their lung pathology and CXCL1 response were transiently different. While highly susceptible TNF-α deficient mice succumbed with overwhelming M . tuberculosis infection, and IL-1RI deficient mice showed intermediate susceptibility, IL-36R-deficient mice controlled the infection, with bacterial burden, lung inflammation and pathology, similar to wild-type controls. Therefore, IL-36R signaling has only limited influence in the control of mycobacterial infection.

BACKGROUND: Several activities of the transmembrane form of TNF (memTNF) in immune responses to intracellular bacterial infection have been shown to be different from those exerted by soluble TNF. Evidence is based largely on studies in transgenic mice expressing memTNF, but precise cellular mechanisms are not well defined and the importance of TNF receptor regulation is unknown. In addition, memTNF activities are defined for a particular modification of the extracellular domain of TNF but a direct comparison of different mutant memTNF molecules has not been done in vivo. METHODOLOGY: To understand the activities of memTNF we compared two commonly used mouse strains lacking soluble TNF but possessing functional and normally regulated membrane-bound TNF knockin (memTNF KI) for their capacity to generate cell-mediated immune responses and resistance to M. bovis BCG infection, and to regulate TNF receptors. PRINCIPAL FINDINGS: M. bovis BCG infection resulted in similar bacterial loads in one strain of memTNF KI (memTNF(Δ1-9,K11E)) and in wild-type mice, in contrast, the other strain of memTNF KI mice (memTNF(Δ1-12)) showed higher sensitivity to infection with high mortality (75%), greater bacterial load and massive lung pathology. The pattern of cytokines/chemokines, inflammatory cells, pulmonary NF-κB phosphorylation, antigen-dependent IFN-γ response, and splenic iNOS was impaired in M. bovis BCG-infected memTNF(Δ1-12) KI mice. Macrophages expressing TNFR2 were reduced but soluble TNFRs were higher in memTNF(Δ1-12) KI mice during the infection. In vitro, M. bovis BCG-induced NF-κB activation and cytokines were also decreased in memTNF(Δ1-12) KI bone marrow-derived macrophages. CONCLUSION: Our data show that two memTNF molecules exerted very different activities upon M. bovis BCG infection resulting in protection or not to bacterial infection. These results suggest a regulatory mechanism of memTNF and TNF receptors being critical in the outcome of the infection and highlight the role of cell-bound and soluble TNFR2 in memTNF-mediated anti-microbial mechanisms.

International audience; TNF is essential to control Mycobacterium tuberculosis infection and cannot be replaced by other proinflammatory cytokines. Overproduction of TNF may cause immunopathology, while defective TNF production results in uncontrolled infection. The critical role of TNF in the control of tuberculosis has been illustrated recently by primary and reactivation of latent infection in some patients under pharmacological anti-TNF therapy for rheumatoid arthritis or Crohn's disease. In this review, we discuss results of recent studies aimed at better understanding of molecular, cellular and kinetic aspects of TNF-mediated regulation of host-mycobacteria interactions. In particular, recent data using either mutant mice expressing solely membrane TNF or specific inhibitor sparing membrane TNF demonstrated that membrane TNF is sufficient to control acute M. tuberculosis infection. This is opening the way to selective TNF neutralization that might retain the desired anti-inflammatory effect but reduce the infectious risk.

International audience; BACKGROUND: Tumor necrosis factor (TNF) is associated with the development of inflammatory pathologies. Antibodies and soluble TNF (solTNF) receptors that neutralize excessive TNF are effective therapies for inflammatory and autoimmune diseases. However, clinical use of TNF inhibitors is associated with an increased risk of infections. METHODS: A novel dominant-negative (DN) strategy of selective TNF neutralization, consisting of blocking solTNF while sparing transmembrane TNF (tmTNF), was tested in mouse models of mycobacterial infection and acute liver inflammation. XENP1595, a DN-TNF biologic, was compared with etanercept, a TNF receptor 2 (TNFR2)-IgG1 Fc fusion protein that inhibits murine solTNF and tmTNF. RESULTS: XENP1595 protected mice from acute liver inflammation induced by endotoxin challenge in Mycobacterium bovis bacillus Calmette-Guérin (BCG)-infected mice, but, in contrast to etanercept, it did not compromise host immunity to acute M. bovis BCG and Mycobacterium tuberculosis infections in terms of bacterial burden, granuloma formation, and innate immune responses. CONCLUSIONS: A selective inhibitor of solTNF efficiently protected mice from acute liver inflammation yet maintained immunity to mycobacterial infections. In contrast, nonselective inhibition of solTNF and tmTNF suppressed immunity to M. bovis BCG and M. tuberculosis. Therefore, selective inhibition of solTNF by DN-TNF biologics may represent a new therapeutic strategy for the treatment of inflammatory diseases without compromising host immunity.

Obesity with insulin resistance and alcohol are the most frequent causes of steatohepatitis. This work investigates the contribution of bioactive TNF and Th1 type cytokines in a mouse model of steatohepatitis induced by FAT alone or FAT+EtOH and endotoxin. The extent of liver injury and cytokine activation induced by endotoxin in chronic FAT-fed mice, FAT+EtOH-fed mice, or mice fed standard chow were analyzed. Endotoxin administration to either FAT-fed or FAT+EtOH-fed mice increased serum ALT and AST compared to standard chow mice. Immunoreactive TNF was strongly activated by LPS in FAT-fed and FAT+EtOH-fed mice which presented the highest levels, but low levels were found in standard chow mice. In contrast, bioactive TNF was only present in serum of FAT-fed and in particular the highest levels were found in FAT+EtOH-fed mice. Moreover, soluble TNFR2 but not TNFR1 was found in lower amounts in serum of FAT+EtOH-fed mice compared to FAT-fed mice. Steatohepatitis was associated with increased IL-6, IFN-gamma, and iNOS mRNA and proteins. Data show that a moderately FAT diet and low-dose EtOH concur to generate steatohepatitis and TNF liver expression after LPS. In this model, changes in the regulation of TNF are associated with increased expression of IL-6, IFN-gamma, and iNOS.

Interleukin (IL)-12p40, a subunit of IL-12p70 and IL-23, has previously been shown to inhibit IL-12p70 activity and interferon-gamma (IFN-gamma) production. However, recent evidence has suggested that the role of IL-12p40 is more complex. To study the contribution of IL-12p40 to immune responses against mycobacterial infections, we have used transgenic (tg) mice overexpressing IL-12p40 under the control of a major histocompatibility complex-II promoter. The IL-12p40 transgene was expressed during steady state at concentrations of 129 +/- 25 ng/ml of serum and 75 +/- 13 ng per spleen, while endogenous IL-12p40 was hardly detectable in control littermates. Bacille Calmette-Guerin (BCG) infection strongly induced the expression of IL-12p40 transgene in infected organs, and IL-12p40 monomeric and dimeric forms were identified in spleen of IL-12p40 tg mice. Excessive production of IL-12p40 resulted in a 14-fold increase in IL-12p70 serum levels in tg mice versus non-transgenic mice. IL-23 was also strongly elevated in the serum and spleens of IL-12p40 tg mice through BCG infection. While IFN-gamma and tumour necrosis factor protein levels were similar in IL-12p40 tg and non-transgenic mice, Th2 type immune responses were reduced in IL-12p40 tg mice. The number of BCG granulomas and macrophage expressing inducible nitric oxide synthase were similar in IL-12p40 tg and non-transgenic mice. IL-12p40 tg mice were as resistant as non-transgenic mice to BCG and Mycobacterium tuberculosis infections as they could efficiently control bacillary growth. These data show that high amounts of IL-12p40 promotes IL-12p70 and IL-23 formation, but that does not affect T helper 1 type immune responses and granuloma function, thus leading to normal mycobacterial clearance in infected organs.

To study the specific role of transmembrane tumor necrosis factor (TmTNF) in host defense mechanisms against bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis infections, we compared the immune responses of TNF/lymphotoxin (LT)-alpha(-/-) mice expressing a noncleavable transgenic TmTNF (TmTNF tg) to those of TNF/LT-alpha(-/-) and wild-type mice. Susceptibility of TNF/LT-alpha(-/-) mice to BCG infection was associated with impaired induction of systemic RANTES but not of monocyte chemoattractant protein 1 (MCP-1), the development of excessive local and systemic Th1-type immune responses, and a substantially reduced inducible nitric oxide synthase (iNOS) activity. Resistance of TmTNF tg mice to BCG infection was associated with efficient activation of iNOS in granulomas and with the regulated release of local and systemic chemokines and Th1-type cytokines. However, M. tuberculosis infection of TmTNF tg mice resulted in longer survival and enhanced resistance compared to TNF/LT-alpha(-/-) mice but higher sensitivity than wild-type mice. TmTNF tg mice exhibited reduced pulmonary iNOS expression and showed an exacerbated cellular infiltration in the lungs despite a modest bacillary content. Our data thus indicate a role for TmTNF in host defense against mycobacteria by contributing to induction and regulation of Th1-type cytokine and chemokine expression leading to development of bactericidal granulomas expressing iNOS, which critically determines susceptibility versus resistance of the host to mycobacterial infections.

BACKGROUND/AIMS: Bacillus Calmette Guerin (BCG) infection causes hepatic injury following granuloma formation and secretion of cytokines which render mice highly sensitive to endotoxin-mediated hepatotoxicity. This work investigates the role of inducible nitric oxide synthase (iNOS) in liver damage induced by BCG and endotoxins in BCG-infected mice. METHODS: Liver injury and cytokine activation induced by BCG and by LPS upon BCG infection (BCG/LPS) were compared in wild-type and iNOS-/- mice. RESULTS: iNOS-/- mice infected with living BCG are protected from hepatic injury when compared to wild-type mice which express iNOS protein in macrophages forming hepatic granulomas. In addition, iNOS-/- mice show a decrease in BCG-induced IFN-gamma serum levels. LPS challenge in BCG-infected mice strongly activates iNOS in the liver and spleen of wild-type mice which show important liver damage associated with a dramatic increase in TNF and IL-6 and also Th1 type cytokines. In contrast, iNOS-/- mice are protected from liver injury after BCG/LPS challenge and their TNF, IL-6 and Th1 type cytokine serum levels raise moderately. CONCLUSIONS: These results demonstrate that nitric oxide (NO) from iNOS is involved in hepatotoxicity induced by both mycobacterial infection and endotoxin effects upon BCG infection and that inhibition of NO from iNOS protects from liver injuries.

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-α (LT-α) locus (Tm TNF tg mice). These mice were as resistant to BCG infection as wild-type mice, whereas TNF/LT-α−/−, TNF−/−, and LT-α−/− 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-α−/− 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-α 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.

The role of nitric oxide (NO) in Mycobacterium bovis Bacillus Calmette Guerin (BCG) infection was investigated using nitric oxide synthase 2 (nos2)-deficient mice, because NO plays a pivotal protective role in M. tuberculosis infection. We demonstrate that nos2-deficient mice were unable to eliminate BCG and succumbed within 8 to 12 weeks to BCG infection (10(6) CFU) with cachexia and pneumonia, whereas all infected wild-type mice survived. The greatest mycobacterial loads were observed in lung and spleen. Nos2-deficient mice developed large granulomas consisting of macrophages and activated T cells and caseous necrotic lesions in spleen. The macrophages in granulomas from nos2-deficient mice had reduced acid phosphatase activities, suggesting that NO is required for macrophage activation. The absence of NOS2 affected the cytokine production of the Th1 type of immune response, except IL-18. Serum amounts of IL-12p40 were increased and IFN-gamma was decreased compared with wild-type mice. The lack of NOS2 resulted in an overproduction of TNF, observed throughout the infection period. Additionally, TNFR1 and TNFR2 shedding was altered compared with wild-type mice. Up-regulation of TNF may be compensatory for the lack of NOS2. The late neutralization of TNF by soluble TNF receptors resulted in heightened disease severity and accelerated death in nos2-deficient mice but had no effect in wild-type mice. In conclusion, the inability of nos2-deficient mice to kill M. bovis BCG resulted in an accumulation of mycobacteria with a dramatic activation of the immune system and overproduction of pro-inflammatory cytokines, which resulted in death.

Patients with chronic granulomatous disease (CGD) lack generation of reactive oxygen species (ROS) through the phagocyte NADPH oxidase NOX2. CGD is an immune deficiency that leads to frequent infections with certain pathogens; this is well documented for S. aureus and A. fumigatus, but less clear for mycobacteria. We therefore performed an extensive literature search which yielded 297 cases of CGD patients with mycobacterial infections; M. bovis BCG was most commonly described (74%). The relationship between NOX2 deficiency and BCG infection however has never been studied in a mouse model. We therefore investigated BCG infection in three different mouse models of CGD: Ncf1 mutants in two different genetic backgrounds and Cybb knock-out mice. In addition, we investigated a macrophage-specific rescue (transgenic expression of Ncf1 under the control of the CD68 promoter). Wild-type mice did not develop severe disease upon BCG injection. In contrast, all three types of CGD mice were highly susceptible to BCG, as witnessed by a severe weight loss, development of hemorrhagic pneumonia, and a high mortality (∼50%). Rescue of NOX2 activity in macrophages restored BCG resistance, similar as seen in wild-type mice. Granulomas from mycobacteria-infected wild-type mice generated ROS, while granulomas from CGD mice did not. Bacterial load in CGD mice was only moderately increased, suggesting that it was not crucial for the observed phenotype. CGD mice responded with massively enhanced cytokine release (TNF-α, IFN-γ, IL-17 and IL-12) early after BCG infection, which might account for severity of the disease. Finally, in wild-type mice, macrophages formed clusters and restricted mycobacteria to granulomas, while macrophages and mycobacteria were diffusely distributed in lung tissue from CGD mice. Our results demonstrate that lack of the NADPH oxidase leads to a markedly increased severity of BCG infection through mechanisms including increased cytokine production and impaired granuloma formation., Author Summary The vaccine Mycobacterium bovis BCG is administrated to prevent early age tuberculosis in endemic areas. BCG is a live vaccine with a low incidence of complications. However, local or disseminated BCG infection may occur, in particular in immunodeficient individuals. Chronic granulomatous disease (CGD), a deficiency in the superoxide-producing phagocyte NADPH oxidase, is a primary immune deficiency and one of the most frequent congenital defects of phagocyte in humans. Here we analyze the role of the phagocyte NADPH oxidase NOX2 in the defense against BCG. An extensive literature review suggested that BCG infection is by far the most common mycobacterial disease in CGD patients (220 published cases). We therefore studied BCG infection in several CGD mouse models showing that these were highly susceptible to BCG infection with a mortality rate of ∼50%. As compared to the wild type, CGD mice showed a markedly increased release of cytokines, an altered granuloma structure, and were unable to restrain mycobacteria within granulomas. Rescue of the phagocyte NADPH oxidase in macrophages was sufficient to protect mice from BCG infection and to sequester the mycobacteria within granulomas. Thus, superoxide generation by macrophages plays an important role for the defense against BCG infection and prevents overshooting release of proinflammatory cytokines.