CD3+ Macrophages Deliver Proinflammatory Cytokines by a CD3- and Transmembrane TNF-Dependent Pathway and Are Increased at the BCG-Infection Site.

Macrophages are essential cells of the innate immune response against microbial infections, and they have the ability to adapt under both pro- and anti-inflammatory conditions and develop different functions. A growing body of evidence regarding a novel macrophage subpopulation that expresses CD3 has recently emerged. Here, we explain that human circulating monocytes can be differentiated into CD3+TCRαß+ and CD3+TCRαß- macrophages. Both cell subpopulations express on their cell surface HLA family molecules, but only the CD3+TCRαß+ macrophage subpopulation co-express CD1 family molecules and transmembrane TNF (tmTNF). CD3+TCRαß+ macrophages secrete IL-1ß, IL-6 IP-10, and MCP-1 by both tmTNF- and CD3-dependent pathways, while CD3+TCRαß- macrophages specifically produce IFN-γ, TNF, and MIP-1ß by a CD3-dependent pathway. In this study, we also used a mouse model of BCG-induced pleurisy and demonstrated that CD3+ myeloid cells (TCRαß+ and TCRαß- cells) are increased at the infection sites during the acute phase (2 weeks post-infection). Interestingly, cell increment was mediated by tmTNF, and the soluble form of TNF was dispensable. BCG-infection also induced the expression of TNF receptor 2 on CD3+ myeloid cells, which increased after BCG-infection, suggesting that the tmTNF/TNFRs axis plays an important role in the presence or function of these cells in tuberculosis.

Myeloid cell TNFR1 signaling dependent liver injury and inflammation upon BCG infection.

TNF plays a critical role in mononuclear cell recruitment during acute Bacillus Calmette-Guérin (BCG) infection leading to an effective immune response with granuloma formation, but may also cause tissue injury mediated by TNFR1 or TNFR2. Here we investigated the role of myeloid and T cell specific TNFR1 and R2 expression, and show that absence of TNFR1 in myeloid cells attenuated liver granuloma formation and liver injury in response to acute BCG infection, while TNFR2 expressed in myeloid cells contributed only to liver injury. TNFR1 was the main receptor controlling cytokine production by liver mononuclear cells after antigenic specific response, modified CD4/CD8 ratio and NK, NKT and regulatory T cell recruitment. Further analysis of CD11b+CD3+ phagocytic cells revealed a TCRαß expressing subpopulation of unknown function, which increased in response to BCG infection dependent of TNFR1 expression on myeloid cells. In conclusion, TNFR1 expressed by myeloid cells plays a critical role in mononuclear cell recruitment and injury of the liver after BCG infection.

Transmembrane TNF and Partially TNFR1 Regulate TNFR2 Expression and Control Inflammation in Mycobacterial-Induced Pleurisy.

Pleural tuberculosis is one of the most frequent forms of extra-pulmonary tuberculosis observed in patients infected with Mycobacterium tuberculosis. Tumor Necrosis Factor (TNF) is a crucial cytokine needed to control tuberculosis infection that remains a leading cause of morbidity and mortality worldwide. TNF blockade compromises host immunity and may increase the risk of reactivation of latent infection resulting in overt pulmonary, pleural and extra-pulmonary tuberculosis. While TNF signaling is mainly considered pro-inflammatory, its requirement for the anti-inflammation process involved in the resolution of infection and tissue repair is less explored. Our study analyzes the role of TNF and TNF receptors in the control of the inflammatory process associated with Bacillus Calmette-Guérin (BCG)-induced pleurisy. This study shows that the absence of TNF causes exacerbated inflammation in the pleural cavity of BCG-infected mice which is controlled by the transmembrane TNF (tmTNF) expression. The lack of TNF is associated with an impaired cellular expression and shedding of TNFR2 in the pleural cavity. The presence of tmTNF restores the normal expression of TNFR2 on myeloid cells during BCG-induced pleurisy. We also show that absence of TNFR1 affects the expression of TNFR2 on pleural cells and inflammation in the pleural cavity of BCG-infected mice. In conclusion, tmTNF but not soluble TNF prevents pleural cavity inflammation leading to attenuation and the resolution of the inflammatory process caused by mycobacterial pleurisy in association with the expression of TNFR2 on myeloid cells.

Transmembrane Tumor Necrosis Factor Controls Myeloid-Derived Suppressor Cell Activity via TNF Receptor 2 and Protects from Excessive Inflammation during BCG-Induced Pleurisy.

Pleural tuberculosis (TB) is a form of extra-pulmonary TB observed in patients infected with Mycobacterium tuberculosis. Accumulation of myeloid-derived suppressor cells (MDSC) has been observed in animal models of TB and in human patients but their role remains to be fully elucidated. In this study, we analyzed the role of transmembrane TNF (tmTNF) in the accumulation and function of MDSC in the pleural cavity during an acute mycobacterial infection. Mycobacterium bovis BCG-induced pleurisy was resolved in mice expressing tmTNF, but lethal in the absence of tumor necrosis factor. Pleural infection induced MDSC accumulation in the pleural cavity and functional MDSC required tmTNF to suppress T cells as did pleural wild-type MDSC. Interaction of MDSC expressing tmTNF with CD4 T cells bearing TNF receptor 2 (TNFR2), but not TNFR1, was required for MDSC suppressive activity on CD4 T cells. Expression of tmTNF attenuated Th1 cell-mediated inflammatory responses generated by the acute pleural mycobacterial infection in association with effective MDSC expressing tmTNF and interacting with CD4 T cells expressing TNFR2. In conclusion, this study provides new insights into the crucial role played by the tmTNF/TNFR2 pathway in MDSC suppressive activity required during acute pleural infection to attenuate excessive inflammation generated by the infection.

Low Dose BCG Infection as a Model for Macrophage Activation Maintaining Cell Viability.

Mycobacterium bovis BCG, the current vaccine against tuberculosis, is ingested by macrophages promoting the development of effector functions including cell death and microbicidal mechanisms. Despite accumulating reports on M. tuberculosis, mechanisms of BCG/macrophage interaction remain relatively undefined. In vivo, few bacilli are sufficient to establish a mycobacterial infection; however, in vitro studies systematically use high mycobacterium doses. In this study, we analyze macrophage/BCG interactions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activation without affecting viability. We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels of proinflammatory cytokines and transcription factors while MOI 0.1 was more efficient for early stimulation of IL-1ß, MCP-1, and KC. Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrial structures. Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 0.1 infection. In conclusion, BCG infection at low dose is an efficient in vitro model to study macrophage/BCG interactions that maintains macrophage viability and mitochondrial structures. This represents a novel model that can be applied to BCG research fields including mycobacterial infections, cancer immunotherapy, and prevention of autoimmunity and allergies.

Tumor Necrosis Factor and Its Receptors Are Crucial to Control Mycobacterium bovis Bacillus Calmette-Guerin Pleural Infection in a Murine Model.

Tumor necrosis factor (TNF) is crucial to control Mycobacterium tuberculosis infection, which remains a leading cause of morbidity and mortality worldwide. TNF blockade compromises host immunity and may cause reactivation of latent infection, resulting in overt pulmonary, pleural, and extrapulmonary tuberculosis. Herein, we investigate the roles of TNF and TNF receptors in the control of Mycobacterium bovis bacillus Calmette-Guerin (BCG) pleural infection in a murine model. As controls, wild-type mice and those with a defective CCR5, a receptor that is crucial for control of viral infection but not for tuberculosis, were used. BCG-induced pleural infection was uncontrolled and progressive in absence of TNF or TNF receptor 1 (TNFR1)/TNFR2 (TNFR1R2) with increased inflammatory cell recruitment and bacterial load in the pleural cavity, and heightened levels of pleural and serum proinflammatory cytokines and chemokines, compared to wild-type control mice. The visceral pleura was thickened with chronic inflammation, which was prominent in TNF(-/-) and TNFR1R2(-/-) mice. The parietal pleural of TNF(-/-) and TNFR1R2(-/-) mice exhibited abundant inflammatory nodules containing mycobacteria, and these mice developed nonresolving inflammation and succumbed from disseminated BCG infection. By contrast, CCR5(-/-) mice survived and controlled pleural BCG infection as wild-type control mice. In conclusion, BCG-induced pleurisy was uncontrolled in the absence of TNF or TNF receptors with exacerbated inflammatory response, impaired bacterial clearance, and defective mesothelium repair, suggesting a critical role of TNF to control mycobacterial pleurisy.

Control of Mycobacterial Infections in Mice Expressing Human Tumor Necrosis Factor (TNF) but Not Mouse TNF.

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.

Much More than M1 and M2 Macrophages, There are also CD169(+) and TCR(+) Macrophages.

Monocytes are considered to be precursor cells of the mononuclear phagocytic system, and macrophages are one of the leading members of this cellular system. Macrophages play highly diverse roles in maintaining an organism's integrity by either directly participating in pathogen elimination or repairing tissue under sterile inflammatory conditions. There are different subpopulations of macrophages and each one has its own characteristics and functions. In this review, we summarize present knowledge on the polarization of macrophages that allows the generation of subpopulations called classically activated macrophages or M1 and alternative activated macrophages or M2. Furthermore, there are macrophages that their origin and characterization still remain unclear but have been involved as main players in some human pathologies. Thus, we also review three other categories of macrophages: tumor-associated macrophages, CD169(+) macrophages, and the recently named TCR(+) macrophages. Based on the literature, we provide information on the molecular characterization of these macrophage subpopulations and their specific involvement in several human pathologies such as cancer, infectious diseases, obesity, and asthma. The refined characterization of the macrophage subpopulations can be useful in designing new strategies, supplementing those already established for the treatment of diseases using macrophages as a therapeutic target.

Limited Contribution of IL-36 versus IL-1 and TNF Pathways in Host Response to Mycobacterial Infection.

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.

Bacillus calmette-guerin infection in NADPH oxidase deficiency: defective mycobacterial sequestration and granuloma formation.

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.

IL-36 signaling amplifies Th1 responses by enhancing proliferation and Th1 polarization of naive CD4+ T cells.

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-36ß 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.

Membrane-bound TNF induces protective immune responses to M. bovis BCG infection: regulation of memTNF and TNF receptors comparing two memTNF molecules.

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.

Soluble TNF, but not membrane TNF, is critical in LPS-induced hepatitis.

BACKGROUND & AIMS: : Bacillus Calmette-Guérin (BCG) infection causes hepatic injury following granuloma formation and secretion of cytokines which renders mice highly sensitive to endotoxin-mediated hepatotoxicity. Tumor necrosis factor (TNF) is required for granuloma formation and is one of the most important cytokines in liver injury. TNF inhibitors are effective therapies for inflammatory diseases. However, clinical use of non-selective TNF inhibitors is associated with an increased risk of infections. This work investigates the differential roles of soluble TNF (solTNF) and membrane TNF (memTNF) in BCG infection, BCG/LPS- and D-GALN/LPS-induced liver injury. METHODS: We have used both genetic and pharmacologic approaches and analyzed liver injury, TLR4, cytokine and iNOS activation induced by BCG, BCG/LPS and D-GALN/LPS. RESULTS: BCG infection-induced liver injury is seen in wild-type mice but not in TNF(-/-), memTNF knock-in (KI), and sTNFR1-Fc transgenic mice. Severity of BCG-induced liver injury is correlated with BCG-granuloma number and hepatic expression of TLR4 and iNOS. In addition, protection from liver damage caused by BCG/LPS or D-GALN/LPS administration was observed in TNF(-/-), memTNF KI and sTNFR1-Fc transgenic mice. To extend the genetic findings, we then evaluated whether selective pharmacological inhibition of solTNF by dominant-negative (DN)-TNF neutralization and non-selective inhibition of solTNF and memTNF by anti-TNF antibodies and etanercept (TNFR2-IgG1) can protect the mice from liver injury. Both selective and non-selective inhibition of solTNF protected mice from BCG/LPS and D-GALN/LPS-induced liver damage. CONCLUSIONS: These data suggest that memTNF is not mediating liver injury and that selective inhibition of solTNF sparing memTNF may represent a new therapeutic strategy to treat immune-mediated inflammatory liver diseases.

Acute and chronic anti-inflammatory properties of the stem bark aqueous and methanol extracts of Sclerocarya birrea (Anacardiaceae).

Sclerocarya birrea is used in folk medicine for the treatment of inflammatory disorders. The effect of the stem bark aqueous and methanol extracts of S. birrea (150 or 300 mg/kg) was evaluated on carrageenan-, histamine- or serotonin-induced paw oedema in rats. The methanol extract of S. birrea (300 mg/kg) being the most active, exhibited a maximum inhibition of 75.45 and 55.31% on carrageenan- and histamine-induced inflammation, respectively. When administered at 300 mg/kg, the methanol extract of S. birrea also exhibited 80.68% inhibition on the 10th day and 54.43% inhibition on the 21st day in formalin- or complete Freund's adjuvant (CFA)-induced paw oedema in rats. GSH level was significantly increased (75.14%), while MAD level was significantly decreased (31.22%) in the liver of CFA rats treated with S. birrea (300 mg/kg). The results suggest that the anti-inflammatory activity of the aqueous and methanol extracts of S. birrea is due to the inhibition of histamine and prostaglandin pathways and to its antioxidant activity.

Dominant-negative tumor necrosis factor protects from Mycobacterium bovis Bacillus Calmette Guérin (BCG) and endotoxin-induced liver injury without compromising host immunity to BCG and Mycobacterium tuberculosis.

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.

Fat diet and alcohol-induced steatohepatitis after LPS challenge in mice: role of bioactive TNF and Th1 type cytokines.

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-12p40 overexpression promotes interleukin-12p70 and interleukin-23 formation but does not affect bacille Calmette-Guérin and Mycobacterium tuberculosis clearance.

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-Guérin (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.

Differential effects of total and partial neutralization of tumor necrosis factor on cell-mediated immunity to Mycobacterium bovis BCG infection.

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.

Contribution of transmembrane tumor necrosis factor to host defense against Mycobacterium bovis bacillus Calmette-guerin and Mycobacterium tuberculosis infections.

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.

Inhibition of inducible nitric oxide synthase protects against liver injury induced by mycobacterial infection and endotoxins.

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.