Maria-Luisa Olleros

TNF in Host Resistance to Tuberculosis Infection

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 Crohns 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.

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.

Roles of soluble and membrane TNF and related ligands in mycobacterial infections: effects of selective and non-selective TNF inhibitors during infection

TNF plays an essential and non-redundant role in host defense mechanisms against Mycobacterium tuberculosis (Mtb) infection. TNF contributes to the development of granulomas, microstructures encasing pathogens and concentrating interactions between phagocytes and lymphocytes, and promotes bactericidal pathways to limit and destroy the invading intracellular pathogen. Production of TNF is associated with the development of human inflammatory diseases, and its inhibition, although an effective therapy, increases the risk of infections including either new or reactivation of tuberculosis infection. Studies on the role of membrane TNF in the absence of secreted TNF using genetic mouse models have shown that membrane TNF protects from M. bovis BCG and acute M. tuberculosis infections but does not induce inflammation in mouse. Pharmacological approaches of selective and non-selective soluble TNF inhibition show that a selective inhibitor of soluble TNF does not suppress host immunity to M. tuberculosis and M. bovis BCG infections, yet protects mice from arthritis and liver inflammatory diseases. This suggests that neutralization of soluble TNF may be effective to inhibit inflammatory diseases and also reduce the infection risks associated with current anti-TNF therapies.

In Vitro Inhibition of Lipopolysaccharide and Mycobacterium Bovis Bacillus Calmette Guérin-Induced Inflammatory Cytokines and in Vivo Protection from D-Galactosamine/Lps -Mediated Liver Injury by the Medicinal Plant Sclerocarya Birrea

Sclerocarya birrea is a medicinal plant used for the treatment of inflammatory- and bacterial-related diseases. The present study investigated in vitro and in vivo the effects of the stem bark methanol extract of S. birrea. Nitrite, TNF, IL-1β, IL-6 and IL-12p40 production by bone marrow-derived macrophages (BMDM) pre-incubated with or without S. birrea, and stimulated with Lipopolysaccharide (LPS) or infected with live Mycobacterium bovis Bacillus Calmette Guérin (BCG) was evaluated. S. birrea extract inhibited, in a concentration-dependent manner, nitrite, TNF, IL-1β, IL-6 and IL-12p40 production by BMDM stimulated with LPS or infected with live BCG. The iNOS expression was reduced by S. birrea after stimulation of BMDM with LPS. In addition, S. birrea inhibited the nuclear factor κB (NF-κB) activation by both LPS and BCG. The effects of the plant extract were also evaluated in an in vivo model of liver injury induced by D-galactosamine/LPS (D-GalN/LPS) administration in mice. S. birrea limited D-GalN/LPS-liver injury as assessed by a reduction in transaminases and TNF, IL-1β, IL-6 serum levels, and translocation of NF-κB to the nucleus. Taken together, our data indicate that stem bark methanol extract of S. birrea possesses anti-inflammatory properties by inhibiting NF-κB activation and cytokine release induced by inflammatory or infectious stimuli.

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.

Limited Role for Lymphotoxin α in the Host Immune Response to Mycobacterium tuberculosis

The contribution of lymphotoxin (LT)α in the host immune response to virulent Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin infections was investigated. Despite their ability to induce Th1 cytokine, IFN-γ, and IL-12 pulmonary response, “conventional” LTα−/− mice succumb rapidly to virulent M. tuberculosis aerosol infection, with uncontrolled bacilli growth, defective granuloma formation, necrosis, and reduced pulmonary inducible NO synthase expression, similar to TNF−/− mice. Contributions from developmental lymphoid abnormalities in LTα−/− mice were excluded because hematopoietic reconstitution with conventional LTα−/− bone marrow conferred enhanced susceptibility to wild-type mice, comparable to conventional LTα−/− control mice. However, conventional LTα−/− mice produced reduced levels of TNF after M. bovis bacillus Calmette-Guérin infection, and their lack of control of mycobacterial infection could be due to a defective contribution of either LTα or TNF, or both, to the host immune response. To address this point, the response of “neo-free” LTα−/− mice with unperturbed intrinsic TNF expression to M. tuberculosis infection was investigated in a direct comparative study with conventional LTα−/− mice. Strikingly, although conventional LTα−/− mice were highly sensitive, similar to TNF−/− mice, neo-free LTα−/− mice controlled acute M. tuberculosis infection essentially as wild-type mice. Pulmonary bacterial burden and inflammation was, however, slightly increased in neo-free LTα−/− mice 4–5 mo postinfection, but importantly, they did not succumb to infection. Our findings revise the notion that LTα might have a critical role in host defense to acute mycobacterial infection, independent of TNF, but suggest a contribution of LTα in the control of chronic M. tuberculosis infection.