expressing human soluble TNFR1-IgG, that expression levels of the TNF neutralizing transgene determined the enhanced protection to M. bovis BCG infection or the higher susceptibility of mice compared to littermate control mice. We found that low levels of soluble receptors were activating TNF expression and macrophage effector mechanisms whilst high levels of soluble receptors were neutralizing both sol and memTNF blocking all protective TNF-mediated activities. The immune cellular activation was attributed to reverse signaling of memTNF binding to solTNFRs. We hypothesize that a precise regulation of memTNF activity can be orchestrated by membrane and soluble TNFRs and an unbalance of this regulatory and complex system can modify the outcome of the disease. We finally investigate if memTNF bone-marrow derived macrophages could show different responses upon M. bovis BCG infection in vitro and observed that macrophages from memTNFD112 KI mice produced lower amounts of cytokines/ chemokines and showed an impaired p65 NF-kB phosphorylation pattern indicating that the deficiency is already on memTNFD112 KI macrophages which will influence the host immune response. These data suggest that the small difference of the two memTNF molecules results in major effects in signalling and memTNFmediated activities. The mechanisms explaining the differences between memTNF molecules with only a minor Lonafarnib chemical information change of 3 amino acids at positions 1012 are still not elucidated. Some hypothesis can be envisaged to understand why such important consequence can occur in vivo. We hypothesize that TACE can still bind to memTNFD19,K11E although unable to digest whereas the deletion of 3 amino acids prevent the 
binding of TACE to memTNFD112. Upon TNF activation, TACE activity is directed to TNFR2 which can be found decreased on macrophages but substantially increased as solTNFR2 form that will neutralize memTNF thus preventing its biological activities. Materials and Methods Mice C57BL/6 mice, memTNFD19,K11E KI mice, memTNFD1 KI mice memTNFD1126TNFR12/2 mice, memTNFD1 12 6TNFR22/2 mice, TNFR16TNFR22/2 mice and TNF2/ 2 mice were maintained under conventional conditions in the animal facility of the Medical Faculty, University of Geneva. All animal experiments were carried out in accordance with institutional guidelines and were approved by the academic ethical committee on animal experimentation and the Cantonal Veterinary Office from Geneva, authorization number: 31.1.1005/ 3202/2. 12 M. bovis BCG infection Mice were infected intravenously with 107 living M. bovis BCG Connaught, or BCG Pasteur GFP . Body weights were monitored during infection, and mice were sacrificed at 2 and 4 weeks post-infection, or 24 weeks post-infection for survival monitoring. Determination of colony forming units from infected organs The number of viable bacteria recovered from frozen organs was evaluated as previously described. Membrane TNF and TNFRs Protection to BCG Infection Histological analyses Histological analyses of liver were performed at 4 weeks after BCG infection. Livers were fixed in 4% buffered formaldehyde and embedded in paraffin for subsequent hematoxylin/eosin staining. Evaluation of the lesions was done with Metamorph sofware. Evaluation PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22187884 of serum, organ and cell culture supernatant levels of cytokines and chemokines For cytokine and chemokine detection, organs were homogenized in 0.04% Tween 80/saline buffer as previously described. IFN-c, IL-12p40, IL-6, MCP-1 RANTES, sTNF