Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-B pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin ␤ receptor (LT␤R) as a prototype, we showed that activation of the alternative, but not the classical, NF-B pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LT␤R essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LT␤R appeared to be required for the activation of the alternative, but not the classical, NF-B pathway. In vivo, ligand-induced internalization of LT␤R in mesenteric lymph node stromal cells correlated with induction of alternative NF-B target genes. Thus, our data shed light on LT␤R cellular trafficking as a process required for specific biological functions of NF-B.

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In the green alga Chlamydomonas reinhardtii, a mutant deprived of complex I enzyme activity presents a 1T deletion in the mitochondrial nd5 gene. The loss of the ND5 subunit prevents the assembly of the 950 kDa whole complex I. Instead, a low abundant 700 kDa subcomplex, loosely associated to the inner mitochondrial membrane, is assembled. The resolution of the subcomplex by SDS-PAGE gave rise to 19 individual spots, sixteen having been identified by mass spectrometry analysis. Eleven, mainly associated to the hydrophilic part of the complex, are homologs to subunits of the bovine enzyme whereas five (including gamma-type carbonic anhydrase subunits) are specific to green plants or to plants and fungi. None of the subunits typical of the beta membrane domain of complex I enzyme has been identified in the mutant. This allows us to propose that the truncated enzyme misses the membrane distal domain of complex I but retains the proximal domain associated to the matrix arm of the enzyme. A complex I topology model is presented in the light of our results. Finally, a supercomplex most probably corresponding to complex I-complex III association, was identified in mutant mitochondria, indicating that the missing part of the enzyme is not required for the formation of the supercomplex.

NF-κB-inducing kinase (NIK) is well-known for its role in promoting p100/NF-κB2 processing into p52, a process defined as the alternative, or non-canonical, NF-κB pathway. Here we reveal an unexpected new role of NIK in TNFR1-mediated RIP1-dependent apoptosis, a consequence of TNFR1 activation observed in c-IAP1/2-depleted conditions. We show that NIK stabilization, obtained by activation of the non-death TNFRs Fn14 or LTβR, is required for TNFα-mediated apoptosis. These apoptotic stimuli trigger the depletion of c-IAP1/2, the phosphorylation of RIP1 and the RIP1 kinase-dependent assembly of the RIP1/FADD/caspase-8 complex.In the absence of NIK, the phosphorylation of RIP1 and the formation of RIP1/FADD/caspase-8 complex are compromised while c-IAP1/2 depletion is unaffected. In vitro kinase assays revealed that recombinant RIP1 is a bona fide substrate of NIK. In vivo, we demonstrated the requirement of NIK pro-death function, but not the processing of its substrate p100 into p52, in a mouse model of TNFR1/LTβR-induced thymus involution. In addition, we also highlight a role for NIK in hepatocyte apoptosis in a mouse model of virus-induced TNFR1/RIP1-dependent liver damage. We conclude that NIK not only contributes to lymphoid organogenesis, inflammation and cell survival but also to TNFR1/RIP1-dependent cell death independently of the alternative NF-κB pathway.

Background and aim Mesenchymal stem cells (MSCs) have anti-inflammatory and anti-fibrotic properties and could be a potential therapy for Crohn’s Disease (CD) strictures. In this phase I–II pilot trial, we assessed safety and efficacy of local MSC injection to treat CD strictures. Methods CD patients with a short (less than 5 cm in length) non-passable stricture accessible by ileocolonoscopy were included. Allogenic bone-marrow derived MSCs were injected in the 4 quadrants of the stricture. Adverse events and clinical scores were evaluated at each followup visit while endoscopy and magnetic resonance enterography were performed at baseline, week (W)12 and W48. The main judgement criterion for efficacy was the complete (defined by the ability to pass the ileocolonoscope) or partial (defined by a diameter increase) resolution of the stricture at W12. Second efficacy criteria included assessment of the stricture at W48 and evolution of clinical scores at W12 and W48. Results We performed 11 MSC injections in 10 CD patients (3 primary and 7 anastomotic strictures; 1 stricture injected twice). MSC injections were well tolerated but 4 hospitalizations for occlusion were reported. At W12, 5 patients presented a complete or partial resolution of the stricture (2 complete and 3 partial). Seven patients were re-evaluated at W48 (1 dilated, 1 operated, and 1 lost to follow-up) and 4 patients had a complete resolution. The evolution of clinical scores between W0, W12 and W48 was not statistically significant. Conclusion MSCs injection in CD stricture was well tolerated and may offer a benefit.