Employing the candidate genetic approach, we previously identified mutations in the gene encoding NEMO (NF- kB essential modulator), an intracellular signaling constituent of the NF-kB pathway, results in ectodermal dysplasia with an immune deficiency. Mutations in the zinc finger domain of NEMO block CD40 mediated activation of NF- kB and prevent B cells from undergoing class switch recombination (CSR) and APCs from synthesizing NF -kB regulated cytokines such as IL -12 or TNF-a when stimulated with CD40 ligand. Interestingly, NF- KB activation via other signaling pathways such as TNF-a or members of the Toll- like receptor family are intact in these patients. ? ? The majority of NEMO mutations occur in exon 10, but biochemical mechanism by which they cause immune dysfunction remains undefined. We investigated the effect of a cysteine to arginine mutation (C417R) found in the NEMO zinc finger domain on dendrtic cell (DC) function. Following CD40 ligand (CD40L) stimulation of dendrtic cells prepared from two unrelated patients with the NEMO C417R mutation, we found that lysine 63 (K63)-linked polyubiquitination of NEMO was absent. As a consequence, CD40 stimulated EDI DCs had relatively reduced or absent cell aggregates and dendtritic extensions, failed to synthesize the c-Rel dependant cytokine IL-12, and failed support allogeneic lymphocyte proliferation in vitro. We utilized gene expression profiling to compare the genes induced by CD40L in normal DCs but not in EDI DCs. We identified the c-Rel target IL-12p35 (IL12A) in this gene set as well as a number of genes that are likely to be critical to the maturation and function of DCs. Amongst these are a set of genes such as LAD1, LAMB3, and SERPINE1, that have functions in adhesion or in the extracellular matrix and likely influence the clumping behavior of DCs, as well as cytoskeletal components such as AVIL, CAPG and SEPT11 that may regulate dendrite formation. In contrast to CD40 stimulation, downstream NF-kB activity, DC maturation, and NEMO ubiquitination were normal in EDI DCs following stimulation with the TLR4 ligand lipopolysaccharide (LPS). These results show for the first time that CD40 and TLR4 use different signaling pathways to ubiquitinate NEMO, and offer insight into a mutation in the zinc finger domain of NEMO leads to pathway specific defects in N F-kB signaling and thus immune deficiency.? ? Ectodermal dysplasisa with immune deficiency has also been associated with a single heterozygous mutation at position Ser32 of the NF-kB inhibitor IkBa, one of two phosphorylation sites that are essential for targeting IkBa for proteosomal degradation and hence for activation of NF-kB. We report a novel heterozygous nonsense mutation in the IKBA gene of a one-year-old male child with EDI that introduces a premature STOP codon at position Glu14. An in-frame methionine downstream of the nonsense mutation allows for reinitiation of translation. The resulting N-terminally truncated protein lacks both serine phosphorylation sites and inhibits NF-kB signalling by functioning as a dominant negative on NF-kB activity in lymphocytes and monocytes. These findings support the scanning model for translation initiation in eukaryotes and confirm the critical role of the NF-kB in the human immune response. ? ? We also found that osteopenia is a prominent and previously unappreciated clinical feature of patients with X-linked hyper-IgM syndrome (XHIM), an inherited immune deficiency disorder caused by mutations in the gene encoding CD40 ligand (CD40L). We therefore conducted studies to determine the relationship between CD40L and osteoclastogenesis. Recognizing that activated T cells express surface RANK ligand (RANKL) and can induce osteoclast differentiation of myeloid cells expressing RANK, we assessed the capacity of wild type T cells and CD40L-/- T cells to induce osteoclastogenesis in vitro. Relative to wild type T cells, activated CD40L-/- T cells from both humans and mice promoted robust osteoclast differentiation of myeloid cells. Whereas activated CD40L-/- T cells had normal expression of RANKL, they were deficient in IFN-g production. In subsequent studies, we cultured activated CD40L-/- T cells in the presence of INF-g and found that the osteoclastic capacity of CD40L -/- T cells could be greatly diminished. These results show for the first time that CD40L can influence RANKL signaling through T cell priming, and thus demonstrate a regulatory role for CD40L in bone mineralization that is absent in patients with XHIM.? ? ? CYLD is a deubiquitinating enzyme that is altered in patients with familial cylindromatosis, a condition characterized by numerous benign adnexal tumors. In mice deficient deficient in CYLD we show that the development of B cells, T cells, and myeloid cells is unaffected in CYLD deficient mice, but that the activation of these cells with mediators of innate and adaptive immunity results in enhanced NF-kB activity and is associated with increased NEMO ubiquitination. CYLD deficient mice are more susceptible to induced colonic inflammation and show a dramatic increase in the incidence of tumors in a colitis associated cancer (CAC) model. These results suggest that CYLD limits inflammation and tumorigenesis by regulating NEMO ubiquitination in vivo.

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National Institute of Allergy and Infectious Diseases (NIAID)
Intramural Research (Z01)
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Salt, Bryn H; Niemela, Julie E; Pandey, Rahul et al. (2008) IKBKG (nuclear factor-kappa B essential modulator) mutation can be associated with opportunistic infection without impairing Toll-like receptor function. J Allergy Clin Immunol 121:976-82
Lopez-Granados, Eduardo; Temmerman, Stephane T; Wu, Lynne et al. (2007) Osteopenia in X-linked hyper-IgM syndrome reveals a regulatory role for CD40 ligand in osteoclastogenesis. Proc Natl Acad Sci U S A 104:5056-61
Temmerman, Stephane T; Ma, Chi A; Borges, Louis et al. (2006) Impaired dendritic-cell function in ectodermal dysplasia with immune deficiency is linked to defective NEMO ubiquitination. Blood 108:2324-31
Zhang, Jun; Stirling, Brigid; Temmerman, Stephane T et al. (2006) Impaired regulation of NF-kappaB and increased susceptibility to colitis-associated tumorigenesis in CYLD-deficient mice. J Clin Invest 116:3042-9
Orange, Jordan S; Jain, Ashish; Ballas, Zuhair K et al. (2004) The presentation and natural history of immunodeficiency caused by nuclear factor kappaB essential modulator mutation. J Allergy Clin Immunol 113:725-33