Annick Itie

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyers patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4

Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, and members of the pro-inflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains called Toll/IL-1R/plant R gene homology (TIR) domains. Intracellular signalling mechanisms mediated by TIRs are similar, with MyD88 (refs 5–8) and TRAF6 (refs 9, 10) having critical roles. Signal transduction between MyD88 and TRAF6 is known to involve the serine-threonine kinase IL-1 receptor-associated kinase 1 (IRAK-1) and two homologous proteins, IRAK-2 (ref. 12) and IRAK-M. However, the physiological functions of the IRAK molecules remain unclear, and gene-targeting studies have shown that IRAK-1 is only partially required for IL-1R and TLR signalling. Here we show by gene-targeting that IRAK-4, an IRAK molecule closely related to the Drosophila Pelle protein, is indispensable for the responses of animals and cultured cells to IL-1 and ligands that stimulate various TLRs. IRAK-4-deficient animals are completely resistant to a lethal dose of lipopolysaccharide (LPS). In addition, animals lacking IRAK-4 are severely impaired in their responses to viral and bacterial challenges. Our results indicate that IRAK-4 has an essential role in innate immunity.

Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b.

The signalling thresholds of antigen receptors and co-stimulatory receptors determine immunity or tolerance to self molecules. Changes in co-stimulatory pathways can lead to enhanced activation of lymphocytes and autoimmunity, or the induction of clonal anergy. The molecular mechanisms that maintain immunotolerance in vivo and integrate co-stimulatory signals with antigen receptor signals in T and B lymphocytes are poorly understood. Members of the Cbl/Sli family of molecular adaptors function downstream from growth factor and antigen receptors. Here we show that gene-targeted mice lacking the adaptor Cbl-b develop spontaneous autoimmunity characterized by auto-antibody production, infiltration of activated T and B lymphocytes into multiple organs, and parenchymal damage. Resting cbl-b -/- lymphocytes hyperproliferate upon antigen receptor stimulation, and cbl-b-/- T cells display specific hyperproduction of the T-cell growth factor interleukin-2, but not interferon-γ or tumour necrosis factor-α. Mutation of Cbl-b uncouples T-cell proliferation, interleukin-2 production and phosphorylation of the GDP/GTP exchange factor Vav1 from the requirement for CD28 co-stimulation. Cbl-b is thus a key regulator of activation thresholds in mature lymphocytes and immunological tolerance and autoimmunity.

Requirement for Casper (c-FLIP) in Regulation of Death Receptor–Induced Apoptosis and Embryonic Development

Casper (c-FLIP) associates with FADD and caspase-8 in signaling complexes downstream of death receptors like Fas. We generated Casper-deficient mice and cells and noted a duality in the physiological functions of this molecule. casper-/- embryos do not survive past day 10.5 of embryogenesis and exhibit impaired heart development. This phenotype is reminiscent of that reported for FADD-/- and caspase-8-/- embryos. However, unlike FADD-/- and caspase-8-/- cells, casper-/- embryonic fibroblasts are highly sensitive to FasL- or TNF-induced apoptosis and show rapid induction of caspase activities. NF-kappaB and JNK/SAPK activation is intact in TNF-stimulated casper-/- cells. These results suggest that Casper has two distinct roles: to cooperate with FADD and caspase-8 during embryonic development and to mediate cytoprotection against death factor-induced apoptosis.

The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses.

We investigated the in vivo function of the B7 family member B7-H3 (also known as B7RP-2) by gene targeting. B7-H3 inhibited T cell proliferation mediated by antibody to T cell receptor or allogeneic antigen-presenting cells. B7-H3-deficient mice developed more severe airway inflammation than did wild-type mice in conditions in which T helper cells differentiated toward type 1 (TH1) rather than type 2 (TH2). B7-H3 expression was consistently enhanced by interferon-γ but suppressed by interleukin 4 in dendritic cells. B7-H3-deficient mice developed experimental autoimmune encephalomyelitis several days earlier than their wild-type littermates, and accumulated higher concentrations of autoantibodies to DNA. Thus, B7-H3 is a negative regulator that preferentially affects TH1 responses.

Deficiency of T2K leads to apoptotic liver degeneration and impaired NF‐κB‐dependent gene transcription

Induction of NF‐κB‐dependent transcription requires phosphorylation and subsequent degradation of I‐κB, an inhibitor of NF‐κB, followed by nuclear translocation and DNA binding of NF‐κB. Tumor necrosis factor receptor‐associated factor 2 (TRAF2) plays a role in NF‐κB activation in response to cytokines such as tumor necrosis factor α (TNFα). In this study, we purified and characterized a novel kinase (T2K, also known as TBK1 or NAK), which associates with TRAF2 and exhibits kinase activity towards I‐κBα in vitro. The physiological function of T2K was investigated using T2K‐deficient mice. Heterozygotes appear normal, but t2k−/− animals die at ∼E14.5 of massive liver degeneration and apoptosis. Never theless, hematopoietic progenitors from T2K‐deficient fetal liver support normal lymphocyte development. Furthermore, t2k−/− embryonic fibroblasts and thymocytes do not display increased sensitivity to TNFα‐induced apoptosis. In response to either TNFα or IL‐1 induction, t2k−/− embryonic fibroblasts exhibit normal degradation of I‐κB and κB‐binding activity. However, NF‐κB‐directed transcription is dramatically reduced. These results demonstrate that, like I‐κB kinase β and the RelA subunit of NF‐κB, T2K is critical in protecting embryonic liver from apoptosis. However, T2K has a unique role in the activation of NF‐κB‐directed transcription, apparently independent of I‐κB degradation and NF‐κB DNA binding.

Generation and Characterization of Smac/DIABLO-Deficient Mice

ABSTRACT The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac−/− ) mice by using homologous recombination in embryonic stem (ES) cells. Smac−/− mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac−/− cells, all types of cultured Smac−/− cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.

Generation and Characterization of B7-H4/B7S1/B7x-Deficient Mice

ABSTRACT Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.

The Apoptotic Protease-Activating Factor 1-Mediated Pathway of Apoptosis Is Dispensable for Negative Selection of Thymocytes

Negative selection is a process to delete potentially autoreactive clones in developing thymocytes. Programmed cell death or apoptosis is thought to play an important role in this selection process. In this study, we investigated the role of apoptotic protease-activating factor 1 (Apaf1), a mammalian homologue of CED-4, in programmed cell death during the negative selection in thymus. There was no developmental abnormality in thymocytes from newborn Apaf1−/− mice in terms of CD4 and CD8 expression pattern and thymocyte number. Clonal deletion by endogenous male H-Y Ag of Apaf1-deficient thymocytes with transgenic expression of H-Y Ag-specific TCRs (H-Y Tg/Apaf1−/− thymocytes) was normally observed in lethally irradiated wild-type mice reconstituted with fetal liver-derived hemopoietic stem cells. Clonal deletion induced in vitro by a bacterial superantigen was also normal in fetal thymic organ culture. Thus, Apaf1-mediated pathway of apoptosis is dispensable for the negative selection of thymocytes. However, H-Y Tg/Apaf1−/− thymocytes showed partial resistance to H-Y peptide-induced deletion in vitro as compared with H-Y Tg/Apaf1+/− thymocytes, implicating the Apaf1-mediated apoptotic pathway in the negative selection in a certain situation. In addition, the peptide-induced deletion was still observed in H-Y Tg/Apaf1−/− thymocytes in the presence of a broad spectrum caspase inhibitor, z-VAD-fmk, suggesting the presence of caspase-independent cell death pathway playing roles during the negative selection. We assume that mechanisms for the negative selection are composed of several cell death pathways to avoid failure of elimination of autoreactive clones.

Characterization of cells and gene-targeted mice deficient for the p53-binding kinase homeodomain-interacting protein kinase 1 (HIPK1)

The tumor suppressor p53 is regulated in part by binding to cellular proteins. We used p53 as bait in the yeast two-hybrid system and isolated homeodomain-interacting protein kinase 1 (HIPK1) as a p53-binding protein. Deletion analysis showed that amino acids 100–370 of p53 and amino acids 885-1093 of HIPK1 were sufficient for HIPK1–p53 interaction. HIPK1 was capable of autophosphorylation and specific serine phosphorylation of p53. The HIPK1 gene was highly expressed in human breast cancer cell lines and oncogenically transformed mouse embryonic fibroblasts. HIPK1 was localized to human chromosome band 1p13, a site frequently altered in cancers. Gene-targeted HIPK1−/− mice were grossly normal but oncogenically transformed HIPK1 −/− mouse embryonic fibroblasts exhibited reduced transcription of Mdm2 and were more susceptible than transformed HIPK1+/+ cells to apoptosis induced by DNA damage. Carcinogen-treated HIPK1 −/− mice developed fewer and smaller skin tumors than HIPK1+/+ mice. HIPK1 may thus play a role in tumorigenesis, perhaps by means of the regulation of p53 and/or Mdm2.