The thymic medulla provides a microenvironment where medullary thymic epithelial cells (mTECs) express autoimmune regulator and diverse tissue-restricted genes, contributing to launching self-tolerance. Positive selection is essential for thymic medulla formation via a previously unknown mechanism. Here we show that the cytokine RANK ligand (RANKL) was produced by positively selected thymocytes and regulated the cellularity of mTEC by interacting with RANK and osteoprotegerin. Forced expression of RANKL restored thymic medulla in mice lacking positive selection, whereas RANKL perturbation impaired medulla formation. These results indicate that RANKL produced by positively selected thymocytes is responsible for fostering thymic medulla formation, thereby establishing central tolerance.

Medullary thymic epithelial cells (mTECs) establish T cell self-tolerance through the expression of autoimmune regulator (Aire) and peripheral tissue-specific self-antigens. However, signals underlying mTEC development remain largely unclear. Here, we demonstrate crucial regulation of mTEC development by receptor activator of NF-kappaB (RANK) and CD40 signals. Whereas only RANK signaling was essential for mTEC development during embryogenesis, in postnatal mice, cooperation between CD40 and RANK signals was required for mTEC development to successfully establish the medullary microenvironment. Ligation of RANK or CD40 on fetal thymic stroma in vitro induced mTEC development in a tumor necrosis factor-associated factor 6 (TRAF6)-, NF-kappaB inducing kinase (NIK)-, and IkappaB kinase beta (IKKbeta)-dependent manner. These results show that developmental-stage-dependent cooperation between RANK and CD40 promotes mTEC development, thereby establishing self-tolerance.

The microenvironments of the thymus are generated by thymic epithelial cells (TECs) and are essential for inducing immune self-tolerance or developing T cells. However, the molecular mechanisms that underlie the differentiation of TECs and thymic compartmentalization are not fully understood. Here we show that deficiency in the tumor necrosis factor receptor-associated factor (TRAF) 6 results in disorganized distribution of medullary TECs (mTECs) and the absence of mature mTECs. Engraftment of thymic stroma of TRAF6(-/-) embryos into athymic nude mice induced autoimmunity. Thus, TRAF6 directs the development of thymic stroma and represents a critical point of regulation for self-tolerance and autoimmunity.

Nuclear factor‐κΒ (NF‐κB) binds specifically to NF‐κB‐binding sites (κB sites, 5′‐GGGRNNYYCC‐3′; R, purine; Y, pyrimidine; N, any nucleotide) present in enhancer regions of various genes. Binding of various cytokines, growth factors and pathogen‐associated molecular patterns to specific receptors activates NF‐κB and expression of genes that play critical roles in inflammation, innate and acquired immunity, bone remodeling and generation of skin appendices. Activation of NF‐κB is also involved in cancer development and progression. NF‐κB is activated in cells that become malignant tumors and in cells that are recruited to and constitute the tumor microenvironment. In the latter scenario, the TLR‐TRAF6‐NF‐kB pathways seem to play major roles, and NF‐κB activation results in production of cytokines, which in turn induce NF‐κB activation in premalignant cells, leading to expression of genes involved abnormal growth and malignancy. Furthermore, NF‐κB activation is involved in bone metastasis. Osteoclasts, whose generation requires the RANK‐TRAF6‐NF‐κB pathways, release various growth factors stored in bone, which results in creation of microenvironment suitable for proliferation and colonization of cancer cells. Therefore, NF‐κB and molecules involved its activation, such as TRAF6, are attractive targets for therapeutic strategies against cancer. (Cancer Sci 2007; 98: 268–274)

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Varney et al. report that that deletion of the TRAF-interacting protein TIFAB contributes to an MDS-like phenotype in mice by up-regulating TRAF6 and contributing to hematopoietic dysfunction.

BackgroundIn response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.Principal FindingsHere we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7.Conclusions/SignificanceThus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.

Lactoferrin (LF) has been implicated in innate immunity. Here we reveal the signal transduction pathway responsible for human LF (hLF)‐triggered nuclear factor‐κB (NF‐κB) activation. Endotoxin‐depleted hLF induces NF‐κB activation at physiologically relevant concentrations in the human monocytic leukemia cell line, THP‐1, and in mouse embryonic fibroblasts (MEFs). In MEFs, in which both tumor necrosis factor receptor‐associated factor 2 (TRAF2) and TRAF5 are deficient, hLF causes NF‐κB activation at a level comparable to that seen in wild‐type MEFs, whereas TRAF6‐deficient MEFs show significantly impaired NF‐κB activation in response to hLF. TRAF6 is known to be indispensable in leading to NF‐κB activation in myeloid differentiating factor 88 (MyD88)‐dependent signaling pathways, while the role of TRAF6 in the MyD88‐independent signaling pathway has not been clarified extensively. When we examined the hLF‐dependent NF‐κB activation in MyD88‐deficient MEFs, delayed, but remarkable, NF‐κB activation occurred as a result of the treatment of cells with hLF, indicating that both MyD88‐dependent and MyD88‐independent pathways are involved. Indeed, hLF fails to activate NF‐κB in MEFs lacking Toll‐like receptor 4 (TLR4), a unique TLR group member that triggers both MyD88‐depependent and MyD88‐independent signalings. Importantly, the carbohydrate chains from hLF are shown to be responsible for TLR4 activation. Furthermore, we show that lipopolysaccharide‐induced cytokine and chemokine production is attenuated by intact hLF but not by the carbohydrate chains from hLF. Thus, we present a novel model concerning the biological function of hLF: hLF induces moderate activation of TLR4‐mediated innate immunity through its carbohydrate chains; however, hLF suppresses endotoxemia by interfering with lipopolysaccharide‐dependent TLR4 activation, probably through its polypeptide moiety.