The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response.

Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.

TNFR-associated factor 6 regulates TCR signaling via interaction with and modification of LAT adapter.

TNFR-associated factor (TRAF)6 is an essential ubiquitin E3 ligase in immune responses, but its function in adaptive immunity is not well understood. In this study, we show that TRAF6 is recruited to the peripheral ring of the T cell immunological synapse in Jurkat T cells or human primary CD4(+) T cells conjugated with staphylococcal enterotoxin E-pulsed B cells. This recruitment depends on TRAF6 interacting with linker for activation of T cells (LAT) via its TRAF domain. Although LAT was indispensable for TCR/CD28-induced TRAF6 ubiquitination and its ligase activity, RNA interference-induced TRAF6 knockdown in T cells decreased TCR/CD28-induced LAT ubiquitination, tyrosine phosphorylation, and association with tyrosine kinase ZAP70. Overexpression of TRAF6 or its catalytically inactive form C70A promoted and decreased, respectively, LAT tyrosine phosphorylation upon stimulation. Moreover, LAT was ubiquitinated at Lys(88) by TRAF6 via K63-linked chain. In addition, TRAF6 was required for and synergized with LAT to promote the TCR/CD28-induced activation of NFAT. These results reveal a novel function and mechanism of TRAF6 action in the TCR-LAT signaling pathway distinct from its role in TCR-induced NF-κB activation, indicating that LAT also plays an adapter role in TCR/CD28-induced activation of TRAF6.

Enhancing CD8 T-cell memory by modulating fatty acid metabolism.

CD8 T cells, which have a crucial role in immunity to infection and cancer, are maintained in constant numbers, but on antigen stimulation undergo a developmental program characterized by distinct phases encompassing the expansion and then contraction of antigen-specific effector (T(E)) populations, followed by the persistence of long-lived memory (T(M)) cells. Although this predictable pattern of CD8 T-cell responses is well established, the underlying cellular mechanisms regulating the transition to T(M) cells remain undefined. Here we show that tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), an adaptor protein in the TNF-receptor and interleukin-1R/Toll-like receptor superfamily, regulates CD8 T(M)-cell development after infection by modulating fatty acid metabolism. We show that mice with a T-cell-specific deletion of TRAF6 mount robust CD8 T(E)-cell responses, but have a profound defect in their ability to generate T(M) cells that is characterized by the disappearance of antigen-specific cells in the weeks after primary immunization. Microarray analyses revealed that TRAF6-deficient CD8 T cells exhibit altered expression of genes that regulate fatty acid metabolism. Consistent with this, activated CD8 T cells lacking TRAF6 display defective AMP-activated kinase activation and mitochondrial fatty acid oxidation (FAO) in response to growth factor withdrawal. Administration of the anti-diabetic drug metformin restored FAO and CD8 T(M)-cell generation in the absence of TRAF6. This treatment also increased CD8 T(M) cells in wild-type mice, and consequently was able to considerably improve the efficacy of an experimental anti-cancer vaccine.

Conditional targeting of tumor necrosis factor receptor-associated factor 6 reveals opposing functions of Toll-like receptor signaling in endothelial and myeloid cells in a mouse model of atherosclerosis.

BACKGROUND: Previous studies implicated Toll-like receptor signaling as a critical pathogenic pathway in atherosclerosis, but the cell-specific mechanisms by which Toll-like receptors act to control atherosclerotic plaque development remain poorly understood. METHODS AND RESULTS: To study the cell-specific role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in atherosclerosis, we generated ApoE(-/-) mice with endothelial cell- or myeloid cell-specific TRAF6 deficiency using Cre/LoxP-mediated gene targeting. Endothelial TRAF6 deficiency reduced atherosclerosis in female ApoE(-/-) mice by inhibiting nuclear factor-κB-dependent proinflammatory gene expression and monocyte adhesion to endothelial cells. In contrast, myeloid cell-specific TRAF6 deficiency caused exacerbated atherosclerosis, with larger plaques containing more necrotic areas in both male and female ApoE(-/-) mice. TRAF6-deficient macrophages showed impaired expression of the antiinflammatory and atheroprotective cytokine interleukin-10, elevated endoplasmic reticulum stress, increased sensitivity to oxidized low-density lipoprotein-induced apoptosis, and reduced capacity to clear apoptotic cells. Thus, the reduced antiinflammatory properties, coupled with increased sensitivity to apoptosis and impaired efferocytosis capacity of TRAF6-deficient macrophages, result in exacerbated atherosclerosis development in TRAF6(MYKO)/ApoE(-/-) mice. CONCLUSION: Toll-like receptor-mediated TRAF6 signaling acts in endothelial cells to promote atherosclerosis but displays atheroprotective, antiinflammatory and prosurvival functions in myeloid cells.

TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of immune homeostasis.

TRAF6 has a key role in the regulation of innate immune responses by mediating signals from both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Here we show that T cell-specific deletion of TRAF6 unexpectedly results in multiorgan inflammatory disease. TRAF6-deficient T cells exhibit hyperactivation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway compared with wild-type T cells and, as a result, become resistant to suppression by CD4+ CD25+ regulatory T cells. These data identify a previously unrecognized role for TRAF6 in the maintenance of peripheral tolerance, and suggest the presence of a T cell-intrinsic control mechanism to render responder T cells susceptible to tolerizing signals.

Differential TRAF3 utilization by a variant human CD40 receptor with enhanced signaling.

CD40 is required for T cell-dependent humoral immunity, but it can also contribute to the pathogenesis of autoimmunity and B cell malignancy. The TNFR-associated factor (TRAF)2 and TRAF6 adaptor proteins are positive regulators of CD40 signaling required to activate downstream kinase cascades and transcription factors. In contrast, TRAF3 can serve as a negative regulator of CD40 signaling, and CD40 signals are amplified in TRAF3(-/-) B cells. We previously reported a gain-of-function polymorphism of the human CD40 receptor, hCD40-P227A, which signals in an amplified manner to B lymphocytes. In this study, we show that hCD40-P227A binds more TRAF3 and TRAF5, as well as certain associated proteins, than wild-type-CD40. Studies in TRAF-deficient B cell lines revealed that hCD40-P227A uses TRAF3 as a positive rather than negative regulator. Although located outside of any known TRAF binding sites, the P227A polymorphism can alter TRAF binding and dramatically changes the role played by TRAF3 in CD40 signaling.

A novel TNF receptor-associated factor 6 binding domain mediates NF-kappa B signaling by the common cytokine receptor beta subunit.

GM-CSF, IL-3, and IL-5 are proinflammatory cytokines that control the production and function of myeloid and lymphoid cells. Their receptors are composed of a ligand-specific alpha subunit and a shared common signal-transducing beta subunit (beta common receptor or GM-CSFR beta [beta(c)]). The pleiotropic nature of biologic outcomes mediated by beta(c) and the presence of large, uncharacterized regions of its cytoplasmic domain suggest that much remains to be learned about its downstream signaling pathways. Although some previous work has attempted to link beta(c) with NF-kappaB activation, a definitive mechanism that mediates this pathway has not been described and, to date, it has not been clear whether the receptor can directly activate NF-kappaB. We demonstrate that NF-kappaB activation by beta(c) is dependent on TNFR-associated factor 6 (TRAF6) and that association of TRAF6 with beta(c) requires a consensus-binding motif found in other molecules known to interact with TRAF6. Furthermore, point mutation of this motif abrogated the ability of beta(c) to mediate NF-kappaB activation and reduced the viability of an IL-3-dependent hematopoietic cell line. Because this receptor plays a key role in hematopoiesis and the beta(c) cytoplasmic domain identified in this work mediates hematopoietic cell viability, this new pathway is likely to contribute to immune cell biology. This work is significant because it is the first description of a TRAF6-dependent signaling pathway associated with a type I cytokine receptor. It also suggests that TRAF6, a mediator of TNFR and TLR signaling, may be a common signaling intermediate in diverse cytokine receptor systems.

Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6.

Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.

Osteoclast differentiation independent of the TRANCE-RANK-TRAF6 axis.

Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor kappaB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE-RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-alpha in the presence of cofactors such as TGF-beta. We provide direct evidence against the current paradigm that the TRANCE-RANK-TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.

Epstein-Barr virus latent membrane protein 1 modulates distinctive NF- kappaB pathways through C-terminus-activating region 1 to regulate epidermal growth factor receptor expression.

Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1) is required for EBV B-lymphocyte transformation, transforms rodent fibroblasts, and can induce lymphoma and epithelial hyperplasia in transgenic mice. Two domains have been identified within the intracellular carboxy terminus that can activate NF-kappaB, C-terminus-activating region 1 (CTAR1) and CTAR2, through interactions with tumor necrosis receptor-associated factors (TRAFs). CTAR1 can activate both the canonical and noncanonical NF-kappaB pathways and has unique effects on cellular gene expression. The epidermal growth factor receptor (EGFR) is highly induced by LMP1-CTAR1 in epithelial cells through activation of a novel NF-kappaB form containing p50 homodimers and Bcl-3. To further understand the regulation of NF-kappaB in CTAR1-induced EGFR expression, we evaluated the ability of CTAR1 to induce EGFR in mouse embryonic fibroblasts (MEFs) defective for different NF-kappaB effectors. CTAR1-mediated EGFR induction required the NF-kappaB-inducing kinase (NIK) but not the IkappaB kinase (IKK) complex components that regulate canonical or noncanonical NF-kappaB pathways. CTAR1-mediated induction of nuclear p50 occurred in IKKbeta-, IKKgamma-, and NIK-defective MEFs, indicating that this induction is not dependent on the canonical or noncanonical NF-kappaB pathways. EGFR and nuclear p50 were expressed at high levels in TRAF2(-/-) fibroblasts and were not induced by CTAR1. In TRAF3(-/-) MEFs, CTAR1 induced nuclear p50 but did not affect basal levels of STAT3 serine phosphorylation or induce EGFR expression. EGFR was induced by LMP1 in TRAF6(-/-) MEFs. These findings suggest that this novel NF-kappaB pathway is differentially regulated by TRAF2 and TRAF3, and that distinct interactions of LMP1 and its effectors regulate LMP1-mediated gene expression.

IL-17 signaling for mRNA stabilization does not require TNF receptor-associated factor 6.

IL-17 alone is a relatively weak inducer of gene expression, but cooperates with other cytokines, including TNF-alpha, to generate a strong response in part via prolongation of mRNA t(1/2). Because TNFR-associated factor 6 (TRAF6) has been reported to be essential for signaling by IL-17, we examined its involvement in IL-17-mediated mRNA stabilization. Although overexpression of TRAF6 in HeLa cells activates NF-kappaB, it does not stabilize transfected KC mRNA. Furthermore, a dominant-negative TRAF6 abrogates NF-kappaB activation, but does not block IL-17-induced chemokine mRNA stabilization. IL-17 can stabilize KC and MIP-2 mRNAs comparably in TNF-alpha-treated mouse embryo fibroblasts from TRAF6(+/+) and TRAF6(-/-) mice. TRAF6 is known to couple upstream signals with activation of p38 MAPK and mitogen activated protein kinase activated protein kinase 2, both of which have been shown to be important for Toll/IL-1R-mediated mRNA stabilization in various cell types. Inhibition of p38 MAPK, however, does not block IL-17-induced KC mRNA stabilization, and IL-17 can stabilize KC mRNA equally in mouse embryo fibroblasts from both wild-type and mitogen activated protein kinase activated protein kinase 2/3 doubly-deficient mice. Finally, IL-17 can amplify the levels of multiple TNF-alpha-stimulated mRNAs in wild-type and TRAF6-deficient cells, but not in cells from Act1(-/-) mice. Collectively, these findings demonstrate the existence of a TRAF6/p38 MAPK-independent pathway that couples the IL-17R with enhanced mRNA stability. Because the most potent effects of IL-17 on gene expression are obtained in cooperation with other cytokines such as TNF-alpha, these findings suggest that this pathway is a major contributing mechanism for response to IL-17.

Involvement of TRAF6 in regulating immune defense and ovarian development in Musca domestica.

The tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a key cytoplasm signaling adaptor that mediates signals activated by TNFR superfamily and the interleukin-1/Toll-like receptor (IL-1/TLR) superfamily. In the present research, a housefly Musca domestica TRAF6 (MdTRAF6) gene is identified and characterized, with a 51.7-kDa protein possessing a RING domain and a conserved C-terminal TRAF homology MATH domain encoded. MdTRAF6 is widely expressed in diverse tissues with high expression levels in gut and fat body, which is of the highest levels in adult in all growth stages. The expression of MdTRAF6 could be remarkably induced by bacterial challenge, and the silencing MdTRAF6 could alter the expressions of NF-κB-like genes (relish and dorsal) and antimicrobial peptide genes (cecropin, diptericin, attacin, muscin), thus leading elevated mortalities of larvae followed by bacterial infection. Inspiringly. MdTRAF6-depleted adult flies display higher mortality, lower fertility and reduced survival of offspring than the controls. Further investigation reveals that knockdown of MdTRAF6 disturbs the ovarian development and impaires the expressions of vitellogenin and vitellogenin receptor genes in the adult females. All these phenotypes show crucial roles of MdTRAF6 in innate immunity via positive regulation of the Toll pathway and negative regulation of the Imd pathway, and in reproduction by maintaining ovarian development.

TRAF6 deficiency promotes TNF-induced cell death through inactivation of GSK3beta.

TNF receptor-associated factor 6 (TRAF6) plays a key role in the regulation of innate immune responses by mediating signals from both TNF receptors (TNFRs) and interleukin-1 receptors (IL-1Rs)/Toll-like receptors (TLRs). Here, we define a new role for TRAF6 in antagonizing cell death during TNF signaling. In TRAF6-deficient 3T3 (T6(-/-) 3T3) cells, TNF stimulation leads to the accumulation of reactive oxygen species (ROS), which in turn results in prolonged c-Jun N-terminal kinase (JNK) activation and accelerated cell death. Furthermore, TNF-induced p65/RelA phosphorylation as well as transcriptional activity of nuclear factor-kappaB (NF-kappaB) was significantly downregulated in T6(-/-) 3T3 cells. Interestingly, TRAF6 deficiency leads to constitutive phosphorylation and inactivation of glycogen synthase kinase 3beta (GSK3beta). Restoration of GSK3beta activity through exogenous expression of a GSK3beta constitutive active form rescued cell death in TRAF6-null 3T3 cells. These data suggest a role for TRAF6 in the maintenance of cell survival by regulating GSK3beta activity in TNF signaling.

TRAF6 is a critical signal transducer in IL-33 signaling pathway.

IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.

TRAF6 maintains mammary stem cells and promotes pregnancy-induced mammary epithelial cell expansion.

Receptor activator of nuclear factor (NF)-κB (RANK) signaling promotes pregnancy-dependent epithelial cell differentiation and expansion for mammary gland development, which requires NF-κB pathway-dependent Cyclin D1 induction and inhibitor of DNA binding 2 (Id2) pathway-dependent anti-apoptotic gene induction. However, the roles of tumor necrosis factor receptor-associated factor 6 (TRAF6) remain unclear despite its requirement in RANK signaling. Here we show that TRAF6 is crucial for both mammary stem cell maintenance and pregnancy-induced epithelial cell expansion. TRAF6 deficiency impairs phosphoinositide 3-kinase (PI3K)/AKT and canonical NF-κB pathways, whereas noncanonical NF-κB signaling remains functional. Therefore, we propose that TRAF6 promotes cell proliferation by activating PI3K/AKT signaling to induce retinoblastoma phosphorylation in concert with noncanonical NF-κB pathway-dependent Cyclin D1 induction. Furthermore, TRAF6 inhibits apoptosis by activating canonical NF-κB signaling to induce anti-apoptotic genes with the Id2 pathway. Therefore, proper orchestration of TRAF6-dependent and -independent RANK signals likely establishes mammary gland formation.

Sensitivity of TLR4- and -7-induced NF kappa B1 p105-TPL2-ERK pathway to TNF-receptor-associated-factor-6 revealed by RNAi in mouse macrophages.

Tumor necrosis factor (TNF)-receptor-associated-factor-6 (TRAF6) is an adaptor protein involved in Toll-like receptor (TLR) signaling. Recent studies using macrophages from TRAF6 knockout mice have revealed that TRAF6 is required for TLR7 signaling. However, an essential role of TRAF6 in TLR4 signaling and cytokine production is slightly controversial. Using an RNAi approach to reduce the cellular levels of TRAF6, we tested the role of this adaptor protein on the sensitivity of the various components of the ERK pathway mediated by TLR4 and -7 in Raw264.7, a mouse macrophage cell line. ERK activation in macrophages by TLR4 and -7 is mediated via a MAP3K, called TPL2/COT, which under unstimulated conditions is associated with NF kappa B1 p105, a member of the I kappa B family of proteins. Upon stimulation with TLR ligands, p105 is phosphorylated by I kappa B kinase (IKK) complex and partially degraded, which releases TPL2. The free TPL2 is active and stimulates the ERK pathway via MEK1/2. The free TPL2, however, is also unstable and is targeted for degradation. We demonstrate here that reduced level of TRAF6 ( approximately 80% decrease) in macrophages does not significantly affect any of the components of the TLR4-stimulated ERK pathway, including p105 phosphorylation, TPL2 degradation and ERK1/2 phosphorylation. Surprisingly, however, TLR4-induced JNK1/2 phosphorylation is significantly blocked by TRAF6 knockdown, suggesting that ERK and JNK pathways are differentially sensitive to TRAF6 levels. Furthermore, although TLR4-mediated IKK-induced p105 phosphorylation is not sensitive to TRAF6 knockdown, I kappa B alpha phosphorylation (also, IKK-induced) is significantly blocked, suggesting that TLR4 activation results in a TRAF6-sensitive and -insensitive IKK activation in macrophages. In contrast to TLR4 signaling, TLR7 activation of ERK, JNK pathways and phosphorylation of p105 and I kappa B alpha are completely inhibited in TRAF6 knockdown cells. Compared to the signaling data, while TLR4-induced TNFalpha mRNA expression is not significantly inhibited by TRAF6 knockdown, TLR7-induced TNFalpha mRNA is significantly blocked. In contrast, both TLR4- and TLR7-induced IL6 mRNA are significantly blocked by TRAF6 knockdown. These results suggest that while TRAF6 is absolutely essential for TLR7 activation of ERK, JNK and NF kappa B pathways, TLR4-induced ERK, JNK pathways and IKK-mediated phosphorylation of I kappa B family members as well as cytokine expression are differentially sensitive to the cellular levels of TRAF6. These results have important implications in terms of therapeutic targeting of TRAF6 complexes in diseases where TLR4 and -7 are involved.

Development of intestinal M cells and follicle-associated epithelium is regulated by TRAF6-mediated NF-κB signaling.

M cells are located in the follicle-associated epithelium (FAE) that covers Peyer's patches (PPs) and are responsible for the uptake of intestinal antigens. The differentiation of M cells is initiated by receptor activator of NF-κB. However, the intracellular pathways involved in M cell differentiation are still elusive. In this study, we demonstrate that the NF-κB pathway activated by RANK is essential for M cell differentiation using in vitro organoid culture. Overexpression of NF-κB transcription factors enhances the expression of M cell-associated molecules but is not sufficient to complete M cell differentiation. Furthermore, we evaluated the requirement for tumor necrosis factor receptor-associated factor 6 (TRAF6). Conditional deletion of TRAF6 in the intestinal epithelium causes a complete loss of M cells in PPs, resulting in impaired antigen uptake into PPs. In addition, the expression of FAE-associated genes is almost silenced in TRAF6-deficient mice. This study thus demonstrates the crucial role of TRAF6-mediated NF-κB signaling in the development of M cells and FAE.

The TLR signaling adaptor TRAM interacts with TRAF6 to mediate activation of the inflammatory response by TLR4.

TLRs act as sentinels in professional immune cells to detect and initiate the innate immune response to pathogen challenge. TLR4 is a widely expressed TLR, responsible for initiating potent immune responses to LPS. TRAM acts to bridge TLR4 with TRIF, orchestrating the inflammatory response to pathogen challenge. We have identified a putative TRAF6-binding motif in TRAM that could mediate a novel signaling function for TRAM in TLR4 signaling. TRAM and TRAF6 association was confirmed by immunoprecipitation of endogenous, ectopically expressed and recombinant proteins, which was ablated upon mutation of a key Glu residue in TRAM (TRAM E183A). TRAF6 and TRAM were observed colocalizing using confocal microscopy following ectopic expression in cells and the ability of TRAM and TRAM E183A to activate luciferase-linked reporter assays was determined in HEK293 and TRAF6-deficient cells. Importantly, TRAM-deficient macrophages reconstituted with TRAM E183A display significantly reduced inflammatory TNF-α, IL-6, and RANTES protein production compared with WT TRAM. These results demonstrate a novel role for TRAM in TLR4-mediated signaling in regulating inflammatory responses via its interaction with TRAF6, distinct from its role as a bridging adaptor between TLR4 and TRIF.