Fish TRAF2 promotes innate immune response to RGNNV infection.

Tumour necrosis factor receptor-associated factors (TRAFs) are key regulatory proteins in the NF-κB signaling pathways. TRAF2 participates in the activation of both canonical and non-canonical NF-κB pathways, which are crucial for cell inflammation and cell survival. To elucidate its function in teleost fish, TRAF2 homologues of yellow grouper (Epinephelus awoara) and golden pompano (Trachinotus ovatus) have been cloned and characterized in this study. The open reading frame (ORF) of grouper TRAF2 (EaTRAF2) consists of 1563 nucleotides encoding a 521 amino acid protein with a predicted molecular mass of 58.70 kDa. The ORF of golden pompano TRAF2 (ToTRAF2) consists of 1563 nucleotides encoding a 521 amino acid protein with a predicted molecular mass of 58.66 kDa EaTRAF2 and ToTRAF2 share 99.23% and 99.42% identity with orange-spotted grouper (Epinephelus coioides) TRAF2 (EcTRAF2), respectively. Quantitative real-time PCR analysis indicated that the expression of EaTRAF2 was increased in grouper spleen (GS) cells after Red-spotted grouper nervous necrosis virus (RGNNV) infection; while the expression of ToTRAF2 was decreased in golden pompano brain (TOGB) cells after RGNNV infection. Both EaTRAF2 and ToTRAF2 were identified as a cytosolic protein and suggested to be associated with vesicles scattering in the cytoplasm. Both EaTRAF2 and ToTRAF2 enhanced RGNNV replication during viral infection in vitro. Further studies showed that EaTRAF2 and ToTRAF2 overexpression decreased the expression levels of interferon associated cytokines and pro-inflammatory factors. Taken together, these results are important for better understanding of the function of TRAF2 in fish and reveal its involvement in host response to immune challenges in RGNNV.

Cutting Edge: The RNA-Binding Protein Ewing Sarcoma Is a Novel Modulator of Lymphotoxin ß Receptor Signaling.

Lymphotoxin ß receptor (LTßR) signaling is crucial for lymphoid tissue organogenesis and immune homeostasis. To identify novel regulatory mechanisms for signaling, we implemented a two-step screen that uses coexpression analysis of human fibroblasts undergoing LTßR stimulation and affinity-purification mass spectrometry for the LTßR signaling protein TNFR-associated factor 3 (TRAF3). We identify Ewing sarcoma (EWS) protein as a novel LTßR signaling component that associates with TRAF3 but not with TNFR-associated factor 2 (TRAF2). The EWS:TRAF3 complex forms under unligated conditions that are disrupted following activation of the LTßR. We conclude that EWS limits expression of proinflammatory molecules, GM-CSF, and ERK-2, promoting immune homeostasis.

TRAF2 and TRAF5 associated with the signal transducing receptor gp130 limit IL-6-driven transphosphorylation of JAK1 through the inhibition of proximal JAK-JAK interaction.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 constitutively bind to glycoprotein 130 kDa (gp130) and inhibit IL-6-driven activation of signal transducer and activator of transcription 3 (STAT3) in CD4+ T cells, which limits the differentiation of pro-inflammatory IL-17-producing helper T cells that require IL-6-receptor (IL-6R) signals for their development. However, it is not known how the interaction between TRAF and gp130 negatively regulates STAT3 activity in the IL-6R complex. We hypothesized that TRAF proteins associated with gp130 might limit the activation of Janus kinase that is needed for the activation of STAT3. To test this, we transfected HEK293T cells to express gp130 and TRAF2 or TRAF5 together with two chimeric JAK1 proteins combined with either the N-terminal or the C-terminal protein fragment of firefly luciferase. Using this luciferase fragment complementation system, we found that the recovery of luciferase enzyme activity was coincident with proximal JAK1-JAK1 interaction and phosphorylation of JAK1 in the IL-6R complex and that the expression of TRAF protein significantly inhibited the recovery of luciferase activity. The binding of TRAF to gp130 via the C-terminal TRAF domain was essential for the inhibition. In accordance with this, upon stimulation of endogenous gp130 with a complex of IL-6 and IL-6R, Traf5-/- CD4+ T cells displayed significantly higher amounts of phosphorylated JAK1 than did their wild-type counterparts. Therefore, our results demonstrate that gp130-associated TRAF2 and TRAF5 inhibit the interaction between two JAK proteins in the IL-6R complex that is essential for initiating the JAK-STAT signaling pathway.

cIAP1/2-TRAF2-SHP-1-Src-MyD88 Complex Regulates Lipopolysaccharide-Induced IL-27 Production through NF-κB Activation in Human Macrophages.

The inhibitors of apoptosis (IAP) proteins, initially described in the context of apoptosis regulation as promoting cell survival, have recently emerged as key regulators of innate immune signaling. As a result, downregulation of IAP via Smac mimetics (SMM) has both survival and immunoregulatory effects. IAPs modulate cytokine production in murine models either as a single agent or in response to LPS. However, the role of SMM and the involvement of IAPs in primary human cells and in particular macrophages with respect to cytokine production and innate immune responses remain largely unknown. IL-27, a member of the IL-12 cytokine family produced by APCs such as macrophages, has broad immunoregulatory properties in both innate and adaptive immune responses. Herein, we show that cellular IAPs (cIAPs) positively regulate LPS-induced IL-27 production in both primary human monocytes and macrophages. Investigations for the signaling mechanism of cIAPs involvement in IL-27 production in human macrophages revealed that LPS-induced IL-27 production is regulated by a novel signaling complex comprising cIAP1/2, TNFR-associated factor 2 (TRAF2), SHP-1, Src, and MyD88 leading to p38, c-Jun N-terminal kinases (JNK) and Akt activation and NF-κB signaling. In cancer cells, SMM induce the production of cytokines by activating the noncanonical alternate NF-κB pathway. However, in human macrophages, SMM do not induce the production of TNF-α and other cytokines while inhibiting LPS-induced IL-27 production by inhibiting the classical NF-κB pathway. These signaling pathways may constitute novel therapeutic avenues for immune modulation of IL-27 and provide insight into the modulatory immune effects of SMM.

Intracellular regulation of TNF activity in health and disease.

Tumor Necrosis Factor alpha (TNFα, TNF) is a key mediator and regulator of mammalian immune responses in healthy organisms and in diseased conditions. TNF governs development of the immune system, cell survival signaling pathways, proliferation and regulates metabolic processes. Whereas TNF-induced NF-κB and MAP pro-survival kinase activities constitute its major biochemical functions, TNF can also stimulate cell death in certain pathological situations. TNF-induced signal transduction pathways are tightly regulated through ubiquitination and phosphorylation of molecules partaking in all TNF-dependent membrane-associated and intracellular protein signaling complexes. Deregulated TNF signaling in individuals carrying naturally occurring genetic mutations in proteins that mediate TNF signaling, or in corresponding genetically modified animal models, results in severe pathologies. In this review we will describe the current knowledge of TNF signaling and its relevance for human health.

A molluscan TNF receptor-associated factor 2 (TRAF2) was involved in host defense against immune challenges.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a member of the TRAF superfamily that acted as a key signal transduction protein and has been implicated in inflammatory and apoptosis processes in mammals. However, identification of TRAF2s in invertebrates is very limited and its function, in particular that under immune challenges, is still unknown. In this report, a molluscan TRAF2 gene (referred to as AwTRAF2) was cloned and characterized from the freshwater bivalve, Anodonta woodiana. The open reading frame (ORF) of AwTRAF2 was 1683 bp in length, which encoded a putative 560 amino acid-protein. The deduced AwTRAF2 sequence shared similar structural characteristics and close evolutionary relationship with mollusk TRAF2s. The tissue-specific expression analysis revealed that AwTRAF2 mRNA was broadly expressed in all tested tissues, with high expression in gill and hepatopancreas. In addition, in vivo injection experiments directly showed that AwTRAF2 mRNA levels in hepatopancreas were significantly up-regulated in response to bacterial pathogen (Vibrio alginolyticus and Staphylococcus aureus) and PAMPs (Lipopolysaccharides and Peptidoglycan) challenges. Moreover, fluorescence microscopy observations revealed that AwTRAF2 was mainly located in cytoplasm of HEK293T cells and its overexpression significantly increased the transcriptional activities of the NF-κB-Luc reporter gene in HEK293T cells. Taken together, this study provided the experimental evidence of the presence of a functional TRAF2 in freshwater bivalves, which revealed its involvement in host response to immune challenges in A. woodiana.

TRAF2 of black carp upregulates MAVS-mediated antiviral signaling during innate immune response.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of the tumor necrosis factor (TNF) mediated signaling of higher vertebrates. To elucidate its function in teleost fish, TRAF2 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. The open reading frame (ORF) of black carp TRAF2 (bcTRAF2) consists of 1611 nucleotides and bcTRAF2 contains 536 amino acids. bcTRAF2 protein migrated around 65 KDa in immunoblot analysis of both EPC and HEK293T cells. bcTRAF2 was identified as a cytosolic protein and suggested to be associated with vesicles scattering in the cytoplasm. NF-κB transcription instead of IFN transcription was activated by bcTRAF2 in reporter assay. It was interesting that bcMAVS-mediated IFN production was up-regulated by bcTRAF2 in a dose dependent manner in reporter assay. Accordingly, EPC cells transfected with both bcMAVS and bcTRAF2 showed enhanced antiviral activity comparing EPC cells only expressing bcMAVS. When co-expressed with bcMAVS, bcTRAF2 was redistributed in the cytoplasm and its subcellular location overlapped with the subcellular location of bcMAVS, which suggested the association between these two molecules. Taken together, the data generated in this paper supported the conclusion that bcTRAF2 was recruited into host innate immune response and positively regulated bcMAVS-mediated antiviral signaling.

Artemisinin inhibits inflammatory response via regulating NF-κB and MAPK signaling pathways.

Artemisinin, isolated from the Chinese plant Artemisia annua, has been used for many years to treat different forms of malarial parasites. In this study, we explored the anti-inflammatory activity of artemisinin and the underlying mechanism of this action. We demonstrated that the anti-inflammatory effects of artemisinin in TPA-induced skin inflammation in mice. Then the artemisinin significantly inhibited the expression of NF-κB reporter gene induced by TNF-α in a dose-dependent manner. Artemisinin also inhibited TNF-α induced phosphorylation and degradation of IκBα, p65 nuclear translocation. Artemisinin also has an impact on upstream signaling of IKK through the inhibition of expression of adaptor proteins, TNF receptor-associated factor 2 (TRAF2) and receptor interacting protein 1 (RIP1). Furthermore, pretreatment of cells with artemisinin prevented the TNF-α-induced expression of NF-κB target genes, such as anti-apoptosis (c-IAP1, Bcl-2, and FLIP), proliferation (COX-2, cyclinD1), invasion (MMP-9), angiogenesis (VEGF), and major inflammatory cytokines (TNF-α, iNOS, and MCP1). We also proved that artemisinin potentiated TNF-α-induced apoptosis. Moreover, artemisinin significantly impaired the ROS production and phosphorylation of p38 and ERK, but did not affect the phosphorylation of JNK. Taken together, artemisinin may be a potentially useful therapeutic agent for inflammatory-related diseases.

Suppressor of cytokine signaling 3 plays an important role in porcine circovirus type 2 subclinical infection by downregulating proinflammatory responses.

Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated diseases and usually evokes a subclinical infection, without any obvious symptoms, in pigs. It remains unclear how PCV2 leads to a subclinical infection. In this study, we found that peripheral blood mononuclear cells (PBMCs) from PCV2-challenged piglets with no significant clinical symptoms exhibited increased expression of suppressor of cytokine signaling (SOCS) 3, but no significant changes in the expression of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α; this differed from piglets that displayed significant clinical symptoms. IL-6- and TNF-α-mediated signalings were inhibited in PBMCs from subclinical piglets. Elevated SOCS3 levels inhibited IL-6- and TNF-α-mediated NF-kappa-B inhibitor alpha degradation in PBMCs and PK-15 cells. SOCS3 production was also increased in PCV2-infected PK-15 porcine kidney cells, and IL-6 and TNF-α production that was induced by PCV2 in PK-15 cells was significantly increased when SOCS3 was silenced by a small interfering RNA. SOCS3 interacted with signal transducer and activator of transcription 3 and TNF-associated receptor-associated factor 2, suggesting mechanisms by which SOCS3 inhibits IL-6 and TNF-α signaling. We conclude that SOCS3 plays an important role in PCV2 subclinical infection by suppressing inflammatory responses in primary immune cells.

Hematopoietic stem cell quiescence and function are controlled by the CYLD-TRAF2-p38MAPK pathway.

The status of long-term quiescence and dormancy guarantees the integrity of hematopoietic stem cells (HSCs) during adult homeostasis. However the molecular mechanisms regulating HSC dormancy remain poorly understood. Here we show that cylindromatosis (CYLD), a tumor suppressor gene and negative regulator of NF-κB signaling with deubiquitinase activity, is highly expressed in label-retaining dormant HSCs (dHSCs). Moreover, Cre-mediated conditional elimination of the catalytic domain of CYLD induced dHSCs to exit quiescence and abrogated their repopulation and self-renewal potential. This phenotype is dependent on the interactions between CYLD and its substrate TRAF2 (tumor necrosis factor-associated factor 2). HSCs expressing a mutant CYLD with an intact catalytic domain, but unable to bind TRAF2, showed the same HSC phenotype. Unexpectedly, the robust cycling of HSCs lacking functional CYLD-TRAF2 interactions was not elicited by increased NF-κB signaling, but instead by increased activation of the p38MAPK pathway. Pharmacological inhibition of p38MAPK rescued the phenotype of CYLD loss, identifying the CYLD-TRAF2-p38MAPK pathway as a novel important regulator of HSC function restricting HSC cycling and promoting dormancy.

Blockade of CD40-TRAF2,3 or CD40-TRAF6 is sufficient to inhibit pro-inflammatory responses in non-haematopoietic cells.

Inhibition of the CD40-CD154 pathway controls inflammatory disorders. Unfortunately, administration of anti-CD154 monoclonal antibodies causes thromboembolism. Blockade of signalling downstream of CD40 may represent an approach to treat CD40-driven inflammatory disorders. Blocking tumour necrosis factor receptor-associated factor 6 (TRAF6) signalling downstream of CD40 in MHC II(+) cells diminishes inflammation. However, CD40-TRAF6 blockade may cause immunosuppression. We examined the role of CD40-TRAF2,3 and CD40-TRAF6 signalling in the development of pro-inflammatory responses in human non-haematopoietic and monocytic cells. Human aortic endothelial cells, aortic smooth muscle cells, renal proximal tubule epithelial cells, renal mesangial cells and monocytic cells were transduced with retroviral vectors that encode wild-type CD40, CD40 with a mutation that prevents TRAF2,3 recruitment (ΔT2,3), TRAF6 recruitment (ΔT6) or both TRAF2,3 plus TRAF6 recruitment (ΔT2,3,6). Non-haematopoietic cells that expressed CD40 ΔT2,3 exhibited marked inhibition in CD154-induced up-regulation of vascular cell adhesion molecule 1, intercellular adhesion molecule 1 (ICAM-1), monocyte chemotactic protein 1 (MCP-1), tissue factor and matrix metalloproteinase 9. Similar results were obtained with cells that expressed CD40 ΔT6. Although both mutations impaired ICAM-1 up-regulation in monocytic cells, only expression of CD40 ΔT6 reduced MCP-1 and tissue factor up-regulation in these cells. Treatment of endothelial and smooth muscle cells with cell-permeable peptides that block CD40-TRAF2,3 or CD40-TRAF6 signalling impaired pro-inflammatory responses. In contrast, while the CD40-TRAF2,3 blocking peptide did not reduce CD154-induced dendritic cell maturation, the CD40-TRAF6 blocking peptide impaired this response. Hence, preventing CD40-TRAF2,3 or CD40-TRAF6 interaction inhibits pro-inflammatory responses in human non-haematopoietic cells. In contrast to inhibition of CD40-TRAF6 signalling, inhibition of CD40-TRAF2,3 signalling did not impair dendritic cell maturation. Blocking CD40-TRAF2,3 signalling may control CD40-CD154-dependent inflammatory disorders.

Hepatitis C virus (HCV)-induced suppressor of cytokine signaling (SOCS) 3 regulates proinflammatory TNF-α responses.

TNF-α is a proinflammatory cytokine, dramatically elevated during pathogenic infection and often responsible for inflammation-induced disease pathology. SOCS proteins are inhibitors of cytokine signaling and regulators of inflammation. In this study, we found that both SOCS1 and SOCS3 were transiently induced by TNF-α and negatively regulate its NF-κB-mediated signal transduction. We discovered that PBMCs from HCV-infected patients have elevated endogenous SOCS3 expression but less TNF-α-mediated IκB degradation and proinflammatory cytokine production than healthy controls. HCV protein expression in Huh7 hepatocytes also induced SOCS3 and directly inhibited TNF-α-mediated IL-8 production. Furthermore, we found that SOCS3 associates with TRAF2 and inhibits TRAF2-mediated NF-κB promoter activity, suggesting a mechanism by which SOCS3 inhibits TNF-α-mediated signaling. These results demonstrate a role for SOCS3 in regulating proinflammatory TNF-α signal transduction and reveal a novel immune-modulatory mechanism by which HCV suppresses inflammatory responses in primary immune cells and hepatocytes, perhaps explaining mild pathology often associated with acute HCV infection.

Activation-induced tumor necrosis factor receptor-associated factor 3 (Traf3) alternative splicing controls the noncanonical nuclear factor κB pathway and chemokine expression in human T cells.

The noncanonical nuclear factor κB (ncNFκB) pathway regulates the expression of chemokines required for secondary lymphoid organ formation and thus plays a pivotal role in adaptive immunity. Whereas ncNFκB signaling has been well described in stromal cells and B cells, its role and regulation in T cells remain largely unexplored. ncNFκB activity critically depends on the upstream NFκB-inducing kinase (NIK). NIK expression is negatively regulated by the full-length isoform of TNF receptor-associated factor 3 (Traf3) as formation of a NIK-Traf3-Traf2 complex targets NIK for degradation. Here we show that T cell-specific and activation-dependent alternative splicing generates a Traf3 isoform lacking exon 8 (Traf3DE8) that, in contrast to the full-length protein, activates ncNFκB signaling. Traf3DE8 disrupts the NIK-Traf3-Traf2 complex and allows accumulation of NIK to initiate ncNFκB signaling in activated T cells. ncNFκB activity results in expression of several chemokines, among them B cell chemoattractant (CxCL13), both in a model T cell line and in primary human CD4(+) T cells. Because CxCL13 plays an important role in B cell migration and activation, our data suggest an involvement and provide a mechanistic basis for Traf3 alternative splicing and ncNFκB activation in contributing to T cell-dependent adaptive immunity.

Inhibition of postprandial hyperglycemia by either an insulin-dependent or -independent drug reduces the expression of genes related to inflammation in peripheral leukocytes of OLETF rats.

Treatment with the dipeptidyl peptidase-4 inhibitor, anagliptin, or with the α-glucosidase inhibitor, miglitol, reduced the oral sucrose load-inducible expression of interleukin (IL)-1ß, IL-18, tumor necrosis factor-α, S100a8, S100a9, S100a11, IL-1R2, IL-1Rn and tumor necrosis factor receptor 2 genes in peripheral leukocytes of Otsuka Long-Evans Tokushima fatty (OLETF) rats at the stage of impaired glucose tolerance. Inhibiting postprandial hyperglycemia reduced the expression of genes related to inflammation in peripheral leukocytes of OLETF rats.

TWEAK inhibits TRAF2-mediated CD40 signaling by destabilization of CD40 signaling complexes.

We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-κB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-κB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)-induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged CD40 expression and normal CD40L-CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L-CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2-cIAP1/2 ubiquitinase complex and did not affect the stability of the immunoprecipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L-CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions.

T cell costimulation with anti-CD137 monoclonal antibodies is mediated by K63-polyubiquitin-dependent signals from endosomes.

Agonist anti-CD137 (4-1BB) mAbs enhance CD8-mediated antitumor immunity. Agonist anti-human CD137 mAbs binding to four distinct epitopes on the CD137 glycoprotein costimulated T cell activation irrespective of the engaged epitope or its interference with CD137L binding. CD137 perturbation with all these agonist mAbs resulted in Ag and Ab internalization toward an endosomal vesicular compartment. Internalization was observed in activated T lymphocytes from humans and mice, not only in culture but also in Ab-injected living animals. These in vivo experiments were carried out upon systemic i.v. injections with anti-CD137 mAbs and showed CD137 internalization in tumor-infiltrating lymphocytes and in activated human T cells transferred to immunodeficient mice. Efficient CD137 internalization required K63 polyubiquitination and endocytosed CD137-containing vesicles recruited TNFR-associated factor (TRAF) 2 and were decorated with K63 polyubiquitins. CD137 stimulation activates NF-κB through a K63-linked polyubiquitination-dependent route, and CD137-associated TRAF2 becomes K63 polyubiquitinated. Consistent with a role for TRAF2 in CD137 signaling, transgenic mice functionally deficient in TRAF2 showed delayed immunotherapeutic activity of anti-CD137 mAbs. As a whole, these findings advance our knowledge of the mechanisms of action of anti-CD137 immunostimulatory mAbs such as those currently undergoing clinical trials in cancer patients.

IκB kinase ε phosphorylates TRAF2 to promote mammary epithelial cell transformation.

NF-κB transcription factors are central regulators of inflammation and when dysregulated contribute to malignant transformation. IκB kinase ε (IKKε; IKKi, encoded by IKBKE) is a breast oncogene that is amplified in 30% of breast cancers and drives transformation in an NF-κB-dependent manner. Here we demonstrate that IKKε interacts with and phosphorylates tumor necrosis factor receptor-associated factor 2 (TRAF2) at Ser11 in vitro and in vivo. This activity promotes Lys63-linked TRAF2 ubiquitination and NF-κB activation and is essential for IKKε transformation. Breast cancer cells that depend on IKKε expression for survival are also dependent on TRAF2. This work defines TRAF2 phosphorylation to be one key effector of IKKε-induced mammary epithelial cell transformation.

Aberrant accumulation of interleukin-10-secreting neutrophils in TRAF2-deficient mice.

Highly coordinated expression of inflammatory and anti-inflammatory cytokines is crucial for maintaining homeostasis of the gut that is constantly exposed to large amounts of commensal bacteria. We have previously reported that tumor necrosis factor (TNF) receptor-associated factor (Traf)2(-/-) mice spontaneously develop severe colitis and that the development of colitis largely depends on TNFα-dependent apoptosis of colonic epithelial cells. However, the detailed molecular mechanisms underlying the immunological disorders of Traf2(-/-) mice are not fully understood. Here we show that interleukin (IL)-10-secreting neutrophils accumulated in peripheral blood and bone marrow (BM) cells from Traf2(-/-) mice compared with those from wild-type mice. Treatment of Traf2(-/-) mice with neutralizing antibody against TNFα or crossing Traf2(-/-) mice with Tnfr1(-/-) mice reduced the percentages of IL-10-secreting neutrophils, suggesting that the development of IL-10-secreting neutrophils largely depended on TNFα signals. Moreover, stimulation of BM cells from wild-type mice with lipopolysaccharide and Pam3CS(K)4, a ligand for Toll-like receptor 4 and 2, respectively, induced differentiation of BM cells into IL-10-secreting neutrophils. These results suggest that the development of IL-10-secreting neutrophils is not restricted to Traf2(-/-) mice, but could be generalized to wild-type mice under certain conditions such as inflammation. Finally, combined treatment of Traf2(-/-) mice with neutralizing antibodies against TNFα and IL-10, but not each antibody alone, substantially ameliorated colitis and prolonged survival. Together, abrogation of immunosuppressive conditions mediated by IL-10-secreting neutrophils might be an alternative strategy to treat chronic inflammatory diseases at least under certain conditions.

Signal transduction by tumor necrosis factor receptors.

Tumor necrosis factor (TNF) is a key mediator in the inflammatory response which is implicated in the onset of a number of diseases. Research on TNF led to the characterization of the largest family of cytokines known until now, the TNF superfamily, which exert their biological effects through the interaction with transmembrane receptors of the TNFR superfamily. TNF itself exerts its biological effects interacting with two different receptors: TNFR1 and TNFR2. TNFR1 presents a death domain on its intracellular region. In contrast to TNFR1, TNFR2 does not have a death domain. Activation of TNFR1 implies the consecutive formation of two different TNF receptor signalling complexes. Complex I controls the expression of antiapoptotic proteins that prevent the triggering of cell death processes, whereas Complex II triggers cell death processes. TNFR2 only signals for antiapoptotic reactions. However, recent evidence indicates that TNFR2 also signals to induce TRAF2 degradation. TRAF2 is a key mediator in signal transduction of both TNFR1 and TNFR2. Thus, this novel signalling pathway has two important implications: on one hand, it represents an auto regulatory loop for TNFR2; on the other hand, when this signal is triggered TNFR1 activity is modified so that antiapoptotic pathways are inhibited and apoptotic reactions are enhanced.

Non-canonical NF-κB signaling activation and regulation: principles and perspectives.

Nuclear-factor κB (NF-κB) transcription factors are activated by a wide variety of stimuli in diverse cell types and control key aspects of immune function and development. Receptor-mediated activation of NF-κB appears to occur through two distinct signaling pathways termed as the canonical and non-canonical NF-κB pathways. Although much work has demonstrated the physiological importance of non-canonical NF-κB signaling to immunity and its involvement in diverse pathologies, such as cancers and autoimmune disease, the architecture and regulation of the pathway is only beginning to be understood. The non-canonical pathway appears to be activated by a select set of receptors within the tumor necrosis factor superfamily, and we discuss the molecular mechanisms that connect ligation of these receptors to pathway activation. It has become increasingly clear that the key regulatory step of the pathway involves modulation of the post-translational degradation of NF-κB-inducing kinase (NIK), the central activating kinase of non-canonical NF-κB signaling. How NIK post-translational stability is controlled before and after receptor ligation is an important aspect of understanding non-canonical NF-κB signaling. Furthermore, how release of NF-κB dimers downstream of the pathway's activation is actually connected to its identified physiological and pathological roles is a key remaining question in the field.