TRAF6 Signaling Research Articles

The potential role of SNHG16/ miRNA-146a/ TRAF6 signaling pathway in the protective effect of zoledronate against colorectal cancer and associated osteoporosis in mouse model

Bone fracture as a consequence of colorectal cancer (CRC) and associated osteoporosis (OP) is considered a risk factor for increasing the mortality rate among CRC patients. SNHG16/ miRNA-146a/ TRAF6 signaling pathway is a substantial contributor to neoplastic evolution, progression, and metastasis. Here, we investigated the effect of zoledronate (ZOL) on the growth of CRC and associated OP in a mouse model. Thirty Balb/c mice were divided into Naïve, azoxymethane (AOM)/dextran sodium sulfate (DSS), and ZOL groups. Body weight and small nucleolar RNA host gene 16 (SNHG16) expression, microRNA-146a, and TRAF6 in bone, colon, and stool were investigated. Samples of colon and bone were collected and processed for light microscopic, immunohistochemical staining for cytokeratin 20 (CK20), nuclear protein Ki67 (pKi-67), and caudal type homeobox transcription factor 2 (CDx2) in colon and receptor activator of nuclear factor kB (RANK) and osteoprotegerin (OPG) in bone. A computerized tomography (CT) scan of the femur and tibia was studied. ZOL produced a significant decrease in the expression of SNHG16 and TRAF6 and an increase in miRNA-146a in the colon and bone. ZOL administration improved the histopathological changes in the colon, produced a significant decrease in CK20 and Ki-67, and increased CDx2 expressions. In bone, ZOL prevented osteoporotic changes and tumour cell invasion produced a significant decrease in RANK and an increase in OPG expressions, alongside improved bone mineral density in CT scans. ZOL could be a promising preventive therapy against colitis-induced cancer and associated OP via modulation expression of SNHG16, miRNA-146a, and TRAF6.

TIFAB regulates the TIFA–TRAF6 signaling pathway involved in innate immunity by forming a heterodimer complex with TIFA

Nuclear factor κB (NF-κB) is activated by various inflammatory and infectious molecules and is involved in immune responses. It has been elucidated that ADP-β-D-manno-heptose (ADP-Hep), a metabolite in gram-negative bacteria, activates NF-κB through alpha-kinase 1 (ALPK1)-TIFA-TRAF6 signaling. ADP-Hep stimulates the kinase activity of ALPK1 for TIFA phosphorylation. Complex formation between phosphorylation-dependent TIFA oligomer and TRAF6 promotes the polyubiquitination of TRAF6 for NF-κB activation. TIFAB, a TIFA homolog lacking a phosphorylation site and a TRAF6 binding motif, is a negative regulator of TIFA-TRAF6 signaling and is implicated in myeloid diseases. TIFAB is indicated to regulate TIFA-TRAF6 signaling through interactions with TIFA and TRAF6; however, little is known about its biological function. We demonstrated that TIFAB forms a complex not with the TIFA dimer, an intrinsic form of TIFA involved in NF-κB activation, but with monomeric TIFA. The structural analysis of the TIFA/TIFAB complex and the biochemical and cell-based analyses showed that TIFAB forms a stable heterodimer with TIFA, inhibits TIFA dimer formation, and suppresses TIFA-TRAF6 signaling. The resultant TIFA/TIFAB complex is a "pseudo-TIFA dimer" lacking the phosphorylation site and TRAF6 binding motif in TIFAB and cannot form the orderly structure as proposed for the phosphorylated TIFA oligomer involved in NF-κB activation. This study elucidated the molecular and structural basis for the regulation of TIFA-TRAF6 signaling by TIFAB.

ADP-Hep-Induced Liquid Phase Condensation of TIFA-TRAF6 Activates ALPK1/TIFA-Dependent Innate Immune Responses.

The ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with fork head-associated domain)-TRAF6 signaling pathway plays a pivotal role in regulating inflammatory processes, with TIFA and TRAF6 serving as key molecules in this cascade. Despite its significance, the functional mechanism of TIFA-TRAF6 remains incompletely understood. In this study, we unveil that TIFA undergoes liquid-liquid phase separation (LLPS) induced by ALPK1 in response to adenosine diphosphate (ADP)-β-D-manno-heptose (ADP-Hep) recognition. The phase separation of TIFA is primarily driven by ALPK1, the pT9-FHA domain, and the intrinsically disordered region segment. Simultaneously, TRAF6 exhibits phase separation during ADP-Hep-induced inflammation, a phenomenon observed consistently across various inflammatory signal pathways. Moreover, TRAF6 is recruited within the TIFA condensates, facilitating lysine (K) 63-linked polyubiquitin chain synthesis. The subsequent recruitment, enrichment, and activation of downstream effectors within these condensates contribute to robust inflammatory signal transduction. Utilizing a novel chemical probe (compound 22), our analysis demonstrates that the activation of the ALPK1-TIFA-TRAF6 signaling pathway in response to small molecules necessitates the phase separation of TIFA. In summary, our findings reveal TIFA as a sensor for upstream signals, initiating the LLPS of itself and downstream proteins. This process results in the formation of membraneless condensates within the ALPK1-TIFA-TRAF6 pathway, suggesting potential applications in therapeutic biotechnology development.

Ozone exposure affects corneal epithelial fate by promoting mtDNA leakage and cGAS/STING activation

Ozone is a common air pollutant associated with various human diseases. The human ocular surface is frequently exposed to ozone in the troposphere, but the mechanisms by which ozone affects the ocular surface health remain unclear. This study aimed to establish a mouse model to investigate the effects of ozone exposure on the ocular surface and the corneal epithelium. The findings revealed that ozone exposure disrupted corneal epithelial homeostasis and differentiation, resulting in corneal squamous metaplasia. Further, ozone exposure induced oxidative damage and cytoplasmic leakage of mitochondrial DNA (mtDNA), thereby activating the cGAS/STING signaling pathway. The activation of the cGAS/STING signaling pathway triggered the activation of downstream NF-κB and TRAF6 signaling pathways, causing corneal inflammation, thereby promoting corneal inflammation and squamous metaplasia. Finally, C-176, a selective STING inhibitor, effectively prevented and treated corneal inflammation and squamous metaplasia caused by ozone exposure. This study revealed the role of mtDNA leakage-mediated cGAS/STING activation in corneal squamous epithelial metaplasia caused by ozone exposure. It also depicted the abnormal expression pattern of corneal epithelial keratin using three-dimensional images, providing new targets and strategies for preventing and treating corneal squamous metaplasia and other ocular surface diseases.

Transmission of NLRP3-IL-1β Signals in Cerebral Ischemia and Reperfusion Injury: from Microglia to Adjacent Neuron and Endothelial Cells via IL-1β/IL-1R1/TRAF6.

The pyrin domain-containing protein 3 (NLRP3) inflammasome drives the profound cerebral ischemia and reperfusion injury (I/R) and mediates the secretion of IL-1β (interleukin-1β), which exerts a subsequent cascade of inflammatory injury. The NLRP3-activated-microglial manipulation in adjacent neuronal and endothelial NLRP3 activation has been confirmed in our previous studies. In the present study, we extended the cognition of how microglia mediated neuronal and endothelial NLRP3-IL-1β signaling during cerebral ischemia and reperfusion injury. In vitro, Neuro-2a and bEND3 cells were cultured alone or co-cultured with BV2 cells and oxygen-glucose deprivation/reoxygenation (OGD/R) was performed. In vivo, transient middle cerebral artery occlusion (tMCAO) rat models and lentiviral silencing targeting IL-1R1 were performed. The NLRP3 inflammasome activation was evaluated by enzyme-linked immunosorbent assay, western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence. In the co-culture system after OGD/R treatment, NLRP3 inflammasomes in neurons and endothelial cells were activated by microglial IL-1β via IL-1β/IL-1R1/TRAF6 signaling pathway, with the basal protein level of NLRP3. In addition, ruptured lysosomes engulfing ASC specks which were possibly secreted from microglia triggered the enhanced NLRP3 expression. In cortices of tMCAO rats at 24h of reperfusion, silencing IL-1R1, mainly presented in neurons and endothelial cells, was efficient to block the subsequent inflammatory damage and leukocyte brain infiltration, leading to better neurological outcome. Neuronal and endothelial NLRP3 inflammasomes were activated by microglia in cerebral ischemia and reperfusion injury mainly via IL-1β/IL-1R1/TRAF6 signaling, which might be therapeutically targetable.

Inhibition of platelet activation suppresses reactive enteric glia and mitigates intestinal barrier dysfunction during sepsis

BackgroundIntestinal barrier dysfunction, which is associated with reactive enteric glia cells (EGCs), is not only a result of early sepsis but also a cause of multiple organ dysfunction syndrome. Inhibition of platelet activation has been proposed as a potential treatment for septic patients because of its efficacy in ameliorating the organ damage and barrier dysfunction. During platelet activation, CD40L is translocated from α granules to the platelet surface, serving as a biomarker of platelet activation a reliable predictor of sepsis prognosis. Given that more than 95% of the circulating CD40L originate from activated platelets, the present study aimed to investigate if inhibiting platelet activation mitigates intestinal barrier dysfunction is associated with suppressing reactive EGCs and its underlying mechanism.MethodsCecal ligation and puncture (CLP) was performed to establish the sepsis model. 24 h after CLP, the proportion of activated platelets, the level of sCD40L, the expression of tight-junction proteins, the intestinal barrier function and histological damage of septic mice were analyzed. In vitro, primary cultured EGCs were stimulated by CD40L and LPS for 24 h and EGCs-conditioned medium were collected for Caco-2 cells treatment. The expression of tight-junction proteins and transepithelial electrical resistance of Caco-2 cell were evaluated.ResultsIn vivo, inhibiting platelet activation with cilostazol mitigated the intestinal barrier dysfunction, increased the expression of ZO-1 and occludin and improved the survival rate of septic mice. The efficacy was associated with reduced CD40L+ platelets proportion, decreased sCD40L concentration, and suppressed the activation of EGCs. Comparable results were observed upon treatment with compound 6,877,002, a blocker of CD40L–CD40–TRAF6 signaling pathway. Also, S-nitrosoglutathione supplement reduced intestinal damage both in vivo and in vitro. In addition, CD40L increased release of TNF-α and IL-1β while suppressed the release of S-nitrosoglutathione from EGCs. These EGCs-conditioned medium reduced the expression of ZO-1 and occludin on Caco-2 cells and their transepithelial electrical resistance, which could be reversed by CD40-siRNA and TRAF6-siRNA transfection on EGCs.ConclusionsThe inhibition of platelet activation is related to the suppression of CD40L-CD40-TRAF6 signaling pathway and the reduction of EGCs activation, which promotes intestinal barrier function and survival in sepsis mice. These results might provide a potential therapeutic strategy and a promising target for sepsis.

A peptide from wheat germ abolishes the senile osteoporosis by regulating OPG/RANKL/RANK/TRAF6 signaling pathway

BackgroundOxidative stress played a key role in the development of bone brittleness and is an important pathogenic factor of senile osteoporosis. A variety of animal and plant-derived peptides have been shown to have significant anti-osteoporosis effects in vivo and in vitro. PurposeIn this study, we aim to explore the possible mechanism of wheat germ peptide ADWGGPLPH on senile osteoporosis. Study designNaturally, aged rats were used as animal models of senile osteoporosis. MethodsWheat germ peptide ADWGGPLPH was administered from 9-months-old to 21-months-old, and the effect of ADWGGPLPH on preventing senile osteoporosis was evaluated by measuring serum biochemical indexes, bone histomorphometry, bone biomechanics, and other indexes to elucidate the mechanism of ADWGGPLPH in delaying senile osteoporosis by detecting the expression of osteoporosis-related proteins. ResultsThe results showed that ADWGGPLPH could effectively reduce the level of oxidative stress and improve the microstructure and bone mineral density in senile osteoporosis rats. In addition, ADWGGPLPH could improve the proliferation and differentiation activity of osteoblasts and effectively inhibit osteoclasts’ differentiation by regulating the OPG/RANKL/RANK/TRAF6 pathway. ConclusionADWGGPLPH from wheat germ exhibited a notably effect on senile osteoporosis and has a high potential in the development of the nutrient regimen to against senile osteoporosis.

Gonadal hormone trigger the dynamic microglial alterations through Traf6/TAK1 axis that correlate with depressive behaviors

Gonadal hormone deficiency is associated with the development of depression, but what mediates this association is unclear. To test the possibility that it reflects neuroimmune and neuroinflammatory processes, we analyzed how gonadal hormone deficiency and replacement affect microglial activation and inflammatory response during the development of depressive symptomatology in gonadectomized male mice. Testosterone level and the ratio of testosterone to estradiol in the serum and brain tissue of mice exposed to 3–35 days of chronic unpredictable stress were much lower than in control animals. Gonadal hormone sustained deficiency in gonadectomized mice and subsequent led to acute inflammation at day 7 following castration. Activating microglia in mice exposed to 7 days of castration subsequently suppressed the proliferation of microglia, such that their numbers in hippocampus and cortex were lower than the numbers in sham-operated mice after 30 days of castration. Here, we showed that gonadal hormone deficiency induces Traf6-mediated microglia activation, a type of inflammatory mediator. Microglia treated in this way for long time showed down-regulation of activation markers, abnormal morphology and depressive-like behaviors. Restoration and maintenance of a fixed ratio of testosterone to estradiol significantly suppressed microglial activation, neuronal necroptosis, dramatically inducing hippocampal neurogenesis and reducing depressive behaviors via the suppression of Traf6/TAK1 pathway. These findings suggest that activated or immunoreactive microglia contribute to gonadal hormone deficiency-induced depression, as well as testosterone and estradiol exert synergistic anti-depressant effects via suppressing microglial activaton in gonadectomized male mice, possibly through Traf6 signaling.

TRAF6 functions as a tumor suppressor in myeloid malignancies by directly targeting MYC oncogenic activity.

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an invivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification.

TRIP6 promotes inflammatory damage via the activation of TRAF6 signaling in a murine model of DSS-induced colitis

BackgroundTRIP6 is a zyxin family member that serves as an adaptor protein to regulate diverse biological processes. In prior reports, TRIP6 was shown to play a role in regulating inflammation. However, its in vivo roles and mechanistic importance in colitis remain largely elusive. Herein, we therefore employed TRIP6-deficient (TRIP6−/−) mice in order to explore the mechanistic importance of TRIP6 in a dextran sodium sulfate (DSS)-induced model of murine colitis.FindingsWild-type (TRIP6+/+) mice developed more severe colitis following DSS-mediated disease induction relative to TRIP6−/− mice, as evidenced by more severe colonic inflammation and associated crypt damage. TRIP6 expression in wild-type mice was significantly elevated following DSS treatment. Mechanistically, TRIP6 binds to TRAF6 and enhances oligomerization and autoubiquitination of TRAF6. This leads to the activation of NF-κB signaling and the expression of pro-inflammatory cytokines such as TNFα and IL-6, in the in vivo mouse model of colitis.ConclusionsThese in vivo data demonstrate that TRIP6 serves as a positive regulator of DSS-induced colitis through interactions with TRAF6 resulting in the activation of inflammatory TRAF6 signaling, highlighting its therapeutic promise as a protein that theoretically can be targeted to prevent or treat colitis.

Inflammation rapidly recruits mammalian GMP and MDP from bone marrow into regional lymphatics.

Innate immune cellular effectors are actively consumed during systemic inflammation, but the systemic traffic and the mechanisms that support their replenishment remain unknown. Here, we demonstrate that acute systemic inflammation induces the emergent activation of a previously unrecognized system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors, but not other progenitors or stem cells, from bone marrow (BM) to regional lymphatic capillaries. The progenitor traffic to the systemic lymphatic circulation is mediated by Ccl19/Ccr7 and is NF-κB independent, Traf6/IκB-kinase/SNAP23 activation dependent, and is responsible for the secretion of pre-stored Ccl19 by a subpopulation of CD205+/CD172a+ conventional dendritic cells type 2 and upregulation of BM myeloid progenitor Ccr7 signaling. Mature myeloid Traf6 signaling is anti-inflammatory and necessary for lymph node myeloid cell development. This report unveils the existence and the mechanistic basis of a very early direct traffic of myeloid progenitors from BM to lymphatics during inflammation.

Echinococcus granulosus cyst fluid suppresses inflammatory responses by inhibiting TRAF6 signalling in macrophages.

Echinococcus granulosus sensu lato has complex defence mechanisms that protect it from the anti-parasitic immune response for long periods. Echinococcus granulosus cyst fluid (EgCF) is involved in the immune escape. Nevertheless, whether and how EgCF modulates the inflammatory response in macrophages remains poorly understood. Here, real-time polymerase chain reaction and enzyme-linked immunosorbent assay revealed that EgCF could markedly attenuate the lipopolysaccharide (LPS)-induced production of pro-inflammatory factors including tumour necrosis factor-α, interleukin (IL)-12 and IL-6 but increase the expression of IL-10 at mRNA and protein levels in mouse peritoneal macrophages and RAW 264.7 cells. Mechanically, western blotting and immunofluorescence assay showed that EgCF abolished the activation of nuclear factor (NF)-κB p65, p38 mitogen-activated protein kinase (MAPK) and ERK1/2 signalling pathways by LPS stimulation in mouse macrophages. EgCF's anti-inflammatory role was at least partly contributed by promoting proteasomal degradation of the critical adaptor TRAF6. Moreover, the EgCF-promoted anti-inflammatory response and TRAF6 proteasomal degradation were conserved in human THP-1 macrophages. These findings collectively reveal a novel mechanism by which EgCF suppresses inflammatory responses by inhibiting TRAF6 and the downstream activation of NF-κB and MAPK signalling in both human and mouse macrophages, providing new insights into the molecular mechanisms underlying the E. granulosus-induced immune evasion.

SUMOylation involves in β-arrestin-2-dependent metabolic regulation in breast cancer cell

β-arrestin-2, a multifunctional adaptor protein, was originally identified as a negative regulator of G protein–mediated signaling. We previously revealed that SUMOylation as a novel mechanism modulates β-arrestin-2-mediated IL-1R/TRAF6 signaling. However, the potential role of β-arrestin-2 SUMOylation in tumor cells was incompletely explored. In this study, we showed that SUMOylation deficiency of β-arrestin-2 resulted in slower migration of breast cancer cells, but little effect on the cell proliferation. Importantly, our data indicated that SUMOylation involves in β-arrestin-2-dependent metabolic regulation, suggesting a potent regulatory pattern for β-arrestin-2-mediated biological functions of tumor cells.

Minocycline reverses IL-17A/TRAF3IP2-mediated p38 MAPK/NF-κB/iNOS/NO-dependent cardiomyocyte contractile depression and death

Minocycline, an FDA-approved second-generation semisynthetic tetracycline, exerts antioxidant, anti-apoptotic and anti-inflammatory effects, independent of its antimicrobial properties. Interleukin (IL)-17A is an immune and inflammatory mediator, and its sustained induction is associated with various cardiovascular diseases. Here we investigated (i) whether IL-17A induces cardiomyocyte contractile depression and death, (ii) whether minocycline reverses IL-17A's negative inotropic effects and (iii) investigated the underlying molecular mechanisms. Indeed, treatment with recombinant mouse IL-17A impaired adult cardiomyocyte contractility as evidenced by a 34% inhibition in maximal velocity of shortening and relengthening after 4 h (P < .01). Contractile depression followed iNOS induction at 2 h (2.13-fold, P < .01) and NO generation at 3 h (3.71-fold, P <.01). Further mechanistic investigations revealed that IL-17A-dependent induction of iNOS occurred via TRAF3IP2, TRAF6, TAK1, NF-κB, and p38MAPK signaling. 1400 W, a highly specific iNOS inhibitor, suppressed IL-17A-induced NO generation and contractile depression, where as the NO donors SNAP and PAPA-NONOate both suppressed cardiomyocyte contractility. IL-17A also stimulated cardiomyocyte IL-1β and TNF-α secretion, however, their neutralization failed to modulate IL-17A-mediated contractile depression or viability. Further increases of IL-17A concentration and the duration of exposure enhanced IL-1β and TNF-α secreted levels, buthad no impact on adult cardiomyocyte viability. However, when combined with pathophysiological concentrations of IL-1β or TNF-α, IL-17A promoted adult cardiomyocyte death. Importantly, minocycline blunted IL-17A-mediated deleterious effects, indicating its therapeutic potential in inflammatory cardiac diseases.

Structural analysis of TIFA: Insight into TIFA-dependent signal transduction in innate immunity

TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA), originally identified as an adaptor protein of TRAF6, has recently been shown to be involved in innate immunity, induced by a pathogen-associated molecular pattern (PAMP). ADP-β-D-manno-heptose, a newly identified PAMP, binds to alpha-kinase 1 (ALPK1) and activates its kinase activity to phosphorylate TIFA. Phosphorylation triggers TIFA oligomerisation and formation of a subsequent TIFA–TRAF6 oligomeric complex for ubiquitination of TRAF6, eventually leading to NF-κB activation. However, the structural basis of TIFA-dependent TRAF6 signalling, especially oligomer formation of the TIFA–TRAF6 complex remains unknown. In the present study, we determined the crystal structures of mouse TIFA and two TIFA mutants—Thr9 mutated to either Asp or Glu to mimic the phosphorylation state—to obtain the structural information for oligomer formation of the TIFA–TRAF6 complex. Crystal structures show the dimer formation of mouse TIFA to be similar to that of human TIFA, which was previously reported. This dimeric structure is consistent with the solution structure obtained from small angle X-ray scattering analysis. In addition to the structural analysis, we examined the molecular assembly of TIFA and the TIFA–TRAF6 complex by size-exclusion chromatography, and suggested a model for the TIFA–TRAF6 signalling complex.

IL-17A neutralizing antibody attenuates eosinophilic meningitis caused by Angiostrongylus cantonensis by involving IL-17RA/Traf6/NF-κB signaling

BackgroundAngiostrongylus cantonensis (A. cantonensis) is a foodborne parasite that can invade the central nervous system (CNS), resulting in eosinophilic meningitis (EM). However, the mechanism by which A. cantonensis causes eosinophilic infiltration into CNS is not well understood. MethodsIn this study eosinophilic infiltration into the CNS caused by A. cantonensis was assessed based on eosinophil counts and evaluation of interleukin (IL)-5 and −13 levels by real-time PCR in brain of Balb/c mice. The expression and activation of IL-17A, IL17 receptor (IL-17R A), and IL-17RC and the related signaling molecules nuclear factor (NF)-κB1, NF-κB2, NF-κB activator (Act)1, tumor necrosis factor receptor-associated factor (Traf)5, and Traf6 during A. cantonensis infection in brain tissue of Balb/c mice were examined by real-time, western blotting and immunofluroence. A. cantonensis-infected Balb/c mice were treated with IL-17A neutralizing antibody to evaluate the role of IL17A in eosinophil accumulation in the CNS. ResultsOur results showed A. cantonensis infection caused eosinophil accumulation and alterations in IL-5 and -13 levels. The expression of IL-17A and −17RA, Act1, and Traf6 but not of IL-17RC and Traf5 was upregulated during infection; this was accompanied by NF-κB1 and -κB2 activation. Importantly, application of IL-17A neutralizing antibody attenuated eosinophil accumulation in CNS and reversed the changes in IL-5 and -13 expression caused by A. cantonensis infection. Additionally, IL-17RA and Traf6 levels decreased, which was accompanied by NF-κB inactivation. ConclusionIL-17A plays an important role in EM caused by A. cantonensis, possibly through activation of NF-κB via the IL-17RA/Traf6 signaling pathway. These findings highlight the potential for using IL-17A neutralizing antibody as a therapeutic strategy for the treatment of EM.

Gonadal Hormone Trigger Traf6-Mediated Dynamic Microglial Alterations that Correlate with Depressive Behaviors

Gonadal hormone deficiency is associated with the development of depression, but what mediates this association is unclear. To test the possibility that it reflects neuroimmune and neuroinflammatory processes, we analyzed how gonadal hormone deficiency and replacement affect microglial activation and inflammatory response during the development of depressive symptomatology in gonadectomized male mice. Testosterone level and the ratio of testosterone to estradiol in the serum and brain tissue of mice exposed to 3-35 days of chronic unpredictable stress were much lower than in control animals. Gonadal hormone sustained deficiency in gonadectomized mice and subsequent led to acute inflammation at day 7 following castration. Activating microglia in mice exposed to 7 days of castration subsequently suppressed the proliferation of microglia, such that their numbers in hippocampus and cortex were lower than the numbers in sham-operated mice after 30 days of castration. Here, we showed that gonadal hormone deficiency induces Traf6-mediated microglia activation, a type of inflammatory mediator. Microglia treated in this way for long time showed down-regulation of activation markers, abnormal morphology and depressive-like behaviors. Restoration and maintenance of a fixed ratio of testosterone to estradiol significantly suppressed microglial activation, neuronal necroptosis, dramatically inducing hippocampal neurogenesis and reducing depressive behaviors via the suppression of Traf6/TAK1 pathway. These findings suggest that activated or immunoreactive microglia contribute to gonadal hormone deficiency-induced depression, as well as testosterone and estradiol exert synergistic anti-depressant effects via suppressing microglial activaton in gonadectomized male mice, possibly through Traf6 signaling. Funding: This study was supported by National Nature Science Foundation of China (No. 81572069, 81501815, 81772265). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: Experiments in ICR male mice were approved by the Animals Ethics and Welfare Committee of Renmin Hospital of Wuhan University. All the experiments were conducted in accordance with the Directives of Wuhan University on animal ethics and welfare.

Immune Control by TRAF6-Mediated Pathways of Epithelial Cells in the EIME (Epithelial Immune Microenvironment).

In the protective responses of epithelial tissues, not only immune cells but also non-immune cells directly respond to external agents. Epithelial cells can be involved in the organization of immune responses through two phases. First, the exogenous harmful agents trigger the primary responses of the epithelial cells leading to various types of immune cell activation. Second, cytokines produced by the immune cells that are activated directly by the external agents and indirectly by the epithelial cell products elicit the secondary responses giving rise to further propagation of immune responses. TRAF6 is a ubiquitin E3 ligase, which intermediates between various types of receptors for exogenous agents or endogenous mediators and activation of subsequent transcriptional responses via NF-kappaB and MAPK pathways. TRAF6 ubiquitously participates in many protective responses in immune and non-immune cells. Particularly, epithelial TRAF6 has an essential role in the primary and secondary responses via driving type 17 response in psoriatic inflammation of the skin. Consistently, many psoriasis susceptibility genes encode the TRAF6 signaling players, such as ACT1 (TRAF3IP2), A20 (TNFAIP3), ABIN1 (TNIP1), IL-36Ra (IL36RN), IkappaBzeta (NFKBIZ), and CARD14. Herein, we describe the principal functions of TRAF6, especially in terms of positive and regulatory immune controls by interaction between immune cells and epithelial cells. In addition, we discuss how TRAF6 in the epithelial cells can organize the differentiation of immune responses and drive inflammatory loops in the epithelial immune microenvironment, which is termed EIME.

Macrophage CD40 signaling drives experimental autoimmune encephalomyelitis.

The costimulatory CD40L–CD40 dyad plays a major role in multiple sclerosis (MS). CD40 is highly expressed on MHCII+ B cells, dendritic cells and macrophages in human MS lesions. Here we investigated the role of the CD40 downstream signaling intermediates TNF receptor‐associated factor 2 (TRAF2) and TRAF6 in MHCII+ cells in experimental autoimmune encephalomyelitis (EAE). Both MHCII–CD40–Traf2 −/− and MHCII–CD40–Traf6 −/− mice showed a reduction in clinical signs of EAE and prevented demyelination. However, only MHCII–CD40–Traf6 −/− mice displayed a decrease in myeloid and lymphoid cell infiltration into the CNS that was accompanied by reduced levels of TNF‐α, IL‐6 and IFN‐γ. As CD40–TRAF6 interactions predominantly occur in macrophages, we subjected CD40 flfl LysM cre mice to EAE. This myeloid‐specific deletion of CD40 resulted in a significant reduction in EAE severity, reduced CNS inflammation and demyelination.In conclusion, the CD40–TRAF6 signaling pathway in MHCII+ cells plays a key role in neuroinflammation and demyelination during EAE. Concomitant with the fact that CD40–TRAF6 interactions are predominant in macrophages, depletion of myeloid CD40 also reduces neuroinflammation. CD40–TRAF6 interactions thus represent a promising therapeutic target for MS. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity

Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P2 (PIP2). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains (ΔIPMK) are presented. Although the KM for PIP2 is identical between ΔIPMK and wild type, ΔIPMK has a 1.8-fold increase in kcat for PIP2, indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 Å crystal structure of ΔIPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative “ATP-clamp” in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the KM for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms.