TRAF6 Research Articles

TRAF3 Alterations Identify a Subgroup of Patients with Adverse Clinical Outcome and Induce Metabolic Reprogramming in Chronic Lymphocytic Leukemia

The TNF receptor-associated factor3 (TRAF3) gene located in 14q32 is frequently altered by deletion and/or mutation in multiple myeloma, lymphoma and other B-cell malignancies such as chronic lymphocytic leukemia (CLL). TRAF3 is a negative regulator of the non-canonical NF-KB signaling, and recent data uncovered additional functions of this gene in inflammation, antiviral immunity or cellular metabolism. Nevertheless, the biological and clinical significance of TRAF3 alterations in CLL is unknown. For that, the aim of this study was to assess the clinical impact of TRAF3 alterations in a CLL cohort as well as its biological role in CLL pathogenesis through CRISPR/Cas9-generated models. TRAF3 alterations (mutations and copy number variations-CNVs) were assessed by NGS in our previously published cohort of 340 CLLs (Pérez Carretero, AJH 2022). A total of 14 patients exhibited a TRAF3 CNV loss, and 10 of them showed additional TRAF3 loss-of-function mutations, indicating a biallelic inactivation of this gene in CLL. Interestingly, the presence of TRAF3 alterations was significantly associated with shorter time to first treatment (6 vs 75 months, p<0.001), being an independent risk factor in the multivariate analysis (p=0.029). To further investigate the biological role of TRAF3, we used the CRISPR/Cas9 system to model TRAF3 loss-of-function mutations in the CLL-derived cell line PGA1, and characterized these cells by transcriptomics, metabolomics and functional studies. RNA-seq analyses identified 54 dysregulated genes enriched in pathways such as NIK/NF-kB signaling (FDR=0.029). Concretely, NIK (a direct target of TRAF3-mediated degradation) was significantly upregulated (Q<0.001) in TRAF3mut clones, which correlated with a higher nuclear activity of the non-canonical NF-kB transcription factors p52 (p=0.05) and RelB (p=0.02). Moreover, we validated these results in primary CLL cells (n=15; 3 TRAF3mut cells). Besides, PGA1-TRAF3mut cells showed increased levels of BAX (p=0.05) and more sensitivity to venetoclax (IC50 3.1 vs 13.8μM, p=0.003). In the metabolomic study, we found an enrichment of metabolites involved in Warburg effect, citric acid cycle (TCA) and glutamate metabolism (FDR<0.001, 0.003, 0.1 resp.), with significant higher levels of α-ketoglutarate (TCA intermediate synthesized from glutamate), pyruvate and lactate (products of glycolysis) (p<0.001, p<0.001, p=0.002) in PGA1-TRAF3mut cells. To better analyze their metabolic specificities, we evaluated the mitochondrial oxygen consumption rate (OCR) extracellular acidification (ECAR) and cell viability at basal state and in response to metabolic inhibitors. Of note, PGA1-TRAF3mut cells exhibited distinct mitochondrial characteristics: higher basal and maximal respiration levels and a more important spare capacity and generation of ATP-linked to mitochondrial respiration. Glycolysis and glycolytic reserve were also enhanced in the TRAF3mut cells. In this way, they were more sensitive to 2-deoxy-D-glucose (glucose uptake inhibitor), and more resistant to oxamate (lactate-dehydrogenase inhibitor, marker of anaerobic glycolysis). Overall, these results indicate, unlike WT, TRAF3mut cells instead utilize a mitochondrial glycolytic metabolism. Surprisingly, by using UK5099 (inhibitor of the mitochondrial pyruvate transporter that regulates mitochondrial glycolysis) mutant cells showed higher maximal respiration levels than WT, suggesting a metabolic reprogramming towards an alternative metabolic way fueling mitochondrial metabolism. We therefore tested the effect of C968 (glutaminolysis inhibitor) and we found that TRAF3mut cells were also more resistant to this drug, what reveals a specific metabolic plasticity (mitochondrial glycolysis/glutaminolysis) in the PGA1-TRAF3mut cells that does not exist in the WT. Interestingly, the combined treatment (UK5099 and C968) induced more cell death in the TRAF3mut than in the WT cells (p=0.03), suggesting a new potential opportunity to treat patients with TRAF3 alterations. In conclusion, TRAF3 deficiency appeared to be an independent prognostic factor of time to first treatment in CLL. This alteration results in a non-canonical NF-KB activation and in a metabolic reprogramming towards an enhanced mitochondrial glycolysis and glutaminolysis, which could be potentially targetable in CLL patients. Fund.: PI21/00983 FI19/00191 FEHH

A Circulating Microbial Metabolite Drives the Clonal Expansion of Pre-Leukemic Cells

Inflammatory mediators, including cytokines and microbial ligands, are implicated in the development of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A consequence of aging and various autoimmune and inflammatory conditions is loss of intestinal barrier integrity and translocation of microbial species and metabolites into circulation. More recent findings revealed that patients with inflammatory bowel disease (IBD) harbor mutant hematopoietic clones and often present with clinical manifestations of MDS/AML. Moreover, intestinal barrier dysfunction is commonly reported at diagnosis in patients with MDS and AML. These observations led us to speculate that microbial-derived signals may contribute to the expansion of pre-leukemic hematopoietic stem and progenitor cells (HSPCs) and development of myeloid malignancies. To determine if loss of intestinal barrier integrity contributes to the expansion of pre-leukemic DNMT3a-mutant HSPCs, we generated a model of IBD by inducing gut epithelial injury using dextran sulfate sodium (DSS). Upon gut epithelial injury, we observed a significant expansion of DNMT3a-mutant HSPCs, but not of wild-type (WT) HSPCs, in vivo. Unexpectedly, DNMT3a-mutant HSPCs do not exhibit a competitive advantage over WT cells in the absence of intestinal barrier dysfunction, such as following DSS or high-dose radiation. Fecal material from DSS-treated WT mice was sufficient to induce expansion of DNMT3a-mutant HSPCs and this expansion was prevented with broad-spectrum antibiotics. These findings indicate that the competitive advantage of DNMT3a-mutant HSPCs following gut epithelial injury was due to alterations in the microbiome. In parallel sequencing studies, we identified a significant enrichment of gram-negative bacterial phyla in stool samples from the IBD mouse model, IBD patients, and treatment-naive AML patients. All gram-negative bacteria uniquely generate a microbial intermediate, ADP-heptose, as part of the lipopolysaccharide biosynthesis pathway. ADP-heptose can enter the blood circulation and freely translocate into cells and activate the ALPK1-TIFA complex ("TIFAsome"). Alpha-protein kinase 1 (ALPK1) and tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with an FHA domain (TIFA) are overexpressed in primary human MDS and AML HSPCs as compared to healthy donors and their expression is correlated with poor prognosis. Gram negative bacteria and corresponding ADP-heptose levels were also significantly increased in the plasma of individuals with intestinal barrier injury, such with IBD, MDS, or upon aging, as compared to young healthy individuals. The circulating levels of ADP-heptose from aged individuals and IBD or MDS patients were sufficient to induce the TIFAsome in leukemic cells. Moreover, administration of disease relevant levels of ADP-heptose resulted in expansion and enhanced function of mouse DNMT3a-mutant HSPCs and of human AML cell lines and primary MDS cells in vivo. Expansion of DNMT3a-mutant HSPCs and human MDS/AML cells was directly related to overexpression of ALPK1, as WT HSPCs or ALPK1-deficient AML cells did not respond to ADP-heptose. To gain deeper mechanistic insight, we examined global transcriptional and epigenetic changes in purified WT and DNTM3a-mutant HSCs treated with ADP-heptose. DNMT3a-mutant HSCs treated with ADP-heptose resulted in epigenetic changes and increased expression of gene programs related to non-canonical NF-kB, ERK/MAPK, cell cycle activation, cell survival, and stem cell function. Indeed, ADP-heptose caused increased cycling and self-renewal of DNMT3a-mutant HSPCs, but not WT HSPCs, via the TIFAsome in vitro and in vivo. Based on the enriched pathways in DNMT3a-mutant HSCs treated with ADP-heptose, we screened small molecule inhibitors of TIFAsome activation. We found that targeting UBE2N abrogated ADP-heptose-mediated signaling via the TIFAsome and expansion of patient-derived AML xenografts. In summary, we report that conditions leading to intestinal barrier dysfunction and circulating ADP-heptose from gram-negative bacteria induce expansion of pre-leukemic and MDS/AML cells via ALPK1-TIFA. Lastly, we provide a potential therapeutic strategy to prevent the competitive advantage of pre-leukemic cells prior to overt malignant transformation.

Metrnl ameliorates diabetic cardiomyopathy via inactivation of cGAS/STING signaling dependent on LKB1/AMPK/ULK1-mediated autophagy

IntroductionMeteorin-like hormone (Metrnl) is ubiquitously expressed in skeletal muscle, heart, and adipose with beneficial roles in obesity, insulin resistance, and inflammation. Metrnl is found to protect against cardiac hypertrophy and doxorubicin-induced cardiotoxicity. However, its role in diabetic cardiomyopathy (DCM) is undefined. ObjectivesWe aimed to elucidate the potential roles of Metrnl in DCM. MethodsGain- andloss-of-function experimentswere utilized to determine the roles of Metrnl in the pathological processes of DCM. ResultsWe found that plasma Metrnl levels, myocardial Metrnl protein and mRNA expressions were significantly downregulated in both streptozotocin (STZ)-induced (T1D) mice and leptin receptor deficiency (db/db) (T2D) mice. Cardiac-specific overexpression (OE) of Metrnl markedly ameliorated cardiac injury and dysfunction in both T1D and T2D mice. In sharp contrast, specific deletion of Metrnl in the heart had the opposite phenotypes. In parallel, Metrnl OE ameliorated, whereas Metrnl downregulation exacerbated high glucose (HG)-elicited hypertrophy, apoptosis and oxidative damage in primary neonatal rat cardiomyocytes. Antibody-induced blockade of Metrnl eliminated the effects of benefits of Metrnl in vitro and in vivo. Mechanistically, Metrnl activated the autophagy pathway and inhibited the cGAS/STING signaling in a LKB1/AMPK/ULK1-dependent mechanism in cardiomyocytes. Besides, Metrnl-induced ULK1 phosphorylation facilitated the dephosphorylation and mitochondrial translocation of STING where it interacted with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase that was responsible for ubiquitination and degradation of STING, rendering cardiomyocytes sensitive to autophagy activation. ConclusionThus, Metrnl may be an attractive therapeutic target or regimen for treating DCM.

Fibronectin-LILRB4/gp49B interaction negatively regulates osteoclastogenesis through inhibition of RANKL-induced TRAF6/TAK1/NF-kB/MAPK signaling.

Dysregulation of osteoclasts, the multinucleated cells responsible for bone resorption, contributes to several degenerative bone disorders. Previously, we showed that blocking the leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4), a kind of inhibitory receptor that plays an important role in immune regulation, promotes osteoclast differentiation in vitro. Here, we explored whether gp49B, the murine ortholog of LILRB4, regulates osteoclastogenesis in vivo, and whether fibronectin (FN), a ligand of LILRB4/gp49B, certainly contributes to LILRB4/gp49B-mediated osteoclastogenesis. In comparison with wild-type mice, gp49B deficiency mice exhibited a loss of trabecular bone number and an increase in osteoclast formation. Gp49B knockout improved the bone resorptive capacity of osteoclasts derived from murine Raw264.7 cells by increasing osteoclast formation. We further revealed that gp49B deficiency increased the receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced signaling transduction by increasing the phosphorylation of transforming growth factor (TGF)-activated kinase 1 (TAK1), NF-κB and mitogen-activated protein kinases (MAPKs). Furthermore, the N-terminal 30 kDa proteolytic fragments of FN promoted gp49B-mediated inhibition of osteoclastogenesis by increasing Src homology-2-containing tyrosine phosphatase 1 (SHP-1) phosphorylation and tumor necrosis factor receptor-associated factor 6 (TRAF6)-SHP-1 association. In summary, the FN-LILRB4/gp49B interaction negatively regulates RANKL-induced TRAF6/TAK1/NF-κB/MAPK signaling in osteoclastogenesis.

Involvement of adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 in diallyl trisulfide-induced cytotoxicity in hepatocellular carcinoma cells.

It has been demonstrated that APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) is involved in the regulation of several growth-related signaling pathways and thus closely associated with the development and progression of some cancers. Diallyl trisulfide (DAT), a garlic-derived bioactive compound, exerts selective cytotoxicity to various human cancer cells through interfering with pro-survival signaling pathways. However, whether and how DAT affects survival of human hepatocellular carcinoma (HCC) cells remain unclear. Herein, we tested the hypothesis of the involvement of APPL1 in DAT-induced cytotoxicity in HCC HepG2 cells. We found that Lys 63 (K63)-linked polyubiquitination of APPL1 was significantly decreased whereas phosphorylation of APPL1 at serine residues remained unchanged in DAT-treated HepG2 cells. Compared with wild-type APPL1, overexpression of APPL1 K63R mutant dramatically increased cell apoptosis and mitigated cell survival, along with a reduction of phosphorylation of STAT3, Akt, and Erk1/2. In addition, DAT administration markedly reduced protein levels of intracellular TNF receptor-associated factor 6 (TRAF6). Genetic inhibition of TRAF6 decreased K63-linked polyubiquitination of APPL1. Moreover, the cytotoxicity impacts of DAT on HepG2 cells were greatly attenuated by overexpression of wild-type APPL1. Taken together, these results suggest that APPL1 polyubiquitination probably mediates the inhibitory effects of DAT on survival of HepG2 cells by modulating STAT3, Akt, and Erk1/2 pathways.

Cross-Regulation of F-Box Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection.

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c → C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ, and TRAF proteins 1-5 compared with isografts. In the acute model, treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3, preserved FBXL2, and substantially reduced T-bet, IFN-γ, and TRAFs 1-5, consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1 > DBA/2J (day 28), we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.

A tetravalent peptide that binds to the RANK-binding region of TRAF6 via a multivalent interaction efficiently inhibits osteoclast differentiation

Inhibition of osteoclast differentiation is a promising approach for the treatment of osteoporosis and rheumatoid arthritis. Receptor activator of nuclear factor kappa B (NF-κB) (RANK), which is an essential molecule for osteoclast differentiation, interacts with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) to transduce downstream signals. Both RANK and TRAF6 have homo-trimeric structures, forming a multivalent interaction between the Pro-X-Glu-X-X-(aromatic/acidic) motif of RANK and the C-terminal domain of TRAF6 (TRAF-C), that markedly increases the binding affinity. Here, we designed a tetravalent peptide, RANK-tet, containing the TRAF-C-binding motif of RANK and found that RANK-tet binds to TRAF-C with high affinity. In contrast, a monomeric form of RANK-tet (RANK-mono) with the same TRAF-C-binding motif did not bind to TRAF-C, clearly indicating the multivalent interaction is strictly required for the high-affinity binding to TRAF-C. RANK-tet did not bind to a series of TRAF-C-mutants with an amino acid substitution in the RANK-binding region, indicating that RANK-tet specifically targets the RANK-binding region of TRAF-C. A cell-permeable form of RANK-tet that has poly-Arg residues at each C-terminal of the TRAF-C-binding motif efficiently inhibited the RANK ligand (RANKL)-induced differentiation of bone marrow cells to osteoclasts. Thus, this compound can be an effective anti-osteoclastogenic agent.

Preventive Mechanism of Lycopene on Intestinal Toxicity Caused by Cyclophosphamide Chemotherapy in Mice by Regulating TLR4-MyD88/TRIF-TRAF6 Signaling Pathway and Gut-Liver Axis.

Cyclophosphamide (CYC) is the first-line chemotherapy drug for cancer in clinical practice, and its intestinal toxicity seriously affects the treatment effect and prognosis of patients. Lycopene (LP) is the main pigment of ripe tomatoes and has strong antioxidant activity. However, the mechanism by which LP prevents CYC-induced intestinal injury remains unclear. The aim of this study was to investigate the mechanism of LP in preventing intestinal toxicity caused by CYC chemotherapy in mice. The results showed that LP significantly prevented spleen and thymus atrophy induced by CYC. In terms of intestinal injury, LP significantly increased the levels of superoxide dismutase (SOD), secretory immunoglobulin A (sIgA), interleukin (IL)-4, IL-12, and interferon (IFN)-γ, decreased the content of lipid oxidation (MDA), upregulated the protein expressions of toll-like receptors 4 (TLR4), myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), toll/IL-1receptor domain containing adaptor protein inducing IFN-β (TRIF), p-P38 MAPK (P38), and p-nuclear factor kappa-B (NF-κB) p65, and improved the small intestine tissue injury induced by CYC. In terms of liver injury, LP significantly increased the content of glutathione (GSH), decreased the contents of MDA, nitric oxide (NO), IL-1β, IL-6, and tumor necrosis factor (TNF)-α, and repaired the liver tissue injury induced by CYC. Importantly, 10 mg/kg LP significantly prevented intestinal microbiota dysregulation in CYC mice. These results suggested that LP significantly prevented intestinal injury induced by CYC in mice by regulating the TLR4-MyD88/TRIF-TRAF6 signaling pathway and gut-liver axis.

Neuroprotective effect of alpha-kinase 1 knockdown against cerebral ischemia through inhibition of the NF-κB pathway and neuroinflammation

BackgroundActivation of the nuclear factor B (NF-κB) signaling pathway by pattern recognition receptors (PRRs) is regarded as a crucial mechanism of neuroinflammation and brain injury after acute ischemic stroke. The stimulation of alpha-kinase 1 (ALPK1), a newly identified PRR, triggers NF-κB activation and an inflammatory response. Longitudinal population-based genetic epidemiological studies suggest that the ALPK1 gene is a susceptible site to ischemic stroke. However, the function of ALPK1 in the central nervous system remains unclear. The present study explored the role of ALPK1 in acute ischemic stroke. MethodsBV2 microglial cells were stimulated with conditioned medium (CM) that was collected from oxygen and glucose deprivation (OGD)-treated HT22 neurons, and a murine brain ischemia model was established to detect the changes of ALPK1 expression. We used lentivirus to knockdown ALPK1 to explore the effects of ALPK1 in cerebral ischemia models in vitro and in vivo. ResultsWe observed a significant increase of ALPK1 expression in BV2 cells that were stimulated with OGD CM. The knockdown of ALPK1 inhibited the phosphorylation of tumor necrosis factor receptor associated factor-interacting protein with a forkhead-associated domain (TIFA), the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), the activation of NF-κB, and the levels of proinflammatory factors in the BV2 cells. We also verified a neuroprotective effect of ALPK1 knockdown against ischemic brain injury through inhibition of the TIFA/TRAF6/NF-κB pathway and neuroinflammation in mice. ConclusionsThis study demonstrates that ALPK1 is implicated in sterile inflammatory injury after acute brain ischemia, which provides first evidence for the therapeutic potential of ALPK1 inhibition in ischemic stroke.

TRAF3/p38-JNK Signalling Crosstalk with Intracellular-TRAIL/Caspase-10-Induced Apoptosis Accelerates ROS-Driven Cancer Cell-Specific Death by CD40

The capacity to induce tumour-cell specific apoptosis represents the most unique feature of the TNF receptor (TNFR) family member CD40. Recent studies on the signalling events triggered by its membrane-presented ligand CD40L (mCD40L) in normal and malignant epithelial cells have started to unravel an exquisite context and cell type specificity for the functional effects of CD40. Here, we demonstrate that, in comparison to other carcinomas, mCD40L triggered strikingly more rapid apoptosis in colorectal carcinoma (CRC) cells, underpinned by its ability to entrain two concurrently operating signalling axes. CD40 ligation initially activates TNFR-associated factor 3 (TRAF3) and subsequently NADPH oxidase (NOX)/Apoptosis signal-regulating kinase 1 (ASK1)-signalling and induction of reactive oxygen species (ROS) to mediate p38/JNK- and ROS-dependent cell death. At that point, p38/JNK signalling directly activates the mitochondrial pathway, and triggers rapid induction of intracellular TNF-related apoptosis-inducing ligand (TRAIL) that signals from internal compartments to initiate extrinsic caspase-10-asscociated apoptosis, leading to truncated Bid (tBid)-activated mitochondrial signalling. p38 and JNK are essential both for direct mitochondrial apoptosis induction and the TRAIL/caspase-10/tBid pathway, but their involvement follows functional hierarchy and temporally controlled interplay, as p38 function is required for JNK phosphorylation. By engaging both intrinsic and extrinsic pathways to activate apoptosis via two signals simultaneously, CD40 can accelerate CRC cell death. Our findings further unravel the multi-faceted properties of the CD40/mCD40L dyad, highlighted by the novel TNFR crosstalk that accelerates tumour cell-specific death, and may have implications for the use of CD40 as a therapeutic target.

Are Marine Heatwaves Responsible for Mortalities of Farmed Mytilus galloprovincialis? A Pathophysiological Analysis of Marteilia Infected Mussels from Thermaikos Gulf, Greece.

Simple SummaryClimate change effects strongly and negatively influence the health and productivity of marine bivalves and particularly bivalve aquaculture. It is therefore of critical importance to study the physiological response of bivalves against marine heatwaves under field conditions. Thus, in the present study, we evaluated the pathophysiological pathways of the Mediterranean mussel originating from mortality events attributed to extreme seawater temperatures in the context of Marteilia refrigens infection. Our study is focused on the Thermaikos Gulf, the most important mussel cultivation area in Greece, comparing the traditional bouchot-like mussel farming system with the modern long-line system. Heatwaves increased all examined molecular, biochemical and pathophysiological markers in M. galloprovincialis mussels, which were reinforced in Marteilia infected individuals. Therefore, these results enlighten us on the biological impacts of heatwaves, providing valuable information regarding the underlying mechanisms. Thus, insights for the future management of the marine aquatic sector could be provided and subsequently address measures for the decrease or elimination of this phenomenon and restoration of marine production.Marine heatwaves (excessive seawater temperature increases) pose high risk to bivalves’ health and farming. The seawater temperature increase is responsible for various pathogen population expansions causing intense stress to marine organisms. Since the majority of knowledge so far derives from laboratory experiments, it is crucial to investigate stress responses in field conditions in order to understand the mechanisms leading to bivalves’ mortality events after exposure to temperature extremes. Thus, we evaluated the pathophysiological response of the Mediterranean mussel Mytilus galloprovincialis originating from mortality events enhanced by intense heatwaves in Thermaikos Gulf, north Greece, along with Marteilia refrigens infection. Mussels that have been exposed to high environmental stressors such as high temperature were examined for various molecular and biochemical markers, such as hsp70, bax, bcl-2, irak4 and traf6 gene expression, as well as the enzymatic activity of the hsp70, hsp90, bax, bcl-2, cleaved caspases, TNFa and ll-6 proteins. Furthermore, histopathology and molecular positivity to Marteilia sp. were addressed and correlated with the gene expression results. Our findings elucidate the molecular and biochemical pathways leading to mortality in farmed mussels in the context of Marteilia infection, which according to the results is multiplied by heatwaves causing a significant increase in pathophysiological markers.

The Tumor Suppressor Protein TRAF3 Modulates GSK3 Activity and Susceptibility of B Lymphoma Cells to GSK3 Inhibition.

Simple SummaryTRAF3 is an adapter protein that regulates signals through many receptors important for B cell differentiation and function. Loss-of-function mutations or deletions of TRAF3 are common in B cell malignancies. Glycogen Synthase Kinase 3 (GSK3) regulates signaling in growth, survival, and metabolism pathways. GSK3 inhibitors have been effective against many solid tumors; the inhibitor used in this work is currently being tested for efficacy against BCLs. We found that TRAF3 and GSK3 associate in multiple BCL cell lines, and that BCLs with low TRAF3 have a higher susceptibility to GSK3 inhibition. In contrast to BCL cell lines, GSK3 inhibition has little effect on TRAF3-sufficient and deficient resting primary B cells. These results suggest TRAF3 level as a predictor of BCL responsiveness to GSK3 inhibitor therapy.TNF receptor-associated factor 3 (TRAF3) is an adapter protein that inhibits many signals that promote B cell survival and activation. Mice with a B cell-specific TRAF3 deficiency and humans with a rare haploinsufficiency in TRAF3 have enhanced development of BCLs as they age. Loss-of-function mutations in TRAF3 are common in B cell malignancies. Recent studies show that pharmacological inhibition of the enzyme glycogen synthase kinase 3 (GSK3), which regulates cellular growth, survival, and metabolism, inhibits growth and survival of BCL-derived B cells. In this study, we found that TRAF3 and GSK3 associated in B cells. The relative levels of TRAF3 in BCL cell lines correlated positively with the ratio of inactive to total GSK3β, and negatively correlated with susceptibility to GSK3 inhibition by the GSK3 inhibitory drug 9-ING-41, currently in clinical trials. Uniquely in BCLs with low TRAF3, GSK3 inhibition caused increased loss of the TRAF3-regulated, anti-apoptotic protein Mcl-1. GSK3 inhibition also blocked hyperresponsiveness to IL-6 receptor signaling in TRAF3-deficient BCL cells. Together, these results support the utility of 9-ING-41 as a treatment for BCL, and suggest that a decrease or loss of TRAF3 in BCLs could act as a biomarker for increased susceptibility to GSK3 inhibitor treatment.

Downregulation of miR-146a-5p Promotes Acute Pancreatitis through Activating the TLR9/NLRP3 Signaling Pathway by Targeting TRAF6 In Vitro Rat Model.

Acute pancreatitis (AP) is mainly caused by acinar cells releasing various inflammatory factors, causing inflammatory storms and leading to severe pancreatitis. Detection methods and treatment targets for pancreatitis are lacking, raising the urgency of identifying diagnostic markers and therapeutic targets for AP. MicroRNAs (miRNAs) have recently been identified as molecular markers for various biological processes such as tumors, immunity, and metabolism, and the involvement of miRNAs in inflammatory responses has been increasingly studied. To explore the role of miRNAs in AP is the primary objective of this study. By using qPCR on our cerulein-induced pancreatitis cell model, it is worth noting that the change of miR-146a-5p expression in inflammation-related miRNAs in AP was predominant. Next, ELISA, CCK8, and flow cytometry were used to inspect the impact of miR-146a-5p on pancreatitis. BiBiServ bioinformatics anticipated binding ability of miR-146a-5p and 3′-untranslated region (3′UTR) of TNF receptor-associated factor 6 (TRAF6), and the dual-luciferase assay verified the combination of the two. TRAF6 knockdown verified the effect of TRAF6 on the progression of pancreatitis. Finally, rescue experiments verified the capability of miR-146a-5p and TRAF6 interaction on the Toll-like receptor 9 (TLR9)/NOD-like receptor protein 3 (NLRP3) signaling pathway and cell function. The expression of miR-146a-5p decreased in cerulein-induced AR42J pancreatic acinar cells. Functional experiments verified that miR-146a-5p facilitated the proliferation of AR42J pancreatic acinar cells and inhibited their apoptosis. Bioinformatic predictions and dual-luciferase experiments verified the actual binding efficiency between miR-146a-5p and 3′UTR of TRAF6. Our study confirmed that knockdown of TRAF6 restrained the progression of pancreatitis, and knockdown of TRAF6 rescued pancreatitis caused by miR-146a-5p downregulation by the TLR9/NLRP3 signaling pathway. Therefore, downregulation of miR-146a-5p in the induced pancreatitis cell model promotes the progression of pancreatitis via the TLR9/TRAF6/NLRP3 signaling pathway. There is potential for miR-146a-5p to serve as a diagnostic marker and therapeutic nucleic acid drug for AP.

Knockdown of circ_0038467 alleviates lipopolysaccharides-induced 16HBE cell injury by regulating the miR-545-3p/TRAF1 axis in neonatal pneumonia

BackgroundNeonatal pneumonia is a common illness in the neonatal period with a high fatality rate. Accumulating proofs have attested to the crucial role of circular RNAs (circRNAs) in pneumonia. This study was intended to expound on the function of circ_0038467 and the underlying mechanism in lipopolysaccharide (LPS)-stimulated 16HBE cell injury in neonatal pneumonia. Methods16HBE cells were exposed to LPS to establish an in vitro neonatal pneumonia cell model. Quantitative real-time polymerase chain reaction (qRT-PCR) was implemented for detecting the levels of circ_0038467, microRNA-545-3p (miR-545-3p), and tumor necrosis factor receptor-associated factor 1 (TRAF1) in neonatal pneumonia serums and LPS-treated 16HBE cells. Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) incorporation, and flow cytometry assays were used to examine cell viability, proliferation, and apoptosis, respectively. The protein abundances of proliferation/apoptosis/inflammation-correlated makers and TRAF1 were tested by Western blot. RNase R and Actinomycin D assays were implemented to determine the features of circ_0038467. The mutual effect between miR-545-3p and circ_0038467 or TRAF1 was affirmed by a dual-luciferase reporter and RNA pull-down assay assays. ResultsCirc_0038467 was upregulated in neonatal pneumonia serum specimens and LPS-triggered 16HBE cells. LPS administration restrained 16HBE cell proliferation and promoted apoptosis and inflammation, whereas circ_0038467 silence recovered these influences. Meanwhile, miR-545-3p was targeted by circ_0038467, and circ_0038467 could modulate LPS-treated 16HBE cell injury through absorbing miR-545-3p. Furthermore, circ_0038467 controlled TRAF1 level via segregating miR-545-3p. Moreover, TRAF1 overexpression relieved the suppressive impact of circ_0038467 silence in LPS-triggered 16HBE cell detriment. ConclusionCirc_0038467 knockdown mitigated LPS-exposed 16HBE cell damage through regulating miR-545-3p/PPARA axis.

Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response inFusarium solani-Induced Keratitis via the NF-κB Signaling Pathway

PurposeThe purpose of this study was to elucidate the effect of methyltransferase-like enzyme 3 (METTL3) on inflammation and the NF-κB signaling pathway in fungal keratitis (FK).MethodsWe established corneal stromal cell models and FK mouse models by incubation with Fusarium solani. The overall RNA N6-methyladenosine (m6A) level was determined using an m6A RNA methylation assay kit. The expression of METTL3 was quantified via real-time quantitative polymerase chain reaction (RT–PCR), Western blotting, and immunofluorescence. Subsequently, the level of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) was identified by Western blotting and immunofluorescence. Moreover, we assessed the effect of METTL3 by transfecting cells with siRNA (in vitro) or adeno-associated virus (in vivo). Hematoxylin and eosin (H&E) staining and slit-lamp biomicroscopy were performed to evaluate corneal damage. Furthermore, the state of NF-κB signaling pathway activation was examined by Western blotting. In addition, RT–PCR and enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate levels of the pro-inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and TNF-ɑ.ResultsOur data demonstrated that the levels of the RNA m6A methylation and METTL3 were dramatically increased and that the NF-κB signaling pathway was activated in Fusarium solani-induced keratitis. Inhibition of METTL3 decreased the level of TRAF6, downregulated the phospho-p65(p-p65)/p65 and phospho-IκB(p-IκB)/IκB protein ratios, simultaneously attenuating the inflammatory response and fungal burden in FK.ConclusionsOur research suggests that the m6A methyltransferase METTL3 regulates the inflammatory response in FK by modulating the NF-κB signaling pathway.

Toll-Like Receptor 9 Signaling Pathway Contributes to Intestinal Mucosal Barrier Injury in Mice With Severe Acute Pancreatitis.

The purpose of this study was to investigate the role and mechanism of toll-like receptor 9 (TLR9) in intestinal mucosal barrier injury in mice with severe acute pancreatitis (SAP). The mice were randomly divided into 3 groups: control group, SAP group, and TLR9 antagonist-treated group. The expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, diamine oxidase, and endotoxin core antibodies were detected by enzyme-linked immunosorbent assay. The protein expression of zonula occluden-1 (ZO)-1, occludin, TLR9, myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), p-nuclear factor (NF)-κB p65, and NF-κB p65 were detected by Western blot. TdT-mediated dUTP nick-end labeling staining was used for detecting intestinal epithelial cell apoptosis. The expression of TLR9 and its related pathway proteins MyD88, TRAF6, and p-NF-κB p65 in the intestinal tract of SAP mice were significantly increased compared with that of control mice. Inhibition of the TLR9 expression could reduce the level of serum proinflammatory cytokines, reduce the apoptosis of intestinal epithelial cells, improve intestinal permeability, and ultimately reduce the damage of intestinal mucosal barrier function in SAP. Toll-like receptor 9/MyD88/TRAF6/NF-κB signaling pathway plays an important role in intestinal mucosal barrier injury of SAP.

Impact of Long Non-Coding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 on the Susceptibility of High Glucose-Treated Cardiomyocytes to Hypoxia/Reoxygenation

To estimate the effect of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on susceptibility of high glucose (HG)-treated cardiomyocytes (CMs) to hypoxia/reoxygenation (H/R). Forty healthy rats were assigned to the control, diabetes mellitus (DM), DM + ischemia/reperfusion injury (IRI) control, and DM IRI groups (n = 10 for each group). Rat CMs (H9C2) were subjected to HG and H/R treatments. LncRNA MALAT1 and cyclic-AMP responsive element modulator (CREM) mRNA levels were measured using quantitative polymerase chain reaction, and protein levels of CREM, myeloid differentiation primary response protein 88 (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor associated factor 6 (TRAF6), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were quantified using western blotting. Flow cytometry was applied for detection of apoptosis, and a cell viability analyzer determined the number of living cells. IRI induced myocardial apoptosis and elevated lncRNA MALAT1, CREM, and MyD88/IRAK1/TRAF6 levels in DM group mice. In H9C2 cells, HG treatment downregulated CREM expression, which resulted in the downregulation of lncRNA MALAT1 expression, reducing the susceptibility to H/R, increasing cell viability and apoptosis, decreasing the activity of the MyD88/IRAK1/TRAF6 signaling pathway, and suppressing TNF-α and IL-6 expression. LncRNA MALAT1 regulates the susceptibility of HG-treated CMs to H/R through the MyD88 signaling pathway.

Expression of the miR-9-5p, miR-125b-5p and its target gene NFKB1 and TRAF6 in childhood-onset systemic lupus erythematosus (cSLE)

Childhood- onset systemic lupus erythematosus (cSLE) is a multisystem inflammatory disease that can lead to severe clinical conditions resulting in early comorbidities. Several genetic, environmental, and immunological factors are known to influence the onset of the disease. MiRNAs have been already considered as potential actors involved in the development and activity of the SLE. Thus, understanding the behavior of these regulators can contribute to clarify the inflammatory process affecting SLE patients. Among miRNAs, miR-125b-5p and miR-9-5p targeting NFKB1 and TRAF6 genes can be involved in the etio-pathogenesis of the disease by modulating inflammation. In this study we evaluated miR-9-5p and miR-125b-5p expression and its target genes NFKB1 and TRAF6 in peripheral blood samples (PBMC) from the 35 cSLE patients and 35 healthy controls. MiRNAs and gene target expression have been evaluated by using RT-PCR with specific TaqMan® probes. Both miR-9-5p [Fold Change (FC) = −2.21; p = 0.002] and miR-125b-5p (FC= −3.30; p < 0.0001) and NFKB1 (FC = −1.84; p < 0.001) were downregulated in cSLE patients, while TRAF6 was upregulated (FC = 1.80; p = 0.006) in cSLE patients when compared to controls. A significant correlation was found between miR-125b-5p and its target gene NFKB1 [Spearman (r) = 0.47; p = 0.023]. Our results showed miR-125b-5p and miR-9-5p differential expression in cSLE patients, possibly contributing to better understanding the role of these regulators in cSLE development and disease pathogenesis.

Downregulation of deubiquitinating enzyme USP25 promotes the development of allergic rhinitis by enhancing TSLP signaling in the nasal epithelium.

Ubiquitin-specific peptidase 25 (USP25) is a key deubiquitylase belonging to the USP superfamily that is primarily involved in inflammation and the immune response. Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine that is regarded as the master switch that initiates and maintains the type 2 immune response in allergic rhinitis (AR). However, the molecular mechanisms by which USP25 regulates TSLP signaling in the nasal epithelium in AR remain unclear. The present study assessed the protein expression levels of USP25 in the nasal epithelium of patients with AR. Moreover, USP25 knockout (KO) and wild-type (WT) mice were treated with ovalbumin (OVA) to establish a model of AR. The results of western blotting and immunohistochemistry in the present study demonstrated that the protein expression levels of USP25 were significantly decreased in the nasal mucosa of patients with AR and AR mice, whereas the protein expression levels of TSLP were significantly increased. Allergic inflammation was more severe in USP25 KO mice compared with WT mice exposed to OVA, as demonstrated by increased nose scratching and sneezing, increased eosinophil infiltration, goblet cell hyperplasia and enhanced T helper type 2 (Th2) cytokine production. The results of in vitro experiments demonstrated that silencing or overexpression of USP25 decreased or increased TNF receptor-associated factor 3 (TRAF3) protein expression levels, respectively, in human nasal epithelial cells, whereas TSLP protein expression levels were negatively associated with the expression of USP25 and TRAF3. In summary, USP25 downregulation enhanced TSLP signaling in the nasal mucosal epithelium via decreased TRAF3 expression, thereby exacerbating inflammation in AR. Therefore, USP25 may act as a novel therapeutic target in AR.

TRAF3 enhances type I interferon receptor signaling in T cells by modulating the phosphatase PTPN22.

Type I interferons (IFNs) are among the most powerful tools that host cells deploy against intracellular pathogens. Their effectiveness is due both to the rapid, directly antiviral effects of IFN-stimulated gene products and to the effects of type I IFN on responding immune cells. Type I IFN signaling through its receptor, IFNAR, is tightly regulated at multiple steps in the signaling cascade, including at the level of IFNAR downstream effectors, which include the kinase JAK1 and the transcriptional regulator STAT1. Here, we found that tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) enhanced the activation of JAK1 and STAT1 specifically in CD4+ T cells by preventing recruitment of the negative regulatory phosphatase PTPN22 to the IFNAR complex. The balance between signals through IFNAR and other cytokine receptors influences CD4+ T cell differentiation and function during infections. Our work reveals TRAF3 and PTPN22 as key regulators of CD4+ T cell activation by type I IFNs.