Inactivation of Tnf-α/Tnfr signaling attenuates progression of intervertebral disc degeneration in mice.

Background: Intervertebral disc degeneration (IVDD) is a major cause of low back pain (LBP), worsened by chronic inflammatory processes associated with aging. Tumor necrosis factor alpha (Tnf-α) and its receptors, Tnf receptor type 1 (Tnfr1) and Tnf receptor type 2 (Tnfr2), are upregulated in IVDD. However, its pathologic mechanisms remain poorly defined. Methods: To investigate the role of Tnfr in IVDD, we generated global Tnfr1/2 double knockout (KO) mice and age-matched control C57BL/6 male mice, and analyzed intervertebral disc (IVD)-related phenotypes of both genotypes under physiological conditions, aging, and lumbar spine instability (LSI) model through histological and immunofluorescence analyses and µCT imaging. Expression levels of key extracellular matrix (ECM) proteins in aged and LSI mice, especially markers of cell proliferation and apoptosis, were evaluated in aged (21-month-old) mice. Results: At 4 months, KO and control mice showed no marked differences of IVDD-related parameters. However, at 21 months of age, the loss of Tnfr expression significantly alleviated IVDD-like phenotypes, including a significant increase in height of the nucleus pulposus (NPs) and reductions of endplates (EPs) porosity and histopathological scores, when compared to controls. Tnfr deficiency promoted anabolic metabolism of the ECM proteins and suppressed ECM catabolism. Tnfr loss largely inhibited hypertrophic differentiation, and, in the meantime, suppressed cell apoptosis and cellular senescence in the annulus fibrosis, NP, and EP tissues without affecting cell proliferation. Similar results were observed in the LSI model, where Tnfr deficiency significantly alleviated IVDD and enhanced ECM anabolic metabolism while suppressing catabolism. Conclusion: The deletion of Tnfr mitigates age-related and LSI-induced IVDD, as evidenced by preserved IVD structure, and improved ECM integrity. These findings suggest a crucial role of Tnf-α/Tnfr signaling in IVDD pathogenesis in mice. Targeting this pathway may be a novel strategy for IVDD prevention and treatment.

Elevated plasma levels of TNF-R1 predict the development of acute ischemic events in coronary patients with diabetes. / Los niveles plasmáticos elevados de TNF-R1 predicen el desarrollo de eventos isquémicos agudos en pacientes coronarios con diabetes.

OBJECTIVES: To examine the relationship between inflammatory biomarkers and the occurrence of cardiovascular events in patients with type 2 diabetes mellitus (DM2) and stable coronary artery disease. METHODS: A total of 964 patients with stable coronary artery disease were included. Plasma levels of inflammatory markers, including tumour necrosis factor receptors 1 and 2 (TNF-R1 and TNF-R2), growth differentiation factor-15 (GDF-15), soluble suppression of tumorigenicity 2 (sST2), and high-sensitivity C-reactive protein (hsCRP) were measured. The primary endpoint was the development of acute ischaemic events (any type of acute coronary syndrome, stroke, or transient ischaemic attack). RESULTS: There were 232 diabetic patients and 732 non-diabetic patients. Patients with coronary artery disease and DM2 (232, 24%) had higher levels of TNF-R1, TNF-R2, GDF-15, sST2 (P<.001), and hsCRP compared to patients without DM2, indicating a higher inflammatory state. After a median follow-up of 5.39 (2.81-6.92) years, patients with DM2 more frequently developed the primary endpoint (15.9% vs 10.8%; P=.035). Plasma levels of TNF-R1 were independent predictors of the primary endpoint in patients with DM2, along with male gender, triglyceride levels, and the absence of treatment with angiotensin-converting enzyme inhibitors. None of these inflammatory markers predicted the development of this event in non-diabetic patients. CONCLUSIONS: Patients with stable coronary artery disease and DM2 exhibit elevated levels of the proinflammatory markers TNF-R1, TNF-R2, GDF-15, and sST2. Moreover, TNF-R1 is an independent predictor of acute ischaemic events only in diabetic patients.

Sheep (Ovis aries) Milk Exosomal miRNAs Attenuate Dextran Sulfate Sodium-Induced Colitis in Mice via TLR4 and TRAF-1 Inhibition.

Mammalian milk exosomal miRNAs play an important role in maintaining intestinal immune homeostasis and protecting epithelial barrier function, but the specific miRNAs and whether miRNA-mediated mechanisms are responsible for these benefits remain a matter of investigation. This study isolated sheep milk-derived exosomes (sheep MDEs), identifying the enriched miRNAs in sheep MDEs, oar-miR-148a, and oar-let-7b as key components targeting TLR4 and TRAF1, which was validated by a dual-luciferase reporter assay. In dextran sulfate sodium-induced colitis mice, administration of sheep MDEs alleviated colitis symptoms, reduced colonic inflammation, and systemic oxidative stress, as well as significantly increased colonic oar-miR-148a and oar-let-7b while reducing toll-like receptor 4 (TLR4) and TNF-receptor-associated factor 1 (TRAF1) level. Further characterization in TNF-α-challenged Caco-2 cells showed that overexpression of these miRNAs suppressed the TLR4/TRAF1-IκBα-p65 pathway and reduced IL-6 and IL-12 production. These findings indicate that sheep MDEs exert gastrointestinal anti-inflammatory effects through the miRNA-mediated modulation of TLR4 and TRAF1, highlighting their potential in managing colitis.

TRAP1 modulates mitochondrial biogenesis via PGC-1α/TFAM signalling pathway in colorectal cancer cells.

Metabolic rewiring promotes cancer cell adaptation to a hostile microenvironment, representing a hallmark of cancer. This process involves mitochondrial function and is mechanistically linked to the balance between mitochondrial biogenesis (MB) and mitophagy. The molecular chaperone TRAP1 is overexpressed in 60-70% of human colorectal cancers (CRC) and its over-expression correlates with poor clinical outcome, being associated with many cancer cell functions (i.e. adaptation to stress, protection from apoptosis and drug resistance, protein synthesis quality control, metabolic rewiring from glycolysis to mitochondrial respiration and vice versa). Here, the potential new role of TRAP1 in regulating mitochondrial dynamics was investigated in CRC cell lines and human CRCs. Our results revealed an inverse correlation between TRAP1 and mitochondrial-encoded respiratory chain proteins both at transcriptional and translational levels. Furthermore, TRAP1 silencing is associated with increased mitochondrial mass and mitochondrial DNA copy number (mtDNA-CN) as well as enhanced MB through PGC-1α/TFAM signalling pathway, promoting the formation of new functioning mitochondria and, likely, underlying the metabolic shift towards oxidative phosphorylation. These results suggest an involvement of TRAP1 in regulating MB process in human CRC cells. KEY MESSAGES: TRAP1 inversely correlates with protein-coding mitochondrial gene expression in CRC cells and tumours. TRAP1 silencing correlates with increased mitochondrial mass and mtDNA copy number in CRC cells. TRAP1 silencing favours mitochondrial biogenesis in CRC cells.

Effects of novel lactoferrin peptides on LPS-induced alveolar bone destruction in a rat model.

To develop novel bovine lactoferrin (bLF) peptides targeting bLF-tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6) binding sites, we identified two peptides that could target bLF-TRAF6 binding sites using structural analysis. Moreover, another peptide that could bind to the TRAF6 dimerization area was selected from the bLF sequence. The effects of each peptide on cytokine expression in lipopolysaccharide (LPS)-stimulated osteoblasts (ST2) and on osteoclastogenesis were examined using an LPS-treated co-culture of primary bone marrow cells (BMCs) with ST2 cells and a single culture of osteoclast precursor cells (RAW-D) treated with soluble receptor activator of NF-κB ligand. Finally, the effectiveness of these peptides against LPS-induced alveolar bone destruction was assessed. Two of the three peptides significantly suppressed LPS-induced TNF-α and interleukin-1ß expression in ST2 cells. Additionally, these peptides inhibited and reversed LPS-induced receptor activator of NF-κB ligand (RANKL) upregulation and osteoprotegerin (OPG) downregulation, respectively. Furthermore, both peptides significantly reduced LPS-induced osteoclastogenesis in the BMC-ST2 co-culture and RANKL-induced osteoclastogenesis in RAW-D cells. In vivo, topical application of these peptides significantly reduced the osteoclast number by downregulating RANKL and upregulating OPG in the periodontal ligament. It is indicated that the novel bLF peptides can be used to treat periodontitis-associated bone destruction.

LncRNA ZFPM2-AS1 promotes phyllodes tumor progression by binding to CDC42 and inhibiting STAT1 activation.

Breast phyllodes tumor (PT) is a rare fibroepithelial neoplasm with potential malignant behavior. Long non-coding RNAs (lncRNAs) play multifaceted roles in various cancers, but their involvement in breast PT remains largely unexplored. In this study, microarray was leveraged for the first time to investigate the role of lncRNA in PT. We identified lncRNA ZFPM2-AS1 was significantly upregulated in malignant PT, and its overexpression endowed PT with high tumor grade and adverse prognosis. Furthermore, we elucidated that ZFPM2-AS1 promotes the proliferation, migration, and invasion of malignant PT in vitro. Targeting ZFPM2-AS1 through nanomaterial-mediated siRNA delivery in patient-derived xenograft (PDX) model could effectively inhibit tumor progression in vivo. Mechanistically, our findings showed that ZFPM2-AS1 is competitively bound to CDC42, inhibiting ACK1 and STAT1 activation, thereby launching the transcription of TNFRSF19. In conclusion, our study provides evidence that ZFPM2-AS1 plays a pivotal role in the pathogenesis of breast PT, and suggests that ZFPM2-AS1 could serve as a prognostic indicator for patients with PT as well as a promising novel therapeutic target.

4-1BB-encoding CAR causes cell death via sequestration of the ubiquitin-modifying enzyme A20.

CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T cells. However, the molecular events associated with the ectopic and constitutive display of either CD28 or 4-1BB in CAR-T cells have been only partially explored. In the current study, we demonstrated that 4-1BB incorporated within the CAR leads to cell cluster formation and cell death in the forms of both apoptosis and necroptosis in the absence of CAR tonic signaling. Mechanistic studies illustrate that 4-1BB sequesters A20 to the cell membrane in a TRAF-dependent manner causing A20 functional deficiency that in turn leads to NF-κB hyperactivity, cell aggregation via ICAM-1 overexpression, and cell death including necroptosis via RIPK1/RIPK3/MLKL pathway. Genetic modulations obtained by either overexpressing A20 or releasing A20 from 4-1BB by deleting the TRAF-binding motifs of 4-1BB rescue cell cluster formation and cell death and enhance the antitumor ability of 4-1BB-costimulated CAR-T cells.

TNFR1/p38αMAPK signaling in Nex + supraspinal neurons regulates estrogen-dependent chronic neuropathic pain.

Upregulation of soluble tumor necrosis factor (sTNF) cytokine signaling through TNF receptor 1 (TNFR1) and subsequent neuronal hyperexcitability are observed in both animal models and human chronic neuropathic pain (CNP). Previously, we have shown that estrogen modulates sTNF/TNFR1 signaling in CNP, which may contribute to female prevalence of CNP. The estrogen-dependent role of TNFR1-mediated supraspinal neuronal circuitry in CNP remains unknown. In this study, we interrogated the intersect between supraspinal TNFR1 mediated neuronal signaling and sex specificity by selectively removing TNFR1 in Nex + neurons in adult mice (NexCreERT2::TNFR1f/f). We determined that mechanical hypersensitivity induced by chronic constriction injury (CCI) decreases over time in males, but not in females. Subsequently, we investigated two downstream pathways, p38MAPK and NF-κB, important in TNFR1 signaling and injury response. We detected p38MAPK and NF-κB activation in male cortical tissue; however, p38MAPK phosphorylation was reduced in NexCreERT2::TNFR1f/f males. We observed a similar recovery from acute pain in male mice following CCI when p38αMAPK was knocked out of supraspinal Nex + neurons (NexCreERT2::p38αMAPKf/f), while chronic pain developed in female mice. To explore the intersection between estrogen and inflammation in CNP we used a combination therapy of an estrogen receptor ß (ER ß) inhibitor with a sTNF/TNFR1 or general p38MAPK inhibitor. We determined both combination therapies lends therapeutic relief to females following CCI comparable to the response evaluated in male mice. These data suggest that TNFR1/p38αMAPK signaling in Nex + neurons in CNP is male-specific and lack of therapeutic efficacy following sTNF inhibition in females is due to ER ß interference. These studies highlight sex-specific differences in pathways important to pain chronification and elucidate potential therapeutic strategies that would be effective in both sexes.

Lower levels of TRAF1 in Brodmann's area 24, but not 46, in bipolar disorders are not detectable in major depressive disorders.

INTRODUCTION: Multiple lines of research implicate inflammation-related pathways in the molecular pathology of mood disorders, with our data suggesting a critical role for aberrant cortical tumour necrosis factor α (TNF)-signaling in the molecular pathology of bipolar disorders (BPD) and major depressive disorders (MDD). METHODS: To extend our understanding of changes in TNF-signaling pathways in mood disorders we used Western blotting to measure levels of tumour necrosis factor receptor associated factor 1 (TRAF1) and transmembrane TNF receptor superfamily member 1B (tmTNFRSF1B) in Brodmann's areas (BA) 24 and 46 from people with BPD and MDD. These proteins are key rate-limiting components within TNF-signaling pathways. RESULTS: Compared to controls, there were higher levels of TRAF1 of large effect size (η = 0.19, Cohen's d = 0.97) in BA 24, but not BA 46, from people with BPD. Levels of TRAF1 were not altered in MDD and levels of tmTNFRSF1B were not altered in either disorder. LIMITATIONS: The cases studied had been treated with psychotropic drugs prior to death which is an unresolvable study confound. Cohort sizes are relatively small but not untypical of postmortem CNS studies. CONCLUSIONS: To facilitate post-synaptic signaling, TRAF1 is known to associate with tmTNFRSF1B after that receptor takes its activated conformation which occurs predominantly after it binds to transmembrane TNF (tmTNF). Simultaneously, when tmTNFRSF1B binds to tmTNF reverse signaling through tmTNF is activated. Hence our findings in BA 24 argues that bidirectional TNF-signaling may be an important component of the molecular pathology of BPD.

TRAF3 regulation of proximal TLR signaling in B cells.

Toll-like receptors are pattern recognition receptors that bridge the innate and adaptive immune responses and are critical for host defense. Most studies of Toll-like receptors have focused upon their roles in myeloid cells. B lymphocytes express most Toll-like receptors and are responsive to Toll-like receptor ligands, yet Toll-like receptor-mediated signaling in B cells is relatively understudied. This is an important knowledge gap, as Toll-like receptor functions can be cell type specific. In striking contrast to myeloid cells, TRAF3 inhibits TLR-mediated functions in B cells. TRAF3-deficient B cells display enhanced IRF3 and NFκB activation, cytokine production, immunoglobulin isotype switching, and antibody production in response to Toll-like receptors 3, 4, 7, and 9. Here, we address the question of how TRAF3 impacts initial B-cell Toll-like receptor signals to regulate downstream activation. We found that TRAF3 in B cells associated with proximal Toll-like receptor 4 and 7 signaling proteins, including MyD88, TRAF6, and the tyrosine kinase Syk. In the absence of TRAF3, TRAF6 showed a greater association with several Toll-like receptor signaling proteins, suggesting that TRAF3 may inhibit TRAF6 access to Toll-like receptor signaling complexes and thus early Toll-like receptor signaling. In addition, our results highlight a key role for Syk in Toll-like receptor signaling in B cells. In the absence of TRAF3, Syk activation was enhanced in response to ligands for Toll-like receptors 4 and 7, and Syk inhibition reduced downstream Toll-like receptor-mediated NFκB activation and proinflammatory cytokine production. This study reveals multiple mechanisms by which TRAF3 serves as a key negative regulator of early Toll-like receptor signaling events in B cells.

Circulating TNF receptor levels are associated with estimated glomerular filtration rate even in healthy individuals with normal kidney function.

The association between serum tumor necrosis factor receptor (TNFRs: TNFR1, TNFR2) levels and estimated glomerular filtration rate (eGFR) observed in patients with diabetes has not been comprehensively tested in healthy subjects with normal kidney function. It also remains unclear whether TNFR levels differ by age and sex, and between healthy subjects and diabetics. We measured serum TNFR levels in 413 healthy subjects and 292 patients with type 2 diabetes. In healthy subjects, TNFR levels did not differ between men and women. Additionally, TNFR2, but not TNFR1, levels increased with age. In multivariate analysis, TNFR1 was associated only with cystatin C-based eGFR (eGFR-CysC), whereas TNFR2 was associated with systolic blood pressure in addition to eGFR-CysC. Both TNFRs were associated with lower eGFR (eGFR-Cys < 90 mL/min/1.73 m2) even after adjustment for relevant clinical factors. Upon combining healthy subjects and patients with diabetes, the presence of diabetes and elevated glycated hemoglobin level were significant factors in determining TNFR levels. TNFR levels were associated with eGFR-CysC, but were not affected by age and sex in healthy subjects with normal kidney function. TNFR levels in patients with diabetes appeared to be higher than in healthy subjects.

Correlation of Increased Soluble Tumor Necrosis Factor Receptor 1 with Mortality and Dependence on Treatment in Non-Small-Cell Lung Cancer Patients: A Longitudinal Cohort Study.

BACKGROUND: Tumor necrosis factor (TNF) is a multipotent cytokine involved in inflammation and anti-tumor activity. TNF-α exerts its function upon binding to TNF-receptor 1 (TNF-R1) and TNF-receptor 2 (TNF-R2). This study investigates the relationship of soluble (s) TNF-R1 levels in non-small-cell lung cancer (NSCLC) patients with treatment and overall survival. METHODS: In total, 134 NSCLC patients treated at the Medical Faculty of Martin Luther University Halle-Wittenberg between 2017 and 2019 were included in this study. Serum levels of sTNF-R1 were measured via ELISA at baseline and during and after treatment. A linear mixed-effects model was used to assess sTNF-R1 changes over time. Linear regression was applied to investigate the association between clinical characteristics and changes in sTNF-R1. Cox regression models were used to estimate associations with overall mortality. RESULTS: The estimated average sTNFR-1 at baseline was 2091.71 pg/mL, with a change of 6.19 pg/mL per day. Cox models revealed that the individual change in sTNF-R1 was more strongly associated with mortality than its baseline value, especially after adjusting for covariates. CONCLUSIONS: This study provides evidence that the individual change in sTNF-R1 levels during and after treatment were associated with the risk of mortality, suggesting the use of the sTNF-R1 trajectory as a prognostic marker.

Functional analyses of TRAF6 gene in Argopecten scallops.

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family has been reported to be involved in many immune pathways. In a previous study, we identified 5 TRAF genes, including TRAF2, 3, 4, 6, and 7, in the bay scallop (Argopecten irradians, Air) and the Peruvian scallop (Argopecten purpuratus, Apu). Since TRAF6 is a key molecular link in the TNF superfamily, we conducted a series of studies targeting the TRAF6 gene in the Air and Apu scallops as well as their hybrid progeny, Aip (Air ♀ × Apu ♂) and Api (Apu ♀ × Air ♂). Subcellular localization assay showed that the Air-, Aip-, and Api-TRAF6 were widely distributed in the cytoplasm of the human embryonic kidney cell line (HEK293T). Additionally, dual-luciferase reporter assay revealed that among TRAF3, TRAF4, and TRAF6, only the overexpression of TRAF6 significantly activated NF-κB activity in the HEK293T cells in a dose-dependent manner. These results suggest a crucial role of TRAF6 in the immune response in Argopecten scallops. To investigate the specific immune mechanism of TRAF6 in Argopecten scallops, we conducted TRAF6 knockdown using RNA interference. Transcriptomic analyses of the TRAF6 RNAi and control groups identified 1194, 2403, and 1099 differentially expressed genes (DEGs) in the Air, Aip, and Api scallops, respectively. KEGG enrichment analyses revealed that these DEGs were primarily enriched in transport and catabolism, amino acid metabolism, peroxisome, lysosome, and phagosome pathways. Expression profiles of 28 key DEGs were confirmed by qRT-PCR assays. The results of this study may provide insights into the immune mechanisms of TRAF in Argopecten scallops and ultimately benefit scallop breeding.

The ever-expanding role of cytokine receptor DR3 in T cells.

Death Receptor 3 (DR3) is a cytokine receptor of the Tumor Necrosis Factor receptor superfamily that plays a multifaceted role in both innate and adaptive immunity. Based on the death domain motif in its cytosolic tail, DR3 had been proposed and functionally affirmed as a trigger of apoptosis. Further studies, however, also revealed roles of DR3 in other cellular pathways, including inflammation, survival, and proliferation. DR3 is expressed in various cell types, including T cells, B cells, innate lymphocytes, myeloid cells, fibroblasts, and even outside the immune system. Because DR3 is mainly expressed on T cells, DR3-mediated immune perturbations leading to autoimmunity and other diseases were mostly attributed to DR3 activation of T cells. However, which T cell subset and what T effector functions are controlled by DR3 to drive these processes remain incompletely understood. DR3 engagement was previously found to alter CD4 T helper subset differentiation, expand the Foxp3+ Treg cell pool, and maintain intraepithelial γδ T cells in the gut. Recent studies further unveiled a previously unacknowledged aspect of DR3 in regulating innate-like invariant NKT (iNKT) cell activation, expanding the scope of DR3-mediated immunity in T lineage cells. Importantly, in the context of iNKT cells, DR3 ligation exerted costimulatory effects in agonistic TCR signaling, unveiling a new regulatory framework in T cell activation and proliferation. The current review is aimed at summarizing such recent findings on the role of DR3 on conventional T cells and innate-like T cells and discussing them in the context of immunopathogenesis.

An immunocytokine consisting of a TNFR2 agonist and TNFR2 scFv enhances the expansion of regulatory T cells through TNFR2 clustering.

Regulatory T cells (Tregs) are lymphocytes that play a central role in peripheral immune tolerance. Tregs are promising targets for the prevention and suppression of autoimmune diseases, allergies, and graft-versus-host disease, and treatments aimed at regulating their functions are being developed. In this study, we created a new modality consisting of a protein molecule that suppressed excessive immune responses by effectively and preferentially expanding Tregs. Recent studies reported that tumor necrosis factor receptor type 2 (TNFR2) expressed on Tregs is involved in the proliferation and activation of Tregs. Therefore, we created a functional immunocytokine, named TNFR2-ICK-Ig, consisting of a fusion protein of an anti-TNFR2 single-chain Fv (scFv) and a TNFR2 agonist TNF-α mutant protein, as a new modality that strongly enhances TNFR2 signaling. The formation of agonist-receptor multimerization (TNFR2 cluster) is effective for the induction of a strong TNFR2 signal, similar to the TNFR2 signaling mechanism exhibited by membrane-bound TNF. TNFR2-ICK-Ig improved the TNFR2 signaling activity and promoted TNFR2 cluster formation compared to a TNFR2 agonist TNF-α mutant protein that did not have an immunocytokine structure. Furthermore, the Treg expansion efficiency was enhanced. TNFR2-ICK-Ig promotes its effects via scFv, which crosslinks receptors whereas the agonists transmit stimulatory signals. Therefore, this novel molecule expands Tregs via strong TNFR2 signaling by the formation of TNFR2 clustering.

Tocilizumab for treating mevalonate kinase deficiency and TNF receptor-associated periodic syndrome: a case series and literature review.

BACKGROUND: Mevalonate kinase deficiency (MKD) and TNF receptor-associated periodic syndrome (TRAPS) are categorized as systemic autoinflammatory diseases (SAIDs), which are rare diseases characterized by early onset, severe conditions, and challenging diagnosis and treatment. Although different SAIDs have varying standard treatments, some SAIDs are poorly controlled after routine treatment, seriously affecting the growth and development of children and their quality of life. This study aims to provide more treatment strategies for SAIDs. CASE PRESENTATION: We present two Chinese patients with MKD and TRAPS who were resistant to TNF- (tumor necrosis factor-) α blockade. After using etanercept, baricitinib, and glucocorticoid, patients with MKD and TRAPS still had periodic fever and rash. Due to the unavailability of IL-1 antagonists in the Chinese Mainland, we started administering intravenous tocilizumab (TCZ) at a dosage of 240 mg every three weeks. They had not experienced fever or rash after receiving one or two doses of TCZ. Before treatment with TCZ in the MKD patient, white blood cell (WBC) count, and TNF-α level were normal, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) increased significantly, and IL-6 increased slightly. After treatment with TCZ, ESR and CRP levels returned to normal; however, IL-6 increased occasionally. In the TRAPS patient, ESR, CRP, WBC, IL-6, and TNF-α levels were increased significantly. After TCZ treatment, ESR, CRP, WBC, IL-6, and TNF-α levels returned to normal. The two patients were treated with TCZ for more than six months and achieved clinical and serological remission. Furthermore, they had no adverse reactions after injection of TCZ. CONCLUSION: In the absence of IL-1 antagonists in mainland China, tocilizumab emerges as an alternative drug in SAIDs that are resistant to TNF-α blockade.

Tumor necrosis factor receptor 2 activation elicits sex-specific effects on cortical myelin proteins and functional recovery in a model of multiple sclerosis.

Multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS), predominately affects females compared to males. Tumor necrosis factor (TNF), a pro-inflammatory cytokine, signaling through TNF receptor 1 contributes to inflammatory disease pathogenesis. In contrast, TNF receptor 2 signaling is neuroprotective. Current anti-TNF MS therapies are shown to be detrimental to patients due to pleiotropic effects on both pro- and anti-inflammatory functions. Using a non-pertussis toxin (nPTX) experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice, we systemically administered a TNFR2 agonist (p53-sc-mTNFR2) to investigate behavioral and pathophysiological changes in both female and male mice. Our data shows that TNFR2 activation alleviates motor and sensory symptoms in females. However, in males, the agonist only alleviates sensory symptoms and not motor. nPTX EAE induction in TNFR2 global knockout mice caused exacerbated motor symptoms in females along with an earlier day of onset, but not in males. Our data demonstrates that TNFR2 agonist efficacy is sex-specific for alleviation of motor symptoms, however, it effectively reduces mechanical hypersensitivity in both females and males. Altogether, these data support the therapeutic promise TNFR2 agonism holds as an MS therapeutic and, more broadly, to treat central neuropathic pain.

IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

PURPOSE: Vascular endothelial growth factor tyrosine kinase inhibitors (TKIs) have been the standard of care for advanced and metastatic clear cell renal cell carcinoma (ccRCC). However, the therapeutic effect of TKI monotherapy remains unsatisfactory given the high rates of acquired resistance to TKI therapy despite favorable initial tumor response. MATERIALS AND METHODS: To define the TKI-resistance mechanism and identify new therapeutic target for TKI-resistant ccRCC, an integrative differential gene expression analysis was performed using acquired resistant cohort and a public dataset. Sunitinib-resistant RCC cell lines were established and used to test their malignant behaviors of TKI resistance through in vitro and in vivo studies. Immunohistochemistry was conducted to compare expression between the tumor and normal kidney and verify expression of pathway-related proteins. RESULTS: Integrated differential gene expression analysis revealed increased interferon-induced transmembrane protein 3 (IFITM3) expression in post-TKI samples. IFITM3 expression was increased in ccRCC compared with the normal kidney. TKI-resistant RCC cells showed high expression of IFITM3 compared with TKI-sensitive cells and displayed aggressive biologic features such as higher proliferative ability, clonogenic survival, migration, and invasion while being treated with sunitinib. These aggressive features were suppressed by the inhibition of IFITM3 expression and promoted by IFITM3 overexpression, and these findings were confirmed in a xenograft model. IFITM3-mediated TKI resistance was associated with the activation of TRAF6 and MAPK/AP-1 pathways. CONCLUSIONS: These results demonstrate IFITM3-mediated activation of the TRAF6/MAPK/AP-1 pathways as a mechanism of acquired TKI resistance, and suggest IFITM3 as a new target for TKI-resistant ccRCC.

CD4+ T-Cell Legumain Deficiency Attenuates Hypertensive Damage via Preservation of TRAF6.

BACKGROUND: T cells are central to the immune responses contributing to hypertension. LGMN (legumain) is highly expressed in T cells; however, its role in the pathogenesis of hypertension remains unclear. METHODS: Peripheral blood samples were collected from patients with hypertension, and cluster of differentiation (CD)4+ T cells were sorted for gene expression and Western blotting analysis. TLGMNKO (T cell-specific LGMN-knockout) mice (Lgmnf/f/CD4Cre), regulatory T cell (Treg)-specific LGMN-knockout mice (Lgmnf/f/Foxp3YFP Cre), and RR-11a (LGMN inhibitor)-treated C57BL/6 mice were infused with Ang II (angiotensin II) or deoxycorticosterone acetate/salt to establish hypertensive animal models. Flow cytometry, 4-dimensional label-free proteomics, coimmunoprecipitation, Treg suppression, and in vivo Treg depletion or adoptive transfer were used to delineate the functional importance of T-cell LGMN in hypertension development. RESULTS: LGMN mRNA expression was increased in CD4+ T cells isolated from hypertensive patients and mice, was positively correlated with both systolic and diastolic blood pressure, and was negatively correlated with serum IL (interleukin)-10 levels. TLGMNKO mice exhibited reduced Ang II-induced or deoxycorticosterone acetate/salt-induced hypertension and target organ damage relative to wild-type (WT) mice. Genetic and pharmacological inhibition of LGMN blocked Ang II-induced or deoxycorticosterone acetate/salt-induced immunoinhibitory Treg reduction in the kidneys and blood. Anti-CD25 antibody depletion of Tregs abolished the protective effects against Ang II-induced hypertension in TLGMNKO mice, and LGMN deletion in Tregs prevented Ang II-induced hypertension in mice. Mechanistically, endogenous LGMN impaired Treg differentiation and function by directly interacting with and facilitating the degradation of TRAF6 (tumor necrosis factor receptor-associated factor 6) via chaperone-mediated autophagy, thereby inhibiting NF-κB (nuclear factor kappa B) activation. Adoptive transfer of LGMN-deficient Tregs reversed Ang II-induced hypertension, whereas depletion of TRAF6 in LGMN-deficient Tregs blocked the protective effects. CONCLUSIONS: LGMN deficiency in T cells prevents hypertension and its complications by promoting Treg differentiation and function. Specifically targeting LGMN in Tregs may be an innovative approach for hypertension treatment.

Glucocorticoid-induced TNF receptor family-related protein functions as a costimulatory molecule for murine eosinophils.

Eosinophils are typical effector cells associated with type 2 immune responses and play key roles in the pathogenesis of allergic diseases. These cells are activated by various stimuli, such as cytokines, chemokines, and growth factors, but the regulatory mechanisms of eosinophil effector functions remain unclear. Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR), a transmembrane protein belonging to the tumor necrosis factor (TNF) receptor superfamily, is a well-known regulatory molecule for T cell activation. Here, we show that GITR is also constitutively expressed on eosinophils and functions as a costimulatory molecule for these cells. Although degranulation was unaffected by GITR engagement of murine bone marrow-derived eosinophils, secretion of inflammatory cytokines such as interleukin (IL)-4, IL-6, and IL-13 from IL-33-activated bone marrow-derived eosinophils was augmented by anti-mouse GITR agonistic antibody (DTA-1). In conclusion, our results provide a new regulatory pathway of cytokine secretion from eosinophils in which GITR functions as a costimulatory molecule.