[Immune Regulation by TNF Receptor-associated Factor 5].

The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family of molecules are intracellular adaptors that regulate cellular signaling through members of the TNFR and Toll-like receptor superfamily. Mammals have seven TRAF molecules numbered sequentially from TRAF1 to TRAF7. Although TRAF5 was identified as a potential regulator of TNFR superfamily members, the in vivo function of TRAF5 has not yet been fully elucidated. We identified an unconventional role of TRAF5 in interleukin-6 (IL-6) receptor signaling involving CD4+ T cells. Moreover, TRAF5 binds to the signal-transducing glycoprotein 130 (gp130) receptor for IL-6 and inhibits the activity of the janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. In addition, Traf5-deficient CD4+ T cells exhibit significantly enhanced IL-6-driven differentiation of T helper 17 (Th17) cells, which exacerbates neuroinflammation in experimental autoimmune encephalomyelitis. Furthermore, TRAF5 demonstrates a similar activity to gp130 for IL-27, another cytokine of the IL-6 family. Additionally, Traf5-deficient CD4+ T cells display significantly increased IL-27-mediated differentiation of Th1 cells, which increases footpad swelling in delayed-type hypersensitivity response. Thus, TRAF5 functions as a negative regulator of gp130 in CD4+ T cells. This review aimed to explain how TRAF5 controls the differentiation of CD4+ T cells and discuss how the expression of TRAF5 in T cells and other cell types can influence the development and progression of autoimmune and inflammatory diseases.

Combination of bazedoxifene with chemotherapy and SMAC-mimetics for the treatment of colorectal cancer.

Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Identification of Serum Biomarkers to Monitor Therapeutic Response in Intestinal-Type Gastric Cancer.

There are a limited number of clinically useful serum biomarkers to predict tumor onset or treatment response in gastric cancer (GC). For this reason, we explored the serum proteome of the gp130Y757F murine model of intestinal-type gastric cancer (IGC). We identified 30 proteins with significantly elevated expression in early gp130Y757F IGC and 12 proteins that were significantly elevated in late gp130Y757F IGC compared to age- and gender-matched wild-type mice. Within these signatures, there was an overlap of 10 proteins commonly elevated in both early- and late-stage disease. These results highlight the potential to identify serum biomarkers of disease stage. Since IGC in the gp130Y757F model can be reversed following therapeutic inhibition of Interleukin (IL)-11, we explored whether the protein signatures we identified could be used to monitor tumor regression. We compared two different therapeutic modalities and found 5 proteins to be uniquely differentially expressed between control animals and animals halfway through treatment, with 10 differentially expressed at the end of treatment. Our findings highlight the potential to identify reliable biomarkers to track IGC tumor regression in response to treatment.

Structural insights into IL-11-mediated signalling and human IL6ST variant-associated immunodeficiency.

IL-11 and IL-6 activate signalling via assembly of the cell surface receptor gp130; however, it is unclear how signals are transmitted across the membrane to instruct cellular responses. Here we solve the cryoEM structure of the IL-11 receptor recognition complex to discover how differences in gp130-binding interfaces may drive signalling outcomes. We explore how mutations in the IL6ST gene encoding for gp130, which cause severe immune deficiencies in humans, impair signalling without blocking cytokine binding. We use cryoEM to solve structures of both IL-11 and IL-6 complexes with a mutant form of gp130 associated with human disease. Together with molecular dynamics simulations, we show that the disease-associated variant led to an increase in flexibility including motion within the cytokine-binding core and increased distance between extracellular domains. However, these distances are minimized as the transmembrane helix exits the membrane, suggesting a stringency in geometry for signalling and dimmer switch mode of action.

Importance of IL-6 trans-signaling and high autocrine IL-6 production in human osteoarthritic chondrocyte metabolism.

OBJECTIVE: Neutralization of Interleukin (IL)-6-signaling by antibodies is considered a promising tool for the treatment of osteoarthritis (OA). To gain further insight into this potential treatment, this study investigated the effects of IL-6-signaling and IL-6 neutralization on chondrocyte metabolism and the release of IL-6-signaling-related mediators by human chondrocytes. DESIGN: Chondrocytes were collected from 49 patients with advanced knee/hip OA or femoral neck fracture. Isolated chondrocytes were stimulated with different mediators to analyze the release of IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130). The effect of IL-6 and IL-6/sIL-6R complex as well as neutralization of IL-6-signaling on the metabolism was analyzed. RESULTS: OA chondrocytes showed high basal IL-6 production and release, which was strongly negatively correlated with the production of cartilage-matrix-proteins. Chondrocytes produced and released sIL-6R and sgp130. The IL-6/sIL-6R complex significantly increased nitric oxide, prostaglandin E2 and matrix metalloproteinase 1 production, decreased Pro-Collagen Type II and mitochondrial ATP production, and increased glycolysis in OA chondrocytes. Neutralization of IL-6-signaling by antibodies did not significantly affect the metabolism of OA chondrocytes, but blocking of glycoprotein 130 (gp130)-signaling by SC144 significantly reduced the basal IL-6 release. CONCLUSION: Although IL-6 trans-signaling induced by IL-6/sIL-6R complex negatively affects OA chondrocytes, antibodies against IL-6 or IL-6R did not affect chondrocyte metabolism. Since inhibition of gp130-signaling reduced the enhanced basal release of IL-6, interfering with gp130-signaling may ameliorate OA progression because high cellular release of IL-6 correlates with reduced production of cartilage-matrix-proteins.

Nonspecific Inhibition of IL6 Family Cytokine Signalling by Soluble gp130.

IL6 is a proinflammatory cytokine that binds to membrane-bound IL6 receptor (IL6R) or soluble IL6R to signal via gp130 in cis or trans, respectively. We tested the hypothesis that sgp130Fc, which is believed to be a selective IL6 trans-signalling inhibitor, is in fact a non-specific inhibitor of gp130 signalling. In human cancer and primary cells, sgp130Fc inhibited IL6, IL11, OSM and CT1 cis-signalling. The IC50 values of sgp130Fc for IL6 and OSM cis-signalling were markedly (20- to 200-fold) lower than the concentrations of sgp130Fc used in mouse studies and clinical trials. sgp130 inhibited IL6 and OSM signalling in the presence of an ADAM10/17 inhibitor and the absence of soluble IL6R or OSMR, with effects that were indistinguishable from those of a gp130 neutralising antibody. These data show that sgp130Fc does not exclusively block IL6 trans-signalling and reveal instead that broad inhibition of gp130 signalling likely underlies its therapeutic effects. This proposes global or modular inhibition of gp130 as a therapeutic approach for treating human disease.

Bazedoxifene Suppresses the Growth of Osteosarcoma Cells by Inhibiting IL-6 and IL-11/GP130 Signaling Pathway.

Osteosarcoma is the most common primary bone tumor. Using the multiple ligands simultaneous docking method, we found that bazedoxifene could bind to the GP130 D1 domain. We then demonstrated that bazedoxifene can decrease cell viability and cell migration of osteosarcoma cells by inhibiting interleukin 6 (IL-6) and IL-11/GP130 signaling. Consistently, treatment with IL-6 or IL-11 antibody or knockdown of GP130 by siRNA silenced the activation of STAT3, ERK, and AKT. Similarly, recombinant IL-6 and IL-11 proteins antagonized the inhibitory effect of bazedoxifene on osteosarcoma cells. Finally, the combinational treatment of temsirolimus and bazedoxifene synergistically suppressed osteosarcoma development in vitro and in vivo. Our findings suggest that bazedoxifene directly prompts the deactivation of GP130 and inhibits the osteosarcoma progression in vitro and in vivo. Therefore, bazedoxifene could be effectively applied as a therapeutic drug for human osteosarcoma in the future.

An epithelial gene signature of trans-IL-6 signaling defines a subgroup of type 2-low asthma.

BACKGROUND: Asthma is stratified into type 2-high and type 2-low inflammatory phenotypes. Limited success has been achieved in developing drugs that target type 2-low inflammation. Previous studies have linked IL-6 signaling to severe asthma. IL-6 cooperates with soluble-IL-6Rα to activate cell signaling in airway epithelium. OBJECTIVE: We sought to study the role of sIL-6Rα amplified IL-6 signaling in airway epithelium and to develop an IL-6+ sIL-6Rα gene signature that may be used to select asthma patients who potentially respond to anti-IL-6 therapy. METHODS: Human airway epithelial cells were stimulated with combinations of IL-6, sIL-6Rα, and inhibitors, sgp130 (Olamkicept), and anti-IL-6R (Tocilizumab), to assess effects on pathway activation, epithelial barrier integrity, and gene expression. A gene signature was generated to identify IL-6 high patients using bronchial biopsies and nasal brushes. RESULTS: Soluble-IL-6Rα amplified the activation of the IL-6 pathway, shown by the increase of STAT3 phosphorylation and stronger gene induction in airway epithelial cells compared to IL-6 alone. Olamkicept and Tocilizumab inhibited the effect of IL-6 + sIL-6Rα on gene expression. We developed an IL-6 + sIL-6Rα gene signature and observed enrichment of this signature in bronchial biopsies but not nasal brushes from asthma patients compared to healthy controls. An IL-6 + sIL-6Rα gene signature score was associated with lower levels of sputum eosinophils in asthma. CONCLUSION: sIL-6Rα amplifies IL-6 signaling in bronchial epithelial cells. Higher local airway IL-6 + sIL-6Rα signaling is observed in asthma patients with low sputum eosinophils.

The Human GP130 Cytokine Receptor and Its Expression-an Atlas and Functional Taxonomy of Genetic Variants.

Genetic variants in IL6ST encoding the shared cytokine receptor for the IL-6 cytokine family GP130 have been associated with a diverse number of clinical phenotypes and disorders. We provide a molecular classification for 59 reported rare IL6ST pathogenic or likely pathogenic variants and additional polymorphisms. Based on loss- or gain-of-function, cytokine selectivity, mono- and biallelic associations, and variable cellular mosaicism, we grade six classes of IL6ST variants and explore the potential for additional variants. We classify variants according to the American College of Medical Genetics and Genomics criteria. Loss-of-function variants with (i) biallelic complete loss of GP130 function that presents with extended Stüve-Wiedemann Syndrome; (ii) autosomal recessive hyper-IgE syndrome (HIES) caused by biallelic; and (iii) autosomal dominant HIES caused by monoallelic IL6ST variants both causing selective IL-6 and IL-11 cytokine loss-of-function defects; (iv) a biallelic cytokine-specific variant that exclusively impairs IL-11 signaling, associated with craniosynostosis and tooth abnormalities; (v) somatic monoallelic mosaic constitutively active gain-of-function variants in hepatocytes that present with inflammatory hepatocellular adenoma; and (vi) mosaic constitutively active gain-of-function variants in hematopoietic and non-hematopoietic cells that are associated with an immune dysregulation syndrome. In addition to Mendelian IL6ST coding variants, there are common non-coding cis-acting variants that modify gene expression, which are associated with an increased risk of complex immune-mediated disorders and trans-acting variants that affect GP130 protein function. Our taxonomy highlights IL6ST as a gene with particularly strong functional and phenotypic diversity due to the combinatorial biology of the IL-6 cytokine family and predicts additional genotype-phenotype associations.

Molecular Design of Novel Inhibitor by Targeting IL-6Rα using Combined Pharmacophore and Experimentally Verified Plant Products with Scaffold-Hopping Techniques: A Dual Therapeutic Strategy for COVID-19 and Cancer.

The IL-6/IL-6R/gp130 complex serves as a significant indicator of cytokine release syndrome in COVID-19 and chronic inflammation, increasing the risk of cancer. Therefore, we identified IL-6Rα as a potential target to block gp130 interaction. Notably, there has been no reception of approval for an orally available drug to serve this purpose, to date. In this study, we targeted IL-6Rα to inhibit IL-6Rα/gp130 interaction. The selection of the lead candidate L821 involved the amalgamation of three drug discovery approaches. This library was screened employing tertiary structure-based pharmacophore models followed by molecular docking models, scaffold-hopping, MM/PBSA as well as MM/GBSA analysis, and assessments of pKi and ADMET properties. After evaluating the binding interactions with key amino acids, 15 potential ligands were chosen, with the top ligand undergoing further investigation by means of molecular dynamics simulations. Considering the stability of the complexes, the strong interactions observed between ligand and residues of IL-6Rα/gp130, and the favorable binding free energy calculations, L821 emerged as the prime candidate for inhibiting IL-6Rα. Notably, L821 exhibited a docking-based binding affinity of -9.5 kcal/mol. Our study presents L821 as a promising inhibitor for future in vitro analysis, potentially combatting SARS-CoV-2-related cytokine storms and serving as an oncogenic drug therapy.

Role of interleukin 6 and its soluble receptor on the diffusion barrier dysfunction of alveolar tissue.

During respiratory infection, barrier dysfunction in alveolar tissue can result from "cytokine storm" caused by overly reactive immune response. Particularly, interleukin 6 (IL-6) is implicated as a key biomarker of cytokine storm responsible for and further progression to pulmonary edema. In this study, alveolar-like tissue was reconstructed in a microfluidic device with: (1) human microvascular lung endothelial cells (HULEC-5a) cultured under flow-induced shear stress and (2) human epithelial cells (Calu-3) cultured at air-liquid interface. The effects of IL-6 and the soluble form of its receptor (sIL-6R) on the permeability, electrical resistance, and morphology of the endothelial and epithelial layers were evaluated. The diffusion barrier properties of both the endothelial and epithelial layers were significantly degraded only when IL-6 treatment was combined with sIL-6R. As suggested by recent review and clinical studies, our results provide unequivocal evidence that the barrier dysfunction occurs through trans-signaling in which IL-6 and sIL-6R form a complex and then bind to the surface of endothelial and epithelial cells, but not by classical signaling in which IL-6 binds to membrane-expressed IL-6 receptor. This finding suggests that the role of both IL-6 and sIL-6R should be considered as important biomarkers in developing strategies for treating cytokine storm.

Bispecific soluble cytokine receptor-nanobody fusions inhibit Interleukin (IL-)6 trans-signaling and IL-12/23 or tumor necrosis factor (TNF) signaling.

At least 0.5% of people in the Western world develop inflammatory bowel disease (IBD). While antibodies that block tumor necrosis factor (TNF) α and Interleukin (IL-)23 have been approved for the treatment of IBD, IL-6 antibodies failed in the phase II clinical trial due to non-tolerable side effects. However, two clinical phase II studies suggest that inhibiting IL-6/soluble IL-6R (sIL-6R)-induced trans-signaling via the cytokine receptor gp130 benefit IBD patients with fewer adverse events. Here we develop inhibitors targeting a combination of IL-6/sIL-6R and TNF or IL-12/IL-23 signaling, named cs130-TNFVHHFc and cs130-IL-12/23VHHFc. Surface plasmon resonance experiments showed that recombinant cs130-TNFVHHFc and cs130-IL-12/23VHHFc bind with high affinity to IL-6/sIL-6R complexes and human TNFα (hTNFα) or IL-12/IL-23, respectively. Immunoprecipitation experiments have verified the higher ordered complex formation of the inhibitors with IL-6/sIL-6R and IL-12. We demonstrated that cs130-TNFVHHFc and cs130-IL-12/23VHHFc block IL-6/sIL-6R trans-signaling-induced proliferation and STAT3 phosphorylation of Ba/F3-gp130 cells, as well as hTNFα- or IL-23-induced signaling, respectively. In conclusion, cs130-TNFVHHFc and cs130-IL-12/23VHHFc represent a class of dimeric and bispecific chimeric cytokine inhibitors that consist of a soluble cytokine receptor fused to anti-cytokine nanobodies.

Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex.

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease, cancer, and fibrosis. All IL-6 family cytokines signal through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct as well as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the IL-11 signaling complex, comparing it with the IL-6 complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear difference between IL-11 and IL-6. Surface plasmon resonance analysis showed that the interaction between IL-11 and IL-11 receptor α (IL-11Rα) is entropy driven, whereas that between IL-6 and IL-6 receptor α (IL-6Rα) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11Rα complex results in entropy loss, but that the interaction of gp130 with the IL-6/IL-6Rα complex results in entropy gain. Our hydrogen-deuterium exchange mass spectrometry experiments suggested that the D2 domain of gp130 was not involved in IL-6-like interactions in the IL-11/IL-11Rα complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface located in its D2 domain of gp130. Our findings suggest that unique interactions of the IL-11 signaling complex with gp130 are responsible for the distinct biological activities of IL-11 compared to IL-6.

IL-6 Trans-Signaling Is Increased in Diabetes, Impacted by Glucolipotoxicity, and Associated With Liver Stiffness and Fibrosis in Fatty Liver Disease.

Many people living with diabetes also have nonalcoholic fatty liver disease (NAFLD). Interleukin-6 (IL-6) is involved in both diseases, interacting with both membrane-bound (classical) and circulating (trans-signaling) soluble receptors. We investigated whether secretion of IL-6 trans-signaling coreceptors are altered in NAFLD by diabetes and whether this might associate with the severity of fatty liver disease. Secretion patterns were investigated with use of human hepatocyte, stellate, and monocyte cell lines. Associations with liver pathology were investigated in two patient cohorts: 1) biopsy-confirmed steatohepatitis and 2) class 3 obesity. We found that exposure of stellate cells to high glucose and palmitate increased IL-6 and soluble gp130 (sgp130) secretion. In line with this, plasma sgp130 in both patient cohorts positively correlated with HbA1c, and subjects with diabetes had higher circulating levels of IL-6 and trans-signaling coreceptors. Plasma sgp130 strongly correlated with liver stiffness and was significantly increased in subjects with F4 fibrosis stage. Monocyte activation was associated with reduced sIL-6R secretion. These data suggest that hyperglycemia and hyperlipidemia can directly impact IL-6 trans-signaling and that this may be linked to enhanced severity of NAFLD in patients with concomitant diabetes. ARTICLE HIGHLIGHTS: IL-6 and its circulating coreceptor sgp130 are increased in people with fatty liver disease and steatohepatitis. High glucose and lipids stimulated IL-6 and sgp130 secretion from hepatic stellate cells. sgp130 levels correlated with HbA1c, and diabetes concurrent with steatohepatitis further increased circulating levels of all IL-6 trans-signaling mediators. Circulating sgp130 positively correlated with liver stiffness and hepatic fibrosis. Metabolic stress to liver associated with fatty liver disease might shift the balance of IL-6 classical versus trans-signaling, promoting liver fibrosis that is accelerated by diabetes.

IL-6 and IL-27 play both distinct and redundant roles in regulating CD4 T-cell responses during chronic viral infection.

The IL-6 cytokine family signals through the common signal transduction molecule gp130 combined with a cytokine-specific receptor. Gp130 signaling on CD4 T cells is vital in controlling chronic infection of mice with lymphocytic choriomeningitis virus clone 13 (LCMV Cl13), but the precise role of individual members of the IL-6 cytokine family is not fully understood. Transcriptional analysis highlighted the importance of gp130 signaling in promoting key processes in CD4 T cells after LCMV Cl13 infection, particularly genes associated with T follicular helper (Tfh) cell differentiation and IL-21 production. Further, Il27r-/-Il6ra-/- mice failed to generate antibody or CD8 T-cell immunity and to control LCMV Cl13. Transcriptomics and phenotypic analyses of Il27r-/-Il6ra-/- Tfh cells revealed that IL-6R and IL-27R signaling was required to activate key pathways within CD4 T cells. IL-6 and IL-27 signaling has distinct and overlapping roles, with IL-6 regulating Tfh differentiation, IL-27 regulating CD4 T cell survival, and both redundantly promoting IL-21.

Stattic ameliorates the cecal ligation and puncture-induced cardiac injury in septic mice via IL-6-gp130-STAT3 signaling pathway.

AIM: Sepsis-induced cardiac dysfunction is the leading cause of higher morbidity and mortality with poor prognosis in septic patients. Our recent previous investigation provides evidence of the hallmarks of signal transducer and activator of transcription3 (STAT3) activation in sepsis and targeting of STAT3 with Stattic, a small-molecule inhibitor of STAT3, has beneficial effects in various septic tissues. We investigated the possible cardioprotective effects of Stattic on cardiac inflammation and dysfunction in mice with cecal ligation and puncture (CLP)-induced sepsis. MAIN METHODS: A polymicrobial sepsis model was induced by CLP in mice and Stattic (25 mg/kg) was intraperitoneally given at one and twelve hours after CLP operation. The cecum was exposed in sham-control mice without CLP. After 18 h of surgery, electrocardiogram (ECG) for anaesthized mice was registered followed by collecting of samples of blood and tissues for bimolecular and histopathological assessments. Myeloperoxidase, a marker of neutrophil infiltration, was assessed immunohistochemically. KEY FINDINGS: CLP profoundly impaired cardiac functions as evidenced by ECG changes in septic mice as well as elevation of cardiac enzymes, and inflammatory markers with myocardial histopathological and immunohistochemical alterations. While, Stattic markedly reversed the CLP-induced cardiac abnormalities and restored the cardiac function by its anti-inflammatory activities. SIGNIFICANCE: Stattic treatment had potential beneficial effects against sepsis-induced cardiac inflammation, dysfunction and damage. Its cardioprotective effects were possibly attributed to its anti-inflammatory activities by targeting STAT3 and downregulation of IL-6 and gp130. Our investigations suggest that Stattic could be a promising target for management of cardiac sepsis and inflammation-related cardiac damage.

[Angiotensin converting enzyme 2 alleviates infectious bronchitis virus-induced cellular inflammation by suppressing IL-6/JAK2/STAT3 signaling pathway].

The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested: the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P < 0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P < 0.05 or P < 0.01); the expression of ACE2 decreased significantly (P < 0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P < 0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P > 0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P < 0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P < 0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P > 0.05), but the IL-6 level and expression of mRNA increased significantly (P < 0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P > 0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.

Buyang Huanwu Decoction promotes the neurological recovery of traumatic spinal cord injury via inhibiting apoptosis, inflammation, and oxidative stress.

BACKGROUND: The incidence rate of spinal cord injury (SCI) is increasing, and the mortality or disability rate caused by SCI remains high in the world. Buyang Huanwu Decoction (BYHWD) is a kind of Traditional Chinese medicine, and it is believed to be effective in several kinds of nervous system diseases. Whether BYHWD could improve SCI and the potential function mechanism remain unclear. METHODS: SCI animal model was established by damaging T10 spinal cord. Animals experiments included five groups as follows: Sham, SCI, SCI+BYHWD, SCI+mesenchymal stromal cells (MSCs), and SCI+BYHWD+MSCs. H2 O2 -treated cells (100 µM, 6 h) were used to simulate SCI damage in vitro, which included five groups as follows: control, H2 O2 , H2 O2 +BYHWD, H2 O2 +MSCs, and H2 O2 +BYHWD+MSCs. The behavioral function was evaluated with Tarlov and inclined plated test score. Western blot analysis and immunohistochemical staining were used to detect protein expression. The levels of superoxide dismutase (SOD), catalase (CAT), malondiadehyde (MDA), interleukin (IL)-1ß, tumor necrosis factor-α, and IL-6 in serum were measured with commercial enzyme-linked immunosorbent assay kits. terminal deoxynucleotidyl transferase dUTP nick end labeling staining and flow cytometry were performed to measure apoptosis in vivo and in vitro levels. Gene expression profiling analysis was performed to analyze differential expression genes. RESULTS: BYHWD suppressed apoptosis and accelerating cell proliferation after SCI. Recovery of neurofunction, inhibition of inflammatory response, and oxidative condition were achieved by BYHWD and MSCs. The expression levels of gp130/Janus kinase/signal transducers and activator of transcription (JAK/STAT) were suppressed by BYHWD and MSCs, both in vivo and in vitro. BYHWD and MSCs markedly promoted cells viability and inhibited apoptosis. Greater gene expression difference was observed between group control and H2 O2 through gene expression profiling analysis. The recovery effects of traumatic SCI by BYHWD were similar to MSCs, and synergies effects were observed in several items. CONCLUSION: BYHWD could increase Tarlov score and Basso, Beatie, and Bresnahan functional score, inhibit apoptosis, inflammatory response, and oxidative condition after SCI. The expression level of gp130/JAK/STAT axis was suppressed by BYHWD. BYHWD might be a new therapeutic strategy for the prevention or treatment of SCI.

IL-6/gp130/STAT3 signaling contributed to the activation of the PERK arm of the unfolded protein response in response to chronic ß-adrenergic stimulation.

Prolonged activation of the PERK branch of the unfolded protein response (UPR) promotes cardiomyocytes apoptosis in response to chronic ß-adrenergic stimulation. STAT3 plays a critical role in ß-adrenergic functions in the heart. However, whether STAT3 contributed to ß-adrenoceptor-mediated PERK activation and how ß-adrenergic signaling activates STAT3 remains unclear. This study aimed to investigate whether STAT3-Y705 phosphorylation contributed to the PERK arm activation in cardiomyocytes and if IL-6/gp130 signaling was involved in the chronic ß-AR-stimulation-induced STAT3 and PERK arm activation. We found that the PERK phosphorylation was positively associated with STAT3 activation. Wild-type STAT3 plasmids transfection activated the PERK/eIF2α/ATF4/CHOP pathway in cardiomyocytes while dominant negative Y705F STAT3 plasmids caused no obvious effect on PERK signaling. Stimulation with isoproterenol produced a significant increase in the level of IL-6 in the cardiomyocyte's supernatants, while IL-6 silence inhibited PERK phosphorylation but failed to attenuate STAT3 activation in response to isoproterenol stimulation. Gp130 silence attenuated isoproterenol-induced STAT3 activation and PERK phosphorylation. Inhibiting IL-6/gp130 pathway by bazedoxifene and inhibiting STAT3 by stattic both reversed isoproterenol-induced STAT3-Y705 phosphorylation, ROS production, PERK activation, IRE1α activation, and cardiomyocytes apoptosis in vitro. Bazedoxifene (5 mg/kg/day by oral gavage once a day) exhibited similar effect as carvedilol (10 mg/kg/day by oral gavage once a day) on attenuating chronic isoproterenol (30 mg/kg by abdominal injection once a day, 7 days) induced cardiac systolic dysfunction, cardiac hypertrophy and fibrosis in C57BL/6 mice. Meanwhile, bazedoxifene attenuates isoproterenol-induced STAT3-Y705 phosphorylation, PERK/eIF2α/ATF4/CHOP activation, IRE1α activation, and cardiomyocytes apoptosis to a similar extend as carvedilol in the cardiac tissue of mice. Our results showed that chronic ß-adrenoceptor-mediated stimulation activated the STAT3 and PERK arm of the UPR at least partially via IL-6/gp130 pathway. Bazedoxifene has great potential to be used as an alternative to conventional ß-blockers to attenuate ß-adrenoceptor-mediated maladaptive UPR.