Tumor Necrosis Factor-alpha Treatment Research Articles

Long Non-coding RNA DANCR Regulates the Proliferation and Migration of Human Adipose-derived Mesenchymal Stromal Cells in Inflammation Conditions

Background: Mesenchymal stromal cells (MSCs) and Dexamethasone (Dex) are both effective methods to treat inflammatory diseases. However, the interaction between inflammatory factors, Dex, and MSCs in repair is not fully understood. The purpose of this study is to clarify the effects and mechanisms of glucocorticoids on the tissue repair characteristics of MSCs in an inflammatory environment. Methods: This is an experimental study. Human adipose-derived mesenchymal stromal cells (hASCs) were cultured, and Long non-coding RNA (lncRNA) differentiation antagonizing nonprotein coding RNA (DANCR) expression was detected after treatment with Dex and inflammation factors. Additionally, DANCR was knockdown or overexpressed before Dex or tumor necrosis factor-alpha (TNF-α) treatments, respectively. hASC proliferation, cell cycle, and migration ability were analyzed to evaluate the effects of DANCR in hASCs treated with Dex or TNF-α. Nuclear factor-kB (NF-κB) pathway inhibitors were used to clarify the signal pathway that DANCR involved. All data are presented as the mean ± standard deviation. The two-tailed Student's t-test or one-way analysis of variance (ANOVA) was used to determine the statistical differences between groups. Results: Dex decreased the proliferation and migration of hASCs and upregulated DANCR expression in a dosage-dependent relationship. The knockdown of DANCR reversed Dex's repression of hASC proliferation. Moreover, DANCR was decreased by inflammatory cytokines, and overexpressing DANCR alleviated the promotion effects of TNF-α on hASC proliferation and migration. Furthermore, mechanistic investigation validated that DANCR was involved in the NF- κB signaling pathway. Conclusions: We identified a lncRNA, DANCR, that was involved in Dex and inflammation-affected hASC proliferation and migration. Dex reduced the proliferation and migration of hASCs through DANCR while exerting its anti-inflammatory effects. Thus, it is suggested to avoid the simultaneous application of hASCs and steroids in clinical practice. These results enrich our understanding of the versatile function of lncRNAs in the crosstalk of inflammation conditions and MSCs.

A Novel Treatment of Crohn’s Disease that is Not Only Safe in Pregnancy but Can Correct Infertility and Recurrent Miscarriages

Rationale: Treatment with dopaminergic drugs e.g., dextroamphetamine sulfate (DS) has provided marked relief of abdominal pain and diarrhea in patients with Crohn's disease who failed to respond to other therapies e.g., corticosteroids and biologic monoclonal antibody therapy directed against pro- inflammatory cytokines e.g., tumor necrosis factor-alpha treatment with DS has provided marked amelioration of Crohn’s disease and dysmenorrhea thus relieving excessive bowel inflammation and pelvic inflammation with subsequent successful conception. The likely mechanism of action is through stimulating the release of dopamine from sympathetic nerve fibers leading to diminished cellular permeability thus limiting absorption of toxic elements into those tissues. There are reports of DS improving fecundity i.e. aiding conception and inhibiting miscarriage. Objective: To see if DS combined with luteal phase progesterone in a woman with Crohn’s disease but no pelvic pain suffering with infertility with diminished oocyte reserve can help her to conceive naturally despite failure with other therapies including in vitro fertilization. Findings: Not only did the DS markedly ameliorate her inflammatory bowel symptoms but she also conceived naturally and has passed the first trimester with a live fetus. Conclusion: At her last consultation with her previous fertility specialist, she was advised that she should proceed with donor oocytes. She probably had excessive endometrial inflammation despite the absence of dysmenorrhea. Thus, inflammation may have also caused damage to her ovarian egg supply. Dopaminergic drugs should be considered to help infertile women to conceive when there is evidence of other autoimmune disorders even with no apparent pelvic pain. DS is much safer to take during pregnancy than corticosteroids or biologics even with monoclonal antibodies especially in the last trimester.

Nur77 mitigates endothelial dysfunction through activation of both nitric oxide production and anti-oxidant pathways

BackgroundNur77 belongs to the member of orphan nuclear receptor 4A family that plays critical roles in maintaining vascular homeostasis. This study aims to determine whether Nur77 plays a role in attenuating vascular dysfunction, and if so, to determine the molecular mechanisms involved. MethodsBoth Nur77 knockout (Nur77 KO) and Nur77 endothelial specific transgenic mice (Nur77-Tg) were employed to examine the functional significance of Nur77 in vascular endothelium in vivo. Endothelium-dependent vasodilatation to acetylcholine (Ach) and reactive oxygen species (ROS) production was determined under inflammatory and high glucose conditions. Expression of genes was determined by real-time PCR and western blot analysis. ResultsIn response to tumor necrosis factor alpha (TNF-α) treatment and diabetes, the endothelium-dependent vasodilatation to Ach was significantly impaired in aorta from Nur77 KO as compared with those from the wild-type (WT) mice. Endothelial specific overexpression of Nur77 markedly prevented both TNF-α- and high glucose-induced endothelial dysfunction. Compared with WT mice, after TNF-α and high glucose treatment, ROS production in aorta was significantly increased in Nur77 KO mice, but it was inhibited in Nur77-Tg mice, as determined by dihydroethidium (DHE) staining. Furthermore, we demonstrated that Nur77 overexpression substantially increased the expression of several key enzymes involved in nitric oxide (NO) production and ROS scavenging, including endothelial nitric oxide synthase (eNOS), guanosine triphosphate cyclohydrolase 1 (GCH-1), glutathione peroxidase-1 (GPx-1), and superoxide dismutases (SODs). Mechanistically, we found that Nur77 increased GCH1 mRNA stability by inhibiting the expression of microRNA-133a, while Nur77 upregulated SOD1 expression through directly binding to the human SOD1 promoter in vascular endothelial cells. ConclusionOur results suggest that Nur77 plays an essential role in attenuating endothelial dysfunction through activating NO production and anti-oxidant pathways in vascular endothelium. Targeted activation of Nur77 may provide a novel therapeutic approach for the treatment of cardiovascular diseases associated with endothelial dysfunction.

P731 Relapse rate following withdrawal of anti-TNF therapy in patients with inflammatory bowel disease: A single-center experience

Abstract Background Discontinuation of anti–tumor necrosis factor alpha treatment (anti-TNFα) in patients with inflammatory bowel disease (IBD) poses a significant risk for relapse. The aim of this study is to analyze relapse rates and to possibly identify factors associated with relapse in IBD patients after treatment cessation with anti-TNFα. Methods This is a prospective study conducted at University Clinic of Gastroenterology in Albania from 2019-2022. IBD patients who discontinued biologic therapy with infliximab (IFX) or adalimumab (ADA) were included. All the patients discontinued therapy due to reimbursement issues. Demographic, clinical,laboratory and endoscopic data were collected for each patient. Relapse rates and predictors for relapse were studied using survival and Cox regression analysis. Results A total of 52 IBD patients, mean age 50.8±14.7 years (46 UC and 6 CD), 25(48%) males, were included in the study. Mean follow-up was 29 months. 47.4% were treated with ADA and 51.5 % with IFX. The mean period of biologic therapy was 33.3 months for all patients. Upon treatment cessation, 55.8% of patients were in complete clinical remission, and 30.8% were in partial clinical remission. Cumulative relapse rates at 6, 12, 24 and 36 months were 23.9%, 39.1%, 44.2% and 69.7% respectively for all UC patients while 66.7% of CD patients relapsed within 6 months. The mean time to relapse was 13 months for UC and 5 months for CD patients. Among all IBD patients in complete clinical remission at the time of treatment discontinuation, the cumulative relapse rates at 6, 12, 24 and 36 months, were: 6.9%, 24.8%, 57.7%, and 58% respectively. Significantly higher cumulative relapse rates were found in patients with partial remission, the at the same time points: 37.5%, 50%, 75%, and 81.2% respectively (p=0.01). Multivariable Cox analysis revealed that the only factor associated with lower risk of relapse was mucosal healing (vs nonmucosal healing: HR, 0.322; 95% CI, 1.26 to 6.677; p=0.012). Conclusion The majority of IBD patients relapsed within 3 years after anti TNFα discontinuation, Mucosal healing was the only factor associated with a lower risk of relapse. These results emphasise the crucial role of mucosal healing in guiding treatment decisions for IBD patients.

Self-assembled and perfusable microvasculature-on-chip for modeling leukocyte trafficking.

Leukocyte recruitment from blood to tissue is a process that occurs at the level of capillary vessels during both physiological and pathological conditions. This process is also relevant for evaluating novel adoptive cell therapies, in which the trafficking of therapeutic cells such as chimeric antigen receptor (CAR)-T cells throughout the capillaries of solid tumors is important. Local variations in blood flow, mural cell concentration, and tissue stiffness contribute to the regulation of capillary vascular permeability and leukocyte trafficking throughout the capillary microvasculature. We developed a platform to mimic a biologically functional human arteriole-venule microcirculation system consisting of pericytes (PCs) and arterial and venous primary endothelial cells (ECs) embedded within a hydrogel, which self-assembles into a perfusable, heterogeneous microvasculature. Our device shows a preferential association of PCs with arterial ECs that drives the flow-dependent formation of microvasculature networks. We show that PCs stimulate basement membrane matrix synthesis, which affects both vessel diameter and permeability in a manner correlating with the ratio of ECs to PCs. Moreover, we demonstrate that hydrogel concentration can affect capillary morphology but has no observed effect on vascular permeability. The biological function of our capillary network was demonstrated using an inflammation model, where significantly higher expression of cytokines, chemokines, and adhesion molecules was observed after tumor necrosis factor-alpha (TNF-α) treatment. Accordingly, T cell adherence and transendothelial migration were significantly increased in the immune-activated state. Taken together, our platform allows the generation of a perfusable microvasculature that recapitulates the structure and function of an in vivo capillary bed that can be used as a model for developing potential immunotherapies.

MP24-06 TNF-ALPHA INHIBITOR THERAPY SUPPRESSES GROWTH OF THE PROSTATE GLAND

You have accessJournal of UrologyCME1 Apr 2023MP24-06 TNF-ALPHA INHIBITOR THERAPY SUPPRESSES GROWTH OF THE PROSTATE GLAND Ra'ad Al-Faouri, Christina Sharkey, Leo L. Tsai, Zongwei Wang, and Aria F. Olumi Ra'ad Al-FaouriRa'ad Al-Faouri More articles by this author , Christina SharkeyChristina Sharkey More articles by this author , Leo L. TsaiLeo L. Tsai More articles by this author , Zongwei WangZongwei Wang More articles by this author , and Aria F. OlumiAria F. Olumi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003249.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous studies have established a relationship between prostate inflammation and the development of BPH. Steroid 5 alpha reductase type 2 (SRD5A2) is a pivotal enzyme in the development and growth of the prostate gland and the critical target for BPH therapy. We previously demonstrated that tumor necrosis factor alpha (TNF-a) regulates the epigenetic change of SRD5A2, leading to suppression of SRD5A2 gene and protein expression. However, little is known whether TNF-a inhibitor therapy affects prostatic growth. Here we aimed to evaluate the effect of TNF-a inhibitor therapy on the growth rate of prostate through analyzing the data of serial pelvic imaging scans. METHODS: In this retrospective cohort study, electronic records at Beth Israel Deaconess Medical Center were searched for men aged 18 and over who had serial pelvic Images (MRI or CT scans) while receiving TNF-a inhibitors. 99 men were included in the treatment cohort after applying exclusion criteria (TNF-a treatment duration <1 year, any pelvic/prostate surgery/radiation, use of 5ARIs, advanced prostate cancer, androgen deprivation therapy, testosterone therapy, poor image quality, or imaging interval <1 year). An age-matched cohort was constructed with the same inclusion/exclusion criteria but absent TNF-a therapy (n=99). The total prostate volume (TPV) was measured and calculated from baseline and follow-up imaging. Clinical data was collected for all men. RESULTS: There were no significant differences between the two groups in age, demographics, BMI or comorbidities. Mean baseline TPV was similar in the TNF-a inhibitor therapy group and control group (27.52±8.9 cc vs 27.3±9.6 cc, p-value: 0.71). For the entire cohort, the average imaging follow up duration was 3.79±0.32 years with no significant difference between the treatment and control groups. The median growth rate for men in the treatment cohort was significantly lower than those in the control group (-0.01 cc/year IQR= 1.2 compared to 0.68 cc/year IQR= 2.8, P-value 0.001). In a multiple regression model adjusting for age, race, initial TPV and BMI, men in the treatment group still had a significantly lower growth rate compared to the control group (-0.03 cc/year IQR: 0.82 vs 0.67 cc/year IQR: 0.93, p-value: 0.001). CONCLUSIONS: TNF-a inhibitors are negatively correlated with prostatic growth. Men with autoimmune diseases who are treated with TNF-a inhibitors may experience fewer urinary symptoms related to prostatic enlargement. Our study suggests that inflammatory mediators regulate prostatic growth. Source of Funding: NIH: R01DK124502 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e319 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ra'ad Al-Faouri More articles by this author Christina Sharkey More articles by this author Leo L. Tsai More articles by this author Zongwei Wang More articles by this author Aria F. Olumi More articles by this author Expand All Advertisement PDF downloadLoading ...

A common Matrix metalloproteinase 8 promoter haplotype enhances the risk for hypertension via diminished interactions with nuclear factor kappa B.

Matrix metalloproteinase 8 (MMP8) has a prominent role in collagen turnover in blood vessels and vascular remodeling. The contribution of regulatory single nucleotide polymorphisms in MMP8 to cardiovascular diseases is unclear. We aimed to delineate the influence of MMP8 promoter variations on hypertension. A case-control study in unrelated individuals ( n = 2565) was carried out. Resequencing of the MMP8 proximal promoter, linkage disequilibrium analysis, genotyping of variants and regression analyses were performed. MMP8 promoter-reporter constructs were generated and expressed in human vascular endothelial cells under various conditions. We identified four single nucleotide polymorphisms (SNPs) in the promoter region of MMP8 : -1089A/G (rs17099452), -815G/T (rs17099451), -795C/T (rs11225395), -763A/T (rs35308160); these SNPs form three major haplotypes. Hap3 (viz., GTTT haplotype) carriers showed significant associations with hypertension in two geographically distinct human populations (e.g., Chennai: odds ratio [OR] = 1.47, 95% confidence interval [CI] = 1.16-1.86, P = 2 × 10 -3 ; Chandigarh: OR = 1.85, 95% CI = 1.21-2.81, P = 4 × 10 -3 ). Hap3 carriers also displayed elevated systolic blood pressure, diastolic blood pressure and mean arterial pressure levels. Hap3 promoter-reporter construct showed lower promoter activity than the wild-type (Hap1) construct. In silico analysis and molecular dynamics studies predicted diminished binding of the transcription factor nuclear factor kappa B (NF-κB) to the functional -815T allele of Hap3 compared to the -815G wild-type allele; this prediction was validated by in-vitro experiments. Hap3 displayed impaired response to tumor necrosis factor-alpha treatment, possibly due to weaker binding of NF-κB. Notably, MMP8 promoter haplotypes were identified as independent predictors of plasma MMP8 and endothelial dysfunction markers (von Willebrand factor and endothelin-1) levels. MMP8 promoter GTTT haplotype has a functional role in reducing MMP8 expression during inflammation via diminished interaction with NF-κB and in enhancing the risk of hypertension.

Inflammation depletes humoral immunity by limiting plasma cell access to the bone marrow survival niche

Abstract The survival of plasma cells (PCs) is directly correlated to the longevity of prophylactic titers against pathogens. The bone marrow contains hematopoietic and non-hematopoietic cells that contribute to the maintenance of PCs. Studies have shown that pathogens contain a variety of mechanisms that result in the depletion of bone marrow PCs and antibody titers. Most, if not all, forms of infection induce inflammation, however, the role of inflammation in bone marrow PC depletion is unclear. Here, we show that acute Tumor Necrosis Factor Alpha (TNFa) treatment can deplete pre-existing antibody titers in a variety of models. We also found that recombinant TNFa treatment limits PC retention in the bone marrow via adoptive transfer experiments. To determine if PC retention in the bone marrow was mediated in a cell-intrinsic or − extrinsic manner, we generated bone marrow chimeras with wild-type and TNFa receptor knockout mice. Results showed that TNFa signaling through TNFa receptor 1 in non-hematopoietic cells regulates PC retention in the bone marrow, indicating that this mechanism is cell-extrinsic. Another cell-extrinsic mechanism that we identified is TNFa regulation of syndecan-1, a cell surface proteoglycan found to regulate PC survival and motility. These findings demonstrate that inflammation limits PC access to the bone marrow survival niche, ultimately leading to the reduction of antibody titers and long-term humoral protection. Supported by grants from the NIH (R01 HLI141491) and the Eric Heyer M.D. Ph.D Translational Research Grant

TNF-α Induced Myotube Atrophy in C2C12 Cell Line Uncovers Putative Inflammatory-Related lncRNAs Mediating Muscle Wasting.

Background: Muscle atrophy is a complex catabolic condition developing under different inflammatory-related systemic diseases resulting in wasting of muscle tissue. While the knowledge of the molecular background of muscle atrophy has developed in recent years, how the atrophic conditions affect the long non-coding RNA (lncRNAs) machinery and the exact participation of the latter in the mediation of muscle loss are still unknown. The purpose of the study was to assess how inflammatory condition developing under the tumor necrosis factor alpha (TNF-α) treatment affects the lncRNAs’ expression in a mouse skeletal muscle cell line. Materials and method: A C2C12 mouse myoblast cell line was treated with TNF-α to develop atrophy, and inflammatory-related lncRNAs mediating muscle loss were identified. Bioinformatics was used to validate and analyze the discovered lncRNAs. The differences in their expression under different TNF-α concentrations and treatment times were investigated. Results: Five lncRNAs were identified in a discovery set as atrophy related and then validated. Three lncRNAs, Gm4117, Ccdc41os1, and 5830418P13Rik, were selected as being significant for inflammatory-related myotube atrophy. Dynamics changes in the expression of lncRNAs depended on both TNF-α concentration and treatment time. Bioinformatics analysis revealed the mRNA and miRNA target for selected lncRNAs and their putative involvement in the molecular processes related to muscle atrophy. Conclusions: The inflammatory condition developing in the myotube under the TNF-α treatment affects the alteration of lncRNAs’ expression pattern. Experimental and bioinformatics testing suggested the prospective role of lncRNAs in the mediation of muscle loss under an inflammatory state.

Syndecan-3 as a Novel Biomarker in Alzheimer's Disease.

Early diagnosis of Alzheimer’s disease (AD) is of paramount importance in preserving the patient’s mental and physical health in a fairly manageable condition for a longer period. Reliable AD detection requires novel biomarkers indicating central nervous system (CNS) degeneration in the periphery. Members of the syndecan family of transmembrane proteoglycans are emerging new targets in inflammatory and neurodegenerative disorders. Reviewing the growing scientific evidence on the involvement of syndecans in the pathomechanism of AD, we analyzed the expression of the neuronal syndecan, syndecan-3 (SDC3), in experimental models of neurodegeneration. Initial in vitro studies showed that prolonged treatment of tumor necrosis factor-alpha (TNF-α) increases SDC3 expression in model neuronal and brain microvascular endothelial cell lines. In vivo studies revealed elevated concentrations of TNF-α in the blood and brain of APPSWE-Tau transgenic mice, along with increased SDC3 concentration in the brain and the liver. Primary brain endothelial cells and peripheral blood monocytes isolated from APPSWE-Tau mice exhibited increased SDC3 expression than wild-type controls. SDC3 expression of blood-derived monocytes showed a positive correlation with amyloid plaque load in the brain, demonstrating that SDC3 on monocytes is a good indicator of amyloid pathology in the brain. Given the well-established role of blood tests, the SDC3 expression of monocytes could serve as a novel biomarker for early AD detection.

Inhibitory Effect of Punicalagin on Inflammatory and Angiogenic Activation of Human Umbilical Vein Endothelial Cells.

Punicalagin, a major ellagitannin isolated from pomegranate, is proved to have various pharmacological activities with an undefined therapy mechanism. The objective of this research was to demonstrate the effect of punicalagin on anti-inflammatory and angiogenic activation in human umbilical vein endothelial cells (HUVECs) and their potential mechanisms. Endothelial-leukocyte adhesion assay was applied to evaluate primary cultures of HUVECs activation following tumor necrosis factor alpha (TNF-α) treatment. The endothelial cell proliferation, migration, permeability and tube formation were assessed by EdU assay, wound migration assay, trans-endothelial electrical resistances (TEER) assay, and capillary-like tube formation assay, respectively. In addition, the expression of relevant proteins was assessed using Western blot analysis. We confirmed that punicalagin could reduce the adhesion of human monocyte cells to HUVECs in vitro and in vivo. Further, punicalagin decreased the expression of mRNA and proteins of ICAM-1 and VCAM-1 in HUVECs. Moreover, punicalagin inhibited permeability, proliferation, migration, and tube formation in VEGF-induced HUVECs, suppressed IKK-mediated activation of NF-κB signaling in TNF-α-induced endothelial cells, and inhibited vascular endothelial growth factor receptor 2 (VEGFR2) activation and downstream p-PAK1. Our findings indicated that punicalagin might have a protective effect on HUVECs activation, which suggested that punicalagin functions through an endothelial mediated mechanism for treating various disorders such as, cancer, rheumatoid arthritis, and cardiovascular disease.

Enhanced autophagy suppresses inflammation-mediated bone loss through ROCK1 signaling in bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) have strong proliferative ability and multi-directional differentiation potential. Osteoarthritis is a degenerative joint disease that is closely related to the loss of osteogenic differentiation function of BMSCs. Autophagy, plays a crucial role in the maintenance of cellular functions, but its regulatory mechanism during the osteogenic differentiation of BMSCs remains unclear. In this study, we analyzed the differential gene networks and pathways during BMSC osteogenesis using bioinformatics, and further validated the regulatory roles of autophagy during the osteogenic differentiation of BMSCs in inflammatory condition in vitro. We found that Tumor necrosis factor alpha (TNF-α) treatment led to actin cytoskeleton rearrangements and inhibited osteogenic differentiation in BMSCs. In addition, TNF-α enhanced Rho-associated protein kinase 1 (ROCK1) expression and decreased autophagy activation. ROCK1 knockdown reduced Endoplasmic Reticulum stress (ER stress) and promoted autophagy, resulting reversion of osteogenic differentiation in BMSCs under inflammatory condition. Rapamycin reversed the TNF-α-induced decrease in osteogenesis of BMSCs, assessed by alkaline phosphatase (ALP) activity and Alizarin staining. Autophagy treated with inhibitor 3-Methyladenine (3-MA) further increased TNF-α-induced osteogenesis inhibition of BMSCs. Collectively, these results indicate that ER stress and dysfunction of autophagy promote inflammation-induced bone loss through the activation of ROCK1 signaling in BMSCs.

TNF Alpha-Induced SOX2 Expression Promotes Hepatic Steatois in Diet-Induced Obesity Model

Diet-induced obesity can cause metabolic or inflammatory damage on liver. Nonalcoholic fatty liver disease (NAFLD) begins with the fat accumulation in hepatocyte, but can lead to hepatocellular carcinoma (HCC). Sex-determining region Y-box 2 (SOX2) is a critical transcription factor involving regeneration and pluripotency. The expression level of Sox2 is correlated with progression of HCC, and anti-inflammatory effects of Sox2 in mesenchymal stem cells have been found. However, the expression of Sox2 by inflammatory cytokines in hepatocyte in NAFLD or the role of SOX2 in fat accumulation has been rarely reported. Here, we found that high-fat diet feeding, with or without high fructose in drinking water, significantly upregulated SOX2 in the livers of mice. In vitro, treatment with free fatty acids (FFAs) and fructose increased SOX2 expression in FL83B cells compared with the vehicle-treated group. Furthermore, overexpression or knockdown of SOX2 in FL83B cells promoted or suppressed, respectively, triglyceride synthesis and lipid accumulation after FFAs stimulation. The expression levels of several lipogenesis-related molecules were found to be altered by SOX2 expression. In addition, among several cytokines, only the treatment of tumor necrosis factor-alpha (TNFα) increased the SOX2 expression compared with the vehicle-treated control. Further, upregulation of (TNFα) by FFA/fructose was observed, and TNFα and FFA/fructose induced SOX2 expression was abolished by pretreatment of a TNFα inhibitor. Collectively, our findings suggest that TNFα-SOX2 signaling pathway in hepatocyte may be one of targets for early prevention of the development of NAFLD.

Tumor Necrosis Factor Alpha inhibits humoral immunity by regulating the antibody secreting cell bone marrow niche

Abstract Humoral immunity is a critical component of the immune memory, and its longevity is partly dependent on the survival of antibody-secreting cells (ASC). Previous studies have shown that several types of infections can deplete pre-existing antibody titers, but the exact mechanism of this process is unclear. Most, if not all, forms of infection induce inflammation including Tumor Necrosis Factor Alpha (TNFa). Here we show that TNFa-mediated inflammation depletes pre-existing antibody titers by limiting ASC retention in the bone marrow (BM) survival niche. Acute recombinant TNFa (rTNFa) treatment induces ASC egress from the BM into the blood and limits engraftment of adoptively transferred ASCs into the BM of recipient mice. To determine if the TNFa-mediated ASC egress from the BM was cell-intrinsic or –extrinsic, we generated BM chimeras with wild-type and TNFa receptor knockout mice. We found that TNFa signaling through TNFa receptor 1 in non-hematopoietic cells induces ASC egress from the BM in a cell-extrinsic manner. Treatment with rTNFa did not induce ASC death in the BM, but it increased glucose uptake and expression of CXCR4, a receptor involved in ASC retention in the BM. In contrast, mobilized ASCs in the blood had reduced glucose uptake, CD98 expression, and CD93 expression suggesting that ASC metabolism is highly dependent on the BM niche. Our findings demonstrate that inflammation alters ASC physiology in both cell-intrinsic and extrinsic manners, leading to reduced antibody titers and long-term immune memory.

Development, Characterization and Potential Applications of a Multicellular Spheroidal Human Blood–Brain Barrier Model Integrating Three Conditionally Immortalized Cell Lines

In vitro blood-brain barrier (BBB) models are essential research tools for use in developing brain-targeted drugs and understanding the physiological and pathophysiological functions of the BBB. To develop BBB models with better functionalities, three-dimensional (3D) culture methods have gained significant attention as a promising approach. In this study, we report on the development of a human conditionally immortalized cell-based multicellular spheroidal BBB (hiMCS-BBB) model. After being seeded into non-attachment culture wells, HASTR/ci35 (astrocytes) and HBPC/ci37 cells (brain pericytes) self-assemble to form a spheroid core that is then covered with an outer monolayer of HBMEC/ci18 cells (brain microvascular endothelial cells). The results of immunocytochemistry showed the protein expression of several cellular junction and BBB-enriched transporter genes in HBMEC/ci18 cells of the spheroid model. The permeability assays showed that the hiMCS-BBB model exhibited barrier functions against the penetration of dextran (5 kDa and 70 kDa) and rhodamine123 (a P-glycoprotein substrate) into the core. On the other hand, facilitation of 2-NBDG (a fluorescent glucose analog) uptake was observed in the hiMCS-BBB model. Furthermore, tumor necrosis factor-alpha treatment elicited an inflammatory response in HBMEC/ci18 cells, thereby suggesting that BBB inflammation can be recapitulated in the hiMCS-BBB model. To summarize, we have developed an hiMCS-BBB model that possesses fundamental BBB properties, which can be expected to provide a useful and highly accessible experimental platform for accelerating various BBB studies.

Deletion of Protocadherin Gamma C3 Induces Phenotypic and Functional Changes in Brain Microvascular Endothelial Cells In Vitro.

Inflammation of the central nervous system (CNS) is associated with diseases such as multiple sclerosis, stroke and neurodegenerative diseases. Compromised integrity of the blood-brain barrier (BBB) and increased migration of immune cells into the CNS are the main characteristics of brain inflammation. Clustered protocadherins (Pcdhs) belong to a large family of cadherin-related molecules. Pcdhs are highly expressed in the CNS in neurons, astrocytes, pericytes and epithelial cells of the choroid plexus and, as we have recently demonstrated, in brain microvascular endothelial cells (BMECs). Knockout of a member of the Pcdh subfamily, PcdhgC3, resulted in significant changes in the barrier integrity of BMECs. Here we characterized the endothelial PcdhgC3 knockout (KO) cells using paracellular permeability measurements, proliferation assay, wound healing assay, inhibition of signaling pathways, oxygen/glucose deprivation (OGD) and a pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) treatment. PcdhgC3 KO showed an increased paracellular permeability, a faster proliferation rate, an altered expression of efflux pumps, transporters, cellular receptors, signaling and inflammatory molecules. Serum starvation led to significantly higher phosphorylation of extracellular signal-regulated kinases (Erk) in KO cells, while no changes in phosphorylated Akt kinase levels were found. PcdhgC3 KO cells migrated faster in the wound healing assay and this migration was significantly inhibited by respective inhibitors of the MAPK-, β-catenin/Wnt-, mTOR- signaling pathways (SL327, XAV939, or Torin 2). PcdhgC3 KO cells responded stronger to OGD and TNFα by significantly higher induction of interleukin 6 mRNA than wild type cells. These results suggest that PcdhgC3 is involved in the regulation of major signaling pathways and the inflammatory response of BMECs.

Chronic exposure to TNF reprograms cell signaling pathways in fibroblast-like synoviocytes by establishing long-term inflammatory memory

Fibroblast-like synoviocytes (FLS) play a critical role in the pathogenesis of rheumatoid arthritis (RA). Chronic inflammation induces transcriptomic and epigenetic modifications that imparts a persistent catabolic phenotype to the FLS, despite their dissociation from the inflammatory environment. We analyzed high throughput gene expression and chromatin accessibility data from human and mouse FLS from our and other studies available on public repositories, with the goal of identifying the persistently reprogrammed signaling pathways driven by chronic inflammation. We found that the gene expression changes induced by short-term tumor necrosis factor-alpha (TNF) treatment were largely sustained in the FLS exposed to chronic inflammation. These changes that included both activation and repression of gene expression, were accompanied by the remodeling of chromatin accessibility. The sustained activated genes (SAGs) included established pro-inflammatory signaling components known to act at multiple levels of NF-kappaB, STAT and AP-1 signaling cascades. Interestingly, the sustained repressed genes (SRGs) included critical mediators and targets of the BMP signaling pathway. We thus identified sustained repression of BMP signaling as a unique constituent of the long-term inflammatory memory induced by chronic inflammation. We postulate that simultaneous targeting of these activated and repressed signaling pathways may be necessary to combat RA persistence.

Development of a novel in vitro insulin resistance model in primary human tenocytes for diabetic tendinopathy research.

BackgroundType 2 diabetes mellitus (T2DM) had been reported to be associated with tendinopathy. However, the underlying mechanisms of diabetic tendinopathy still remain largely to be discovered. The purpose of this study was to develop insulin resistance (IR) model on primary human tenocytes (hTeno) culture with tumour necrosis factor-alpha (TNF-α) treatment to study tenocytes homeostasis as an implication for diabetic tendinopathy.MethodshTenowere isolated from human hamstring tendon. Presence of insulin receptor beta (INSR-β) on normal tendon tissues and the hTeno monolayer culture were analyzed by immunofluorescence staining. The presence of Glucose Transporter Type 1 (GLUT1) and Glucose Transporter Type 4 (GLUT4) on the hTeno monolayer culture were also analyzed by immunofluorescence staining. Primary hTeno were treated with 0.008, 0.08, 0.8 and 8.0 µM of TNF-α, with and without insulin supplement. Outcome measures include 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) assay to determine the glucose uptake activity; colourimetric total collagen assay to quantify the total collagen expression levels; COL-I ELISA assay to measure the COL-I expression levels and real-time qPCR to analyze the mRNA gene expressions levels of Scleraxis (SCX), Mohawk (MKX), type I collagen (COL1A1), type III collagen (COL3A1), matrix metalloproteinases (MMP)-9 and MMP-13 in hTeno when treated with TNF-α. Apoptosis assay for hTeno induced with TNF-α was conducted using Annexin-V FITC flow cytometry analysis.ResultsImmunofluorescence imaging showed the presence of INSR-β on the hTeno in the human Achilles tendon tissues and in the hTeno in monolayer culture. GLUT1 and GLUT4 were both positively expressed in the hTeno. TNF-α significantly reduced the insulin-mediated 2-NBDG uptake in all the tested concentrations, especially at 0.008 µM. Total collagen expression levels and COL-I expression levels in hTeno were also significantly reduced in hTeno treated with 0.008 µM of TNF-α. The SCX, MKX and COL1A1 mRNA expression levels were significantly downregulated in all TNF-α treated hTeno, whereas the COL3A1, MMP-9 and MMP-13 were significantly upregulated in the TNF–α treated cells. TNF-α progressively increased the apoptotic cells at 48 and 72 h.ConclusionAt 0.008 µM of TNF-α, an IR condition was induced in hTeno, supported with the significant reduction in glucose uptake, as well as significantly reduced total collagen, specifically COL-I expression levels, downregulation of candidate tenogenic markers genes (SCX and MKX), and upregulation of ECM catabolic genes (MMP-9 and MMP-13). Development of novel IR model in hTeno provides an insight on how tendon homeostasis could be affected and can be used as a tool for further discovering the effects on downstream molecular pathways, as the implication for diabetic tendinopathy.

THU0378 DO COMORBIDITIES DECREASE THE FIRST TNF-INHIBITOR RETENTION AND TREATMENT RESPONSE IN AXIAL SPONDYLOARTHRITIS PATIENTS? DATA FROM TURKBIO

Background:The frequency of comorbidities has increased in spondyloarthritis patients compared to the general population. The effect of comorbidities on tumour necrosis factor alpha inhibitor (TNFi) drug retention and treatment response has not been well evaluated.Objectives:The purpose of this study to assess the impact of comorbidities on the first TNFi drug survival and treatment response in patients with axial spondyloarthritis (axSpA) registered in theTURKBIOdatabase.Methods:In this study, the frequency of comorbidities, disease activity scores at baseline and month 6 and drug retention were recorded in AxSpA patients iniating first TNFi treatment between 2011 and 2019. Kaplan Meier plot and log rank tests were used for drug survival analysis. Cox regression analysis with HR was performed to evaluate the correlation between comorbidities and drug survival.Results:There were 2428 patients with AxSpA (39.3% female) who used their first TNFi during the study period. Among them, a total of 770 (31%) had at least one comorbid disease. Hypertension was the most common comorbidity (9.7%), followed by the affective disorders (8%) and chronic lung disease (5.8%). The baseline characteristics of patients are shown in Table 1.The presence of any comorbidity did not impact the first TNFi retention (Figure 1). When comorbidities were analysed seperately, we found that only history of cerebrovascular event was negatively associated with drug retention rate (HR: 6.9, p:0.008). There was no statistically significant difference in Bath AS Disease Activity Index 50% (BASDAI50) response between patients with and without comorbidity at 6 months. Less axSpA patients with comorbidity achieved a ASDAS score ≤ 2.1 compared to patients without comorbidity at 6 months.Table 1.Baseline Characteristics of PatientsRadiographic Spondyloarthritis, n (%)2318 (95.5)Female, n(%)954 (39.3)Age, year42.2±11.8Age at diagnosis, years32.5± 11.3Age at initial TNFi, years39.4 ± 11.1Symptom duration, years9.7± 7.5Time to initial TNFi, years7±6.8HLA-B27- positivity, n (%)1144 (47.1)Smokers, n (%)1068 (44)Baseline BASDAI35.5±22.2Baseline ASDAS-CRP2.8±1.1Baseline CRP (mg/L)15.7±24.4VAS global patient46.6±28.7-Quantitative variables are presented as mean ± SD, and qualitative variables are presented as frequency and percentage-ASDAS-CRP, Ankylosing Spondylitis Disease Activity Score using C-reactive protein VAS, visual analogue scaleConclusion:The results of this study demonstrated that the presence of previous cerebrovascular event decreased the first TNFi survival in patients with axSpA. It also suggested that comorbidities might decrease TNFi treatment response.Disclosure of Interests:None declared

Global expression and CpG methylation analysis of primary endothelial cells before and after TNFa stimulation reveals gene modules enriched in inflammatory and infectious diseases and associated DMRs.

Endothelial cells are a primary site of leukocyte recruitment during inflammation. An increase in tumor necrosis factor-alpha (TNFa) levels as a result of infection or some autoimmune diseases can trigger this process. Several autoimmune diseases are now treated with TNFa inhibitors. However, genomic alterations that occur as a result of TNF-mediated inflammation are not well understood. To investigate molecular targets and networks resulting from increased TNFa, we measured DNA methylation and gene expression in 40 human umbilical vein endothelial cell primary cell lines before and 24 hours after stimulation with TNFa via microarray. Weighted gene co-expression network analysis identified 15 gene groups (modules) with similar expression correlation patterns; four modules showed a strong association with TNFa treatment. Genes in the top TNFa-associated module were all up-regulated, had the highest proportion of hypomethylated regions, and were associated with 136 Disease Ontology terms, including autoimmune/inflammatory, infectious and cardiovascular diseases, and cancers. They included chemokines CXCL1, CXCL10 and CXCL8, and genes associated with autoimmune diseases including HLA-C, DDX58, IL4, NFKBIA and TNFAIP3. Cardiovascular and metabolic disease genes, including APOC1, ACLY, ELOVL6, FASN and SCD, were overrepresented in a module that was not associated with TNFa treatment. Of 223 hypomethylated regions identified, several were in promoters of autoimmune disease GWAS loci (ARID5B, CD69, HDAC9, IL7R, TNIP1 and TRAF1). Results reveal specific gene groups acting in concert in endothelial cells, delineate those driven by TNFa, and establish their relationship to DNA methylation changes, which has strong implications for understanding disease etiology and precision medicine approaches.