Identification of susceptibility loci for Takayasu arteritis through a large multi-ancestral genome-wide association study.

Takayasu arteritis is a rare inflammatory disease of large arteries. We performed a genetic study in Takayasu arteritis comprising 6,670 individuals (1,226 affected individuals) from five different populations. We discovered HLA risk factors and four non-HLA susceptibility loci in VPS8, SVEP1, CFL2, and chr13q21 and reinforced IL12B, PTK2B, and chr21q22 as robust susceptibility loci shared across ancestries. Functional analysis proposed plausible underlying disease mechanisms and pinpointed ETS2 as a potential causal gene for chr21q22 association. We also identified >60 candidate loci with suggestive association (p < 5 × 10-5) and devised a genetic risk score for Takayasu arteritis. Takayasu arteritis was compared to hundreds of other traits, revealing the closest genetic relatedness to inflammatory bowel disease. Epigenetic patterns within risk loci suggest roles for monocytes and B cells in Takayasu arteritis. This work enhances understanding of the genetic basis and pathophysiology of Takayasu arteritis and provides clues for potential new therapeutic targets.

Pro-fibrotic effect of the susceptible gene PCSK5 in vascular fibrosis of Takayasu arteritis via TGF-ß and SMAD3 signaling pathway activation.

BACKGROUND: Vascular fibrosis directly causes vascular thickening in Takayasu arteritis (TAK), in which sustained transforming growth factor beta (TGF-ß) activation is critical. Understanding TGF-ß activation regulation and blocking it might yield a therapeutic effect in TAK. Proprotein convertase subtilisin/kexin type 5 (PCSK5) rs6560480 (T/C) is associated with TAK development. In this study, we assessed the association between the PCSK5 rs6560480 genotype and PCSK5 expression in TAK and explored its molecular role in TGF-ß activation and vascular fibrosis development. METHODS: In TAK patients, PCSK5 and TGF-ß expression in plasma and aortic tissue was examined by ELISA and immunohistochemical staining, and PCSK5 rs6560480 was genotyped. The correlation between PCSK5 and extracellular matrix (ECM) expression was examined by Western blotting (WB) and immunohistochemistry staining. Detection by co-immunoprecipitation was performed to detect the interaction between PCSK5 and TGF-ß in adventitial fibroblasts (AAFs). Downstream signaling pathways were detected by WB and validated with appropriate inhibitors. Potential immunosuppressive agents to inhibit the effects of PCSK5 were explored in cell culture and TAK patients. RESULTS: Patients with PCSK5 rs6560480 TT patients had significantly higher PCSK5 levels and more thickened vascular lesions than patients with PCSK5 rs6560480 CT. PCSK5 expression was significantly increased in alpha smooth muscle actin (α-SMA)-positive myofibroblasts in TAK vascular lesions. Overexpressing PCSK5 facilitated TGF-ß and downstream SMAD2/3 activation and ECM expression in AAFs and aorta in in-vitro culture. The mechanistic study supported that PCSK5 activated precursor TGF-ß (pro-TGF-ß) to the mature form by binding the pro-TGF-ß cleavage site. Leflunomide inhibited PCSK5 and pro-TGF-ß binding, decreasing TGF-ß activation and ECM expression, which was also partially validated in leflunomide-treated patients. CONCLUSION: The findings revealed a novel pro-fibrotic mechanism of PCSK5 in TAK vascular fibrosis via TGF-ß and downstream SMAD2/3 pathway activation. Leflunomide might be anti-fibrotic by disrupting PCSK5 and pro-TGF-ß binding, presenting a new TAK treatment approach.

SERPINA3, FGA, AGP1, ITIH3 and SAA1 as novel biomarkers for eosinophilic granulomatosis with polyangiitis diagnosis and activity assessment.

OBJECTIVE: To identify novel biomarkers for diagnosis and prediction of active eosinophilic granulomatosis with polyangiitis (EGPA) through data-independent acquisition (DIA) analysis. METHODS: Plasma from 11 EGPA patients and 10 healthy controls (HCs) were analyzed through DIA to identify potential biomarkers. The results were validated in 32 EGPA patients, 24 disease controls (DCs), and 20 HCs using enzyme-linked immunosorbent assay (ELISA). The receiver operating characteristic (ROC) curve was used to assess the diagnostic value of candidate biomarkers. RESULTS: Thirty-five differentially expressed proteins (DEPs) (24 upregulated and 11 downregulated) were screened between EGPA and HC groups. Five proteins, including serine proteinase inhibitor A3 (SERPINA3), alpha-fibrinogen (FGA), alpha-1 acid glycoprotein 1(AGP1), inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3), and serum amyloid A1 (SAA1), were significantly upregulated in EGPA compared with HCs. Apart from SAA1, all proteins were also higher in EGPA patients compared with DCs. Furthermore, a panel of SERPINA3 and SAA1 exhibited potential diagnostic value for EGPA with an area under the curve (AUC) of 0.953, while a panel of SERPINA3, FGA, AGP1, and ITIH3 showed good discriminative power to differentiate EGPA from DCs with AUC of 0.926. Moreover, SERPINA3, FGA, and AGP levels were significantly higher in active EGPA and correlated well with disease activity. A combination of SERPINA3 and AGP1 exhibited an excellent AUC of 0.918 for disease activity assessment. CONCLUSION: SERPINA3, FGA, AGP1, ITIH3 and SAA1 were identified as potential biomarkers for EGPA diagnosis and disease activity assessment. Among them, as a single biomarker, SERPINA3 has the best diagnostic performance.

Association between Life's Essential 8 and all-cause or cardiovascular-specific mortality in patients with rheumatoid arthritis.

OBJECTIVES: Patients with rheumatoid arthritis (RA) have been found to have a higher cardiovascular disease (CVD) burden. We aimed to examine the associations between Life's Essential 8 (LE8), a metric of cardiovascular health (CVH) recently proposed by the American Heart Association, and all-cause and CVD mortality in RA patients. METHODS: This prospective cohort study analysed RA patients from the National Health and Nutrition Examination Survey 2005-2018 with linked mortality data through December 31, 2019. Total LE8 scores were calculated and divided into the high- (LE8 80-100), moderate- (LE8 50-79), and low-CVH (LE8 0-49) groups. Weighted multivariable Cox regression, logistic regression and restricted cubic spline models were applied to explore the association between LE8 and outcomes. RESULTS: A total of 1424 RA patients were enrolled with a weighted mean age of 57.87 years and female proportion of 58.94%. During a median follow-up of 82 months, 270 all-cause (85 CVD) deaths were recorded. Compared with the high-CVH group, participants in the moderate- and low-CVH groups had an 85.8% and 129.5% increased risk of all-cause mortality, respectively. After adjustment for potential confounders, each 1 point decrease in LE8 score was associated with a 2.6% increased risk of CVD mortality. Subgroup analyses showed significant interactions between LE8 score and non-Hispanic white population with risk of all-cause mortality. The results were robust for all-cause mortality, but not for CVD mortality in the sensitivity analysis. CONCLUSIONS: CVH measured by the LE8 score is a robust and independent predictor of all-cause mortality among U.S. RA patients.

Bibliometric analysis and visualisation of research hotspots and frontiers in Takayasu's arteritis.

OBJECTIVES: Takayasu's arteritis (TAK) is an uncommon granulomatous large-vessel vasculitis associated with significant morbidity and mortality. This study endeavours to comprehend the research status and future frontiers through a bibliometric analysis. METHODS: Relevant original articles published in English were acquired from the Science Citation Index Expanded of the Web of Science Core Collection. Analysis of countries/regions, institutions, authors, co-cited references, and keywords were done using Citespace and VOSviewer software. RESULTS: The final analysis included 2215 documents contributed by 9091 scholars from 2053 institutions in 83 countries, with the United States being the largest contributor globally. Institutional and author collaboration analysis showed that collaborations are scattered and lack stable and intensive collaborative relationships. The journal 'Clinical and Experimental Rheumatology' was the most prolific journal. Anti-endothelial cell antibodies, interleukin-6, adalimumab, colour Doppler ultrasonography, and stents and prosthesis were the main research areas in TAK.Double-blind multicentre clinical trials, disease activity evaluation and cytokines were identified to be recent keyword bursts. CONCLUSIONS: Although the area of TAK research is growing rapidly, intensive institutional and author collaboration has to be fostered in the future to fuel TAK research and information dissemination. Future research on TAK may revolve around cytokines, disease activity evaluation and clinical trials.

Exploration of molecular signatures associated with different clinical features of Takayasu arteritis based on a prospective cohort study.

Takayasu arteritis (TAK) is complicated disorder without reliable biomarkers. Here, we aimed to explore TAK-associated factor panels and their changes after biologic treatment. Five factor panels were identified: 1. systemic inflammation: C3, ESR, CRP, PLT, IL-6, C4, and IgG; 2. vascular inflammation: YKL40, IL-16, PTX3, and CCL2; 3. immune regulation panel: IL-10, IFN-γ, CCL5, and MMP1; 4. angiogenesis and fibrosis: FGF, PDGFAB, and VEGF; and 5. vascular remodeling: CD19+ B cell ratio, MMP3, and leptin. Panel 1 parameters were closely related to disease activity, while Panel 5 parameters, particularly CD19+ B cell ratio and leptin, were significantly higher in ischemic patients. After treatment, tocilizumab had a stronger inhibitory effect on Panel 1 parameters, PTX3, and YKL-40, while adalimumab led to an increase in IL-16, CCL2, and leptin levels. Altogether, these data expanded our knowledge regarding molecular background in TAK development and shed light on precise treatment in future studies.

A microRNA sponge, LINC02193, promotes neutrophil activation by upregulating ICAM1 and is correlated with ANCA-associated vasculitis.

OBJECTIVE: To investigate the pathogenic role and underlying mechanisms of lncRNAs in antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). METHODS：: RNA-sequencing (RNA-seq) was applied to screen the expression profile of lncRNAs in peripheral leukocytes from 5 AAV patients and 5 healthy controls (HC). Candidate lncRNAs were preliminarily verified in peripheral leukocytes from 46 AAV patients and 35 HC by qRT-PCR. Then, the identified LINC02193 was further validated in peripheral neutrophils from 67 AAV patients, 45 HC and 64 disease controls. Correlation between LINC02193 levels and disease activity was analyzed. Then, a loss-of-function study was conducted to investigate the role of LINC02193 in neutrophils activation. Furthermore, bioinformatics analysis, dual luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to explore the mechanism of LINC02193 regulating neutrophils activation. RESULTS: A total of 467 upregulated and 412 downregulated lncRNAs were identified in AAV patients. From top 5 upregulated lncRNAs, an elevation of LINC02193 was validated in a larger sample of AAV patients, and positively correlated with disease activity. Knockdown of LINC02193 inhibited ROS and NO production, NETs release and adhesion to endothelial cells of differentiated human promyelocytic leukaemia HL­60 cells (dHL-60), whereas overexpression of ICAM1 counteracted these effects. Mechanistic analysis demonstrated that LINC02193 acted as a miR-485-5p sponge to relieve the repressive effect of miR-485-5p on ICAM1, thus promoting ICAM1 expression. CONCLUSION: LINC02193, a novel lncRNA identified in AAV could function as competing endogenous RNAs (ceRNA) for miR-485-5p to promote ICAM1 expression and neutrophils activation, suggesting its potential as a therapeutic target of AAV.

Treatment efficacy and safety of adalimumab versus tocilizumab in patients with active and severe Takayasu arteritis: an open-label study.

OBJECTIVE: This study aimed to compare the efficacy and safety of adalimumab (ADA) versus tocilizumab (TCZ) in patients with Takayasu arteritis (TAK). METHODS: This was a randomized, controlled, open-label study. Forty patients with active and severe TAK were enrolled. They were treated with ADA (n = 21) combined with glucocorticoids (GCs) and methotrexate (MTX) or TCZ (n = 19) combined with GCs and MTX. The planned follow-up duration was 12 months. The primary end point was the efficacy rate (ER) at 6 months. The secondary endpoints included ER at 9 and 12 months, relapse rate, GC tapering, adverse effects, and life quality changes during treatment. RESULTS: In the intention-to-treat (ITT) population, the ER at 6 months was higher in the ADA group (85.71% vs 52.63%, P= 0.02). A similar direction of effect was noted in the per-protocol set (89.47% vs 62.50%, P= 0.06). The percentages of patients who achieved a GC dose of ≤ 10 mg/day at 6 months were similar between the ADA and TCZ groups (47.37% vs 43.75%, P= 0.83). The ERs at 9 and 12 months were similar between the two groups (P> 0.05). During the first 12 months of treatment, the relapse rate and adverse event incidence were comparable between the two groups (ADA vs TCZ: 9.52% vs 10.53%, P= 0.96; 38.10% vs 47.37%, P= 0.55, respectively). CONCLUSION: ADA combined with GCs and MTX may be more efficacious than TCZ combined with GCs and MTX among patients with active and severe TAK. TRIAL REGISTRATION: Clinicaltrials.gov; NCT04300686.

Single-Cell RNA-Sequencing Reveals Peripheral T Helper Cells Promoting the Development of IgG4-Related Disease by Enhancing B Cell Activation and Differentiation.

Abnormal B cell differentiation plays a critical role in IgG4-related disease (IgG4-RD), but the underlying mechanism remains largely unknown. We investigated the cell landscape from three IgG4-RD retroperitoneal tissues and three control tissues using single-cell RNA-sequencing. Critical cell type or markers were further validated in the peripheral blood from the patients with IgG4-RD and healthy controls via flow cytometry as well as in the IgG4-RD and control tissue via immunofluorescence staining. The increases in B cells, plasma cells, and CD4+ T cells were found in IgG4-RD retroperitoneal tissue. Importantly, among CD4+ T cells, an increase in CD4+CXCR5-PD1hi peripheral T helper (Tph) cells with a high expression of IL-21 and TIGIT was discovered in IgG4-RD tissue, which was further validated in peripheral blood of the patients with IgG4-RD. The Tph cell and TIGIT+ Tph cell proportion were remarkably higher in active IgG4-RD patients and correlated with disease activity. Moreover, TIGIT+CD4+ cells were able to promote B cell differentiation via IL-21. Our study revealed that Tph cells are increased in IgG4-RD and probably play critical roles in B cell differentiation through TIGIT-IL-21 axis. Peripheral Tph cell and TIGIT+Tph cell are potential markers for IgG4-RD disease activity.

Treatment efficacy and safety of tofacitinib versus methotrexate in Takayasu arteritis: a prospective observational study.

OBJECTIVE: To compare the treatment efficacy and safety of tofacitinib (TOF) versus methotrexate (MTX) in Takayasu arteritis (TAK). METHODS: Fifty-three patients with active disease from an ongoing prospective TAK cohort in China were included in this study. Twenty-seven patients were treated with glucocorticoids (GCs) and TOF, and 26 patients were treated with GCs with MTX. The observation period was 12 months. Complete remission (CR), inflammatory parameter changes, GCs tapering and safety were assessed at the 6th, 9th and 12th month. Vascular lesions were evaluated at the 6th and 12th month, and relapse was analysed during 12 months. RESULTS: The CR rate was higher in the TOF group than in the MTX group (6 months: 85.19% vs 61.54%, p=0.07; 12 months: 88.46% vs 56.52%, p=0.02). During 12 months' treatment, patients in the TOF group achieved a relatively lower relapse rate (11.54% vs 34.78%, p=0.052) and a longer median relapse-free duration (11.65±0.98 vs 10.48±2.31 months, p=0.03). Average GCs dose at the 3rd, 6th and 12th month was lower in the TOF group than that in the MTX group (p<0.05). A difference was not observed in disease improvement or disease progression on imaging between the two groups (p>0.05). Prevalence of side effects was low in both groups (3.70% vs 15.38%, p=0.19). CONCLUSION: TOF was superior to MTX for CR induction, a tendency to prevent relapse and tapering of the GCs dose in TAK treatment. A good safety profile for TOF was also documented in patients with TAK.

FABP3 overexpression promotes vascular fibrosis in Takayasu's arteritis by enhancing fatty acid oxidation in aorta adventitial fibroblasts.

OBJECTIVE: To identify the role of fatty acid binding protein 3 (FABP3) in vascular fibrosis in Takayasu's arteritis (TAK) and to explore the underlying molecular mechanism. METHODS: The expression of FABP3 and extracellular matrix proteins (ECMs) were detected in aorta tissues from TAK patients (n = 12) and healthy controls (n = 8) by immunohistochemistry. The concentration of serum proteins was determined by ELISA. CCK8 and Ki67 staining were used to measure aorta adventitial fibroblast (AAF) proliferation. Widely targeted lipidomic profiling was used to screen for associated metabolic pathways. Changes in ECMs and fatty acid oxidation (FAO)-related enzymes were determined by RT-qPCR and Western blot. The interactions between FABP3 and these enzymes were explored with a co-immunoprecipitation (Co-IP) assay. RESULTS: The expression of FABP3 was increased in the thickened adventitia of TAK patients and was positively correlated with the serum expression of ECMs. FABP3 knockdown inhibited AAF proliferation and ECM production, whereas FABP3 overexpression enhanced these processes. Further analysis revealed that FABP3 upregulation promoted carnitine palmitoyltransferase 1A and carnitine/acylcarnitine carrier protein (CACT) expression, two key enzymes in FAO, as well as adenosine triphosphate (ATP) levels. FABP3 and CACT were co-localized in the adventitia and bound to each other in AAFs. Etomoxir reversed the enhanced FAO, ATP production, AAF proliferation and ECM production mediated by FABP3 upregulation. Treatment with 60 g/day curcumin granules for 3 months reduced the level of serum FABP3. Curcumin also inhibited vascular fibrosis by reducing FABP3-enhanced FAO in AAFs. CONCLUSION: Elevated FABP3 expression accelerated vascular fibrosis in TAK, which was likely mediated by promoting FAO in AAFs.

A novel model to assess disease activity in Takayasu arteritis based on 18F-FDG-PET/CT: a Chinese cohort study.

OBJECTIVE: To investigate the utility of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) in assessing disease activity in Takayasu arteritis (TA). METHODS: Ninety-one patients with TA were recruited from a Chinese cohort. Clinical data, acute-phase reactants and 18F-FDG-PET/CT findings were simultaneously recorded. The value of using 18F-FDG-PET/CT to identify active disease was evaluated, using ESR as a reference. Disease activity assessment models were constructed and concordance index (C-index), net reclassification index (NRI), and integrated discrimination index (IDI) were evaluated to compare the benefits of the new modes with ESR and the Kerr score. RESULTS: In total, 64 (70.3%) cases showed active disease. Higher levels of ESR and CRP, and lower IL-2 receptor (IL-2R) levels were observed in active cases. 18F-FDG-PET/CT parameters measured by determining the standard uptake value (SUV), including SUVmean, SUVratio1, SUVratio2, sum of SUVmean and sum of SUVmax, were significantly higher in active disease groups. The C-index threshold of ESR to indicate active disease was 0.78 (95% CI: 0.69, 0.88). The new activity assessment model combining ESR, sum of SUVmean and IL-2R showed significant improvement in C-index over the ESR method (0.96 vs 0.78, P < 0.01; NRI 1.63, P < 0.01; and IDI 0.48, P < 0.01). The new model also demonstrated modest superiority to the Kerr score assessment (0.96 vs 0.87, P = 0.03; NRI 1.19, P < 0.01; and IDI 0.33, P < 0.01). CONCLUSIONS: A novel 18F-FDG-PET/CT-based method that involves combining the sum of SUVmean with ESR score and IL-2R levels demonstrated superiority in identifying active TA compared with conventional methods.

Pulmonary findings on high-resolution computed tomography in Takayasu arteritis.

OBJECTIVE: This study aimed to describe pulmonary high-resolution CT (HRCT) findings in Takayasu arteritis (TA) and to determine possible causes. METHODS: A total of 243 TA patients were enrolled from a prospective cohort after excluding patients with other pulmonary disorders or incomplete data. Patients were divided into two groups: those with normal lung HRCT and those with abnormal lung HRCT. Clinical characteristics were compared between groups and binary logistic regression analysis was applied to identify possible causes of the lung lesions. Follow-up HRCT (obtained in 64 patients) was analysed to study changes in pulmonary lesions after treatment. RESULTS: Of the 243 patients, 107 (44.0%) had normal lung HRCT while 136 (56.0%) had abnormal lung HRCT, including stripe opacity (60.3%), nodules (44.9%), patchy opacity (25.0%), pleural thickening (15.4%), pleural effusion (10.3%), ground-glass opacity (8.1%), pulmonary infarction (6.6%), mosaic attenuation (4.4%), bronchiectasis (3.7%) and pulmonary oedema (2.2%). Patients with abnormal HRCT were significantly more likely to have type II arterial involvement (25% vs 12.2%, P = 0.04), pulmonary arterial involvement (PAI; 21.3% vs 5.6%, P < 0.001), pulmonary hypertension (20.6% vs 8.4%, P = 0.01) and abnormal heart function (27.9% vs 7.6%, P < 0.001). Logistic regression analysis demonstrated that PAI, worsened heart function and age were associated with presence of pulmonary lesions. Pulmonary infarction, pleural effusion and patchy opacities improved partially after treatment. CONCLUSION: Pulmonary lesions are not rare in patients with TA. Age, PAI and worsened heart function are potential risk factors for presence of pulmonary lesions in TA.

Curcumin alleviates inflammation in Takayasu's arteritis by blocking CCL2 overexpression in adventitial fibroblasts.

OBJECTIVES: Takayasu's arteritis (TAK) is a chronic inflammatory disease with several challenges in treatment. Curcumin is known for its anti-inflammatory effects, whereas its effect in the treatment of TAK remains unclear. In this study, we aimed to investigate the effect of curcumin in the treatment of TAK and its underlying mechanisms. METHODS: 16 TAK patients were treated with curcumin granules at a dose of 15 g/day for three months. Kerr score was explored to assess disease activity. Serum levels of inflammatory factors were measured by ELISA. Immunohistochemical and immunofluorescence staining were used to detect the expression of CCL2 (also known as MCP-1) in aortic adventitia. RT-qPCR, ELISA and western blot were used to determine the regulatory effect of curcumin on CCL2 expression in aortic adventitia fibroblasts (AAFs) and its mechanism. RESULTS: Curcumin treatment significantly lowered Kerr score and the levels of serum CCL2 in TAK patients. The expression of CCL2 in TAK aortic adventitia was increased and colocalised with CD68. Serum levels of CCL2 was increased in subjects with Kerr score ≥2. After curcumin treatment, the changes in CCL2 were positively associated with the changes in IL-6. In further analysis, it showed that CCL2 was co-localised with CD90 and α-SMA, markers of adventitia fibroblasts. In vitro, HSP65, an agonist of TLR4, could induce CCL2 expression in AAFs via phosphorylating and activating the JAK2/AKT/STAT3 pathway. Nevertheless, curcumin could reverse the HSP65-induced CCL2 upregulation through restraining JAK2/AKT/STAT3 pathway. The inhibitory effect of curcumin on the JAK2/AKT/STAT3 pathway was even more obvious than that of methotrexate and tofacitinib. CONCLUSIONS: Curcumin alleviated inflammation in TAK by downregulating CCL2 overexpression in AAFs through inhibiting the JAK2/AKT/STAT3 signalling pathway.

The potential role of leflunomide in inhibiting vascular fibrosis by down-regulating type-II macrophages in Takayasu's arteritis.

OBJECTIVES: Inflammatory fibrosis of aortic lesions promoted by type II macrophages (M2) is one of the most serious incidents in Takayasu's arteritis (TAK), and the currently available therapies can not effectively block the inflammatory fibrosis. Here we explored whether leflunomide (LEF) could improve the fibrosis by down-regulating M2 in TAK. METHODS: Peripheral blood mono-nuclear cells (PBMCs) from 16 TAK patients were treated by leflunomide, and the ratio of M1/M2 macrophages and apoptosis of M2 were detected by flow cytometry. Supernatant levels of cytokines and chemokines secreted by M2 were measured by ELISAs. mRNA expression of profibrotic factors in M2 were analysed by real time PCR. Western blotting was used to analyse the activation of signal transducer activator of transcription (STAT)-6. RESULTS: LEF could inhibit M2 polarisation by curtailing STAT6 phosphorylation. LEF could also promote apoptosis of M2 and reduce the release of M2-derived CCL22 as well as the expression of profibrotic cytokines including CCL22 and TGF-ß in M2. CONCLUSIONS: LEF could potentially reduce vascular fibrosis by down-regulating the number and function of M2, which, eventually, could alleviate inflammatory fibrosis of aortic lesions in TAK patients.

CYR61/TGF-ß axis promotes adventitial fibrosis of Takayasu's arteritis in the IL-17 mediated inflammatory microenvironment.

OBJECTIVES: Takayasu's arteritis (TAK) is characterised by inflammation and fibrosis in the aortas, but its pathogenesis remains unclear. The aim of the study is to demonstrate the role of cysteine-rich protein 61 (CYR61), a novel proinflammatory factor, in the inflammation and fibrosis of TAK vessels. METHODS: CYR61 expression in the aortic vessel was compared between TA tissues and healthy samples by immunohistochemistry staining. The effect of CYR61 on the proliferation, migration and activation of adventitial fibroblasts (AFs) in the IL-17-mediated inflammatory microenvironment was studied in vitro. RESULTS: Here we found higher expression of CYR61 in the aortic adventitia in TAK patients than in healthy donors by immunohistochemistry staining. In vitro, recombinant human CYR61 (rhCYR61) significantly upregulated the proliferation of primary human aortic adventitial fibroblasts (AFs) and their expression of extracellular matrix (ECM) proteins such as collagen I, collagen III and fibronectin at the mRNA and protein levels, but rhCYR61 partly inhibited the migration of AFs. The integrin αvß1 was identified as a membrane receptor of CYR61 in AFs, and its downstream Erk1/2 pathway was found activated by detecting its phosphorylation level. Pretreatment with PD98059, an inhibitor of Erk1/2, down-regulated the mRNA and protein expression of ECM proteins in the rhCYR61-stimulated AFs. Furthermore, rhCYR61 up-regulated the expression of TGF-ß, and TGF-ß siRNA transfection obviously attenuated the profibrotic effect ofrhCYR61. Finally, to clarify the cooperation between CYR61 and classical proinflammatory factors, IL-17 was chosen as a co-stimulator in the culture of AFs. rhIL-17 promoted the mRNA and protein expression of CYR61 in AFs, and the collaboration of rhIL-17 and rhCYR61 dramatically boosted the synthesis of ECM and TGF-ß. CONCLUSIONS: Our findings suggest that CYR61 played a profibrotic role through the TGF-ß pathway and it enhanced IL-17-mediated inflammation and fibrosis in the mechanism of vascular impairment in TAK.

Takayasu arteritis risk locus in IL6 represses the anti-inflammatory gene GPNMB through chromatin looping and recruiting MEF2-HDAC complex.

OBJECTIVE: Previous work has revealed a genetic association between Takayasu arteritis and a non-coding genetic variant in an enhancer region within IL6 (rs2069837 A/G). The risk allele in this variant (allele A) has a protective effect against chronic viral infection and cancer. The goal of this study was to characterise the functional consequences of this disease-associated risk locus. METHODS: A combination of experimental and bioinformatics tools were used to mechanistically understand the effects of the disease-associated genetic locus in IL6. These included electrophoretic mobility shift assay, DNA affinity precipitation assays followed by mass spectrometry and western blotting, luciferase reporter assays and chromosome conformation capture (3C) to identify chromatin looping in the IL6 locus. Both cell lines and peripheral blood primary monocyte-derived macrophages were used. RESULTS: We identified the monocyte/macrophage anti-inflammatory gene GPNMB,~520 kb from IL6, as a target gene regulated by rs2069837. We revealed preferential recruitment of myocyte enhancer factor 2-histone deacetylase (MEF2-HDAC) repressive complex to the Takayasu arteritis risk allele. Further, we demonstrated suppression of GPNMB expression in monocyte-derived macrophages from healthy individuals with AA compared with AG genotype, which was reversed by histone deacetylase inhibition. Our data show that the risk allele in rs2069837 represses the expression of GPNMB by recruiting MEF2-HDAC complex, enabled through a long-range intrachromatin looping. Suppression of this anti-inflammatory gene might mediate increased susceptibility in Takayasu arteritis and enhance protective immune responses in chronic infection and cancer. CONCLUSIONS: Takayasu arteritis risk locus in IL6 might increase disease susceptibility by suppression of the anti-inflammatory gene GPNMB through chromatin looping and recruitment of MEF2-HDAC epigenetic repressive complex. Our data highlight long-range chromatin interactions in functional genomic and epigenomic studies in autoimmunity.

The E3 ubiquitin ligase RNF185 facilitates the cGAS-mediated innate immune response.

The cyclic GMP-AMP synthase (cGAS), upon cytosolic DNA stimulation, catalyzes the formation of the second messenger 2'3'-cGAMP, which then binds to stimulator of interferon genes (STING) and activates downstream signaling. It remains to be elucidated how the cGAS enzymatic activity is modulated dynamically. Here, we reported that the ER ubiquitin ligase RNF185 interacted with cGAS during HSV-1 infection. Ectopic-expression or knockdown of RNF185 respectively enhanced or impaired the IRF3-responsive gene expression. Mechanistically, RNF185 specifically catalyzed the K27-linked poly-ubiquitination of cGAS, which promoted its enzymatic activity. Additionally, Systemic Lupus Erythematosus (SLE) patients displayed elevated expression of RNF185 mRNA. Collectively, this study uncovers RNF185 as the first E3 ubiquitin ligase of cGAS, shedding light on the regulation of cGAS activity in innate immune responses.

Autophagy promotes aortic adventitial fibrosis via the IL-6/Jak1 signaling pathway in Takayasu's arteritis.

BACKGROUND: Autophagy is a ubiquitous and evolutionarily conserved self-rescue process. Studies have shown that autophagy is involved in the pathogenesis of multiple diseases; however, whether autophagy is associated with the pathogenesis of Takayasu's arteritis (TA), a large vessel idiopathic inflammatory disease characterized by vascular fibrosis, remains unclear. Moreover, although IL-6 is believed to be a direct target for TA treatment, anti-IL-6 treatment could not block TA-associated fibrosis in some cases, which impairs the aortic function of patients and can result in death. Thus, identify the mechanisms associated with TA is extremely important. Based on the relationship between autophagy and IL-6, we investigated the role of autophagy in the vascular fibrosis of TA induced by IL-6. METHODS: Autophagy proteins (LC3 and Atg3), IL-6, and markers of fibrosis (collagen 1 and α-SMA) were detected in tissues with TA lesions via immunochemistry, immunofluorescence, and Western blot, respectively. Different stages of autophagy were analyzed by the specific inhibitors, 3-methyladenosine (early stage), hydroxychloroquine sulfate (late stage), and bafilomycin A1 (late stage). Autophagosomes were detected using electron microscopy and a viral-vector transfection assay. The fibrosis profiles induced by IL-6-dependent autophagy was assessed with an ELISA. RESULTS: The expression of autophagy, IL-6, and fibrosis markers were elevated and correlated with each other in the adventitia tissues of TA patients. Furthermore, exogenous IL-6/IL-6Rα could significantly increase autophagy and fibrosis in vitro. An autophagy inhibitor was found to significantly block both autophagy and fibrosis induced by IL-6. Finally, IL-6 was found to significantly promote autophagy-induced fibrosis through the activation of the Jak1 pathway. CONCLUSIONS: IL-6-induced autophagy plays an important role in vascular fibrosis of TA. Targeting autophagy pathways might represent a novel therapeutic option for the treatment of TA.