Arthritis Research & Therapy

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Article
MRI-detected inflammation around the extensor tendons of metacarpophalangeal (MCP-) joints is prevalent in RA and poses a markedly increased risk of RA development when present in arthralgia patients. Such inflammation is called ‘peritendinitis’ since anatomy literature reports no presence of a tenosynovial sheath at these tendons. However, the presence or absence of tenosynovium at these extensor tendons has never been studied. Therefore, an anatomical and histological study of extensor tendons at the MCP-joints of three embalmed human hands was performed. Immunohistochemical staining showed the presence of markers for synovial macrophages and fibroblast-like synoviocytes bordering a natural dorsal space next to the extensor tendon, suggesting the presence of a synovial lining. This implies that contrast-enhancement on MRI around extensor tendons at MCP-joints observed in early RA and pre-RA likely represents tenosynovitis and that inflammation of this synovial tissue is an early feature of RA.
 
Study design. i AAV diagnosis (cohorts 1 and 2). ii AAV relapse (cohort 2). i CW-D-UVB at diagnosis was calculated using the participant's location and date of symptom onset if known, or the date of diagnosis minus 77 days. Seventy-seven days represents the undefined prodromal period [36] informed by the RKD registry analysis (see supplementary material). ii In this prospective n-of-1 component, each participant was a 'case' during period(s) of disease relapse and a 'control' during period(s) of remission [37]. The case window started at the date of relapse diagnosis minus 30 days (to account for the diagnostic delay) and ended 135 days later (see Additional file 1: Supplementary Methods). To improve the statistical power, 5 control dates were identified per patient, where possible
AAV relapse. a Latitude (degrees), b average winter vitD-UVB (kJ/m 2 ), c average winter CW-D-UVB (kJ/m 2 ) and d average annual vitD-UVB (kJ/ m 2 ) stratified by disease activity (active vs. remission) in the entire cohort 2
Article
Background The aetiology of ANCA-associated vasculitis (AAV) and triggers of relapse are poorly understood. Vitamin D (vitD) is an important immunomodulator, potentially responsible for the observed latitudinal differences between granulomatous and non-granulomatous AAV phenotypes. A narrow ultraviolet B spectrum induces vitD synthesis (vitD-UVB) via the skin. We hypothesised that prolonged periods of low ambient UVB (and by extension vitD deficiency) are associated with the granulomatous form of the disease and an increased risk of AAV relapse. Methods Patients with AAV recruited to the Irish Rare Kidney Disease (RKD) ( n = 439) and UKIVAS ( n = 1961) registries were studied. Exposure variables comprised latitude and measures of ambient vitD-UVB, including cumulative weighted UVB dose (CW-D-UVB), a well-validated vitD proxy. An n -of-1 study design was used to examine the relapse risk using only the RKD dataset. Multi-level models and logistic regression were used to examine the effect of predictors on AAV relapse risk, phenotype and serotype. Results Residential latitude was positively correlated (OR 1.41, 95% CI 1.14–1.74, p = 0.002) and average vitD-UVB negatively correlated (0.82, 0.70–0.99, p = 0.04) with relapse risk, with a stronger effect when restricting to winter measurements (0.71, 0.57–0.89, p = 0.002). However, these associations were not restricted to granulomatous phenotypes. We observed no clear relationship between latitude, vitD-UVB or CW-D-UVB and AAV phenotype or serotype. Conclusion Our findings suggest that low winter ambient UVB and prolonged vitD status contribute to AAV relapse risk across all phenotypes. However, the development of a granulomatous phenotype does not appear to be directly vitD-mediated. Further research is needed to determine whether sufficient vitD status would reduce relapse propensity in AAV.
 
CONSORT diagram showing flow of patients through the ZEBRA 1 sub-study. The diagram provides a summary of the flow of patients through the ZEBRA 1 sub-study including screened cases and 13 patients randomised
Representative primary and secondary endpoint data for ZEBRA 1 sub-study. A Endpoint data for serum VCAM-1 and eGFR in ZEBRA 1. Upper panels show the serum VCAM-1 levels at baseline, 26 weeks, and 52 weeks for the patients in placebo and zibotentan groups. Mean and SD are indicated. There was no apparent difference between treatment groups. The lower panel shows that eGFR (ml/min/1.73m2) decreased in the placebo arm and increased in the zibotentan arm at 52 weeks. B Candidate CKD-SSc urinary biomarker data for ZEBRA 1. Secondary endpoint data for candidate urinary biomarkers of SSc-CKD identified in a previous cohort study [8]. For urinary ICAM-1 to creatinine ratio, there is no difference between treatment groups or timepoints. For MCP-1 to creatinine ratio, the placebo group shows increasing level at 52 weeks compared to a numerical reduction for the zibotentan group although distribution of data is wide as shown by SD for each time point
Endpoint data for ZEBRA 1 trial at baseline, 26 weeks, and 52 weeks
Article
Background We report results from a phase II randomised placebo-controlled trial assessing zibotentan, a highly selective endothelin receptor antagonist (ERA), in chronic kidney disease (CKD) secondary to systemic sclerosis (SSc). Methods This trial included three sub-studies: ZEBRA 1—a randomised placebo-controlled, double-blind trial of zibotentan in SSc patients with CKD2 or CKD3 (and glomerular filtration rate (GFR) >45 ml/min) over 26 weeks; ZEBRA 2A—a 26-week placebo-controlled, single-blind trial of zibotentan in scleroderma renal crisis patients not requiring dialysis; and ZEBRA 2B—an open label pharmacokinetic study of zibotentan in patients on haemodialysis. Results Sixteen patients were screened for ZEBRA 1. Of these, 6 patients were randomised to zibotentan and 7 to placebo. In ZEBRA 1, there were 47 non-serious adverse events (AE) during the trial. Twenty-seven occurred in the placebo group and 20 in the zibotentan group. One serious adverse event (SAE) occurred during ZEBRA1, in the placebo arm. Descriptive statistics did not suggest an effect of study drug on serum sVCAM1. Estimated GFR numerically declined in patients treated with placebo at 26 weeks and 52 weeks. In contrast, average eGFR increased in zibotentan-treated cases. The 4 patients in ZEBRA 2A experienced 8 non-serious AEs, distributed equally between placebo and zibotentan. There was one SAE each in placebo and zibotentan groups, both unrelated to study medication. ZEBRA 2B recruited 8 patients, 6 completed first dosing, and 2 completed a second dosing visit. Pharmacokinetic analysis confirmed zibotentan levels within the therapeutic range. Three patients experienced 3 non-serious AEs. One SAE occurred and was unrelated to study drug. Conclusions Zibotentan was generally well-tolerated. ZEBRA 1 did not show any effect of zibotentan on serum sVCAM-1 but was associated with numerical improvement in eGFR at 26 weeks that was more marked at 52 weeks. ZEBRA 2B suggested a feasible dose regimen for haemodialysis patients. Trial registration EudraCT no: 2013-003200-39 (first posted January 28, 2014) ClinicalTrials.gov Identifier: NCT02047708 Sponsor protocol number: 13/0077
 
Article
With great interest, we have read the recent article “The risk of malignancy in patients with IgG4-related disease: a systematic review and meta-analysis” by Yu et al. While we have a great appreciation for the work conducted by the authors there are some methodological issues need to be considered. First, the period of articles included in the study, almost before 2013, implied that most follow-up days in these articles were earlier than the established date of a unified definition of IgG4-RD, 2011. Thus, it may lead to misclassification bias in the study. Second, IgG4-RD is a fibrous-inflammatory process that often involves multiple organs; however, malignant tumors related to IgG4-RD proposed in the study were only confined to four diseases. Therefore, we suggest adding subgroup analysis for more malignancies depending on the prevalence of IgG4-RD involved organs to ensure better clinical practice. Third, the causation between IgG4-RD and malignancy remains obscure currently. The time course for development in different malignancies varies significantly so that we cannot infer that malignancies discovered after IgG4-RD are directly relevant. With problems mentioned above, we recommend solutions to make this article more convincing.
 
Article
Current understanding of IL-23 biology, with its link to other pro-inflammatory cytokines, for example, IL-17 and granulocyte macrophage-colony stimulating factor (GM-CSF), is primarily focused on T lymphocyte-mediated inflammation/autoimmunity. Pain is a significant symptom associated with many musculoskeletal conditions leading to functional impairment and poor quality of life. While the role of IL-23 in arthritis has been studied in mouse models of adaptive immune-mediated arthritis using targeted approaches (e.g., monoclonal antibody (mAb) neutralization), the literature on IL-23 and arthritis pain is limited. Encouragingly, the anti-IL-23p19 mAb, guselkumab, reduces pain in psoriatic arthritis patients. Recent evidence has suggested a new biology for IL-23, whereby IL-23 is required in models of innate immune-mediated arthritis and its associated pain with its action being linked to a GM-CSF-dependent pathway (the so-called GM-CSF➔CCL17 pathway). This Commentary discusses the current understanding of potential cytokine networks involving IL-23 in arthritis pain and provides a rationale for future clinical studies targeting IL-23p19 in arthritis pain.
 
Downregulation of CD64/FcγRI on monocytes after exposure to abatacept, as shown by flow cytometry. Peripheral blood monocytes were cultured for 24 h in the absence (mock) or presence of abatacept (ABT) or CD28-Ig and the expression of CD64/FcγRI (A) and CD80, CD86, and CXCR2 (B) was examined by flow cytometry. Peripheral blood monocytes were obtained from 20 patients with RA and 8 controls and used for a validation experiment. The cell surface expression is expressed as the mean fluorescence intensity (MFI) ratio. A representative histogram showing the expression of CD64/FcγRI in a patient with RA and a control is also shown. The results are shown in boxplots, and statistical comparisons between the two groups were made using the Mann–Whitney U test
Downregulation of CD64/FcγRI on monocytes after exposure to abatacept, as shown by immunoblotting. Peripheral blood samples were obtained from 11 patients with RA and 5 controls and used for immunoblotting. The protein expression of CD64/FcγRI, CD80, CD86, CD32a/FcγRIIa, CD32b/FcγRIIb, and CD16/FcγRIII in whole-cell extracts of monocytes cultured for 24 h in the absence (mock) or presence of abatacept or CD28-Ig. A representative immunoblot image showing a patient with RA in the right panel with the positions of molecular weight markers (A). The signal intensity of individual protein bands was quantified using the ImageJ software and is shown as the intensity ratio, which was calculated by dividing the intensity of the band of interest by the intensity of the β-actin band (B). The results are shown in boxplots, and the statistical comparisons between the two groups were made using the Mann–Whitney U test
Downregulation of CD64/FcγRI via abatacept binding to CD86 on monocytes. The effects of anti-CD80, anti-CD86, both mAbs, or isotype-matched mAbs on abatacept-induced downregulation of CD64/FcγRI were examined in a short-term monocyte culture. Peripheral blood samples were obtained from 5 patients with RA and were used. The cell surface expression of CD64/FcγR is expressed as the mean fluorescence intensity (MFI) ratio. The results are shown in boxplots, and statistical comparisons between the two groups were made using the Mann–Whitney U test. A representative result of 3 independent experiments is shown
Effects of abatacept on cytokine/chemokine production in monocytes stimulated with the ACPA immune complex. Peripheral blood samples were obtained from 17 patients with rheumatoid arthritis and used. The concentrations of IL-1β, IL-6, TNF-α, IL-8, and CCL2 were measured by multiplex bead arrays. Statistical comparisons between the two groups were made using the Mann–Whitney U test
Article
  • Ryosuke FukueRyosuke Fukue
  • Yuka OkazakiYuka Okazaki
  • Takahisa GonoTakahisa Gono
  • Masataka KuwanaMasataka Kuwana
Background Abatacept is a recombinant fusion protein composed of the extracellular domain of cytotoxic T-lymphocyte antigen 4 and the Fc portion of immunoglobulin (Ig) G. The mechanism of action of abatacept in rheumatoid arthritis (RA) is believed to be competitive inhibition of T cell costimulation mediated by the binding of CD28 to CD80/CD86 on antigen-presenting cells, and recent studies have shown that abatacept induces reverse signaling in macrophages and osteoclast precursors in a T cell-independent manner. This study aimed to investigate the therapeutic effects of abatacept on circulating monocytes that contribute to RA pathogenesis. Methods Purified circulating monocytes derived from RA patients and controls were cultured in the absence or presence of abatacept or CD28-Ig for 24 h. The recovered cells were subjected to flow cytometry to evaluate the expression levels of cell surface molecules, and cytokines and chemokines in the culture supernatant were measured by multiplex bead arrays. The expression of candidate molecules was further examined by immunoblotting using total cellular extracts of the cultured monocytes. Finally, the effects of abatacept on cytokine production in monocytes stimulated with the immune complex of anti-citrullinated peptide antibodies (ACPAs) were examined. Results CD64/FcγRI was identified as a monocyte-derived molecule that was downregulated by abatacept but not CD28-Ig. This effect was observed in both RA patients and controls. The abatacept-induced downregulation of CD64/FcγRI was abolished by treatment with anti-CD86 antibodies but not anti-CD80 antibodies. Abatacept suppressed the production of interleukin (IL)-1β, IL-6, C-C motif chemokine ligand 2, and tumor necrosis factor-α in cultured monocytes stimulated with the ACPA immune complex. Conclusions The therapeutic effects of abatacept on RA are mediated, in part, by the downregulation of CD64/FcγRI on circulating monocytes via direct binding to CD86 and the suppression of immune complex-mediated inflammatory cytokine production.
 
Patient’s flowchart
DFR behavior of the 23 cases
ROC for patient’s follow-up PT cutoff to predict DFR
Article
Background Medication adherence is suboptimal in rheumatoid arthritis (RA) patients and impacts outcomes. DMARD-free remission (DFR) is a sustainable and achievable outcome in a minority of RA patients. Different factors have been associated with DFR, although persistence in therapy (PT), a component of the adherence construct, has never been examined. The study’s primary aim was to investigate the impact of PT’s characteristics on DFR in a cohort of Hispanic patients with recent-onset RA. Methods A single data abstractor reviewed the charts from 209 early (symptoms duration ≤ 1 year) RA patients. All the patients had prospective assessments of disease activity and PT and at least 1 year of follow-up, which was required for the DFR definition. DFR was defined when patients achieved ≥ 1 year of continuous Disease Activity Score-28 joints evaluated ≤ 2.6, without DMARDs and corticosteroids. PT was defined based on pre-specified criteria and recorded through an interview from 2004 to 2008 and thereafter through a questionnaire. Cases (patients who achieved ≥ 1 DFR status) were paired with controls (patients who never achieved DFR during their entire follow-up) according to ten relevant variables (1:2). Cox regression analysis estimated hazard ratios (HRs) for DFR according to two characteristics of PT: the % of the patient follow-up PT and early PT (first 2 years of patients’ follow-up). Results In March 2022, the population had 112 (55–181) patient/years follow-up. There were 23 patients (11%) with DFR after 74 months (44–122) of follow-up, and the DFR status was maintained during 48 months (18–82). Early PT was associated with DFR, while the % of the patient follow-up PT was not: HR = 3.84 [1.13–13.07] when the model was adjusted for cumulative N of DMARDs/patient and 3.16 [1.14–8.77] when also adjusted for baseline SF-36 physical component score. A lower N of cumulative DMARDs/patient was also retained in the models. Receiving operating curve to define the best cutoff of patient follow-up being PT to predict DFR was 21 months: sensitivity of 0.739, specificity of 0.717, and area under the curve of 0.682 (0.544–0.821). Conclusions DFR status might be added to the benefits of adhering to prescribed treatment.
 
a Time to VTE episode in CMV-seropositive versus CMV-seronegative UHBFT AAV patients. b Time to VTE episode in CMV-seropositive versus CMV-seronegative UNC AAV patients. Time to VTE event was examined by Kaplan-Meier curve analysis (log rank test). CMV seropositive patients are shown in the solid line and CMV seronegative patients in the dashed line. Numbers of patients at risk for each time point displayed below the curve
Article
Background Venous thromboembolism (VTE) is a common complication in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) and confers significant morbidity and mortality. Both acute and past cytomegalovirus (CMV) infection have been identified as risk factors for VTE in immunocompetent and immunosuppressed individuals. Here, we examine whether past exposure to CMV is a risk factor for VTE amongst patients with AAV. Methods We retrospectively analysed outcomes of patients with a new diagnosis of AAV from a UK cohort. All confirmed cases of VTE where CMV IgG serology was available were recorded. Retrospective collection of the same data for patients at a North American centre was used as a validation cohort. Results VTE was common with 12% of patients from the study cohort (total 259 patients) developing an event during the median follow-up period of 8.5 years of which 60% occurred within the first 12 months following diagnosis. Sixteen percent of CMV seropositive patients developed a VTE compared with 5% of patients who were seronegative ( p = 0.007) and CMV seropositivity remained an independent predictor of VTE in multivariable analysis (HR 2.96 [1.094–8.011] p = 0.033). CMV seropositivity at diagnosis was confirmed as a significant risk factor for VTE in the American validation cohort ( p = 0.032). Conclusions VTE is common in patients with AAV, especially within the first year of diagnosis. Past infection with CMV is an independent risk factor associated with VTE in AAV.
 
Flow diagram of study subject selection
Screening and follow-up time points in each cohort
Article
Background Tumor necrosis factor (TNF) inhibitors use in patients with rheumatoid arthritis (RA) has raised safety concerns about cancer risk, but study results remain controversial. This largest nationwide study to date compared cancer risk in TNF inhibitor users to non-biologic disease-modifying anti-rheumatic drug (nbDMARD) users in Korean patients with RA. Methods Data on all the eligible patients diagnosed with RA between 2005 and 2016 were retrieved from the Korean National Health Information Database. The one-to-one matched patients consisted of the matched cohort. The risks for developing all-type and site-specific cancers were estimated using incidence and incidence rate (IR) per 1000 person-years. Adjusted hazard ratio (HR) and 95% confidence interval (CI) were estimated using a Cox regression model. Results Of the 22,851 patients in the before matching cohort, 4592 patients were included in the matched cohort. Treatment with TNF inhibitors was consistently associated with a lower risk of cancer than in the nbDMARD cohort (IR per 1000 person-years, 6.5 vs. 15.6; adjusted HR, 0.379; 95% CI, 0.255–0.563). The adjusted HR (95% CI) was significantly lower in the TNF inhibitor cohort than the nbDMARD cohort for gastrointestinal cancer (0.432; 0.235–0.797), breast cancer (0.146; 0.045–0.474), and genitourinary cancer (0.220; 0.059–0.820). Conclusions The use of TNF inhibitors was not associated with an increased risk of cancer development, and rather associated with a lower cancer incidence in Korean patients with RA. Cautious interpretation is needed not to oversimplify the study results as cancer-protective effects of TNF inhibitors. A further study linking claims and clinical data is needed to confirm our results.
 
Annual trends and changes in RA treatment outcomes from 2011–2020 for each age group. A Annual change of mean DAS28-CRP values, for all patients and for each age group. B Annual change of mean JHAQ scores, for all patients and for each age group. C Annual change of mean EQ5D values, for all patients and for each age group. Abbreviations: DAS28, 28-joint Disease Activity Score; JHAQ, Japan Health Assessment Questionnaire; EQ5D, EuroQol-5 Dimensions questionnaire
Changes of RA treatment outcomes by age groups. A Changes in mean DAS28-CRP values, by age group, for the entire period and for each year. B Changes in mean JHAQ scores, by age group, for the entire period and for each year. C Changes in mean EQ5D values, by age group, for the entire period and for each year. Abbreviations: DAS28, 28-joint Disease Activity Score; JHAQ, Japan Health Assessment Questionnaire; EQ5D, EuroQol-5 Dimensions questionnaire
Article
Background We conducted a single-center cohort study of rheumatoid arthritis (RA) patients from 2011 to 2020 to understand their real world treatment and outcomes, especially changes in physical function and quality of life (QOL) in elderly patients, including those aged ≥ 80 years. Methods For RA patients attending our outpatient clinic, we annually recorded tender and swollen joint counts, laboratory findings, therapeutic drugs, and scores from the Japanese Health Assessment Questionnaire and EuroQoL-5 Dimensions questionnaire. We examined changes in treatment and outcomes over time, by age group, in patients enrolled over a 10-year period, from 2011 to 2020. Results One thousand eight hundred thirty RA patients were enrolled and data were recorded once a year, and a total of 9299 patient records were evaluated. The average age of patients increased by 3.7 years during the study period; the patients aged rapidly. Intensive pharmacological treatment was more frequent in younger patients. Disease activity, physical function, and QOL showed improvement in all age groups over the study period. Physical function and QOL showed greater changes with aging, compared with disease activity. This may be due to the effects of accumulated RA damage, disability due to aging, and depression. Conclusions Intensive pharmacological treatment contributes to not only control of disease activity but also the improvement of physical activity and QOL, even in elderly patients. Relieving age-related physical impairment and depression may improve the QOL of very elderly RA patients.
 
CONSORT flowchart of the infliximab in juvenile-onset SpA trial
Mean active joint counts, swollen joint counts, tender joint counts, and tender enthesis counts registered during the entire duration of the study (RCT + OLE phases) by treatment group according to randomization. A Mean active joint count (primary outcome). B–D Mean number of swollen joints, tender joints, and tender enthesis, respectively. All comparisons showed a significant difference between infliximab and placebo by week 12. In the open-label extension, in which all patients received infliximab, the mean of each outcome showed a sustained response to infliximab
Mean level of high-sensitive C-reactive protein (hsCRP) and Childhood Health Assessment Questionnaire score (CHAQ) registered during the entire duration of the study (RCT + OLE phases) by treatment group according to randomization. A Mean hsCRP serum levels in milligrammes per decilitre. B Mean CHAQ scores. Lines showed a significant and sustained positive effect of infliximab over time
Percentage of patients reaching the American College of Rheumatology (ACR) Paediatric 30 (Pedi 30), 50, 70, 90, and 100 response criteria per treatment group at week 12 (end RCT phase)
Percentage of patients reaching the Assessment of Spondyloarthritis international Society (ASAS) 20, 40, partial remission, and 5/6 response criteria per treatment group at week 12 (end RCT phase)
Article
Objective To assess the efficacy and safety of infliximab versus placebo in the treatment of patients with juvenile-onset spondyloarthritis (JoSpA). Methods Phase III, randomized, double-blind, placebo-controlled trial of 12 weeks that included patients ≤ 18 years old with JoSpA not responding to nonsteroidal anti-inflammatory drugs, sulfasalazine, or methotrexate. Patients were randomly assigned 1:1 to the infusion of infliximab 5mg/kg or placebo; completers entered then an open-label extension (OLE) period of 42 weeks. The primary endpoint was the number of active joints. Secondary outcomes included the assessment of disease activity, tender entheses, spinal mobility, serum C-reactive protein (CRP), the Bath Ankylosing Spondylitis Disease Activity and Functional Index, and the Childhood Health Assessment Questionnaire (CHAQ). Results We randomized 12 patients to infliximab and 14 to placebo. No significant differences were found between groups at baseline. At week 12, the mean number of active joints was 1.4 (SD 2.4) in the infliximab group and 4.1 (SD 3.0) in the placebo group ( p = 0.0002). A repeated-measures mixed model analysis that included all endpoints in the study demonstrated sustained favourable outcomes of infliximab for active joints, tender joints, swollen joints, and tender enthesis counts, as well as for CHAQ and CRP ( p < 0.01). Adverse events were more frequent in the infliximab group, including infections and infusion reactions, but none of them was serious. Conclusion Infliximab is efficacious for patients with JoSpA with an inadequate response to conventional treatment. No serious adverse events with the use of infliximab were observed.
 
Article
Background Osteoarthritis is highly heritable and genome-wide studies have identified single nucleotide polymorphisms (SNPs) associated with the disease. One such locus is marked by SNP rs11732213 (T > C). Genotype at rs11732213 correlates with the methylation levels of nearby CpG dinucleotides (CpGs), forming a methylation quantitative trait locus (mQTL). This study investigated the regulatory activity of the CpGs to identify a target gene of the locus. Methods Nucleic acids were extracted from the articular cartilage of osteoarthritis patients. Samples were genotyped, and DNA methylation was quantified by pyrosequencing at 14 CpGs within a 259-bp interval. CpGs were tested for enhancer effects in immortalised chondrocytes using a reporter gene assay. DNA methylation at the locus was altered using targeted epigenome editing, with the impact on gene expression determined using quantitative polymerase chain reaction. Results rs11732213 genotype correlated with DNA methylation at nine CpGs, which formed a differentially methylated region (DMR), with the osteoarthritis risk allele T corresponding to reduced levels of methylation. The DMR acted as an enhancer and demethylation of the CpGs altered expression of TMEM129 . Allelic imbalance in TMEM129 expression was identified in cartilage, with under-expression of the risk allele. Conclusions TMEM129 is a target of osteoarthritis genetic risk at this locus. Genotype at rs11732213 impacts DNA methylation at the enhancer, which, in turn, modulates TMEM129 expression. TMEM129 encodes an enzyme involved in protein degradation within the endoplasmic reticulum, a process previously implicated in osteoarthritis. TMEM129 is a compelling osteoarthritis susceptibility target.
 
Article
Background The upregulation of interferon (IFN)-stimulated genes induced by type I IFNs (namely type I IFN signature) in rheumatoid arthritis (RA) patients had implications in early diagnosis and prediction of therapy responses. However, factors that modulate the type I IFN signature in RA are largely unknown. In this study, we aim to explore the involvement of VGLL3, a homologue of the vestigial-like gene in Drosophila and a putative regulator of the Hippo pathway, in the modulation of type I IFN signature in the fibroblast-like synoviocytes (FLS) of RA patients. Methods FLS were isolated from RA and osteoarthritis (OA) patients. Expression of VGLL3 in the synovial tissues and FLS was analyzed by immunohistochemistry and PCR. RNA sequencing was performed in RA-FLS upon VGLL3 overexpression. The expression of IFN-stimulated genes was examined by PCR and Western blotting. Results VGLL3 was upregulated in the RA synovium and RA-FLS compared to OA. Overexpression of VGLL3 promoted the expression of IFN-stimulated genes in RA-FLS. The expression of STAT1 and MX1 was also upregulated in RA synovium compared to OA and was associated with the expression of VGLL3 in RA and OA patients. VGLL3 promoted the IRF3 activation and IFN-β1 expression in RA-FLS. Increased IFN-β1 induced the expression of IFN-stimulated genes in RA-FLS in an autocrine manner. VGLL3 also modulated the expression of the Hippo pathway molecules WWTR1 and AMOTL2, which mediated the regulation of IRF3 activation and IFN-β1 production by VGLL3 in RA-FLS. Conclusions VGLL3 drives the IRF3-induced IFN-β1 expression in RA-FLS by inhibiting WWTR1 expression and subsequently promotes the type I IFN signature expression in RA-FLS through autocrine IFN-β1 signaling.
 
Gene expression profiles of various immune cells show a correlation with clinical features of BS. Gene modules consisting of genes with similar co-expression patterns in each cell subset were identified using WGCNA. Each circle represents a module with the colors indicating the cell subset of the module and the numbers indicating the module number. Modules with significant correlation with clinical parameters are visualized. Blue lines represent the negative correlation, and red lines represent the positive correlation with line widths indicating the absolute value of correlation coefficients. The squares indicated clinical parameters. Int/Vasc/Neuro, BS patients with intestinal, vascular, and neurological involvement compared to BS patients without those organs involved. Duration: disease duration
Pathways of inflammatory chemokines and cytokines are activated in antigen-presenting cells of BS patients. A Eigengenes of modules with a positive correlation of BS in healthy controls and BS patients. B Pathway analysis of modules with positive correlation with the diagnosis of BS
BS risk SNP rs2617170 modulates the expression of YBX3. A Relationship of rs2617170 with nearby genes. B eQTL effect of rs2617170 on YBX3. Residuals after normalization are plotted by genotype
A memory CD8+ T cell module with IL-17-associated genes shows a correlation with HLA-B51 positivity. A An enlarged view of modules showing correlation with HLA-B51 positivity in BS patients. B Tc17 score in HLA-B51-positive patients (n = 7) compared to HLA-B51-negative patients (n = 17). C Pathway analysis of “MCD8_08,” the module with the strongest positive correlation with HLA-B51 positivity. Pathways with absolute z scores ≧ 2 are shown. D Pathway analysis of “NCD8_06,” the module with the strongest negative correlation with HLA-B51 positivity. Pathways with absolute z scores ≧ 5 are shown
Article
Background Behçet’s syndrome (BS) is an immune-mediated disease characterized by recurrent oral ulcers, genital ulcers, uveitis, and skin symptoms. HLA-B51, as well as other genetic polymorphisms, has been reported to be associated with BS; however, the pathogenesis of BS and its relationship to genetic risk factors still remain unclear. To address these points, we performed immunophenotyping and transcriptome analysis of immune cells from BS patients and healthy donors. Methods ImmuNexUT is a comprehensive database consisting of RNA sequencing data and eQTL database of immune cell subsets from patients with immune-mediated diseases and healthy donors, and flow cytometry data and transcriptome data from 23 BS patients and 28 healthy donors from the ImmuNexUT study were utilized for this study. Differential gene expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to identify genes associated with BS and clinical features of BS. eQTL database was used to assess the relationship between genetic risk factors of BS with those genes. Results The frequency of Th17 cells was increased in BS patients, and transcriptome analysis of Th17 cells suggested the activation of the NFκB pathway in Th17 cells of BS patients. Next, WGCNA was used to group genes into modules with similar expression patterns in each subset. Modules of antigen-presenting cells were associated with BS, and pathway analysis suggested the activation of antigen-presenting cells of BS patients. Further examination of genes in BS-associated modules indicated that the expression of YBX3 , a member of a plasmacytoid dendritic cell (pDC) gene module associated with BS, is influenced by a BS risk polymorphism, rs2617170, in pDCs, suggesting that YBX3 may be a key molecule connecting genetic risk factors of BS with disease pathogenesis. Furthermore, pathway analysis of modules associated with HLA-B51 indicated that the association of IL-17-associated pathways in memory CD8 ⁺ T cells with HLA-B51; therefore, IL-17-producing CD8 ⁺ T cells, Tc17 cells, may play a critical role in BS. Conclusions Various cells including CD4 ⁺ T cells, CD8 ⁺ T cells, and antigen-presenting cells are important in the pathogenesis of BS. Tc17 cells and YBX3 may be potential therapeutic targets in BS.
 
Visualization of inclusion/exclusion criteria employed to obtain the main cohort, the two exploratory analysis cohorts as well as the two sensitivity analysis cohorts. CRP, C-reactive protein; DAS, disease activity score; ESR, erythrocyte sedimentation rate; MTX, methotrexate; NPR, National Patient Register; PDR, Prescribed Drug Register; RA, rheumatoid arthritis; SRQ, Swedish Rheumatology Quality Register
Article
Objectives To assess whether persistence to treatment with methotrexate (MTX) in early rheumatoid arthritis (RA) is shared among first-degree relatives with RA and to estimate any underlying heritability. Methods First-degree relative pairs diagnosed with RA 1999–2018 and starting MTX (in monotherapy) as their first disease-modifying anti-rheumatic drug (DMARD) treatment were identified by linking the Swedish Rheumatology Quality Register to national registers. Short- and long-term persistence to MTX was defined as remaining on treatment at 1 and 3 years, respectively, with no additional DMARDs added. We assessed familial aggregation through relative risks (RR) using log-binomial regression with robust standard errors and estimated heritability using tetrachoric correlations. We also explored the familial aggregation of EULAR treatment response after 3 and 6 months. To mimic the clinical setting, we also tested the association between having a family history of MTX persistence and persistence within the index patient. Results Familial persistence was not associated with persistence at 1 (RR=1.02, 95% CI 0.87–1.20), only at 3 (RR=1.41, 95% CI 1.14–1.74) years. Heritability at 1 and 3 years was estimated to be 0.08 (95% CI 0–0.43) and 0.58 (95% CI 0.27–0.89), respectively. No significant associations were found between family history and EULAR response at 3 and 6 months, neither overall nor in the clinical setting analysis. Conclusions Our findings imply a familial component, including a possible genetic element, within the long-term persistence to MTX following RA diagnosis. Whether this component is reflective of characteristics of the underlying RA disease or determinants for sustained response to MTX in itself will require further investigation.
 
Adjusted associations of CDAI tertiles with baseline E/e′. Adjusted means and 95% CIs are depicted. All adjusted analyses account for age, gender, BMI, and troponin-I
Adjusted associations of A BNP tertiles with baseline LAVI. B Troponin-I Tertiles with baseline E/e′. Adjusted means and 95% CIs are depicted; all adjusted analyses account for age, gender, and BMI
Article
Background Diastolic dysfunction (DD) is more prevalent in patients with rheumatoid arthritis (RA) compared to the general population. However, its evolution over time and its significant clinical predictors remain uncharacterized. We report on baseline and prospective changes in diastolic function and its associated RA and cardiovascular (CV) predictors. Methods In this study, 158 RA patients without clinical CV disease (CVD) were enrolled and followed up at 4 to 6 years, undergoing baseline and follow-up echocardiography to assess for DD, as well as extensive characterization of RA disease activity and CV risk factors. Novel measures of myocardial inflammation and perfusion were obtained at baseline only. Using baseline and follow-up composite DD (E/e′, Left Atrial Volume Index (LAVI) or peak tricuspid regurgitation (TR) velocity; ≥ 1 in top 25%) as the outcome, multivariable regression models were constructed to identify predictors of DD. Results DD was prevalent in RA patients without clinical heart failure (HF) (40.7% at baseline) and significantly progressed on follow-up (to 57.9%). Baseline composite DD was associated with baseline RA disease activity (Clinical Disease Activity Index; CDAI) (OR 1.39; 95% CI 1.02–1.90; p=0.034). Several individual diastolic parameters (baseline E/e′ and LAVI) were associated with troponin-I and brain natriuretic peptide (BNP). Baseline and follow-up composite DD, however, were not associated with myocardial inflammation, myocardial microvascular dysfunction, or subclinical atherosclerosis. Conclusions DD is prevalent in RA patients without clinical HF and increases to >50% over time. Higher RA disease activity at baseline predicted baseline composite DD. Future longitudinal studies should explore whether adverse changes in diastolic function lead to clinical HF and are attenuated by disease-modifying antirheumatic drugs (DMARDs).
 
SRM with 95% CI for possible score combinations and DAS28 after 3 and 6 months
Reduced (US7) score. In red: joint regions included in the original US7 score; in green: reduced (US7) score; illustration adapted from Backhaus et al. [19]; MCP metacarpophalangeal, PIP proximal interphalangeal, MTP metatarsophalangeal, EDC extensor digitorum communis tendon (extensor compartment lV), ECU extensor carpi ulnaris tendon (extensor compartment Vl)
SRM with 95% CI after 6 months - early vs. established RA
Article
Background: There is no international consensus on an optimal ultrasound score for monitoring of rheumatoid arthritis (RA) on patient-level yet. Our aim was to reassess the US7 score for the identification of the most frequently pathologic and responsive joint/tendon regions, to optimize it and contribute to an international consensus. Furthermore, we aimed to evaluate the impact of disease duration on the performance of the score. Methods: RA patients were assessed at baseline and after 3 and 6 months of starting/changing DMARD therapy by the US7 score in greyscale (GS) and power Doppler (PD). The frequency of pathologic joint/tendon regions and their responsiveness to therapy were analyzed by Friedman test and Cochrane-Q test respectively, including the comparison of palmar vs. dorsal regions (chi-square test). The responsiveness of different reduced scores and the amount of information retained from the original US7 score were assessed by standardized response means (SRM)/linear regression. Analyses were also performed separately for early and established RA. Results: A total of 435 patients (N = 138 early RA) were included (56.5 (SD 13.1) years old, 8.2 (9.1) years disease duration, 80% female). The dorsal wrist, palmar MCP2, extensor digitorum communis (EDC) and carpi ulnaris (ECU) tendons were most frequently affected by GS/PD synovitis/tenosynovitis (wrist: 45%/43%; MCP2: 35%/28%; EDC: 30%/11% and ECU: 25%/11%) and significantly changed within 6 months of therapy (all p ≤0.003 by GS/PD). The dorsal vs. palmar side of the wrist by GS/PD (p < 0.001) and the palmar side of the finger joints by PD (p < 0.001) were more frequently pathologic. The reduced US7 score (GS/PD: palmar MCP2, dorsal wrist, EDC and ECU, only PD: dorsal MCP2) showed therapy response (SRM 0.433) after 6 months and retained 76% of the full US7 score's information. No major differences between the groups of early and established RA could be detected. Conclusions: The wrist, MCP2, EDC, and ECU tendons were most frequently pathologic and responsive to therapy in both early and established RA and should therefore be included in a comprehensive score for monitoring RA patients on patient-level.
 
The BMAL1 expression of NP specimens in human donors, rat NP tissues, and cells. a, b Immunohistochemical staining images and quantification of BMAL1 in human NP tissues from the grade II and grade V groups. We selected 3 random fields in per individual for quantitative analysis (scale bar: 250 μm). n = 3, **P < 0.01. c, d Immunofluorescence staining images and quantification of BMAL1 in human NP tissues from the Grade II and Grade V groups. We selected 3 random fields in per individual for quantitative analysis (scale bar: 125 μm). n = 4, **P < 0.01. e, f Immunofluorescence staining images and quantification of BMAL1 in rat NP tissues from Control and puncture groups cultured 0 days, 7 days, and 14 days (scale bar: 500 μm). n = 3, NS, P>0.01, **P < 0.01. g The mRNA levels of Bmal1 and Clock in control and IL-1β. n = 3, *P<0.05, **P < 0.01
Knockdown of Bmal1 led to the degeneration of NP cells in vitro. a The mRNA levels of Bmal1 in the control and si-Bmal1 groups. b The mRNA levels of Acan and Mmp13 in the control and si-Bmal1 groups. n = 3, *P<0.05, **P < 0.01. c–f Immunofluorescence staining images and quantification of Aggrecan and MMP13 in the control and si-Bmal1 groups (scale bar: 25 μm). n = 3, **P < 0.01
Si-Bmal1 decreased the expression of NRF2 and increased the ROS, inflammatory response, and apoptosis. a, b Immunofluorescence staining images and quantification of NRF2 in the control and si-Bmal1 groups (scale bar: 25 μm). n = 3, **P < 0.01. c, d DCFDA staining images and quantification in the control and si-Bmal1 groups. n = 3, **P < 0.01. e The mRNA levels of Il-1β, Tnf-α, and Il-6 in the control and si-Bmal1 groups. n = 3, *P<0.05, **P < 0.01. f, g TUNEL staining and quantification in the control and si-Bmal1 groups (scale bar: 25 μm). n = 3, **P < 0.01
SFN attenuated si-Bmal1-induced the dysfunction of NP cells. The NP cells were divided into four groups, with no treatment, incubated with 5 μM SFN, si-Bmal1, and si-Bmal1 + 5 μM SFN. a Western blotting of BMAL1, NRF2, Aggrecan, and MMP13 protein expression (normalized to GAPDH expression). b Immunofluorescence staining of NRF2, Aggrecan, and MMP13 staining in Control, SFN, si-Bmal1, and si-Bmal1 + 5 μM SFN group (scale bar: 25 μm). c The levels of TNF-α and IL-1β protein. n = 3, NS, not significant difference, *P<0.05, **P < 0.01. d, e DCFDA staining images and quantification (scale bar: 50 μm). f, g TUNEL staining images and quantification (scale bar: 25 μm)
SFN attenuated the degeneration of rat NP tissues in an organotypic tissue-explant model. The NP tissues were divided into four groups, control, needle-punctured, needle-punctured + 5 μM SFN, and needle-punctured + 10 μM SFN. a Immunofluorescence staining of BMAL1 in rat NP tissues cultured 7 days and 14 days (scale bar: 500 μm). b Immunofluorescence staining of NRF2 in rat NP tissues cultured for 7 days and 14 days (scale bar: 500 μm). c HE and safranin O staining of the rat IVDs cultured for 7 days and 14 days (scale bar: 500 μm)
Article
Background Intervertebral disc (IVD) is a highly rhythmic tissue, which experiences a diurnal cycle of high/low mechanical loading via the changes of activity/rest phase. There are signs that disruption of the peripheral IVD clock is related to the process of intervertebral disc degeneration (IDD). However, it is still unclear whether inflammation could disturb the IVD clock and thus induce the process of IDD. Methods and results In this study, we used IL-1β, a commonly used inflammatory factor, to induce IDD and found that the IVD clock was dampened in degenerated human nucleus pulposus specimens, rat nucleus pulposus (NP) tissues, and cells. In this study, we found that the circadian clock of NP cells was totally disrupted by knockdown of the core clock gene brain and muscle arnt-like protein-1 ( Bmal1 ), which thus induced the dysfunction of NP cells. Next, we explored the mechanism of dampened clock-induced IDD and found that knockdown of Bmal1 decreased the expression of nuclear factor erythroid2-related factor 2 (NRF2), a downstream target gene of Bmal1 , and increased inflammatory response, oxidative stress reaction, and apoptosis of NP cells. In addition, NRF2 activation attenuated the dysfunction of NP cells induced by the dampened IVD clock and the degenerative process of NP tissues in an organotypic tissue-explant model. Conclusions Taken together, our study extends the relationship between peripheral clock and IVD homeostasis and provides a potential therapeutic method for the prevention and recovery of IDD by targeting the clock-controlled gene Nrf2 .
 
Patient inclusion flowchart. Participants who were prescribed with the first-line bDMARDs during 2011 and 2019 were included after excluding other rheumatoid arthritis or juvenile rheumatoid arthritis, development of herpes zoster within 1 month after bDMARDs, and recurrent herpes zoster that occurred within 6 months since the date of the previous herpes zoster infection
bDMARD-dependent proportion of herpes zoster events in rheumatoid arthritis patients. Periods, including 12–24, 24–36, 36–48, and 48–60 months, were divided by 4 for the normalization of event rates to 3-month periods. Each dot is located in the middle of the month, which represents between-period event rates. The index date refers to the initial date of first-line bDMARD treatment. Patients with censored follow-up investigation were excluded from the calculation of the corresponding event rate
Article
Background There is limited information regarding disease-modifying antirheumatic drug (DMARD)-dependent risks of overall, incident, and recurrent herpes zoster (HZ) during first-line biologic DMARD (bDMARD) or targeted synthetic DMARD (tsDMARD) treatment among patients with seropositive rheumatoid arthritis (RA) in terms of HZ risk. Methods A total of 11,720 patients with seropositive RA who were prescribed bDMARD or tofacitinib between January 2011 and January 2019 from the Korean Health Insurance Review & Assessment Service database were studied. A multivariate Cox proportional hazards regression model was adopted to evaluate the adjusted hazard ratio (aHR) with 95% confidence interval (CI) for the risk of HZ dependent on the choice of first-line bDMARDs or tsDMARD, including etanercept, infliximab, adalimumab, golimumab, tocilizumab, rituximab, tofacitinib, and abatacept. Results During the 34,702 person-years of follow-up, 1686 cases (14.4%) of HZ were identified, including 1372 (11.7%) incident and 314 (2.7%) recurrent HZs. Compared with that of the abatacept group, tofacitinib increased the overall risk (aHR, 2.46; 95% CI, 1.61–3.76; P<0.001), incidence (aHR, 1.99; 95% CI, 1.18–3.37; P=0.011), and recurrence (aHR, 3.69; 95% CI, 1.77–7.69; P<0.001) of HZ. Infliximab (aHR, 1.36; 95% CI, 1.06–1.74; P=0.017) and adalimumab (aHR, 1.29; 95% CI, 1.02–1.64; P=0.032) also increased the overall HZ risk. Moreover, a history of HZ was found to be an independent risk factor for HZ (aHR, 1.54; 95% CI, 1.33–1.78; P<0.001). Conclusions HZ risk is significantly increased in RA patients with a history of HZ after the initiation of bDMARDs or tsDMARD. The risk of incident and recurrent HZ was higher after tofacitinib treatment in patients with RA than that after treatment with bDMARDs. Individualized characteristics and history of HZ should be considered when selecting bDMARDs or tsDMARD for RA patients considering HZ risks.
 
Flowchart of eligible and ineligible participants. A total of 573 LUNA participants were followed up after 1 year, and 394 patients were excluded because of insufficient follow-up duration. The total number of excluded patients was 64 (50 patients who were undergoing remission therapy with PSL >15 mg; 14 patients with missing data on the number of days of glucocorticoid administration). There were no cases lacking data on glucocorticoid dose and infection occurrence. The final number of eligible participants was 509
Forest plot of the adjusted hazard ratio of infection occurrence. Age, sex, and concurrent immunosuppressant use were used as covariates
Hazard ratio for infection occurrence in patients who were not administered hydroxychloroquine, adjusted by age, sex, and immunosuppressant use
Hazard ratio for infection occurrence in patients with fixed PSL dose for 1 year
Article
Background Infection is a major cause of mortality in patients with systemic lupus erythematosus (SLE). Therefore, minimizing the risk of infection is an important clinical goal to improve the long-term prognosis of SLE patients. Treatment with ≥7.5 mg prednisolone (PSL) or equivalent has been reported to increase the risk of infections. However, it remains unclear whether <7.5 mg PSL or equivalent dose affects the risk of infection in SLE patients. This study evaluated the association between the occurrence of infection in patients with SLE and low-dose glucocorticoid (GC) usage, especially <7.5 mg PSL or equivalent, to explore the GC dose that could reduce infection occurrence. Methods This prospective cohort study included patients from the Japanese multicenter registry of patients with SLE (defined as ≥4 American College of Rheumatology 1997 revised criteria) over 20 years of age. The PSL dose was categorized as PSL 0–2.5, 2.6–5.0, 5.1–7.5, and 7.6–15.0 mg. The primary outcome was infection requiring hospitalization. We conducted a multivariable analysis using time-dependent Cox regression analysis to assess the hazard ratio of infection occurrence compared with a dose of 0–2.5 mg PSL or equivalent in the other three PSL dose groups. Based on previous reports and clinical importance, the covariates selected were age, sex, and concurrent use of immunosuppressants with GC. In addition, two sensitivity analyses were conducted. Results The mean age of the 509 SLE patients was 46.7 years; 89.0% were female, and 77.2% used multiple immunosuppressants concomitantly. During the observation period, 52 infections requiring hospitalization occurred. The incidence of infection with a PSL dose of 5.0–7.5 mg was significantly higher than that in the PSL 0–2.5 mg group (adjusted hazard ratio: 6.80, 95% confidence interval: 2.17–21.27). The results of the two sensitivity analyses were similar. Conclusions Our results suggested that the use of 5.0–7.5 mg PSL or equivalent could pose an infection risk in SLE patients. This finding indicates that PSL dose should be reduced to as low as possible in SLE patients to avoid infection.
 
Article
Background Studies on the association between coffee, a modifiable lifestyle factor, and rheumatoid arthritis (RA), a chronic autoimmune disease primarily affecting the joints, have been conflicting. The aim of the present study was to study the association between coffee consumption and risk of RA in the context of different lifestyle factors. Methods We included 2184 cases (72% women, mean age 55 years) newly diagnosed with RA during 2005–2018 in Sweden and 4201 controls matched on age, sex, and residential area. Data on coffee consumption was collected through a food frequency questionnaire and categorized into < 2 (reference), 2–< 4, 4–< 6, and ≥ 6 cups/day. We calculated odds ratios (OR) with 95% confidence intervals (CI) for coffee consumption and risk of RA, in a crude model (taking matching factors into account), and then adjusted first for smoking and further for BMI, educational level, alcohol consumption, and physical activity. We also stratified analyses on sex, smoking, rheumatoid factor, and anti-CCP2 status. Results In the crude model, high coffee consumption was associated with increased risk of RA (OR = 1.50, 95% CI 1.20–1.88 for ≥ 6 cups/day compared to < 2 cups). After adjusting for smoking, the OR decreased and was no longer statistically significant (OR = 1.16, 95% CI 0.92–1.46) and decreased further in the full model (OR = 1.14 95% CI 0.89–1.45). This pattern held true in all strata. Conclusion The findings from this large, population-based case-control study did not support a significant association between coffee consumption and risk of RA as a whole nor within different subgroups.
 
Oral dextran sodium sulfate (DSS) administration elicits SpA features in SKG mice. a Experimental scheme of oral DSS administration during the experiment. Mice were given 1% DSS in their drinking water for 2 weeks, followed by regular water. Ten mice were used in each group. Weeks are indicated by w. b The body weight change in BALB/c and SKG mice over the experiment. c–f Arthritis incidence rates and scores and enthesitis incidence rates and scores were recorded every 2 weeks. Values are expressed as mean ± standard error of the mean (SEM). g Representative photographs of hind paws for the enthesitis evaluation method. h Representative haematoxylin and eosin-stained sections of the peripheral enthesis (Achilles tendon) and the axial enthesis (caudal vertebrae). Only DSS-treated SKG mice showed marked cell infiltration around the Achilles tendon and plantar aponeurosis (arrows), and slight cell infiltration around but not inside the annulus fibrosus of the intervertebral disk (arrows). Scale bar = 200μm. Statistical analyses were performed by using the Mann-Whitney U test (*P <0.05)
Oral DSS activates Th1 and Th17 immunity in the spleens of SKG mice. a Representative flow cytometry (FCM) plots of splenic CD4⁺T cells at weeks 0 and 12. b, c IFN-γ (b) and IL-17A (c) positivity of splenic CD4⁺ T cells in BALB/c and SKG mice at weeks 0 and 12. Statistical analyses were performed by using the Mann-Whitney U test (*P <0.05, **P <0.01)
The antibiotics, MEPM and VCM, but not the anti-fungal agent AMPH-B, ameliorate DSS-induced peripheral SpA of SKG mice. a Experimental scheme of mouse treatment with oral antibiotics (MEPM (1 g/L)+VCM (0.5 g/L)) or the anti-fungal agent (AMPH-B (0.3 g/L)) and DSS. Agents were administered in their drinking water with 6% DMSO for 1 week, followed by regular water for 1 week. Then, 1% DSS was administered in their drinking water for 2 weeks, followed by regular water. Ten mice were used in each group. b The body weight change in each group over the experiment. c–f Arthritis incidence rates and scores, enthesitis incidence rates and scores were recorded every 2 weeks. Values are the mean ± standard error of the mean (SEM). g, h FCM analysis of IFN-γ (g) and IL-17A (h) positivity of splenic CD4⁺ T cells in SKG mice administered with DMSO and DMSO plus antibiotics (MEPM+VCM). Five mice per group were used. Statistical analyses were performed by using the Mann-Whitney U test (*P <0.05, **P <0.01)
Oral DSS administration increases the amount of circulating bacterial DNA and the number of common species in the groups. a The concentration of bacterial DNA in whole blood measured by absolute qPCR, 0, 1, 7, and 14 days after oral DSS treatment initiation. Five mice per group were used. Values are presented as mean ± SEM. b The concentration of bacterial DNA measured by qPCR in whole blood after DSS treatment with and without antibiotics. Five mice per group were used. c Venn diagrams showing the number of bacterial species in circulating bacterial DNA on days 0 and 14. Five mice were examined per group. Identical numbers of mice are shown by #. Twenty mice were examined as a whole. d. The Shannon index before and after DSS administration. Five mice were examined per group. Statistical analyses were performed using the Mann-Whitney U test (*P <0.05, **P <0.01)
LEfSe analysis of circulating bacterial DNA in the whole blood of BALB/c and SKG mice with and without oral DSS administration. The cutoff was set as LDA score > 4.0
Article
Background Spondyloarthritis (SpA) is an autoimmune and autoinflammatory musculoskeletal disease characterised by systemic enthesitis. Recent research has focused on subclinical inflammatory bowel disease (IBD) in SpA pathogenesis. SKG mice, harbouring the Zap70 W163C mutation, increase autoreactive Th17 cells intrinsically, and in a conventional environment, they exhibit spontaneous arthritis with fungal factors. Under SPF conditions, they show SpA features, including enteritis, after peritoneal injection of β-1,3-glucan. This study aimed to clarify whether oral dextran sulfate sodium (DSS) administration, utilised in IBD model mice, can provoke SpA features in SKG mice under SPF conditions, focusing on the relationship between gut microorganisms and SpA pathogenesis. Methods BALB/c and SKG mice were administered oral DSS, and their body weights, arthritis, and enthesitis scores were recorded. In another cohort, antibiotics (meropenem and vancomycin) or an anti-fungal agent (amphotericin B) was administered orally before DSS administration. The splenic Th1 and Th17 cell populations were examined before and after DSS administration using flow cytometry. Furthermore, the amount of circulating bacterial DNA in whole blood was measured by absolute quantitative polymerase chain reaction (qPCR), and the number and characteristics of bacterial species corresponding to these circulating DNA were analysed by next-generation sequencing (NGS). Results Ankle enthesitis as a peripheral SpA feature was elicited in half of DSS-administered SKG mice, and none of the BALB/c mice. Pre-administration of antibiotics suppressed enthesitis, whilst an anti-fungal agent could not. Th1 and Th17 cell levels in the spleen increased after DSS administration, and this was suppressed by pre-administration of antibiotics. SKG mice have a larger amount of bacterial DNA in whole blood than BALB/c mice before and 1 day after the initiation of DSS administration. The number of bacterial species in whole blood increased after DSS administration in BALB/c and SKG mice. Some genera and species significantly specific to the DSS-treated SKG mouse group were also detected. Conclusion Oral DSS administration alone elicited peripheral enthesitis in SKG mice with bacterial translocation accompanied by increased splenic Th1 and Th17 cell levels. Pre-administration of antibiotics ameliorated these DSS-induced SpA features. These findings suggest that intestinal bacterial leakage plays a pivotal role in SpA pathogenesis.
 
Metabolomic profiling at time zero. A PCA examining samples at time zero. B Heat map based on Pearson and Ward for determining distance and clustering identified 2 clusters at time zero. C Characteristics of patients in both clusters. Continuous variables were expressed as mean ± standard deviation (SD) and categorical variables as number (N) and percentage. D Top 50 metabolites different between clusters
XOI-based ULT effects on serum metabolomic pathway analysis. A Two-way repeated measures ANOVA identified 115 metabolites (89 decreased and 26 increased) significantly differing between baseline and 24 weeks ULT titration to target. A summary of the numbers of metabolites that achieved statistical significance (p ≤ 0.05), as well as those approaching significance (0.05 < p < 0.10), is shown. B, C Pathway analysis was conducted with the 105 metabolites (p < 0.1) significant at 12 weeks ULT (B) and with the 165 metabolites (p < 0.1) at 24 weeks (C). At 12 weeks ULT, pathways were significantly enriched with changes in the AA metabolism (arrows, B). However, at 24 weeks ULT, significant altered pathways were mostly related to FA and polyamine metabolism (arrows, C)
XOI-based ULT effects on AA metabolism. A Samples collected specially at 12 and 24 weeks of treatment showed significant alterations in aromatic AA, branched-chain AA, G-glutamyl AA. Green: indicates significant difference (p ≤ 0.05) between the groups shown, metabolite ratio of < 1.00. Light green: narrowly missed statistical cutoff for significance 0.05 < p < 0.10, metabolite ratio of < 1.00. Red: indicates significant difference (p ≤ 0.05) between the groups shown, metabolite ratio of ≥ 1.00. Light red: narrowly missed statistical cutoff for significance 0.05 < p < 0.10, metabolite ratio of ≥ 1.00. Blue: indicates significant (p ≤ 0.05) ANOVA. Light blue: indicates 0.05 < p < 0.10 ANOVA effect. B PCA at different time points using only microbiome-related metabolites. C Correlation between metabolites derived from AA that were significant at 12 weeks of ULT titration to target (Y-axis) and metabolites shown to predict microbiome diversity (X-axis). D Correlation between metabolites derived from AA that were not significant at 12 weeks of ULT titration to target (Y-axis) and metabolites shown to predict microbiome diversity (X-axis). Pearson correlation (r) in C and D with a cutoff value of 0.5. The orange color indicates a positive correlation > 0.5, and the dark blue indicates negative correlation ≤0.5
XOI-based ULT effects in lipolysis. A Samples collected at 24 weeks of treatment showed significant alterations in medium chain FAs and long chain FAs. Green: indicates significant difference (p ≤ 0.05) between the groups shown, metabolite ratio of < 1.00. Light green: narrowly missed statistical cutoff for significance 0.05 < p < 0.10, metabolite ratio of < 1.00. Red: indicates significant difference (p ≤ 0.05) between the groups shown, metabolite ratio of ≥1.00. Light red: narrowly missed statistical cutoff for significance 0.05 < p < 0.10, metabolite ratio of ≥ 1.00. Blue: indicates significant (p ≤ 0.05) ANOVA. Light blue: indicates 0.05 < p < 0.10 ANOVA effect. B Free FA release into the media over 30 min following administration of 10 μM CL-316,243 [32] or vehicle control to 3T3-L1 adipocytes pretreated with febuxostat (F, 50 μM) and/or colchicine (C, 10 nM) for 72 h. C TNF treatment at 17 ng/mL was administered for 36 h before the addition of febuxostat (50 μM) and/or colchicine 10 nM. Lipolysis was assessed 72 h later by measuring free FA secreted into the cell culture media over 60 min. Data in B and C presented as mean ± SEM. #p < 0.05, by Holm-Sidak post hoc test after significant 2-way ANOVA for the control versus CL (B) or TNF (C) treated groups, within vehicle, F, C, or F and C treated group. *p < 0.05, by Holm-Sidak post hoc test after significant 2-way ANOVA for the vehicle versus F, C, or F and C treated groups, within CL (B) or TNF (C) treated group
XOI-based ULT effects on FA metabolism. A PLS-DA separation between patients with different pattern of FA levels after ULT therapy: 17 patients did not decrease FA [1], and 17 patients significantly decreased FA [2]. B The variables important in projection (VIP) in discriminate both groups, where a VIP score ≥ 1 was considered as important. C Characteristics of patients in both clusters. Continuous variables were expressed as mean ± standard deviation (SD) and categorical variables as number (N) and percentage
Article
Objective Linked metabolic and cardiovascular comorbidities are prevalent in hyperuricemia and gout. For mechanistic insight into impact on inflammatory processes and cardiometabolic risk factors of xanthine oxidase inhibitor urate-lowering therapy (ULT) titration to target, we performed a prospective study of gout serum metabolomes from a ULT trial. Methods Sera of gout patients meeting the 2015 ACR/EULAR gout classification criteria ( n = 20) and with hyperuricemia were studied at time zero and weeks 12 and 24 of febuxostat or allopurinol dose titration ULT. Ultrahigh performance liquid chromatography-tandem mass spectroscopy acquired the serum spectra. Data were assessed using the Metabolon and Metaboloanalyst software. Lipolysis validation assays were done in febuxostat and/or colchicine-treated 3T3-L1 differentiated adipocytes. Results Serum urate decreased from time zero (8.21 ±1.139 SD) at weeks 12 (5.965 ± 1.734 SD) and 24 (5.655 ±1.763 SD). Top metabolites generated by changes in nucleotide and certain amino acid metabolism and polyamine pathways were enriched at 12 and 24 weeks ULT, respectively. Decreases in multiple fatty acid metabolites were observed at 24 weeks, linked with obesity. In cultured adipocytes, febuxostat significantly decreased while colchicine increased the lipolytic response to β-adrenergic-agonism or TNF. Conclusion Metabolomic profiles linked xanthine oxidase inhibitor-based ULT titration to target with reduced serum free fatty acids. In vitro validation studies revealed that febuxostat, but not colchicine, reduced lipolysis in cultured adipocytes. Since soluble urate, xanthine oxidase inhibitor treatment, and free fatty acids modulate inflammation, our findings suggest that by suppressing lipolysis, ULT could regulate inflammation in gout and comorbid metabolic and cardiovascular disease.
 
Signalling mechanisms leading to senescence. Mechanical stress, DNA damage, ROS, oxidative stress and other adverse conditions induce cellular senescence. p53-p21-CDK2 and p16-CDK4/6 are two pathways involved in senescence. CDK2/4/6 inhibit RB and promote S phase entry, leading to cell cycle arrest and cellular senescence. ROS, reactive oxygen species; CDK, cyclin-dependent kinases; RB, retinoblastoma protein
The effects of mechanical stress on chondrocytes. Under mechanical stress conditions, Piezo1 and TRPV4 channels are activated, leading to Ca²⁺ influx into the cytoplasmand triggering endoplasmic reticulum stress and mitochondrial dysfunction. mtROS and mtDNA are released from damaged mitochondria, which results in DNA damage, active catabolism and cartilage degeneration. NF-κB is usually activated during senescence and promotes SASP factor transcription. Mechanical stress inhibits FBXW7-dependent MKK7 degradation, which leads to JNK pathway activation and cellular senescence. JNK also has anti-senescence effect by regulating p16. The Rac1-ROS pathway participates in NF-κB activation under mechanical stress and promotes the production of gremlin-1. Gremlin-1 in turn activates NF-κB via VEGF2 in an autocrine or paracrine manner. TRPV4, transient receptor potential vanilloid 4; mtROS, mitochondrial reactive oxygen species; NF-κB, nuclear factor kappa-B; SASP, senescence-associated secretory phenotype; FBXW7, F-box and WD repeat domain containing 7; MKK7, mitogen-activated protein kinase kinase 7; JNK, mitogen-activated protein kinase; Rac1, Ras-related C3 botulinum toxin substrate 1; VEGF2, vascular endothelial growth factor 2
The relationship between energy shortage and chondrocyte senescence. Energy deficiency caused by damaged mitochondria activates AMPK, and SIRTs including SIRT1, SIRT3 and SIRT6. SIRT1 protects cartilage by promoting the transcription of Sox9 and collagen 2. Several factors such miR-34a and leptin inhibit SIRT1 and exacerbate chondrocyte senescence and cartilage damage. Damaged mitochondria are eliminated by p62-mediated autophagy. Activated in an inflammatory environment, NF-κB promotes SASP factor transcription. SASP factors activate NF-κB in an autocrine manner, forming a positive feedback loop. Energy deficiency activates mTOR via AMPK. mTOR inhibits ZFP36L1 by activating MKK. ZFP36L1 and some miRNAs, such as miR-204 participate in the degradation of SASPs. Decreased NAD + /NADH also activates SIRT3 and SIRT6 in addition to SIRT1. SIRT3 deacetylates SOD2 and increases SOD2-specific activity, thus protecting chondrocytes against oxidative stress. SIRT6 can inhibit DNA damage and cellular senescence. SIRT, sirtuin; AMPK, adenosine 5′-monophosphate (AMP)-activated protein kinase; mTOR, mammalian target of rapamycin; ZFP36L1, ZFP36 ring finger protein like 1; SOD2, superoxide dismutase 2
Interactions between NK cells and senescent cells. In response to DNA damage, MICA is selectively expressed on senescent cells rather than proliferative cells. MICA interacts with NKG2D and activates NK cells via ITAM. Activated NK cells produce and secrete granzyme B and perforin to kill senescent cells. Shedding MICA from the senescent cell membrane surface leads to NK cells off-target and senescent cell evasion. HLA-E is upregulated by the p38 pathway. HLA-E binds to NKG2A and inhibits NK cell activation via the ITIM on the NKG2A intracellular segment. CD155 exerts dual effects as its combination with DNAM-1 activates NK cells while its combination with CD94 or TIGHT inhibits NK cell activation. Shedding CD155 participates is involved in the evasion of senescent cells as its binding affinity to DNAM-1 is higher than that to TIGHT and CD94. The expression of MICA and CD155 is directly regulated by transcriptional factor E2F1. uPAR is specifically expressed on the senescent cell membrane surface, and CAR-T therapy targeting uPAR has been designed to eliminate senescence. DPP4 has been treated as a target of immunotherapy via ADCC. NKG2D, natural killer group 2, member D; HLA-E, human leukocyte antigen-E; IL-6, interleukin-6; CAR-T cell, chimeric antigen receptor T cell; uPAR, urokinase-type plasminogen activator receptor; ADCC, antibody-dependent cell-mediated cytotoxicity; DPP4, dipeptide peptidase 4; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif
Article
Osteoarthritis (OA) is an age-related cartilage degenerative disease, and chondrocyte senescence has been extensively studied in recent years. Increased numbers of senescent chondrocytes are found in OA cartilage. Selective clearance of senescent chondrocytes in a post-traumatic osteoarthritis (PTOA) mouse model ameliorated OA development, while intraarticular injection of senescent cells induced mouse OA. However, the means and extent to which senescence affects OA remain unclear. Here, we review the latent mechanism of senescence in OA and propose potential therapeutic methods to target OA-related senescence, with an emphasis on immunotherapies. Natural killer (NK) cells participate in the elimination of senescent cells in multiple organs. A relatively comprehensive discussion is presented in that section. Risk factors for OA are ageing, obesity, metabolic disorders and mechanical overload. Determining the relationship between known risk factors and senescence will help elucidate OA pathogenesis and identify optimal treatments.
 
Study flow-chart. The graph provides information on patient-flow, baseline haemodynamics and follow-up. mPAP, mean pulmonary artery pressure; P(A)H, pulmonary (arterial) hypertension; PAWP, pulmonary arterial wedge pressure; PVD, pulmonary vascular disease; PVR, pulmonary vascular resistance
Kaplan-Meier survival analysis of echocardiographic right ventricular function. Patients with a tricuspid annular plane systolic excursion <18 mm assessed by echocardiography, b any impairment of RV function or with c TAPSE/sPAP ratio ≤0.6 mm/mmHg had significantly worse survival than patients with tricuspid annular plane systolic excursion ≥18 mm, normal RV function, or TAPSE/sPAP ratio >0.6 mm/mmHg
Kaplan-Meier analysis of invasively determined right ventricular function. Patients with a pulmonary vascular resistance ≥2 Wood Units, b pulmonary artery compliance <2 ml/mmHg, c cardiac index increase <2 l/min/m², d peak cardiac index <5.5 l/min/m² and/or e RV pulmonary vascular reserve (defined as the increase of mean pulmonary artery pressure/increase of cardiac output during exercise) ≥3 mmHg/(l/min) showed worse survival than SSc patients above the respective thresholds
Kaplan-Meier analysis of multivariable risk set. Multivariable Cox regression analysis identified TAPSE/sPAP ratio ≤0.6 mm/mmHg and diffusion capacity for carbon monoxide of the lung (DLCO) ≤65% predicted as independent prognostic predictors of survival. Patients with none of these risk factors had significantly better survival than patients with one or two risk factors
Article
Background The objective of this study was to investigate the prognostic impact of right ventricular (RV) function at rest and during exercise in patients with systemic sclerosis (SSc) presenting for a screening for pulmonary hypertension (PH). Methods In this study, data from SSc patients who underwent routinely performed examinations for PH screening including echocardiography and right heart catheterization at rest and during exercise were analysed. Uni- and multivariable analyses were performed to identify prognostic parameters. Results Out of 280 SSc patients screened for PH, 225 were included in the analysis (81.3% female, mean age 58.1±13.0 years, 68% limited cutaneous SSc, WHO-FC II–III 74%, 24 manifest PH). During the observation period of 3.2±2.7 (median 2.6) years 35 patients died. Tricuspid annular plane systolic excursion (TAPSE) at rest <18 mm (p=0.001), RV output reserve as increase of cardiac index (CI) during exercise <2 l/min (p<0.0001), RV pulmonary vascular reserve (Δ mean pulmonary artery pressure/Δ cardiac output) ≥3 mmHg/l/min (p<0.0001), peak CI <5.5 l/min/m² (p=0.001), pulmonary arterial compliance <2 ml/mmHg (p=0.002), TAPSE/systolic pulmonary arterial pressure (sPAP) ratio ≤0.6 ml/mmHg (p<0.0001) and echocardiographic qualitative RV function at rest (p<0.0001) significantly predicted worse survival. In the multivariable analysis TAPSE/sPAP ratio and diffusion capacity for carbon monoxide ≤65% were identified as independent prognostic predictors and had 75% sensitivity and 69% specificity to predict future development of pulmonary vascular disease (PVD) during follow-up. Conclusions This study demonstrates that assessment of RV function at rest and during exercise may provide crucial information to identify SSc patients who are at a high risk of poor outcome and for the development of PH and/or PVD.
 
The concept of charge-based targeting of cartilage by using cationic peptide carrier (CPC) to deliver a pro-anabolic growth factor, IGF-1. A CPC +14N is comprised of 14 positively charged arginine (R) residues symmetrically distributed along the peptide length with hydrophilic asparagine (N) used as spacers. Its hydrophilic property minimizes its competitive binding within the synovial fluid. CPC-IGF-1 can rapidly penetrate through the full thickness of cartilage creating a drug depot to provide therapeutic drug doses over several days. B IGF-1 promotes chondrocyte proliferation, proteoglycan synthesis, and cell survival by activating the IGF-1 receptor. IGF-1 also suppresses IL-1-induced catabolism by downregulating the NF-kB pathway
Scheme for the synthesis of CPC-IGF-1 formulation and MALDI mass spectrometry confirming conjugation of CPC to IGF-1. A IGF-1 was first modified with maleimide (IGF-1-Maleimide) through reaction with a bifunctional NHS-PEG2-Maleimide linker via targeting the primary amines on IGF-1, thereby adding the maleimide to IGF-1 through stable amide bonds. The maleimide-modified IGF-1 (IGF-1-Maleimide) was then reacted with CPC +14N consisting of a cysteine residue to form CPC-IGF-1 conjugate through a stable thioether bond. B MALDI mass spectrometry confirming conjugation of CPC to IGF-1
A Intra-cartilage uptake ratios of CPCs in healthy, 20%, and 90% GAG depleted cartilage explants (* vs healthy explant) and their % retention within cartilage over a 7-day desorption period in 1× and 10× PBS. B Half cartilage disks are placed in a custom-designed transport chamber. 1-D diffusion of fluorescent solutes is allowed through the cartilage explant from the superficial zone (SZ) to the deep zone (DZ) for 24 h. A 100 μm slice is cut from the center of the explant and imaged in the X-Y plane to estimate the depth of penetration of solutes from SZ to DZ. The equilibrated explants are also desorbed in 1× PBS for 24 h and imaged to estimate retention of solutes in cartilage. Adapted with permission from [7, 13]. C Confocal images showing 1-D depth of penetration of free IGF-1, CPC, and dual-labeled CPC-IGF-1 conjugate in cartilage after 24 h absorption followed by 24 h desorption in 1× PBS with their respective penetration concentration profiles. The green channel shows IGF-1-FITC and the red channel shows CPC-Cy5. SZ is the superficial zone and DZ and deep zone
Experimental design to study biological effectiveness of CPC-IGF-1 using post-traumatic OA cartilage explant culture models. A Explants were treated with IL-1α (2 ng/ml) for the 16-day culture duration and received either (i) a single dose of 100 ng/ml (S) of free IGF-1, (ii) a continuous dose of 100 ng/ml (C) of free IGF-1, (iii) a single dose 1000 ng/ml (S) of free IGF-1, or (iv) a one-time dose of 1000 ng/ml (S) CPC-IGF-1 conjugate. (v) An untreated control, (vi) explants with single dose of CPC, and (vii) explants with IL-1α dosage only were also added as comparisons. B Percentage cumulative GAG loss to media over 16 days. C Cellular metabolism measured at days 8 and 16 using AlamarBlue assay. D Nitrite release determined by Griess assay at days 2, 8, and 16 (* vs control, # vs IL-1α, $ vs single dose 1000 ng/ml IGF-1 condition. In curve B, the statistical markers are color coordinated and all the data points enclosed within similar markers are statistically significant)
Chondrocyte viability after 8 and 16 days of culture. Viable cells are stained green while non-viable cells are marked red
Article
Background Insulin-like growth factor-1 (IGF-1) has the potential to be used for osteoarthritis (OA) treatment but has not been evaluated in clinics yet owing to toxicity concerns. It suffers from short intra-joint residence time and a lack of cartilage targeting following its intra-articular administration. Here, we synthesize an electrically charged cationic formulation of IGF-1 by using a short-length arginine-rich, hydrophilic cationic peptide carrier (CPC) with a net charge of +14, designed for rapid and high uptake and retention in both healthy and arthritic cartilage. Methods IGF-1 was conjugated to CPC by using a site-specific sulfhydryl reaction via a bifunctional linker. Intra-cartilage depth of penetration and retention of CPC-IGF-1 was compared with the unmodified IGF-1. The therapeutic effectiveness of a single dose of CPC-IGF-1 was compared with free IGF-1 in an IL-1α-challenged cartilage explant culture post-traumatic OA model. Results CPC-IGF-1 rapidly penetrated through the full thickness of cartilage creating a drug depot owing to electrostatic interactions with negatively charged aggrecan-glycosaminoglycans (GAGs). CPC-IGF-1 remained bound within the tissue while unmodified IGF-1 cleared out. Treatment with a single dose of CPC-IGF-1 effectively suppressed IL-1α-induced GAG loss and nitrite release and rescued cell metabolism and viability throughout the 16-day culture period, while free IGF at the equivalent dose was not effective. Conclusions CPC-mediated depot delivery of IGF-1 protected cartilage by suppressing cytokine-induced catabolism with only a single dose. CPC is a versatile cationic motif that can be used for intra-cartilage delivery of other similar-sized drugs.
 
Article
Objective Neutrophils and aberrant NETosis have been implicated in the pathogenesis of diverse autoimmune diseases; however, their roles in primary Sjögren’s syndrome (pSS) remain unclear. We aimed to reveal the potential roles of neutrophils and neutrophil extracellular traps (NETs) in pSS. Methods pSS patients were enrolled and NETosis markers were measured in plasma and labial glands using ELISA and immunofluorescence. The gene signatures of neutrophils were assessed by RNA-Seq and RT-PCR. Reactive oxygen species (ROS), mitochondrial ROS (MitoSOX) production, and JC-1 were measured by flow cytometry. Results NETosis markers including cell-free DNA (cf-DNA) and myeloperoxidase (MPO) in plasma and labial glands from pSS patients were significantly higher than healthy controls (HCs) and were associated with disease activity. RNA sequencing and RT-qPCR revealed activated type I IFN signaling pathway and higher expression of genes related to type I interferon in pSS neutrophils. Further stimulating with IFN-α 2a in vitro significantly induced ROS production and JC-1 monomer percentage in pSS neutrophils. Conclusions Our data suggest the involvement of neutrophils and enhanced NETosis in pSS patients. Further mechanism study in vitro revealed that type I IFN activation in pSS neutrophils led to mitochondrial damage and related ROS production which finally result in the generation of NETs.
 
Search and inclusion/exclusion criteria
In general analysis, the treatment with DMARD was not able to increase lean mass in patients
Positive mean of tocilizumab treatment and TNFi treatment
Regardless of the increase of the mean appendicular lean mass, treatment with DMARD showed no significant change in appendicular lean mass delta
Article
Introduction Rheumatoid arthritis (RA) is an autoimmune disease, characterized by chronic and systemic inflammation. Besides, it is known that RA patients may present several comorbidities, such as sarcopenia, a condition where patients present both muscle mass and muscle quality impairment. RA treatment is mostly pharmacological and consists in controlling systemic inflammation and disease activity. Despite that, the effect of pharmacological treatment on sarcopenia is not well characterized. Objective To summarize the effects of disease-modifying anti-rheumatic drugs (DMARDs) on skeletal muscle tissue in rheumatoid arthritis (RA) patients. Methods A systematic review of randomized clinical trials and observational studies was conducted using MEDLINE, Embase, Cochrane Library, and Web of Science. We selected studies with rheumatoid arthritis patients treated with disease-modifying anti-rheumatic drugs (DMARDs) that analyzed muscle mass parameters such as lean mass and appendicular lean mass. Methodological quality was assessed using the Newcastle-Ottawa Quality Assessment Scale. Standardized mean difference (SMD) and 95% confidence intervals (CI) were set. A meta-analysis of observational studies was performed using the R software, and we considered significant statistics when p < 0.05. Results Nine studies were included in this systematic review. In the meta-analysis, DMARD treatment had no positive difference ( p = 0.60) in lean mass. In the same way, in the appendicular lean mass parameter, our results showed that DMARDs did not have changes between baseline and post-treatment analysis ( p = 0.93). Conclusion There is no evidence of a significant effect of DMARD therapy, either synthetic or biological, on muscle mass. However, this association should be investigated with more studies.
 
Analgesia of established MIA-OA pain by DRG-AAV6-3.2iPA2 in male rats. Animal protocol is schematically outlined (A). The time courses for the group averages of sensitivity to vF, Pin, Heat, Cold, and weight bearing (Wb) before and after DRG injection of either AAV6-3.2iPA2 or AAV6-3.2NP (B–F); *p < 0.05, **p < 0.01, and ***p < 0.001 for comparisons to tBL within the group and #p < 0.05, ##p < 0.01, and ###p < 0.001 for comparisons between the groups post-AAV injection. Repeated measures parametric two-way ANOVA for vF, Heat, and Wb followed by Tukey post hoc, and non-parametric Friedman ANOVA for pin and cold tests and Dunn’s post hoc. Right panels of B–F show tAUCs calculated using the measures 14-day post-MIA and immediate before vector injection as tBL; **p < 0.01 and ***p < 0.001, comparisons of tAUCs between the groups (unpaired, two-tailed Student’s t tests for vF, Heat, and Wb and Mann–Whitney U tests for pin and cold). G The time courses (3 h) of sensitivity to vF and Pin after GBP (100 mg/kg, i.p.) in MIA+AAV-3.2PN rats, performed at the time point as indicated in A–C; **p < 0.01 and ***p < 0.001 vs. before GBP, repeated measures one-way ANOVA for vF with Tukey post hoc, and ###p < 0.001 for pin Friedman and Dunn’s post hoc. H Comparison of the efficacy between AAV-3.2iPA2 analgesia in MIA and GBP (i.p.) in 3.2NP rats by vF and pin tests, ***p < 0.001, and unpaired two-tailed Student’s t test for vF and Mann–Whitney U test for Pin. I Results of the CPP difference scores (seconds) of saline-paired chamber and the GBP-paired chamber between AAV-3.2iPA2 and AAV-3.2NP, ***p < 0.001 (unpaired, two-tailed Student’s t test)
IHC characterization of 3.2iPA2 expression in vivo (male). Representative IHC montage images show GFP-3.2iPA2 expression (green), colabeled with Tubb3 (A, red), CGRP (B, red), CaV3.2 (C, red), and GFAP (D, red). GFP-3.2iPA2 signal (green) is not detected in GFAP-positive glial cells (D, red). Scale bar, 100 μm for all images
Analgesia of DRG-AAV6-3.2iPA2 injection in female MIA rats. Analogous figures to Fig. 1 show significant analgesia after DRG delivery of AAV6-3.2iPA2 to the established MIA pain behavior in female rats. **p < 0.0.1 and ***p < 0.001 for comparisons to the treatment baseline (tBL) within the group and #p < 0.05, ##p < 0.01, and ###p < 0.001 for comparisons between the groups post-AAV injection (A–E). Repeated measures parametric two-way ANOVA for vF, Heat, and Wb followed by Tukey post hoc, and non-parametric Friedman ANOVA for pin and cold tests and Dunn’s post hoc. Right panels of A–E show tAUCs calculated using the measures 14-day post MIA and before vector injection as tBL; **p < 0.0.1 and ***p < 0.001, comparisons of tAUCs between the groups (unpaired, two-tailed Student’s t tests for vF, Heat, and Wb, and Mann–Whitney U tests for pin and cold). CPP difference scores (s) of saline-paired chamber and the GBP-paired chamber between AAV-3.2iPA2 and AAV-3.2NP, **p < 0.01 (unpaired, two-tailed Student’s t test) (F). Representative IHC montage images of GFP3.2iPA2 with Tubb3 show neuronal expression profile after AAV-3.2iPA2 injection (G). GFP-3.2iPA2 signal (green) is not detected in GFAP-positive glial cells (H, red). Scale bar, 100 μm for all images
Current-clamp analysis of AAV6-3.2iPA2 transduction on PSN excitability (male). Representative AP traces elicited by 250 ms depolarizing current of 200 pA (A) and 500 pA (B) (same cells) from rest membrane potential (RMP) were recorded on PSNs dissociated from the rats of saline, MIA, and GFP-expressing neurons in MIA rats treated with AAV6-3.2NP or AAV6-3.2iPA2, as indicated. Comparison of responses (number of APs evoked by a 250-ms stimulus) for the PSNs in different groups across a range of step current injections from 100 to 500 pA (C); ***p < 0.001, two-way ANOVA of main effects of the groups with Bonferroni post hoc. Scatter plots with bars show analyses of the RMP (D), rheobases (E), and AP numbers evoked by input current at 250 pA (F) and 500 pA (F1) from RMP, respectively. The number in each group is the number of analyzed neurons per group. ** and *** denote p < 0.01 and < 0.001, respectively. One-way ANOVA and Turkey post hoc
Article
Background Peripheral and central nociceptive sensitization is a critical pathogenetic component in osteoarthritis (OA) chronic pain. T-type calcium channel 3.2 (Ca V 3.2) regulates neuronal excitability and plays important roles in pain processing. We previously identified that enhanced T-type/Ca V 3.2 activity in the primary sensory neurons (PSNs) of dorsal root ganglia (DRG) is associated with neuropathic pain behavior in a rat model of monosodium iodoacetate (MIA)-induced knee OA. PSN-specific T-type/Ca V 3.2 may therefore represent an important mediator in OA painful neuropathy. Here, we test the hypothesis that the T-type/Ca V 3.2 channels in PSNs can be rationally targeted for pain relief in MIA-OA. Methods MIA model of knee OA was induced in male and female rats by a single injection of 2 mg MIA into intra-knee articular cavity. Two weeks after induction of knee MIA-OA pain, recombinant adeno-associated viruses (AAV)-encoding potent Ca V 3.2 inhibitory peptide aptamer 2 (Ca V 3.2iPA2) that have been characterized in our previous study were delivered into the ipsilateral lumbar 4/5 DRG. Effectiveness of DRG-Ca V 3.2iPA2 treatment on evoked (mechanical and thermal) and spontaneous (conditioned place preference) pain behavior, as well as weight-bearing asymmetry measured by Incapacitance tester, in the arthritic limbs of MIA rats were evaluated. AAV-mediated transgene expression in DRG was determined by immunohistochemistry. Results AAV-mediated expression of Ca V 3.2iPA2 selective in the DRG-PSNs produced significant and comparable mitigations of evoked and spontaneous pain behavior, as well as normalization of weight-bearing asymmetry in both male and female MIA-OA rats. Analgesia of DRG-AAV-Ca V 3.2iPA1, another potent Ca V 3.2 inhibitory peptide, was also observed. Whole-cell current-clamp recordings showed that AAV-mediated Ca V 3.2iPA2 expression normalized hyperexcitability of the PSNs dissociated from the DRG of MIA animals, suggesting that Ca V 3.2iPA2 attenuated pain behavior by reversing MIA-induced neuronal hyperexcitability. Conclusions Together, our results add therapeutic support that T-type/Ca V 3.2 in primary sensory pathways contributes to MIA-OA pain pathogenesis and that Ca V 3.2iPAs are promising analgesic leads that, combined with AAV-targeted delivery in anatomically segmental sensory ganglia, have the potential for further development as a peripheral selective T-type/Ca V 3.2-targeting strategy in mitigating chronic MIA-OA pain behavior. Validation of the therapeutic potential of this strategy in other OA models may be valuable in future study.
 
Characterization of the extracellular vesicles (EVs) for B cell treatment. A Representative plots of EV size according to the FSC-A and SSC-A parameters defined by using beads with reference diameters (0.1, 1, 3, and 6 µm) (left panel) or granulocyte, monocyte lymphocyte, and platelet populations (right panel). B EVs are sensitive to detergent treatment. Representative CD41a versus FSC-A dot plots and CD41a histograms of EVs untreated and treated with 0.05% Triton X-100. CD41a positivity was rapidly altered after Triton X-100 addition. C The vesicular nature and size of EVs. Representative microphotographs of scanning transmission electron microscopy of EVs. These images demonstrate the vesicular shape and size expected for m/lEVs. D Representative IgG expression of m/lEVs from HD (HD-m/lEVs), patients with RA (PRA-m/lEVs), and those from patients with RA that form immune complexes (PRA-m/lEV-ICs). Representative results of at least three independent experiments are shown for all the experiments
Low frequency of CD69⁺ and CD86⁺ cells and proliferation of in vitro-activated B cells exposed to m/lEVs. A Representative contour plots of CD69 expression in B cells from HD cultured for 24 h without stimulation (unstimulated, Unst) or with anti-BCR in the absence or presence of m/lEVs from HD (HD-m/lEVs), patients with RA (PRA-m/lEVs), and patients with RA-forming immune complexes (PRA-m/lEV-ICs). B Frequency of CD69⁺ (left panel), CD80⁺ (center panel), and CD86⁺ (right panel) B cells, cultured for 24 h without stimulation or with anti-BCR in the absence or presence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEV-ICs. The data of five HD donors and medians are shown. C The frequency of CD69⁺ (left panel), CD80⁺ (center panel), and CD86.⁺ (right panel) B cells, cultured for 72 h without stimulation or with anti-BCR and CpG in the absence or presence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEV-ICs. The data of eight HD donors and medians are shown. D Representative histogram of CellTracer expression on B cells cultured for 72 h (left panel), consolidated data of proliferating B cells (center panel), and the index of division (right panel) of B cells cultured for 72 h without stimulation or with anti-BCR + CpG in the absence or presence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEV-ICs. Data of 6 − 8 HD donors and median are shown. B–D Kruskal − Wallis test with Dunn’s post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
Less calcium mobilization of activated B cells exposed to m/lEVs. A The kinetic of calcium mobilization in total B cells based on the ratio of Indo 1 AM bound/unbound. The B cells were kept unstimulated for 30 s and then treated with anti-BCR in the presence or absence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEV-ICs for 210 s (indicated with an arrow). The mean of 5 HD is shown. In the inserted small figure, the linear slopes of calcium mobilization at 30 − 90-s period (linear) are shown. Data of five HD and median are shown. B The area under the curve of calcium mobilization in total B cells at 0 − 30-s (left panel), 30 − 120-s (center panel), and 120 − 240-s (right panel) periods. The data of 5 HD and median are shown. C The area under the curve of calcium mobilization in the 30 − 120-s (top panel) and 120 − 240-s (bottom panel) periods for transitional, CD27⁻IgM⁺, CD27⁺IgM⁺, CD27⁺IgM⁻, and CD27⁻IgM⁻ B cell subsets. Data of 5 HD and median are shown. D Representative histogram of tyrosine phosphorylation (p-Tyr, left panel) in B cells without stimulation or those treated with anti-IgM in the presence or absence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEV-ICs. The mean fluorescence intensity of p-Tyr (right panel) in B cells of 7 HD donors and median are shown. A–D Kruskal − Wallis test with Dunn’s post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
Regulatory effect of m/lEVs in B cells from patients with RA. A Representative contour plots of the CD86 expression in B cells from patients with RA, cultured for 24 h without stimulation (unstimulated, Unst) or with anti-BCR in the absence or presence of m/lEVs from HD (HD-m/lEVs), patients with RA (PRA-m/lEVs), and patients with RA-forming immune complexes (PRA-m/lEV-ICs). B The frequency of CD69⁺ (left panel), CD80⁺ (center panel), and CD86⁺ (right panel) B cells, cultured for 24 h without stimulation or with anti-BCR in the absence or presence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEVs-ICs. Data of five patients with RA and median are shown. C The frequency of CD69⁺ (left panel), CD80⁺ (center panel), and CD86.⁺ (right panel) B cells, cultured for 72 h without stimulation or with anti-BCR and CpG in the absence or presence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEVs-ICs. Data of six patients with RA and median are shown. D The frequency of proliferating B cells cultured of 72 h without stimulation or with anti-BCR and CpG in the absence or presence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEVs-ICs. Data of six patients with RA and median are shown. E Kinetic of calcium mobilization of total B cells based on the ratio of Indo 1 AM bound/unbound in total B cells. After 30 s of acquisition, the B cells were stimulated with anti-BCR in the presence or absence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEVs-ICs (indicated with an arrow). The mean of five patients with RA is shown. In the inserted small figure, the linear slopes of calcium mobilization in total B cells at 30 − 90-s period (linear) are shown. Data of five patients with RA and median are shown. F Area under the curve of calcium mobilization at the baseline (left panel), incremental (center panel), and post-incremental (right panel) phases of B cells. Data of five patients with RA and median are shown. G The median fluorescence intensity (MFI) of tyrosine phosphorylation (p-Tyr) in B cells without stimulation or with anti-IgM in the presence or absence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEV-ICs. Data of five patients with RA and median are shown. B–G Kruskal − Wallis test with Dunn’s post-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
m/lEVs can directly increase B cell production of antibodies and indirectly activate autologous B cells from patients with RA by innate immune cells. A Representative contour plot of plasmablast population CD27⁺CD38hi in enriched B cells cultured with CD40L and IL-21 for 7 days. Plasmablasts were analyzed inside of lymphocyte, live/dead negative, and CD19 + regions. B Frequency of plasmablast cells as explained in A, in the absence or presence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEVs-ICs. Data of five HD, six patients with RA, and median are shown. C, D Levels of C IgM and D IgG in supernatant of culture of B cells as explained in A, in the absence or presence of HD-m/lEVs, PRA-m/lEVs, or PRA-m/lEVs-ICs. Line indicates lowest detection limit of the ELISA (2 ng/mL). E Monocyte-derived macrophages (MDM) from HD and patients with RA were exposed or not to PRA-m/lEVs or PRA-m/lEVs-ICs for 6 h. Autologous B cells were added to previously washed MDM (B cells: MDM ratio of 1-2:1) and incubated for 96 h. F Frequency of CD69⁺, CD80⁺, and CD86.⁺ B cells from cocultures with MDM unstimulated (Unst) or treated with PRA-m/lEVs or PRA-m/lEV-ICs, as detailed in E. G MDM from patients with RA were exposed or not to PRA-m/lEV-ICs for 6 h. Autologous B cells were cultured with the supernatant of exposed MDM and incubated for 96 h. H Frequency of CD69+, CD80+, and CD86+ B cells cultured with the supernatant of MDM unstimulated (Unst) or treated with PRA-m/lEV-ICs, as detailed in G. Data of six HD and six patients with RA are shown. B–D, F Two-way ANOVA test with Šidák post-test. H Wilcoxon test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
Article
Background Extracellular vesicles are involved in the intercellular communication of the immune system. In rheumatoid arthritis (RA), these structures are considered a source of autoantigens that drive proinflammatory responses of innate immune cells. A high concentration of circulating medium/large size extracellular vesicles (m/lEVs) and m/lEVs forming immune complexes (m/lEV-ICs) have been associated with disease activity and systemic inflammation in patients with RA. B cells are central components of RA immunopathology because of their involvement in the production of autoantibodies, antigen presentation, and cytokine production. However, the effect of m/lEVs on B cell function in the context of RA and other autoimmune diseases remains unknown. Methods We evaluated the effect of m/lEVs obtained from healthy donors (HD) and patients with RA on B cell responses in vitro. In addition, we evaluated the effect of pre-exposition of monocyte-derived macrophages (MDM) to m/lEVs on activation of autologous B cells from HD and patients. Results The presence of m/lEVs reduced the frequency of CD69 ⁺ and CD86 ⁺ B cells from HD activated by an agonist of antigen receptor. This regulation of the B cell activation markers by m/lEVs was partially dependent on phosphatidylserine binging. These m/lEVs also reduced the proliferation, calcium mobilization, and global phosphorylation of tyrosine. Similar responses were observed in B cells from patients with RA. However, the presence of m/lEVs promoted high antibody levels in B cells cultured with T cell-dependent stimuli by 7 days. In addition, despite the direct inhibitory effect of m/lEVs on early B cell responses, when B cells were cocultured with autologous MDM previously exposed to m/lEVs or m/lEV-ICs, an increased frequency of CD69 ⁺ B cells from patients with RA was observed, albeit not with cells from HD. Conclusions These data together suggest that m/lEVs have a direct modulatory effect in early responses of B cells through B cell receptor that can potentially fail in patients with RA because of the impact of these vesicles over cells of the innate immune system. This phenomenon can potentially contribute to the loss of tolerance and disease activity in patients with RA.
 
Abbreviations CG: Cryoglobulinemia; CryoVas: Cryoglobulinemic vasculitis; SLE: Systemic lupus erythematosus; Ig: Immunoglobulin; RF: Rheumatoid factor; ANA: Antinuclear antibodies; HIV: Human immunodeficiency virus; HBV: Hepatitis B Virus; HCV: Hepatitis C Virus; SLEDAI: Systemic lupus erythematosus activity index; SLICC: Systemic lupus international collaborating clinics; EULAR: European League Against Rheumatism.
Clinical signs associated with cryoglobulinemia
Article
Objectives The clinical value of cryoglobulinemia (CG) in systemic lupus erythematosus (SLE) is largely unknown. The aim of this retrospective study was to describe the characteristics of CG in SLE, its impact on SLE phenotype, and the features associated with cryoglobulinemic vasculitis (CryoVas) in SLE patients. Methods This retrospective study conducted in a French university hospital reviewed the data from 213 SLE patients having been screened for CG between January 2013 and December 2017. SLE patients positive for CG were compared to SLE patients without CG. Patients were classified as CryoVas using the criteria of De Vita et al. Results Of the 213 SLE patients included (mean age 29.2 years, female sex 85%), 142 (66%) had at least one positive CG in their history, 67% of them having a persistent CG at follow-up. CG was type III in 114 (80%) cases and type II in 27 (19%) cases. The mean concentration of the cryoprecipitate was 40mg/L (range 0-228). Patients with CG had significantly more C4 consumption. Among patients with CG, 21 (15%) developed a CryoVas. The clinical manifestations of patients with CryoVas were mainly cutaneous (purpura, ulcers, digital ischemia) and articular, without any death at follow-up. Severe manifestations of CG included glomerulonephritis in 1/21 (5%) patients and central nervous system involvement in 4/21 (19%) patients. A response to first-line treatments was observed in 12/13 (92%) patients, but relapses were observed for 3 of them. Conclusion CG is frequent in SLE, but mostly asymptomatic. CryoVas features involve mostly joints, skin, and general symptoms. CryoVas in SLE appears to be a specific condition, with a low prevalence of neuropathy, membranoproliferative glomerulonephritis, and severe manifestations.
 
Network analysis of synovial RNA sequencing in early RA reveals gene-gene interactions uniquely linked to the lympho-myeloid pathotype. A Analytical pipeline using network approach to extract informative networks and predictive gene pairs from RNA-seq profiles. Having defined subgroups of patients, an extensive network of interactions is built using merged KEGG pathways enriched with micro-RNAs and transcription factors. The network is replicated for each subgroup and the average expression level of each gene in a subgroup is used to infer a weight on each network node. A first filtering step removes, from each network, nodes (genes) whose weight (subgroup average expression level) is below an optimal threshold obtained via percolation analysis. The second filtering step pull out links (gene-gene interactions) overlapping two or more networks. Robust linear regression with interaction term is used to extract significant gene-gene links. A logistic regression model is built for each significant gene-gene pair to predict response. Ability to predict response is tested by receiver operating characteristic (ROC) curve analysis. B Network of unique active interactions in the lympho-myeloid pathotype. Clusters LM1-LM4. Selected clusters of interest. Labels are determined by gene ontology (GO)/pathway enrichment analysis. Percentages indicate the number of cluster genes included in the associated GO/pathway term. Cluster LM1. Cluster of chemokines needed for leukocyte recruitment (93.5% enrichment). Cluster LM2. Antigen processing and presentation with T cell activation genes (100% enrichment). Cluster LM3. Group of focal adhesion genes comprising collagens, integrins and laminins (93.9% enrichment). Cluster LM4. TNF signaling through mTOR (48.8% enrichment). Cluster LM5. Interferon regulation signaling (87.5% enrichment). Cluster LM6. Genes of the intrinsic and extrinsic apoptotic pathways (50.8% enrichment). C Correlation plots showing differential gene-gene correlations with interactions associated with pathotype. Statistical analysis by robust linear regression model. p-value of the gene to pathotype interacting term is shown. Correlation plots of gene pairs CD28 and PIK3R1, CD79A and LYN, and TNC and ITGB7 across different pathotypes
PPAR-γ signaling is key driver of the diffuse-myeloid pathotype while Wnt/Notch signaling pathways characterize the pauci-immune fibroid pathotype. A Network of unique active interactions in the diffuse-myeloid pathotype. Cluster DM1. Extracellular matrix genes for focal adhesion (75.6% enrichment). Cluster DM2. Cluster of PPAR signaling pathway (78.6% enrichment). B Network of unique active interactions in the pauci-immune fibroid pathotype. Cluster PF1. Group of focal adhesion genes comprising collagens, integrins and laminins (93.3% enrichment). Cluster PF2. Cluster of genes of the Ras signaling pathway (76.6% enrichment). Cluster PF3. Clusters of Notch-, Wnt- and TGF-beta signaling (95.8% enrichment). Cluster PF4. Cytokine-cytokine interaction of pro-inflammatory genes (100% enrichment) Cluster PF5. Vescular permeability genes (57.1% enrichment). C Correlation plots showing differential gene-gene correlations with interactions associated with pathotype. Statistical analysis by robust linear regression model. p-value of the gene to pathotype interacting term is shown. Regression plot of ITGAV and LAMA3, WNT11 and SFRP2 in different pathotypes
Apoptosis and SOCS/STAT signaling differentiate responders to methotrexate-based therapy from non-responders. A Network of unique active interactions in conventional synthetic DMARD responders. Cluster R1. Cell survival genes part of the PI3K-Akt signaling pathway (53.1% enrichment). Cluster R2. Extracellular matrix receptor genes (100% enrichment). Cluster R3. Chemokines receptors binding chemokines (100% enrichment). B Robust linear regression of SOCS2 and STAT2 with interaction term associated with response. p-value of the gene to response interacting term is shown. C Logistic regression of response as a function of SOCS2 and STAT2. p-value of the response to gene interacting term is shown. Expression of STAT2 is dichotomized at ± 1 standard deviation. D Receiver operating characteristic curve analysis of the response prediction ability of the robust linear model incorporating the ratio term (black line) or not (dotted blue line)
Gene pair interactions linked to endothelial activation and Akt signaling enhance prediction of response to methotrexate-based therapy. A Network of unique active interactions in conventional synthetic DMARD poor responders. Cluster NR1. Antigen processing and presentation cluster (100% enrichment). Cluster NR2. Genes of the chemokine signaling pathway (100% enrichment). Cluster NR3. Cluster associated to Wnt signaling pathway (74.2% enrichment). Cluster NR4. Cluster linked to VEGFR2 mediated vascular permeability (38.8% enrichment). Red boxes highlight predictive gene pairs. B–E, H, L Robust linear regression with interaction term associated with response for BGNAI3 and CXCR5CNOS3 and CAMK1, DNOS3 and AKT3EAKT1 and PPP2R3B, HNOS3 and CAMK2D, LATP1B1 and PIK3CD. p-values of the interacting terms are shown. F, I, M Logistic regression of response as a function of FAKT1 and PPP2R3B, INOS3 and CAMK2D, MATP1B1 and PIK3CD. p-values of the response to gene interacting term are shown. Expression of the second gene is dichotomized at ± 1 standard deviation. G, J, N Receiver operating characteristic (ROC) curve analysis of the of robust linear model ability to predict response using GAKT1 and PPP2R3BJNOS3 and CAMK2DNATP1B1 and PIK3CD. All plots show a ROC curve for both the model including the gene-gene ratio interaction term (in black) and the equivalent model excluding the ratio
Baseline demographics of treatment-naïve RA patients recruited into the Pathobiology of Early Arthritis Cohort (PEAC)
Article
Background To determine whether gene-gene interaction network analysis of RNA sequencing (RNA-Seq) of synovial biopsies in early rheumatoid arthritis (RA) can inform our understanding of RA pathogenesis and yield improved treatment response prediction models. Methods We utilized four well curated pathway repositories obtaining 10,537 experimentally evaluated gene-gene interactions. We extracted specific gene-gene interaction networks in synovial RNA-Seq to characterize histologically defined pathotypes in early RA and leverage these synovial specific gene-gene networks to predict response to methotrexate-based disease-modifying anti-rheumatic drug (DMARD) therapy in the Pathobiology of Early Arthritis Cohort (PEAC). Differential interactions identified within each network were statistically evaluated through robust linear regression models. Ability to predict response to DMARD treatment was evaluated by receiver operating characteristic (ROC) curve analysis. Results Analysis comparing different histological pathotypes showed a coherent molecular signature matching the histological changes and highlighting novel pathotype-specific gene interactions and mechanisms. Analysis of responders vs non-responders revealed higher expression of apoptosis regulating gene-gene interactions in patients with good response to conventional synthetic DMARD. Detailed analysis of interactions between pairs of network-linked genes identified the SOCS2/STAT2 ratio as predictive of treatment success, improving ROC area under curve (AUC) from 0.62 to 0.78. We identified a key role for angiogenesis, observing significant statistical interactions between NOS3 (eNOS) and both CAMK1 and eNOS activator AKT3 when comparing responders and non-responders. The ratio of CAMKD2/NOS3 enhanced a prediction model of response improving ROC AUC from 0.63 to 0.73. Conclusions We demonstrate a novel, powerful method which harnesses gene interaction networks for leveraging biologically relevant gene-gene interactions leading to improved models for predicting treatment response.
 
Dietary magnesium deficiency aggravated DMM - induced articular cartilage damage. A Schematic representation of in vivo experimental protocols. B Representative images of Safranin O/Fast Green–stained sections of knee joints from sham and DMM-induced OA mice fed with diets containing different amounts of magnesium (500 mg/kg, 300 mg/kg, 100 mg/kg). The low magnesium groups presented with severe cartilage damage and less Safranin O staining. Magnified images of regions marked by black boxes. Scale bar: 100 μm. C, D The severity of articular cartilage damage of mice groups above was quantified with OARSI scoring. The maximum OARSI scores (the maximum score of four compartments) and summed scores (sum score of four compartments) were presented (n = 9). Data were expressed as the mean ± SD and analyzed by one-way ANOVA test. (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001)
Reduced autophagy contributed to pro-catabolic and anti-anabolic effects after the exposure to magnesium deficient conditions. A Mouse chondrocytes were cultured with TNF-α under different magnesium conditions (0.7 mM, 0.4 mM, and 0.1 mM). Protein levels of MMP13 was detected by western blot. B The quantification of protein expression of MMP13 was done by densitometry analysis of the protein bands. Values were normalized to GAPDH (n = 5). C Protein level of LC3-II and Beclin-1 under different magnesium conditions were analyzed by western blot. D The quantification of protein expression of LC3-II and Beclin-1 was done by densitometry analysis of the protein bands. Values were normalized to β-tubulin (n = 5). E Chondrocyte imaging with DALGreen staining under different magnesium conditions. Autolysosomes were marked with white arrows. Scale bar: 5 μm. F Number of autolysosomes per cell was quantitated. G TEM analysis of autophagosomes in chondrocytes under different magnesium conditions. Autophagosomes were marked with white arrows. Scale bar: 5 μm. H Mouse chondrocytes were cultured with TNF-α under different magnesium conditions with or without rapamycin (Rapa) pretreatment. Protein levels of MMP13 was detected by western blot. I The quantification of protein expression of MMP13 was done by densitometry analysis of the protein bands. Values were normalized to GAPDH (n = 3). (J) Sox9, Col2a1, Acan, and Mmp-13 genes expression were assessed by qRT-PCR (n = 3). β-actin was used as housekeeping gene. Data were expressed as the mean ± SD and analyzed by one-way ANOVA test. (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001)
Magnesium deficiency activated Wnt/β-catenin signaling in chondrocytes. Mouse chondrocytes were cultured under different magnesium conditions (0.7 mM, 0.4 mM and 0.1 mM). Protein levels of β-catenin in total cell lysates (A, B) and nuclear fractions (C, D) were detected by western blot. The quantitation of protein expression of β-catenin was done by densitometry analysis of the protein bands. Values were normalized to β-tubulin and TATA-binding protein (TBP), respectively (n = 8 for β-catenin in total cell lysates; n = 4 for nuclear β-catenin). E mRNA levels of Catenin, Wnt3a, Wnt5a, Wnt5b, and Wnt16 were investigated by qRT-PCR (n = 3, 4 or 5). β-actin was used as housekeeping gene. F, G Immunostaining and quantitative analysis of cells positive for β-catenin in sham and DMM-induced OA mice fed with diets containing different amounts of magnesium (500 mg/kg, 300 mg/kg, 100 mg/kg) (n = 9). Scale bar: 100 μm. Data were expressed as the mean ± SD and analyzed by one-way ANOVA test. (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001)
Activation of Wnt/β-catenin signaling contributed to reduction in autophagy. A Mouse chondrocytes were cultured under different magnesium conditions with or without XAV939 pretreatment. Protein levels of LC3-II and Beclin-1 under different magnesium conditions were analyzed by western blot. B The quantification of protein expression of LC3-II and Beclin-1 was done by densitometry analysis of the protein bands. Values were normalized to β-tubulin (n = 5). C Chondrocyte imaging with DALGreen staining. Autolysosomes were marked with white arrows. Scale bar: 5 μm. D Number of autolysosomes per cell was quantified. Data were expressed as the mean ± SD and analyzed by one-way ANOVA test. (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001)
Impaired magnesium homeostasis induced by magnesium deficiency was mediated by TRPM7. A Representative fluorescence images showing the concentration of intracellular Mg²⁺ of chondrocytes cultured under different magnesium conditions (0.7 mM, 0.4 mM, and 0.1 mM) for 48 h. B Intracellular Mg²⁺ concentration quantified by measuring the intensity of fluorescence (n = 10). C Protein level of TRPM7 under different magnesium conditions for 48 h were analyzed by western blot. D The quantification of protein expression of TRPM7 was done by densitometry analysis of the protein bands. Values were normalized to β-tubulin (n = 3). E, F Immunostaining and quantitative analysis of cells positive for TRPM7 in sham and DMM-induced OA mice fed with diets containing different amounts of magnesium (500 mg/kg, 300 mg/kg, 100 mg/kg) (n = 9). Scale bar: 100 μm. Data were expressed as the mean ± SD and analyzed by one-way ANOVA test (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001). G Summary of the present study: Extracellular magnesium deficiency downregulates the expression of TRPM7, resulting in reduced intracellular magnesium. Subsequently, intracellular magnesium deficiency inhibits autophagy of chondrocytes by the activation of the Wnt/β-catenin signaling pathway and thus aggravates cartilage damage
Article
Background Dietary magnesium deficiency, which is common in modern diet, has been associated with osteoarthritis (OA) susceptibility. Despite this clinical association, no study has addressed if dietary magnesium deficiency accelerates OA development, especially at molecular level. This study aimed to explore aggravating effects of dietary magnesium deficiency on cartilage damage in an injury-induced murine OA model and to determine the underlying mechanism. Methods Twelve-week-old C57BL/6J mice subject to injury-induced OA modeling were randomized into different diet groups in which the mice were fed a diet with daily recommended magnesium content (500 mg/kg) or diets with low magnesium content (100 or 300 mg/kg). Articular cartilage damage was evaluated using the OARSI score. To determine molecular mechanisms in vitro, mouse chondrocytes were treated with media of low magnesium conditions at 0.1 and 0.4 mM, compared with normal magnesium condition at 0.7 mM as control. Anabolic and catabolic factors, autophagy markers, β-catenin, Wnt ligands, and a magnesium channel transient receptor potential cation channel subfamily member 7 (TRPM7) were analyzed by quantitative real-time PCR and immunoblotting. Autolysosomes were detected by DALGreen staining via fluorescence microscopy and autophagosomes were evaluated by transmission electron microscopy. Autophagy markers, β-catenin, and TRPM7 were assessed in vivo in the mouse cartilage, comparing between dietary magnesium deficiency and normal diet, by immunohistochemistry. Results Dietary magnesium deficiency aggravated injury-induced cartilage damage, indicated by significant higher OARSI scores. Autophagy markers LC3-II and Beclin-1 were decreased both in low magnesium diet-fed mice and low magnesium-treated chondrocytes. The number of autolysosomes and autophagosomes was also reduced under low magnesium conditions. Moreover, magnesium deficiency induced decreased anabolic and increased catabolic effect of chondrocytes which could be restored by autophagy activator rapamycin. In addition, reduced autophagy under low magnesium conditions is mediated by activated Wnt/β-catenin signaling. The expression of TRPM7 also decreased in low magnesium diet-fed mice, indicating that downstream changes could be regulated through this channel. Conclusions Dietary magnesium deficiency contributes to OA development, which is mediated by reduced autophagy through Wnt/β-catenin signaling activation. These findings indicated potential benefits of adequate dietary magnesium for OA patients or those individuals at high risk of OA.
 
Article
Background Male HLA-B27-positive radiographic-axial spondyloarthritis (r-axSpA) patients are prone to have severe spinal radiographic progression, but the underlying mechanisms are unclear. We recently showed that persistently elevated Lipocalin 2 (LCN2; L) reflects sacroiliac joint (SIJ) inflammation. LCN2 binds to MMP9. Concomitant elevation of L and LCN2-MMP9 (LM) was detected in many inflammatory diseases. We asked whether L and LM play similar roles in r-axSpA pathogenesis. Methods We analyzed 190 axSpA patients (123 radiographic and 67 non-radiographic axSpA) who had no detectable circulating Oncostatin M, to avoid complications due to cross-talk between pathways. L and LM levels from a single blood sample of each patient were measured and were correlated with MRI and modified stoke AS (mSASS) scoring. Association of elevated L (L+) or concurrent L+ and elevated LM (LM+) patterns with B27 status and gender were assessed. Results In L+LM+ axSpA patients, both L and LM levels correlated with MRI SPARCC SIJ scores, but only LM levels correlated with MRI Berlin Spine Scores, suggesting LM is a biomarker for both SIJ and spinal inflammation. Among patients with minimal spinal ankylosis (mSASSS < 10), 65% of male r-axSpA patients are L+LM+, while 30% and 64% of female patients are L+LM+ and L+, respectively, supporting the role of LM with disease progression. In B27+ L+LM+ male patients, both L and LM (but not CRP) levels correlate with mSASSS. B27 positivity and maleness have additive effects on spondylitis progression, suggesting concurrent high L and LM elevations are associated with B27+ male patients having more significant radiographic damage. L+ B27-negative male patients or L+ female patients are more likely to have milder disease. Conclusion L and LM are informative biomarkers for SIJ and spinal inflammation, as well as for ankylosing development in r-axSpA patients. Distinctive L+LM+ or L+ patterns not only could distinguish clinically aggressive vs milder course of disease, respectively, but also provide an explanation for B27-positive male patients being the most susceptible to severe ankylosis.
 
Comparison of AS patients, IBD patients, AS patients with concomitant IBD, and healthy controls in the Australian cohort, sampled from the terminal ileum, rectum, right colon and stool, whilst accounting for repeated sampling. A sPLSDA visualisation of overall microbiome composition (beta diversity). B PERMANOVA significance testing of beta diversity. C Comparison of species richness (alpha diversity). D ROC analysis for the detection of IBD in the stools of AS patients
Comparison of stool samples of AS patients from three distinct geographic regions: Australia, Italy and Sweden. A sPLSDA visualisation of microbiome composition (beta diversity). B PERMANOVA significance testing of beta diversity. C Comparison of species richness (alpha diversity)
Comparison of microbiome composition in the Australian cohort, sampled from terminal ileum, rectum, right colon and stool, whilst accounting for repeated sampling. Composition was measured according to BASDAI. A sPLSDA visualisation of microbiome composition (beta diversity) according to BASDAI. B PERMANOVA significance testing of beta diversity according to BASDAI. C Comparison of species richness (alpha diversity) according to BASDAI
Comparison of microbiome composition in the Australian cohort, sampled from terminal ileum, rectum, right colon and stool, whilst accounting for repeated sampling. Composition was measured according to FCP levels. A sPLSDA visualisation of microbiome composition (beta diversity) according to FCP level. B PERMANOVA significance testing of beta diversity according to FCP level. C Comparison of species richness (alpha diversity) according to FCP level
Bacterial genera significantly associated (p < 0.05) with multiple clinical attributes. The numbered boxes correspond to the abundance coefficient, with enrichment displayed in green and depletion displayed in red
Article
Background Multiple studies have confirmed dysbiosis in ankylosing spondylitis (AS) and inflammatory bowel disease (IBD); however, due to methodological differences across studies, it has not been possible to determine if these diseases have similar or different gut microbiomes. Results In this study, faecal and intestinal biopsies were obtained from 33 Australian AS patients (including 5 with concomitant IBD, ‘AS-IBD’), 59 IBD patients and 105 healthy controls. Stool samples were also obtained from 16 Italian AS patients and 136 Swedish AS patients. Focusing on the Australian cohort, AS, AS-IBD and IBD patients differed from one another and from healthy controls in both alpha and beta diversity. AS patients with and without clinical IBD could be distinguished from one another with moderate accuracy using stool microbiome (AUC=0.754). Stool microbiome also accurately distinguished IBD patients from healthy controls (AUC=0.757). Microbiome composition was correlated with disease activity measured by BASDAI and faecal calprotectin (FCP) levels. Enrichment of potentially pathogenic Streptococcus was noted in AS, AS-IBD and IBD patients. Furthermore, enrichment of another potentially pathogenic genus, Haemophilus, was observed in AS, AS-IBD, IBD, AS patients with increased BASDAI, and IBD patients with faecal calprotectin >100 μg/mg. Apart from these genera, no other taxa were shared between AS and IBD patients. Conclusions In conclusion, the distinct gut microbiome of AS and AS-IBD patients compared to IBD patients and healthy controls is consistent with immunological and genetic evidence suggesting that the gut plays a different role in driving AS compared with IBD. However, enrichment of two potentially pathogenic genera in both diseases suggests that the presence of a shared/common microbial trigger of disease cannot be discounted.
 
Two patient class trajectories identified with latent class modeling of DAS28-ESR (N = 775)
Receiver-operating characteristic (ROC) curves of algorithms validated on the test set for the “DAS28-ESR” model (N = 410). The area under the curve (AUC) for the least absolute shrinkage and selection operator (LASSO; dashed) and random forests (RF; solid) methods validated on the test set were 0.79 and 0.68, respectively. LASSO, least absolute shrinkage and selection operator; RF, random forests
Feature importance plots of characteristics for A LASSO and B random forests. Feature importance plots for A the DAS28-ESR model with LASSO algorithm, B the components of DAS28-ESR model with LASSO algorithm, C the DAS28-ESR model with random forests methods, and D the components of DAS28-ESR model with random forests methods are provided below. Feature importance was determined based on standardized LASSO coefficients and the Gini score for random forests. The most important feature was set to 100, and the rest is relative to that feature. DAS28ESR, Disease Activity Score with 28-joint count with erythrocyte sedimentation rate; RA, rheumatoid arthritis; SJC66, 66 Swollen Joint Count; ESR, erythrocyte sedimentation rate; ACPA, anti-citrullinated protein antibodies; TJC68, 68 Tender Joint Count; CRP, C-reactive protein; HAQ, Health Assessment Questionnaire Score; PhGA, Physician’s Global Assessment of Disease Activity; PtGA, Patient’s Global Assessment of Disease Activity; MI, missing indicator
Article
Background Methotrexate is the preferred initial disease-modifying antirheumatic drug (DMARD) for rheumatoid arthritis (RA). However, clinically useful tools for individualized prediction of response to methotrexate treatment in patients with RA are lacking. We aimed to identify clinical predictors of response to methotrexate in patients with rheumatoid arthritis (RA) using machine learning methods. Methods Randomized clinical trials (RCT) of patients with RA who were DMARD-naïve and randomized to placebo plus methotrexate were identified and accessed through the Clinical Study Data Request Consortium and Vivli Center for Global Clinical Research Data. Studies with available Disease Activity Score with 28-joint count and erythrocyte sedimentation rate (DAS28-ESR) at baseline and 12 and 24 weeks were included. Latent class modeling of methotrexate response was performed. The least absolute shrinkage and selection operator (LASSO) and random forests methods were used to identify predictors of response. Results A total of 775 patients from 4 RCTs were included (mean age 50 years, 80% female). Two distinct classes of patients were identified based on DAS28-ESR change over 24 weeks: “good responders” and “poor responders.” Baseline DAS28-ESR, anti-citrullinated protein antibody (ACPA), and Health Assessment Questionnaire (HAQ) score were the top predictors of good response using LASSO (area under the curve [AUC] 0.79) and random forests (AUC 0.68) in the external validation set. DAS28-ESR ≤ 7.4, ACPA positive, and HAQ ≤ 2 provided the highest likelihood of response. Among patients with 12-week DAS28-ESR > 3.2, ≥ 1 point improvement in DAS28-ESR baseline-to-12-week was predictive of achieving DAS28-ESR ≤ 3.2 at 24 weeks. Conclusions We have developed and externally validated a prediction model for response to methotrexate within 24 weeks in DMARD-naïve patients with RA, providing variably weighted clinical features and defined cutoffs for clinical decision-making.
 
Patient flow diagram
Mean percentage change in BMD of the lumbar spine (L1–L4) (a), total hip (b), and femoral neck (c). Error bars show standard error. The plot is based on the PP set
Forest plot for comparing Arylia versus Prolia® in terms of mean percent changes in BMD of the lumbar spine (L1–L4), total hip, and femoral neck at 18 months of the study. Forest plot demonstrating both t test analysis and ANCOVA model for PP set
Medians and IQRs for biochemical markers of bone metabolism at 18 months of the trial
Article
Background/objective Osteoporosis is a global health concern with an increasing prevalence worldwide. Denosumab is an antiresoptive agent that has been demonstrated to be effective and safe in osteoporotic patients. This study aimed to compare the efficacy and safety of the biosimilar denosumab candidate (Arylia) to the originator product (Prolia®) in postmenopausal osteoporotic patients. Methods In this randomized, double-blind, active-controlled, noninferiority trial, postmenopausal osteoporotic patients received 60 mg of subcutaneous Arylia or Prolia® at months 0, 6, and 12 and were followed up for 18 months. The primary endpoint was the noninferiority of the biosimilar product to the reference product in the percentage change of bone mineral density (BMD) in 18 months at the lumbar spine (L1-L4), total hip, and femoral neck. The secondary endpoints were safety assessment, the incidence of new vertebral fractures, and the trend of bone turnover markers (BTMs). Results A total of 190 patients were randomized to receive either biosimilar (n = 95) or reference (n = 95) denosumab. In the per-protocol (PP) analysis, the lower limits of the 95% two-sided confidence intervals of the difference between Arylia and Prolia® in increasing BMD were greater than the predetermined noninferiority margin of − 1.78 at the lumbar spine, total hip, and femoral neck sites (mean differences [95% CIs] of 0.39 [− 1.34 to 2.11], 0.04 [− 1.61 to 1.69], and 0.41 [− 1.58 to 2.40], respectively). The two products were also comparable in terms of safety, new vertebral fractures, and trend of BTMs. Conclusion The efficacy of the biosimilar denosumab was shown to be noninferior to that of the reference denosumab, with a comparable safety profile at 18 months. Trial registration ClinicalTrials.gov, NCT03293108; Registration date: 2017–09-19.
 
Levels of neutrophil activation marker calprotectin in patients with AAV and LVV. Plasma levels of A calprotectin were measured by ELISA in healthy controls (HC), and patients with microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), giant cell arteritis (GCA), and Takayasu’s arteritis (TAK) in remission. Plasma levels of B calprotectin were related to disease activity in patients in remission (rem) and matching patients with active disease (active) as assessed by physician global assessment (PGA) in MPA, GPA, GCA, and TAK. Comparison of plasma levels of C calprotectin, among patients with GCA in the presence or absence of overlapping diagnosis of polymyalgia rheumatica (PMR): (PMR+, and PMR−, respectively). Statistical analyses were done using Mann-Whitney U test (A ,C), and Wilcoxon signed-rank test (B) with *p < 0.05, **p < 0.01, and ***p < 0.001. Unless otherwise indicated, all analyses are compared to healthy controls. Each circle represents an individual sample, with the bar representing the median of the group. The dotted line represents the 95th percentile of the HC
Levels of N formyl methionine peptides (fMET) in patients with AAV and LVV. Levels of A fMET were analyzed by ELISA in HC, and patients with MPA, GPA, GCA, and TAK in remission. Plasma levels of fMET (B) were related to disease activity in patients in remission (rem) and matching patients with active disease (active) as assessed by PGA in MPA, GPA, GCA, and TAK. Plasma levels of fMET correlated with CRP (C) and ESR (D). Statistical analyses were done using Mann-Whitney U test and Wilcoxon signed-rank test, with *p < 0.05, **p < 0.01, and ***p < 0.001. Each circle represents an individual sample, with the bar representing the median of the group. The dotted line represents the 95th percentile of the HC
fMET-mediated release of calprotectin. Comparison of plasma levels of A calprotectin in patients with either high or low levels of fMET, as determined by the 95th percentile of HC. Neutrophils were activated by fMLP in vitro (B) and assessed for calprotectin release by ELISA. Statistical analyses were done using Mann-Whitney U test (A) and Wilcoxon signed-rank test (B), with *p < 0.05 and **p < 0.01. Each circle represents an individual sample, with the bar representing the median of the group
fMET activates neutrophils in an FPR1-dependent manner. Neutrophils from a healthy donor were incubated with A plasma from healthy individuals (HC) or patients with vasculitis (Vasc) or B medium and fMLP in presence or absence of Cyclosporine H (CsH) and analyzed for ROS induction using flow cytometry. C Neutrophils were pre-incubated with inhibitors scavenging mitochondrial ROS (MitoTEMPO) and inhibiting NADPH oxidase (DPI) and analyzed for capacity to induce ROS production upon stimulation with media, TLR8 agonist R848, fMLP, and/or plasma from patients with vasculitis (n=10). Statistical analyses were done using Wilcoxon signed-rank test with * p < 0.05 and **p < 0.01. Each circle represents an individual sample, with the bar representing the median of the group
Article
Objective To assess markers of neutrophil activation such as calprotectin and N-formyl methionine (fMET) in anti-neutrophil cytoplasmic autoantibody-associated vasculitis (AAV) and large-vessel vasculitis (LVV). Methods Levels of fMET, and calprotectin, were measured in the plasma of healthy controls (n=30) and patients with AAV (granulomatosis with polyangiitis (GPA, n=123), microscopic polyangiitis (MPA, n=61)), and LVV (Takayasu’s arteritis (TAK, n=58), giant cell arteritis (GCA, n=68)), at times of remission or flare. Disease activity was assessed by physician global assessment. In vitro neutrophil activation assays were performed in the presence or absence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporine H. Results Levels of calprotectin, and fMET were elevated in patients with vasculitis as compared to healthy individuals. Levels of fMET correlated with markers of systemic inflammation: C-reactive protein (r=0.82, p<0.0001), and erythrocyte sedimentation rate (r=0.235, p<0.0001). The neutrophil activation marker, calprotectin was not associated with disease activity. Circulating levels of fMET were associated with neutrophil activation (p<0.01) and were able to induce de novo neutrophil activation via FPR1-mediated signaling. Conclusion Circulating fMET appears to propagate neutrophil activation in AAV and LVV. Inhibition of fMET-mediated FPR1 signaling could be a novel therapeutic intervention for systemic vasculitides.
 
Changes in the effectiveness of remission induction therapy measured through four factors over 6 months. A BVAS. B VDI. C Peripheral eosinophil counts. D Concomitant CS doses. BVAS, Birmingham Vasculitis Activity Score; VDI, Vasculitis Damage Index; CS, corticosteroid; MPZ, mepolizumab; IVCY, intravenous cyclophosphamide. P values were determined by the Wilcoxon signed-rank test. *P < 0.05: baseline (month 0) vs. each observation points (months 1, 3, and 6)
Comparison of the effectiveness of remission induction therapy over 6 months. A Decrease in BVAS. B Increase in VDI. C Reduction in peripheral eosinophil counts. D Concomitant CS dose. E Reduction rate of concomitant CS dose. F Percentage of cases by concomitant CS dose. BVAS, Birmingham Vasculitis Activity Score; VDI, Vasculitis Damage Index; CS, corticosteroid; MPZ, mepolizumab; IVCY, intravenous cyclophosphamide. P values were determined by the Wilcoxon rank-sum test. *P < 0.05: MPZ group vs. IVCY group
Article
Objectives To investigate the safety and effectiveness of mepolizumab (MPZ), an anti-interleukin-5 antibody, as remission induction therapy for severe eosinophilic granulomatosis with polyangiitis (EGPA). Methods The clinical courses of patients with severe EGPA over 6 months were retrospectively investigated and compared between patients treated with high-dose corticosteroid (CS) plus MPZ therapy (MPZ group, n = 7) and those treated with high-dose CS plus intravenous cyclophosphamide (IVCY) pulse therapy (IVCY group, n = 13). The primary endpoints were the MPZ retention rate and the IVCY completion rate. The secondary endpoints were adverse events and changes in the Birmingham Vasculitis Activity Score (BVAS), Vascular Damage Index (VDI), eosinophil counts, and concomitant CS doses, and the extent and rates of these changes were compared between the MPZ and IVCY groups. Results Regarding the primary endpoints, the MPZ retention rate was 100%, and the IVCY completion rate was 61.5%. Regarding the secondary endpoints, adverse events were detected in 2/7 patients (28.6%) in the MPZ group and 7/13 patients (53.8%) in the IVCY group. BVAS and eosinophil counts significantly decreased in both groups at and after month 1, but there was no significant difference in the magnitude of changes between the two groups. VDI scores did not significantly increase in either group, and the degree of changes did not significantly differ between the two groups. Although concomitant CS doses significantly decreased at and after month 1 in both groups, the rates of decrease in CS doses at and after month 3 were significantly higher in the MPZ group. Conclusions This study suggested that the use of MPZ as remission induction therapy for severe EGPA might be safe and effective for controlling disease activity and reducing CS doses.
 
Body growth of normal and OA rats under LD and DL cycle. A LD and DL cycle protocol. B Body weight gain of rats at the end of study. Two-way ANOVA: LD factor: F(1, 24) = 51.81, P < 0.0001; model factor: F(1, 24) = 9.408, P = 0.0053; n = 7 per group. C Linear regression of body weight gain and weeks in each group. D Safranin O and fast green staining for tibia growth plates of normal and OA rats. RZ, resting zone; PZ, proliferating zone; HZ, hypertrophy zone. Scale bar, 50 μm. Two-way ANOVA: Total length: LD factor: F(1, 8) = 25.52, P = 0.0010; RZ: LD factor: F(1, 8) = 13.94, P = 0.0058; PZ: LD × model: F(1, 8) = 8.507, P = 0.0194; HZ: LD factor: F(1, 8) = 38.25, P = 0.0003; n = 3 per group
Effects of LD and DL cycle on cartilage and synovium pathology and inflammation in rats. A Concentration of pro-inflammatory factors of rat serum by ELISA. Two-way ANOVA: IL-1β: model factor: F(1, 28) = 10.79, P = 0.0027; LD × model: F(1, 28) = 75, P < 0.0001; IL-6: LD × model: F(1, 28) = 62.04, P < 0.0001; TNF-α: model factor: F(1, 28) = 5.127, P = 0.0315; LD × model: F(1, 28) = 46.94, P < 0.0001; iNOS: LD factor: F(1, 28) = 24.05, P < 0.0001; model factor: F(1, 28) = 15.34, P = 0.0005; LD × model: F(1, 28) = 10.89, P = 0.0026; n = 8 per group. B, C Safranin O and fast green staining of entire right knee joint and OARSI cartilage score. Scale bar, 1000 μm and 50 μm. Kruskal-Wallis test with Dunn’s correction: n = 3 to 6 per group. D Serum levels of COMP and CTX-II. Two-way ANOVA: COMP: model factor: F(1, 28) = 24.27, P < 0.0001; LD × model: F(1, 28) = 10.68, P = 0.0029; CTX-II: LD × model: F(1, 28) = 43.8, P < 0.0001; n = 8 per group. E, F Immunohistochemical analysis in tibial cartilage. Scale bar, 50 μm. Two-way ANOVA: MMP-3: LD factor: F(1, 8) = 33.23, P = 0.0004; group factor: F(1, 8) = 22.55, P = 0.0014; LD × model: F(1, 8) = 43.19, P = 0.0002; MMP-13: LD factor: F(1, 10) = 6.197, P = 0.0320; group factor: F(1, 10) = 29.78, P = 0.0003; LD × model: F(1, 10) = 8.117, P = 0.0173; ADAMTS-4: LD factor: F(1, 14) = 36.19, P < 0.0001; group factor: F(1, 14) = 43.47, P < 0.0001; LD × model: F(1, 14) = 35.41, P < 0.0001; n = 3 to 5 per group. G, H Safranin O and fast green staining of synovium. Scale bar, 100 μm. Kruskal-Wallis test with Dunn’s correction: n = 3 to 6 per group. I, J Immunofluorescence staining of F4/80⁺ and CD206⁺macrophages in synovium of OA rats. S, synovium; T, tibia; F, femur; M, meniscus. Scale bar, 100 μm and 500 μm. n = 3 per group
Effects of bone metabolism of rats under LD and DL cycle. A Femur and tibia lengths. n = 4 per group. B, C μCT scan and 3D images of femur and tibia epiphyses. Green boxed areas indicate the ROI selected for measurement and 3D microCT reconstruction. BV/TV, Tb.Th, Tb.N, Tb.Sp, and BMD were measured. Two-way ANOVA: BV/TV: LD factor: F(1, 13) = 8.841, P = 0.0108; model factor: F(1, 13) = 5.106, P = 0.0417; LD × model: F(1, 13) = 8.672, P = 0.0114; Tb.N: model factor: F(1, 13) = 5.445, P = 0.0363; LD × model: F(1, 13) = 7.538, P = 0.0167; Tb.Sp: model factor: F(1, 13) = 11.41, P = 0.0049; LD × model: F(1, 13) = 14.32, P = 0.0023; BMD: LD factor: F(1, 13) = 8.137, P = 0.0136; model factor: F(1, 13) = 13.33, P = 0.0029; n = 4 to 5 per group. D, E TRAP staining and histomorphometric of rats. Scale bar, 1000 μm and 100 μm. Two-way ANOVA: LD factor: F(1, 11) = 7.732, P = 0.0179; model factor: F(1, 11) = 14.15, P = 0.0031; LD × model: F(1, 11) = 8.118, P = 0.0158; n = 3 to 5 per group. F Von Kossa staining of undecalcified sections of tibias. Scale bar, 50 mm. G Concentration of TRACP-5b, β-CTx, P1NP and BALP in rat serum. Two-way ANOVA: TRACP-5b: LD factor: F(1, 26) = 16.08, P = 0.0005; model factor: F(1, 26) = 6.982, P = 0.0138; β-CTx: LD factor: F(1, 26) = 5.751, P = 0.0240; n = 4 to 12 per group. H OPG⁺ osteoblasts in the epiphyseal trabecular bones of OA rats. Scale bar, 50 mm. n = 3 per group. I Immunofluorescence staining of bone marrow stromal cells in OA rats. Green, OPG⁺ cells; red, RANKL⁺ cells; blue, nucleus. Scale bar, 20 μm. J Levels of MT in OA rat serum. n = 5-7
Significant activity of the bone marrow MPS after LD reversal. A Presentative images of proliferation of tibial marrow cells stained by Ki67 (white arrow) and DAPI. Scale bar, 50 μm and 20 μm. Two-way ANOVA: LD factor: F(1, 8) = 108.6, P < 0.0001; model factor: F(1, 8) = 5.332, P = 0.0498; n = 3 per group. B Presentative images of F4/80⁺ macrophages (white arrow) of tibia. Scale bar, 50 μm. Two-way ANOVA: LD factor: F(1, 8) = 21.78, P = 0.0016; model factor: F(1, 8) = 28.44, P = 0.0007; LD × model: F(1, 8) = 87.11, P < 0.0001; n = 3 per group. C, D Concentration of MCP-1, GM-CSF and M-CSF in rat serum. Two-way ANOVA: MCP-1:LD × model: F(1, 14) = 6.652, P = 0.0218; M-CSF: LD factor: F(1, 28) = 23.57, P < 0.0001; GM-CSF: LD factor: F(1, 28) = 12.88, P = 0.0013; n = 4 to 11 per group
Changes in expression of clock proteins in representative tissues. A Expression of BMAL1, NR1D1, CRY1, and PER3 protein in heart, liver, spleen, lung, and kidney of OA rats via WB. Unpaired two-tailed Student’s t test or with Welch’s correction, or using the Mann-Whitney test: n = 3 per group. B Expression of BMAL1 protein of tibial cartilage, synovium, and bone marrow of OA rats via IHC staining. Scale bar, 50 μm. n = 3 per group
Article
Background Light alteration affects the internal environment and metabolic homeostasis of the body through circadian rhythm disorders (CRD). CRD is one of the factors that induce and accelerate osteoarthritis (OA). Therefore, the aim of this study was to evaluate the effects of continuous dark-light (DL) cycle on joint inflammation, bone structure, and metabolism in normal and OA Sprague-Dawley (SD) rats. Methods Interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS), and tumor necrosis factor (TNF)-α were used to evaluate the systemic inflammation in rats. The pathological changes and inflammatory reactions of the cartilage and synovium of the knee joint in rats were evaluated by Safranin O-fast green and immunological staining. Bone turnover was assessed by histomorphometry and μCT scanning, as well as bone metabolism markers and proteins. The expression changes of clock proteins BMAL1, NR1D1, PER3, and CRY1 in representative tissues were detected by western blotting. Results DL cycle significantly inhibited body weight gain in normal and OA rats. The levels of proinflammatory factors in the peripheral blood circulation and degradation enzymes in the cartilage were significantly decreased in OA+DL rats. DL cycle significantly destroyed the structure of subchondral bone in hindlimbs of OA rats and reduced trabecular bone numbers. The decrease of bone mineral density (BMD), percent bone volume with respect to total bone volume (BV/TV), trabecular number (TB.N), osteoclast number, and mineralization could also be found. The ratio of the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG) in the bone marrow of OA rats was markedly increased under DL, along with the activation of the mononuclear/phagocyte system. The expression of representative clock proteins and genes BMAL1, PER3, and CRY1 were markedly changed in the tissues of OA+DL rats. Conclusions These results suggested that DL cycle dampened the arthritis and promoted bone resorption and bone mass loss. Graphical abstract DL cycle affects bone turnover by regulating osteoclast production in osteoarthritic rats.
 
Flowchart of patient selection. Patients with rheumatoid arthritis (RA) who started tumor necrosis factor (TNF) inhibitors or tocilizumab between December 2013 and April 2018 were selected from a nationwide database maintained by the Health Insurance Review and Assessment service. According to the exclusion criteria, 6099 patients were selected for the analysis (the incidence of tuberculosis [TB] in these patients including switchers are presented in the Supplement). To avoid the unclear association between biologic agents and TB in patients who switched biologic drugs, we analyzed the incidence of TB in patients who were treated with only one type of TNF inhibitor or tocilizumab during the follow-up. Further, patients who developed TB at > 3 months after stopping the biologic drug were excluded from the analysis because TB was considered to be irrelevant to biologic therapy in these patients. A total of 4736 patients with RA were included for the final analysis RA, rheumatoid arthritis; TNF, tumor necrosis factor; TB, tuberculosis; LTBI, latent tuberculosis infection
Cumulative incidence of tuberculosis (TB) in patients with rheumatoid arthritis (RA) who received biologic therapy. The cumulative incidence rate of TB was evaluated using the Kaplan–Meier method, and the differences among biologic therapies were compared using the log-rank test with multiple comparison adjustment. Infliximab showed a significantly higher risk of TB than etanercept (P = 0.04). The table shows the incidence rate of TB in patients with RA stratified by follow-up duration (< 0.5, 0.5–1, 1–3, and ≥ 3 years) CI, confidence interval
Article
Background Tumor necrosis factor (TNF) inhibitors increase the risk of tuberculosis (TB) in patients with rheumatoid arthritis (RA). This study compared the incidence of TB after treatment with TNF inhibitors and tocilizumab in patients with RA, separately in those who were treated for latent tuberculosis infection (LTBI) and those without evidence of LTBI. Methods This study included patients with RA who initiated TNF inhibitors and tocilizumab between December 2013 and August 2018. Patient data were collected from the nationwide database of the Health Insurance Review and Assessment service in South Korea. The incidence of TB was compared among different biologic drugs in patients with or without LTBI treatment. Results Of 4736 patients, 1168 were treated for LTBI and 48 developed TB (554.9 per 100,000 person-years). When compared based on etanercept, infliximab showed a higher risk of TB (adjusted incidence rate ratio 2.71, 95% confidence interval 1.05–7.01), especially in patients without evidence of LTBI. Other TNF inhibitors and tocilizumab showed a comparable incidence of TB, regardless of treatment for LTBI. There was no significant difference in TB incidence after biologic therapy between patients with and without LTBI treatment (627.9/100,000 vs. 529.5/100,000 person-years). In patients treated for LTBI, no differential risk of TB was observed among biologic drugs. Conclusions The incidence of TB was not significantly different among biologic drugs in the current era, except for infliximab in patients who were not treated for LTBI. Treatment of LTBI might alleviate the drug-specific risk of TB in patients with RA.
 
Article
Background Muscle weakness and decreased fatigue resistance are key manifestations of systemic autoimmune myopathies (SAMs). We here examined whether high-intensity interval training (HIIT) improves fatigue resistance in the skeletal muscle of experimental autoimmune myositis (EAM) mice, a widely used animal model for SAM. Methods Female BALB/c mice were randomly assigned to control (CNT) or EAM groups ( n = 28 in each group). EAM was induced by immunization with three injections of myosin emulsified in complete Freund’s adjuvant. The plantar flexor (PF) muscles of mice with EAM were exposed to either an acute bout or 4 weeks of HIIT (a total of 14 sessions). Results The fatigue resistance of PF muscles was lower in the EAM than in the CNT group ( P < 0.05). These changes were associated with decreased activities of citrate synthase and cytochrome c oxidase and increased expression levels of the endoplasmic reticulum stress proteins (glucose-regulated protein 78 and 94, and PKR-like ER kinase) ( P < 0.05). HIIT restored all these alterations and increased the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and the mitochondrial electron transport chain complexes (I, III, and IV) in the muscles of EAM mice ( P < 0.05). Conclusions HIIT improves fatigue resistance in a SAM mouse model, and this can be explained by the restoration of mitochondria oxidative capacity via inhibition of the ER stress pathway and PGC-1α-mediated mitochondrial biogenesis.
 
Random forest-type machine learning algorithm. SHAP summary graph
(continued)
Baseline characteristics of the sample
Functioning of the random forest model trained to predict minimal activity. Confusion matrix
Article
Background: Very few data are available on predictors of minimal disease activity (MDA) in patients with recent-onset psoriatic arthritis (PsA). Such data are crucial, since the therapeutic measures used to change the adverse course of PsA are more likely to succeed if we intervene early. In the present study, we used predictive models based on machine learning to detect variables associated with achieving MDA in patients with recent-onset PsA. Methods: We performed a multicenter observational prospective study (2-year follow-up, regular annual visits). The study population comprised patients aged ≥18 years who fulfilled the CASPAR criteria and less than 2 years since the onset of symptoms. The dataset contained data for the independent variables from the baseline visit and from follow-up visit number 1. These were matched with the outcome measures from follow-up visits 1 and 2, respectively. We trained a random forest-type machine learning algorithm to analyze the association between the outcome measure and the variables selected in the bivariate analysis. In order to understand how the model uses the variables to make its predictions, we applied the SHAP technique. We used a confusion matrix to visualize the performance of the model. Results: The sample comprised 158 patients. 55.5% and 58.3% of the patients had MDA at the first and second follow-up visit, respectively. In our model, the variables with the greatest predictive ability were global pain, impact of the disease (PsAID), patient global assessment of disease, and physical function (HAQ-Disability Index). The percentage of hits in the confusion matrix was 85.94%. Conclusions: A key objective in the management of PsA should be control of pain, which is not always associated with inflammatory burden, and the establishment of measures to better control the various domains of PsA.
 
Proportion of patients reporting improvements ≥ MCIDa in PROs at weeks 12 (A) and 24 (B). aMCID was defined as reduction of ≥10 mm for PtGA and pain, ≥1 for severity of AM stiffness, reduction of ≥0.22 units for HAQ-DI, increase of ≥4 points for FACIT-F, proxied at one-half standard deviation for duration of AM stiffness, increase of ≥0.05 points for EQ-5D-5L, reduction of 7% in score for WPAI, and increase of ≥2.5 points for SF-36 PCS and MCS. bABA IV at day 1 and weeks 2, 4, 8, 12, 16, and 20 (<60 kg: 500 mg; 60–100 kg: 750 mg; >100 kg: 1,000 mg). cNNTs are for UPA vs ABA. ABA abatacept, AM morning, EQ-5D-5L(index score), EQ-5D 5-Level, FACIT-F Functional Assessment of Chronic Illness Therapy-Fatigue, HAQ-DI Health Assessment Questionnaire Disability Index, IV intravenous, MCID minimal clinically important difference, MCS Mental Component Summary. NNT number needed to treat, PCS Physical Component Summary, PRO patient-reported outcome, PtGA Patient Global Assessment of Disease Activity, SF-36 36-Item Short Form Health Survey, UPA upadacitinib, VAS visual analog scale, WPAI Work Productivity and Activity Impairment. *P<0.05 for UPA vs ABA. P values represent statistical significance between treatment groups
Proportion of patients reporting improvements ≥ MCIDa in SF-36 at weeks 12 (A) and 24 (B). aMCID was defined as increase ≥5.0 for all SF-36 domains. bABA IV at day 1 and weeks 2, 4, 8, 12, 16, and 20 (<60 kg: 500 mg; 60–100 kg: 750 mg; >100 kg: 1000 mg). cNNTs are for UPA vs ABA. ABA abatacept, BP bodily pain, GH general health, IV intravenous, MCID minimal clinically important difference, MH mental health, PF physical functioning, RE role emotional, RP role physical, SF social functioning, SF-36 36-Item Short Form Health Survey, UPA upadacitinib, VT vitality. *P<0.05 for UPA vs ABA. P values represent statistical significance between treatment groups
Article
Background: In previous clinical trials, patients with active rheumatoid arthritis (RA) treated with upadacitinib (UPA) have improved patient-reported outcomes (PROs). This post hoc analysis of SELECT-CHOICE, a phase 3 clinical trial, evaluated the impact of UPA vs abatacept (ABA) with background conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) on PROs in patients with RA with inadequate response or intolerance to biologic disease-modifying antirheumatic drugs (bDMARD-IR). Methods: Patients in SELECT-CHOICE received UPA (oral 15 mg/day) or ABA (intravenous). PROs evaluated included Patient Global Assessment of Disease Activity (PtGA) by visual analog scale (VAS), patient's assessment of pain by VAS, Health Assessment Questionnaire Disability Index (HAQ-DI), morning stiffness duration and severity, 36-Item Short Form Health Survey (SF-36), Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F), Work Productivity and Activity Impairment (WPAI), and EQ-5D 5-Level (EQ-5D-5L) index score. Least squares mean (LSM) changes from baseline to weeks 12 and 24 were based on an analysis of covariance model. Proportions of patients reporting improvements ≥ minimal clinically important differences (MCID) were compared using chi-square tests. Results: Data from 612 patients were analyzed (UPA, n=303; ABA, n=309). Mean age was 56 years and mean disease duration was 12 years. One-third received ≥2 prior bDMARDs and 72% received concomitant methotrexate at baseline. At week 12, UPA- vs ABA-treated patients had significantly greater improvements in PtGA, pain, HAQ-DI, morning stiffness severity, EQ-5D-5L, 2/4 WPAI domains, and 3/8 SF-36 domains and Physical Component Summary (PCS) scores (P<0.05); significant differences persisted at week 24 for HAQ-DI, morning stiffness severity, SF-36 PCS and bodily pain domain, and WPAI activity impairment domain. At week 12, significantly more UPA- vs ABA-treated patients reported improvements ≥MCID in HAQ-DI (74% vs 64%) and SF-36 PCS (79% vs 66%) and 4/8 domain scores (P<0.05). Conclusions: At week 12, UPA vs ABA treatment elicited greater improvements in key domains of physical functioning, pain, and general health and earlier improvements in HAQ-DI. Overall, more UPA- vs ABA-treated patients achieved ≥MCID in most PROs at all timepoints; however, not all differences were statistically significant. These data, however, highlight the faster response to UPA treatment. Trial registration: NCT03086343 , March 22, 2017.
 
Article
Objectives To estimate associations between fine particulate matter (PM2.5) and ozone and the onset of systemic autoimmune rheumatic diseases (SARDs). Methods An open cohort of over 6 million adults was constructed from provincial physician billing and hospitalization records between 2000 and 2013. We defined incident SARD cases (SLE, Sjogren’s syndrome, scleroderma, polymyositis, dermatomyositis, polyarteritis nodosa and related conditions, polymyalgia rheumatic, other necrotizing vasculopathies, and undifferentiated connective tissue disease) based on at least two relevant billing diagnostic codes (within 2 years, with at least 1 billing from a rheumatologist), or at least one relevant hospitalization diagnostic code. Estimated PM2.5 and ozone concentrations (derived from remote sensing and/or chemical transport models) were assigned to subjects based on residential postal codes, updated throughout follow-up. Cox proportional hazards models with annual exposure levels were used to calculate hazard ratios (HRs) for SARDs incidence, adjusting for sex, age, urban-versus-rural residence, and socioeconomic status. Results The adjusted HR for SARDS related to one interquartile range increase in PM2.5 (3.97 µg/m³) was 1.12 (95% confidence interval 1.08–1.15), but there was no clear association with ozone. Indirectly controlling for smoking did not alter the findings. Conclusions We found associations between SARDs incidence and PM2.5, but no relationships with ozone. Additional studies are needed to better understand interplays between the many constituents of air pollution and rheumatic diseases.
 
Article
Background Axial spondyloarthritis (axSpA) is a common chronic inflammatory disease, associated with extracellular matrix (ECM) remodeling of the cartilage, bone, and connective tissues. The primary symptom of axSpA is back pain, caused by inflammation. However, there is a medical need to truly identify patients with axSpA from other subjects with buttock or low back pain attributable to other reasons. We aimed to investigate circulating biomarkers of ECM/inflammation (MMP-degraded type I (C1M), II (C2M, T2CM), III (C3M), IV (C4M), VI (C6M), and X (C10C, COL10NC) collagens, CRPM, PROM and VICM) and ECM formation of type II (PRO-C2), III (PRO-C3), IV (PRO-C4), and VI (PRO-C6) collagens as potential biomarkers to identify patients with axSpA. Methods We measured biomarkers from a cross-sectional study with 204 participants by enzyme-linked immunosorbent assay (ELISA). The study included axSpA patients (N = 41), women with postpartum buttock/pelvic pain (N = 46), disc herniation (N = 25), and a group of healthy subjects (including women without postpartum pelvic pain (N = 14), subjects with various types of physical strain (cleaning staff (N = 26) long-distance runners (N = 23)), and healthy men (N = 29)). Differences between the groups were calculated by ANCOVA and AUC, while Spearman’s correlations were performed with ECM biomarkers and clinical scores. Results Patients with axSpA expressed significantly higher levels of C1M, C4M, and VICM (p < 0.05-p < 0.0001) compared to all the non-axSpA control groups. Further, C6M and PRO-C4 were significantly higher in patients with axSpA (both p < 0.0001) compared to women with postpartum pelvic pain and healthy subjects, whereas PRO-C3 was significantly lower compared to healthy subjects (p = 0.01). The best ECM common biomarker to differentiate between axSpA and the non-axSpA control groups was PRO-C4 (AUC ≥ 0.75; specificity ≥ 0.79, sensitivity = 0.65). Mild correlations were observed between collagen turnover and inflammation biomarkers and CRP and MRI (ρ ≥ 0.3; p < 0.05-p < 0.001). Conclusions Biomarkers of type I, IV, and VI collagen and biomarkers of inflammation showed an altered turnover in patients with axSpA compared with the non-axSpA control groups. Such biomarkers may be useful in combination with MRI or independently to separate patients with axSpA from other back pain conditions.
 
Top-cited authors
Christine Le Maitre
  • Sheffield Hallam University
Judith Hoyland
  • The University of Manchester
Anthony John Freemont
  • The University of Manchester
Graham Peter Riley
  • University of East Anglia
Xinyuan Chang
  • The University of Manchester