Additional file 3: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Plasmid constructs of Per2/Bik promoter. D-box(+), plasmid constructs containing D-box. D-box(−), plasmid constructs without D-box. D-box motifs of Per2 promoter were mutated from 5′-TTATGTAA-3′ to 5′-CGCCAGGC-3′ (−372 to −365), and 5′-TTACGTAA-3′ to 5′-CAGCGTAA-3′ (−47 to −40). Human Bik promoter containing D-box (−780 to +176) and human Bik promoter without D-box (−260 to +323) constructed. (PDF 215 kb

Additional file 5: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

The mRNA expression of circadian clock genes over time. mRNA expression of circadian clock genes measured at –4 h (before synchronization), 0 h (just before MTX stimulation), 24 h, 32 h, and 48 h. Controls and 10/100 nM of MTX showed almost the same expression rhythms, and MTX influenced their expression levels. (PDF 264 kb

Additional file 2: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Detailed information of patients with RA enrolled in this study. Joint samples obtained from 10 different patients to establish primary cultured synovial fibroblast cell lines. Age, sex, disease duration, CRP, DAS28-ESR, and treatment with MTX, PSL, and other DMARDs shown in the table. (PDF 252 kb

Additional file 1: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

The interactions of circadian clock genes and its relative factors. BMAL/CLOCK and PER/CRY create the circadian rhythm via E-box, and DBP, TEF, HLF, E4BP4 via D-box. BMAL and CLOCK heterodimerize and bind to E-box elements on promoter regions of Per and Cry genes to induce their transcription. Thereafter, PER and CRY proteins heterodimerize to inhibit activities of their own and other E-box regulated promoters. (PDF 214 kb

Additional file 6: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Two different transcriptional pathways by which MTX induces apoptosis to synovial fibroblasts: PAR bZIP–Per2 transcriptional pathway and PAR bZIP–Bik transcriptional pathway. We propose MTX induces apoptosis in synovial cells through activated binding of PAR bZIP to D-box in two different genes, Per2 and Bik, and these dual pathways work independently but synergistically. (PDF 206 kb

Additional file 4: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Cell viability of MTX-treated fibroblasts. Cell viability of RA synovial fibroblasts measured by WST-8 assay after 24 h of stimulation of MTX (1 pM to 1 μM). MTX (1 and 10 nM) significantly decreased cell viability as shown in Fig. 1, while 1–100 pM of MTX did not. (PDF 274 kb

DataSheet_1_Increased Circulating Cell-Free DNA in Eosinophilic Granulomatosis With Polyangiitis: Implications for Eosinophil Extracellular Traps and Immunothrombosis.docx

BackgroundEndogenous DNA derived from nuclei or mitochondria is released into the blood circulation as cell-free DNA (cfDNA) following cell damage or death. cfDNA is associated with various pathological conditions; however, its clinical significance in antineutrophil cytoplasmic antibody-associated vasculitis (AAV) remains unclear. This study aimed to evaluate the clinical significance of cfDNA in AAV.MethodsWe enrolled 35 patients with AAV, including 10 with eosinophilic granulomatosis with polyangiitis (EGPA), 13 with microscopic polyangiitis, and 12 with granulomatosis with polyangiitis. Serum cf-nuclear DNA (cf-nDNA) and cf-mitochondrial DNA (cf-mtDNA) levels were measured by quantitative polymerase chain reaction before and after the initiation of immunosuppressive therapy. Tissue samples from EGPA patients were examined by immunofluorescence and transmission electron microscopy. The structure of eosinophil extracellular traps (EETs) and neutrophil extracellular traps (NETs) and stability against DNase were assessed in vitro. Platelet adhesion of EETs were also assessed.ResultsSerum cf-nDNA and cf-mtDNA levels were significantly higher in AAV than in healthy controls, with the highest levels in EGPA; however, serum DNase activities were comparable among all groups. cf-nDNA and cf-mtDNA decreased after treatment and were associated with disease activity only in EGPA. Blood eosinophil count and plasma D-dimer levels were significantly correlated with cf-nDNA in EGPA and cf-mtDNA. EGPA tissue samples showed lytic eosinophils and EETs in small-vessel thrombi. The structure of EETs showed bolder net-like chromatin threads in vitro and EETs showed greater stability against DNase than NETs. EETs provided a scaffold for platelet adhesion.ConclusioncfDNA was increased in EGPA, associated with disease activity. The presence of DNase-resistant EETs in small-vessel thrombi might contribute to higher concentration of cfDNA and the occurrence of immunothrombosis in EGPA.</p

Video_1_Increased Circulating Cell-Free DNA in Eosinophilic Granulomatosis With Polyangiitis: Implications for Eosinophil Extracellular Traps and Immunothrombosis.mp4

BackgroundEndogenous DNA derived from nuclei or mitochondria is released into the blood circulation as cell-free DNA (cfDNA) following cell damage or death. cfDNA is associated with various pathological conditions; however, its clinical significance in antineutrophil cytoplasmic antibody-associated vasculitis (AAV) remains unclear. This study aimed to evaluate the clinical significance of cfDNA in AAV.MethodsWe enrolled 35 patients with AAV, including 10 with eosinophilic granulomatosis with polyangiitis (EGPA), 13 with microscopic polyangiitis, and 12 with granulomatosis with polyangiitis. Serum cf-nuclear DNA (cf-nDNA) and cf-mitochondrial DNA (cf-mtDNA) levels were measured by quantitative polymerase chain reaction before and after the initiation of immunosuppressive therapy. Tissue samples from EGPA patients were examined by immunofluorescence and transmission electron microscopy. The structure of eosinophil extracellular traps (EETs) and neutrophil extracellular traps (NETs) and stability against DNase were assessed in vitro. Platelet adhesion of EETs were also assessed.ResultsSerum cf-nDNA and cf-mtDNA levels were significantly higher in AAV than in healthy controls, with the highest levels in EGPA; however, serum DNase activities were comparable among all groups. cf-nDNA and cf-mtDNA decreased after treatment and were associated with disease activity only in EGPA. Blood eosinophil count and plasma D-dimer levels were significantly correlated with cf-nDNA in EGPA and cf-mtDNA. EGPA tissue samples showed lytic eosinophils and EETs in small-vessel thrombi. The structure of EETs showed bolder net-like chromatin threads in vitro and EETs showed greater stability against DNase than NETs. EETs provided a scaffold for platelet adhesion.ConclusioncfDNA was increased in EGPA, associated with disease activity. The presence of DNase-resistant EETs in small-vessel thrombi might contribute to higher concentration of cfDNA and the occurrence of immunothrombosis in EGPA.</p