Identification of Specific Joint-Inflammatogenic Cell-Free DNA Molecules From Synovial Fluids of Patients With Rheumatoid Arthritis

Figures

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  Figure 1. Elevated cell-free DNA (cfDNA) isolated from the synovial fluid (SFcfDNA) concentrations in RA patients. (A) cfDNA concentrations in plasma of healthy donors (HDs) and rheumatoid arthritis (RA) patients. (B) cfDNA levels of synovial fluid in RA and osteoarthritis (OA) patients. Data was calculated by non-parametric Mann–Whitney test; P < 0.05 is recognized as statistically significant. ***P < 0.001. Data are presented as median with interquartile range.

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  Figure 2. Cellular stimulatory function of SFcfDNA from RA patients. (A) Expression of TNF-α by THP-1 cells stimulated with SFcfDNA from OA or RA patients at two concentrations. The data shown are representative of three independent experiments with the same tendency. (B) The percentage of primary SFMICs from RA patients expressing intracellular TNF-α upon stimulation with SFcfDNA or CpG 2006. The data on the left is a representative sample and on the right is the mean of 12 samples. (C) TNF-α secretion by SFMICs (n = 6) stimulated with CpG 2006 or SFcfDNA (0.5 μg/2 × 105 cells) using the same procedure. (D) IL-6 and IL-8 expression on primary FLSs from RA patients (n = 5) stimulated with SFcfDNA from RA patients. Statistical significance was calculated by one-way ANOVA (A), Kruskal–Wallis Dunn's multiple comparison test (B,C), two-tailed paired T testing (D). *0.01 < P < 0.05, **0.001 < P < 0.01, ***P < 0.001. Data are presented as the mean with SD (A), and (B,C) data are presented as the median with interquartile range.

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  Figure 3. SFcfDNA distribution and CpG-motif-rich (CMR) sequences in synovial fluid. (A) Distribution of total cfDNA density across chromosomes. Briefly, Manhattan plot with –logarithm P-values for the distribution comparison of total cfDNA density across chromosomes. Sequence density of 400-bp chromosome bins across the human genome was used for the measurement of overall genomic distribution of SFcfDNA molecules. Totally 7,590,771 chromosome bins were included and compared. (B) Circular Manhattan plot for the distribution comparison of SFcfDNA at CpG-motif regions. Features aligned onto the main chromosomes as concentric rings (from inner to outer) correspond to (1) Between-group difference (RA–OA) of sequence density of CpG-island regions. The values were coded by color, and the key legend was shown in the center. (2) Manhattan plot with –logarithm P-values for the comparison of sequence density of CpG regions. Totally 28,691 CpG sequences were compared, and the Bonferroni-corrected cut-off with minus logarithm scale is about 5.8. (3) CpG-rich locations identified by sequence density comparison at CpG regions. The larger the circle is, the higher significance it has. (4) The chromosome…

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  Figure 4. SFcfDNA with CpG motif-rich sequences from RA patients induces TNF-α expression in THP-1 cells. (A) TNF-α regulation after stimulation with CMRs and CpG-motif free (CMFs) (250 ng/2 × 105 cells, respectively). Fold change of THP-1 expression was normalized against the control group. (B) Percentage of THP-1 cells expressing TNFα expression following stimulation of CMRs from SFcfDNA. (C) TNF-α expression induced by CMRs containing different numbers of CpG groups. (A,C) Statistical significance was calculated by non-parametric Mann–Whitney test, *0.01 < P < 0.05, **0.001 < P < 0.01. Data are presented as median with interquartile range.

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  Figure 5. Challenge of BALB/c mice with high-frequency CMR sequences. (A) Swelling of the right ankle joint as measured by the change in ankle diameter (n = 5). (B) Photos of right hindpaws on day 3 after challenge with specific sequences. (C) Representative 3D micro-CT images of the ankle joints of mice after administration for 6 days (resolution: 19 μm). Red arrows indicate bone destruction. (D) Representative T2-weighted MRI images of ankle joints of different groups at day 6. The effusion in the joint is highlighted by red arrows. Bone erosion in the CMR-3 group is highlighted with a red asterisk. (E) Representative HE staining of ankle joints of mice 6 days after administration (×200). Inflammatory cell infiltration in the articular cavity is indicated with black arrows. Bone destruction in mice treated with CMR-3 is highlighted with an asterisk. (F) mRNA level of related cytokines and MMP-3 of shinbones at day 6 after immunization. Relative mRNA expression was normalized to β-Actin. (A,F) Significance was calculated by one-way ANOVA. #0.01 < P < 0.05, ##0.001 < P < 0.01, ###P < 0.001 vs. PBS group. *0.01 < P < 0.05, **0.001 < P < 0.01, ***P < 0.001 between two groups. Data are…