Integrating Mendelian randomization and enzyme-linked immunosorbent assay validation identifies candidate mediators across the rheumatoid arthritis disease continuum

Figure 1 Description of the study design in this Mendelian randomization study. A: Mendelian randomization analyses depend on 3 core assumptions; B: Schematic of the study design. IVs: Instrumental variables; IVW: Inverse-variance weighted; kb: Kilobase; MR: Mendelian randomization; PRESSO: Pleiotropy RESidual Sum and Outlier; R²: Coefficient of determination; SNPs: Single-nucleotide polymorphisms.

Figure 2 Forest plot of Mendelian randomization estimates for the causal effects of C-C motif chemokine ligand 28, CD40, interleukin-18 receptor 1, and matrix metalloproteinase-1 on rheumatoid arthritis risk. Effect sizes (odds ratios) and 95% confidence intervals are shown for the inverse-variance weighted, Mendelian randomization-Egger, weighted median, simple mode, and weighted mode estimators. CCL28: C-C motif chemokine ligand 28; CI: Confidence interval; IL-18R1: Interleukin-18 receptor 1; MMP-1: Matrix metalloproteinase-1; MR: Mendelian randomization; nsnp: Number of single-nucleotide polymorphisms; OR: Odds ratio.

Figure 3 Comparison of serum inflammatory cytokine levels between patients with active rheumatoid arthritis and healthy controls. Serum concentrations were measured by enzyme-linked immunosorbent assay in 41 patients with rheumatoid arthritis and 41 matched controls. All 4 cytokines were significantly elevated in the rheumatoid arthritis group compared with the control group: CD40: 341.78 ± 69.99 pg/mL vs 207.12 ± 72.46 pg/mL, mean difference = 134.66 pg/mL [95% confidence interval (CI): 103.35-165.97; P < 0.0001]; C-C motif chemokine ligand 28: 3562.13 ± 699.25 pg/mL vs 1891.77 ± 523.32 pg/mL, mean difference = 1670.36 pg/mL (95%CI: 1398.91-1941.80; P < 0.0001); matrix metalloproteinase-1: 8.67 ± 1.68 ng/mL vs 5.42 ± 1.29 ng/mL, mean difference = 3.26 ng/mL (95%CI: 2.60-3.92; P < 0.0001); interleukin-18 receptor 1: 238.37 ± 47.47 pg/mL vs 140.40 ± 40.41 pg/mL, mean difference = 97.97 pg/mL (95%CI: 78.59-117.34; P < 0.0001). A: CD40; B: C-C motif chemokine ligand 28 (CCL28); C: Matrix metalloproteinase-1 (MMP-1); D: Interleukin-18 receptor 1 (IL-18R1). RA: Rheumatoid arthritis.

Figure 4 Correlation analysis of serum inflammatory cytokine levels in 41 patients with rheumatoid arthritis and 41 healthy controls (n = 82). Pearson correlation coefficients showed significant positive correlations among all pairs (r = 0.436-0.609, all P < 0.0001). A: Correlation between CD40 and C-C motif chemokine ligand 28 (CCL28); B: Correlation between CD40 and matrix metalloproteinase-1 (MMP-1); C: Correlation between CCL28 and MMP-1; D: Correlation between CD40 and interleukin-18 receptor 1 (IL-18R1); E: Correlation between CCL28 and IL-18R1; F: Correlation between MMP-1 and IL-18R1. CI: Confidence interval.

Figure 5 Proposed integrated spatiotemporal model of inflammatory mediator pathways potentially involved in rheumatoid arthritis. Combining Mendelian randomization (MR) analysis (which supports causal inference only under its core assumptions) with clinical enzyme-linked immunosorbent assay (ELISA) validation suggests CD40, C-C motif chemokine ligand 28 (CCL28), and matrix metalloproteinase-1 (MMP-1) as candidate mediators, and interleukin-18 receptor 1 (IL-18R1) as a context-dependent factor. These molecules are hypothesized to drive a compartmentalized 3-stage cascade: (1) CD40 may contribute to adaptive immune activation in lymphoid tissues, (2) CCL28 may amplify local synovial inflammation by recruiting immune effectors (e.g., monocytes, Th22 cells), and (3) MMP-1 may mediate cartilage matrix degradation and erosion. IL-18R1 may exert context-dependent modulatory effects. This proposed “immune-inflammation-tissue destruction” axis offers a hypothesis-generating framework for exploring stage- and tissue-associated biological mechanisms. As with all MR-based inferences, these findings should be considered supportive, and the experimental validation demonstrates consistency with, rather than proof of, the candidate relationships. APC: Antigen-presenting cell; APPC: Activated antigen-presenting cell; RA: Rheumatoid arthritis; SNPs: Single-nucleotide polymorphisms.