Fig 2: Model describing the dysregulation of endogenous inflammation by PK2 in arthritis. In OA, exposure to proinflammatory cytokines upregulates the expression of PKR1. PK2 secreted from OA-SF acts on PKR1 in an autocrine or paracrine manner and reduces the secretion of IL-6 and MMP-3 from OA-SF (A). In contrast, in RA-SF, exposure to proinflammatory cytokines downregulates the expression of PK2 and PKR1 (B). Therefore, PK2 exhibits a weaker anti-inflammatory effect in RA-SF compared with OA-SF. Indeed, the expression of PKR1 in RA synovial tissue was decreased compared with OA synovial tissue. This dysregulation in the endogenous inflammation-mediated modulation of PK2 in RA-SF may be associated with the chronicity of inflammation in the pathogenesis of RA. PK2 prokineticin 2, PKR1 prokineticin receptor 1, OA osteoarthritis, RA rheumatoid arthritis, SF synovial fibroblasts, IL-1ß interleukin-1ß, TNFa tumor necrosis factor a, IL-6 interleukin-6, MMP-3 matrix metalloproteinase 3, pNF?B phospho nuclear factor kappa-light-chain-enhancer of activated B cells.

Fig 3: The PK2 concentration in plasma and synovial fluid from OA and RA patients. (A) Comparison of plasma (N = 20) and synovial fluid (N = 50) PK2 concentrations between OA and RA patients. Data were presented as box plots, the description of which is given in the legend to Fig. 2. For statistical analysis, the Mann–Whitney test was used. *p < 0.05, **p < 0.01. (B) Comparison of PK2 concentrations between plasma and synovial fluid in OA and RA patients. N = 20. For statistical analysis, the Wilcoxon signed-rank test was used. (C) Correlation of PK2 concentrations between plasma and synovial fluid in OA and RA patients. N = 20. The linear regression line, Spearman rank correlation coefficient (R rank), and respective p-value are given. PK2 prokineticin 2, SF synovial fluid, OA osteoarthritis, RA rheumatoid arthritis, NS not significant.

Fig 4: PK2, PKR1, and PKR2 expression in OA and RA synovial tissues. (A) Immunostaining of PK2 (left panel), PKR1 (middle panel), and PKR2 (right panel) proteins in synovial tissue from OA and RA patients. Brown staining indicates PK2- (red arrow), PKR1- (red arrowhead), or PKR2 (red open headed arrow)-positive cells (× 400) Staining with an isotype control as the primary antibody in RA synovial tissue is also demonstrated. (B) Semiquantitative results of PK2, PKR1, and PKR2 expression. Semiquantification was performed using a scoring method (0–4) in the lining and sublining layers of synovial tissue. Data were presented as box plots, in which the boxes demonstrate the 25th and 75th percentiles, the lines within the boxes demonstrate the median, and the lines outside the boxes demonstrate the 10th and 90th percentiles. N = 8–10. For statistical analysis, the Mann–Whitney test was used. *p < 0.05, **p < 0.01. PK2 prokineticin 2, PKR1 prokineticin receptor 1, PKR2 prokineticin receptor 2, OA osteoarthritis, RA rheumatoid arthritis, Iso isotype control, SL sublining layer, LL lining layer.

Fig 5: The induction of phospho-NF?B p65 by TNFa was attenuated in PK2 prestimulated OA-SF. Phospho-NF?B p65 protein in PK2 (10–11 M) or control (water) prestimulated OA- or RA-SF was examined by cell-based ELISAs 0, 5, 15, 30, 60, and 120 min after stimulation with TNFa (10 ng/ml). (A) Control prestimulated OA-SF. (B) PK2 prestimulated OA-SF. (C) Control prestimulated RA-SF. (D) PK2 prestimulated RA-SF. (E) Comparisons of phospho-NF?B p65 expression between PK2 or control prestimulated OA- or RA-SF 30 min after stimulation with TNFa. N = 5–8. Data were given as box plots, the description of which is given in the legend to Fig. 2. Each dot in the figure represents a different cell line derived from a different patient. For statistical analysis, the one-sample Wilcoxon signed-rank test was used for comparisons with time 0 as the control, representing the 100% line. In comparisons among the groups at 30 min after stimulation with TNFa (E), the Mann–Whitney test was used. *p < 0.05, **p < 0.01. NF?B nuclear factor kappa-light-chain-enhancer of activated B cells, PK2 prokineticin 2, CTL control, OA osteoarthritis, RA rheumatoid arthritis, SF synovial fibroblasts, TNFa tumor necrosis factor a.