Fig 2: CRP induces RA-associated chromatin dysregulation via FRA2. a Heatmap showing changes in chromatin accessibility in monocytes stimulated with the presence or absence of CRP for 12 h (p < 0.01 and |fold change| > 1.5). ATAC-seq was used to assess five independent biological replicates. b The upregulated peaks of CRP-stimulation were enriched for transcription factors using HOMER and ranked by p. c Volcano plot showing changes of gene expression in monocytes stimulated with CRP (10 μg/mL) in the presence or absence for 12 h; red dots correspond to upregulated genes with significant (p < 0.05 and |fold change| > 1.5). RNA-seq was performed in three independent biological replicates (monocytes derived from three different patients with OA). d Expression levels of genes FOSL2 were confirmed by real-time RT-PCR in control and CRP stimulation group. The p value was estimated from unpaired Student’s t test. ****p < 0.0001. Error bar represent standard error of the mean (SEM). e Visualization of ATAC-seq footprint for FRA2 motifs in control and CRP stimulation. The ATAC-seq signal across all the motif binding sites in the genome were aligned on the motif and averaged. f Barplot shows expression level of genes with containing FRA2 binding sites during control and CRP stimulation. Error bar represent standard error of the mean (SEM). The p value was estimated from unpaired Student’s t test. ****p < 0.0001. g, h Normalized ATAC-seq profiles at the OCSTAMP(g) and IL-1B(h) locus in control and CRPstim. The shaded regions are more accessible representative peaks containing the FRA2 binding site after CRP stimulation. i, j Expression levels of genes OCSTAMP (i) and IL-1B (j) were confirmed by real-time RT-PCR in control and CRP stimulation group. Error bar represent standard error of the mean (SEM). The p value was estimated from unpaired Student’s t test. *p < 0.05, **p < 0.01. CRP, C-reaction protein; CRPstim, CRP stimulation

Fig 3: CRP stimulation induced RA-associated chromatin dysregulation in monocytes. a Schematic depicting the experimental design for CRP stimulation in vitro. PBMCs of OA patients were co-cultured for 12 h with or without CRP, and then monocytes were sorted and used to generate ATAC-seq and RNAseq libraries. b Barplot showing RAASs before and after CRP stimulation in monocytes. The p value was estimated from paired Student’s t test. **p < 0.01. Error bar represent standard error of the mean (SEM). c Diagrams for the average ATAC-seq signal intensity for C1-C3 peaks of control and CRP stimulation in monocyte. The values of p were calculated for the comparison of the average signals within 10 bp of the peak centres using unpaired t-test. d, f GSEA enrichment plots highlighting RNA-seq signals for proinflammation (d), regulation of OC-differentiation (d), and genes related to RA-associated dysregulation (f) of the running enrichment scores (ES) and positions of gene set members among the rank-ordered list from the GSEA. e, g The barplot shows the gene expression of JAK2 (e), SIGLEC15 (e), and TNFRSF1B (g) in the control and CRP stimulation. The p value was estimated from unpaired Student’s t test. *p < 0.05. CRP, C-reaction protein; OA, osteoarthritis; OC, osteoclast; PBMC, peripheral blood mononuclear cell; CRPstim, CRP stimulation; C3, cluster 3; RAAS, RA-associated ATAC-seq score

Fig 4: CRP-induced NLRP3 inflammasome activation and phosphorylation of Smad3 in HK-2 tubular epithelial cells under high-glucose conditionsWestern blot exhibited CRP-induced NLRP3 expression in HK-2 cells in a time-dependent manner, being significant as early as 6 h and peaking at 12 h (A). qPCR demonstrated that CRP induces NLRP3, IL-1β, and TNF-α mRNA expression level in a time-dependent manner, being significant at around 6 h (B). High-glucose (HG)+CRP conditions significantly enhanced NLRP3 inflammasome-related indexes of protein expression level, including NEK7, C-caspase-1, and IL-1β (C). qPCR showed that HG+CRP conditions significantly enhanced the mRNA expression level of NLRP3, NEK7, IL-1β, and TNF-α (D). Expression levels of NLRP3 and caspase-1 were increased in the HG+CRP group measured by immunofluorescence (E). Scale bars, 20 μm. Phosphorylation of Smad3 significantly increased in the HG+CRP group (F). Each bar represents the mean SEM from three independent experiments in vitro. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus the indicated group.

Fig 5: CRP-induced NLRP3 inflammasome activation was blocked by CRP receptor CD32 antibodyWestern blot showed that the addition of CD32 antibody (CD32Ab) significantly inhibited NLRP3 inflammasome-related indexes of protein expression level, including NEK7, C-caspase-1, and IL-1β in HK-2 cells (A) and mouse mesangial cells (E). qPCR demonstrated that CRP-induced NLRP3, NEK7, IL-1β, and TNF-α mRNA expression levels in HK-2 cells were significantly inhibited with the addition of CD32Ab in HK-2 cells (B) and mouse mesangial cells (F). Expression levels of NLRP3 and caspase-1 were decreased with the addition of CD32Ab, measured by immunofluorescence in HK-2 cells (C) and mouse mesangial cells (G). Scale bars, 20 μm. Phosphorylation of Smad3 significantly decreased in the HG+CRP+CD32Ab group in HK-2 cells (D) and mouse mesangial cells (H). Each bar represents the mean SEM from three independent experiments in vitro. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus the indicated group.