Circulating and Synovial Pentraxin-3 (PTX3) Expression Levels Correlate With Rheumatoid Arthritis Severity and Tissue Infiltration Independently of Conventional Treatments Response

Abstract

Aims: To determine the relationship between PTX3 systemic and synovial levels and the clinical features of rheumatoid arthritis (RA) in a cohort of early, treatment naïve patients and to explore the relevance of PTX3 expression in predicting response to conventional-synthetic (cs) Disease-Modifying-Anti-Rheumatic-Drugs (DMARDs) treatment.

Methods: PTX3 expression was analyzed in 119 baseline serum samples from early naïve RA patients, 95 paired samples obtained 6-months following the initiation of cs-DMARDs treatment and 43 healthy donors. RNA-sequencing analysis and immunohistochemistry for PTX3 were performed on a subpopulation of 79 and 58 synovial samples, respectively, to assess PTX3 gene and protein expression. Immunofluorescence staining was performed to characterize PTX3 expressing cells within the synovium.

Results: Circulating levels of PTX3 were significantly higher in early RA compared to healthy donors and correlated with disease activity at baseline and with the degree of structural damages at 12-months. Six-months after commencing cs-DMARDs, a high level of PTX3, proportional to the baseline value, was still detectable in the serum of patients, regardless of their response status. RNA-seq analysis confirmed that synovial transcript levels of PTX3 correlated with disease activity and the presence of mediators of inflammation, tissue remodeling and bone destruction at baseline. PTX3 expression in the synovium was strongly linked to the degree of immune cell infiltration, the presence of ectopic lymphoid structures and seropositivity for autoantibodies. Accordingly, PTX3 was found to be expressed by numerous synovial cell types such as plasma cells, fibroblasts, vascular and lymphatic endothelial cells, macrophages, and neutrophils. The percentage of PTX3-positive synovial cells, although significantly reduced at 6-months post-treatment as a result of global decreased cellularity, was similar in cs-DMARDs responders and non-responders.

Conclusion: This study demonstrates that, early in the disease and prior to treatment modification, the level of circulating PTX3 is a reliable marker of RA activity and predicts a high degree of structural damages at 12-months. In the joint, PTX3 associates with immune cell infiltration and the presence of ectopic lymphoid structures. High synovial and peripheral blood levels of PTX3 are associated with chronic inflammation characteristic of RA. Additional studies to determine the mechanistic link are required.

Keywords: inflammation; pathotypes; pentraxin-3; rheumatoid arthritis; synovial tissue.

Figure 1

Levels of circulating PTX3 correlate with disease severity in early untreated RA patients, but not with clinical response to cs-DMARDs treatment. (A) PTX3 serum levels in patients with early treatment-naïve RA (n=119) and age- and gender-matched healthy donors (n=43). ****p < 0.0001, as assessed by parametric unpaired t-test. (B) PTX3 serum levels in healthy donors (n=43) and in paired patients at baseline (Bas) (n=119) and at 6-months (n = 95). ns, non-significant, as assessed by the parametric paired t-test comparing RA baseline and 6-months samples. (C) Left panel, PTX3 serum levels at 6-months in patients presenting a low (≤3.23 ng/mL) or high (>3.23 ng/mL) PTX3 serum levels at baseline, ****p < 0.0001 assessed by the parametric unpaired t-test. Right panel, correlation between PTX3 serum levels at baseline and at 6-months, p-value and r coefficient were calculated according to the Pearson correlation test. (D) Heatmap representing the correlation between PTX3 serum levels at baseline or 6-months (6-m) after cs-DMARDs treatment with clinical parameters: erythrocyte sedimentation rate (ESR), C-Reactive Protein (CRP), Disease Activity Score 28 (DAS28), Tender and swollen joint counts (TJC/28, SJC/28), patient (Pt) and physician (Phys) global health assessment by Visual Analogue Scale (VAS) and Health Assessment Questionnaire (HAQ). The red scale represents the spearman r coefficient. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as assessed by the Pearson correlation test. (E) Correlation between PTX3 serum levels at baseline and the van der Heijde modified Sharp score (SHSS) assessed at 12-months, p-value and r coefficient were calculated according to the Pearson correlation test. (F) Table presenting the clinical characteristics of responders (good and moderate) and non-responders patients to cs-DMARD treatments at 6-months, according to EULAR criteria. (G) PTX3 serum levels at baseline (Bas) or at 6-months (6-m) in non-responders (NR, red lines), moderate responders (MR, green lines) and good responders (R, blue lines) patients to cs-DMARD treatments. (A–C Left panel) Data are represented as median +/- interquartile range.

Figure 2

Synovial PTX3 gene expression correlates with disease activity and local inflammation. (A) Correlation between synovial transcript levels of PTX3 at baseline assessed by bulk RNA-seq and CRP, ESR and DAS28. (B) Heatmap representing the correlation between PTX3 synovial transcript levels at baseline and acute phase reactants, selected chemokines, cytokines and related signaling pathways mediators involved in inflammation, tissue remodeling and bone resorption. The red/blue scale represents the spearman r coefficient. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (A, B) p-values and r coefficients were calculated using Pearson correlation test. (C) 2D polar plot of transcript modules containing PTX3 in synovial tissue presenting a lympho-myeloid (lympho), diffuse-myeloid (Diffuse), and pauci-immune fibroid (Pauci) pathotypes (4). Different colors show pairwise comparisons between the three pathotypes: upregulation in one group only (Diffuse: red, Pauci: green and Lympho: blue) or in two groups (Diffuse/Pauci: yellow, Lympho/Diffuse: purple). (D) Pathways and diseases gene set enrichment analysis of the PTX3 module, composed of the 30 genes encoding for the protein integrated in the PTX3 string network, performed using the R interface to the EnrichR database (https://CRAN.R-project.org/package=enrichR). (E) PTX3 module expression in RA synovium classified as pauci-immune (n = 16), diffuse-myeloid (n = 20) and lympho-myeloid (n = 43). Data are represented as median +/- interquartile range. * = p < 0.05, as assessed by the Kruskal–Wallis test with Dunn’s post-test; ns, non-significant.

Figure 3

Synovial PTX3 expression associates with immune cell infiltration and the presence of ectopic lymphoid structure. (A) Sections of RA synovium were stained for PTX3. Representative images are shown for each pathotype (pauci-immune, diffuse-myeloid and lympho-myeloid). Scale bar = 40μm. (B) Digital image analysis was performed on RA synovium sections (n = 58) classified as pauci-immune (n = 10), diffuse-myeloid (n = 16) and lympho-myeloid (n=32). Synovial PTX3+ cells quantification was determined using QuPath software (28). ***p < 0.001, ****p<0.0001, as assessed by the Kruskal–Wallis test with Dunn’s post-test. (C) % of PTX3 positive cells in rheumatoid factor (RF)/cyclic citrullinated peptide (CCP) seropositive/negative patients. *p<0.05, as assessed by the Mann Whitney test. Data are represented as median +/- interquartile range. (D) Correlation between the percentage of PTX3 positive cells and the semi-quantitative scores for inflammatory cells markers (CD3-T cells; CD20-B cells; CD138-plasma cells; CD68L-macrophages of the synovium lining layer; CD68SL-macrophages of the sublining layer) (2, 3). p-values were calculated using Pearson correlation test. (E) Double immunostaining of PTX3 (yellow) with CD55, CD138, LYVE-1, vWF, CD68 and NE (green) in the synovium of RA patients. Nuclei were counterstained with DAPI (blue). White arrows indicate double-positive cells. Representative images are shown. Scale bar = 40μm.

Figure 4

Synovial PTX3 expression is significantly decreased after cs-DMARD treatment but independently of clinical response. (A) % of PTX3 positive synovial cells in paired patients at baseline and at 6-months after cs-DMARD treatment. Data are represented as median +/- interquartile range. ****p < 0.00001, as assessed by the Wilcoxon test. (B) Heatmap representing the correlation between dPTX3 synovial expression (calculated as the difference of expression between baseline and 6-months) and difference between semi-quantitative scores for inflammatory cells markers at baseline and at 6-months (dCD3, dCD20, dCD138, dCD68L, dCD68SL). The red scale represents the spearman r coefficient. *p < 0.05, ***p < 0.001, ns, non-significant. p-values and r coefficients were calculated using Pearson correlation test. (C) % of PTX3 positive synovial cells at baseline (Bas) and at 6-months (6-m) in non-responders (NR, red lines), moderate responders (MR, green lines) and good responders (R, blue lines) patients to cs-DMARD treatment.