Interleukin-36 family dysregulation drives joint inflammation and therapy response in psoriatic arthritis

Abstract

Objectives: IL-36 agonists are pro-inflammatory cytokines involved in the pathogenesis of psoriasis. However, their role in the pathogenesis of arthritis and treatment response to DMARDs in PsA remains uncertain. Therefore, we investigated the IL-36 axis in the synovium of early, treatment-naïve PsA, and for comparison RA patients, pre- and post-DMARDs therapy.

Methods: Synovial tissues were collected by US-guided biopsy from patients with early, treatment-naïve PsA and RA at baseline and 6 months after DMARDs therapy. IL-36 family members were investigated in synovium by RNA sequencing and immunohistochemistry, and expression levels correlated with DMARDs treatment response ex vivo. Additionally, DMARDs effects on IL-36 were investigated in vitro in fibroblast-like synoviocytes.

Results: PsA synovium displayed a reduced expression of IL-36 antagonists, while IL-36 agonists were comparable between PsA and RA. Additionally, neutrophil-related molecules, which drive a higher activation of the IL-36 pathway, were upregulated in PsA compared with RA. At baseline, the synovial expression of IL-36α was significantly higher in PsA non-responders to DMARDs treatment, with the differential expression being sustained at 6 months post-treatment. In vitro, primary PsA-derived fibroblasts were more responsive to IL-36 stimulation compared with RA and, importantly, DMARDs treatment increased IL-36 expression in PsA fibroblasts.

Conclusion: The impaired balance between IL-36 agonists-antagonists described herein for the first time in PsA synovium and the decreased sensitivity to DMARDs in vitro may explain the apparent lower efficacy of DMARDs in PsA compared with RA. Exogenous replacement of IL-36 antagonists may be a novel promising therapeutic target for PsA patients.

Keywords: cytokines; early arthritis; inflammation; interleukin-36; psoriatic arthritis; rheumatoid arthritis; synovitis.

Fig. 1

Expression of IL-36α, IL-36RA and IL-38 in synovium from early DMARDs-naïve PsA and RA patients mRNA expression of IL-36A (A), IL-36RN (B) and IL-1F10 (C) in synovium of PsA and RA patients was assessed by RNA sequencing; n=14 PsA and 18 RA. Sections of human PsA and RA synovium were stained for IL-36α (A), IL-36RA (B) and IL-38 (C). Representative images are shown. Scale bar=100 μm. Enlarged images correspond to the respective boxed areas. Digital image analysis was performed on all PsA (n=27) and RA (n=19) synovium sections. IL-36α+ (A), IL-36RA+ (B) and IL-38+ (C) surfaces were determined using ImageJ software and are presented as % of the synovium surface. (D) Ratios between agonist (IL-36α) and antagonists (IL-36RA and IL-38) expressions are shown. (A–D) Results are presented as mean (s.e.m). *P < 0.05, **P < 0.01 as assessed by Mann–Whitney U test.

Fig. 2

PsA synovium is characterized by a strong neutrophil signature (A) Volcano plot representation of differential expression of neutrophils-associated genes in PsA and RA synovium. Green points mark the genes with significantly increased expression respectively in PsA compared with RA synovium. The x-axis shows log2-fold changes in expression and the y-axis the log odds of a gene being differentially expressed. (B, D) Sections of human PsA and RA synovium were stained for NE (B) and Cathepsin G (D). Representative images are shown. Scale bar = 100 μm. Enlarged images correspond to the respective boxed areas. (C, E) Digital image analysis was performed on all PsA (n=27) and RA (n=19) synovium sections. NE (C) and Cathepsin G (E) positive staining surfaces were determined using ImageJ software and are presented as % of the synovium surface. Results are presented as mean (s.e.m). *P < 0.05, ***P < 0.001 as assessed by Mann–Whitney U test. NE: Neutrophil elastase.

Fig. 3

Association between IL-36α, IL-36RA and IL-38 expression and the synovial histomorphology in early DMARDs-naïve PsA and RA patients (A, C) Sections of PsA and RA synovium were stained for IL-36α, IL-36RA and IL-38. In RA synovium, three main histological pathotypes have been described previously: follicular (F), characterized by the presence of B/T cells frequently forming highly organized ELS, plasma cells and macrophages in the sublining; diffuse (D), marked by a predominant infiltration of CD68+ macrophages without distinctly organized follicular structures; and pauci-immune (P), defined by a scant immune cells infiltrate and a fibroblast-rich stroma. Representative images are shown for each pathotypes: F, D and P. Scale bar = 100 μm. (B, D) Digital image analysis was performed on PsA (n=27) and RA (n=19) synovium sections for each pathotypes (F, D and P). IL-36α+, IL-36RA+ and IL-38+ surfaces were determined using ImageJ software and are presented as % of the synovium surface. *P < 0.05 as assessed by Kruskal–Wallis with Dunn’s post-test. (E) Correlations between IL-36α, IL-36RA, IL-38 and 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) are shown. Positive significant correlations at the tissue protein expression level are presented in green. The green scale indicates the P-values, calculated by Spearman’s bivariate correlation analysis. Non-significant correlations are presented in white. (F) Double immunostaining of IL-36α with CD138, CD68, CD3, CD20 and TE7 in the synovium of PsA patients. IL-36α is shown in red; CD3, CD20, CD138, CD68 or TE7 are shown in green. Nuclei were counterstained with 40, DAPI (blue). Co-localizations of IL-36 with the cellular markers appear in yellow/light green (Merge). Representative images are shown. Scale bar = 20 μm. DAPI: 6-diamidino-2-phenylindole; ELS: ectopic lymphoid structures.

Fig. 4

Relationship between synovial and systemic expression of IL-36α and their antagonists (A) IL-36α, IL-36RA and IL-38 protein levels (pg/ml), as assessed by ELISA in plasma of early DMARDs-naïve PsA (n=38) and RA (n=38) patients. The results are shown as individual values, mean (s.e.m). ****P < 0.0001 as assessed by unpaired Student’s t test. (B) Correlation between the ESR and IL-36α expression in the plasma of PsA and RA patients is represented. The P-value is indicated, as assessed by Spearman’s bivariate correlation analysis. (C) Sections of human PsA and RA synovium were stained for IL-36R (IL-1Rrp2-specific chain). Representative images are shown. Scale bar = 100 μm. Enlarged images correspond to the respective boxed areas. Digital image analysis was performed and is shown in supplementary Fig. S3, available at Rheumatology online. (D) Triple immunostaining of IL-36α with IL-36R and IL-36RA in the synovium of PsA and RA patients. IL-36α is shown in red, IL-36R in light blue and IL-36RA in green. Nuclei were counterstained with DAPI and are shown in blue. Representative images are shown. Enlarged images correspond to the respective white boxed areas. Scale bar = 100 μm. (E) Percentage of synovial IL-36R+ cells from early PsA and RA patients positive for IL-36α or IL-36RA. Cells double-positive for IL-36R and IL-36α (in white) or IL-36R and IL-36RA (in black) were manually counted using the cell count plugin of ImageJ. (F) Correlation between IL-36α expression in the plasma and IL-36RA in the synovium is shown. P-values (shown) were calculated by Spearman’s bivariate correlation analysis. DAPI: 6-diamidino-2-phenylindole.

Fig. 5

IL-36α is highly expressed in the synovium of DMARDs non-responders PsA patients (A, C) Matched baseline and post-DMARDs (6 months) synovial tissue sections of R and NR PsA or RA patients were stained for IL-36α. Representative images are shown. Scale bar = 100 μm. Enlarged images correspond to the respective boxed areas. (B, D) Digital image analysis was performed on PsA (NR: n = 6 at baseline and n = 4 at 6 months; R: n = 10 at baseline and n = 6 at 6 months) and RA (NR: n = 6 at baseline and n = 3 at 6 months; R: n = 9 at baseline and n = 4 at 6 months) synovium sections. *P < 0.05 as assessed by Mann–Whitney U test, ns. (E) PsA (n = 7, in grey) or RA (n = 8, in black) FLS were stimulated with IL-1β and TNF-α (25 ng/ml each) with either MTX or SP (1 mM) and RNA expression of IL-36A, IL-36RN and IL-1F10 (IL-38 gene) was assessed by RT-quantitative PCR after 24 h of stimulation. (F) Immunocytochemistry staining of IL-36α in FLS stimulated with IL-1β and TNF-α (25 ng/ml each) together with 1 mM of MTX or SP for 36 h. IL-36α is shown in red, nuclei are stained with DAPI and represented in blue. DAPI: 4′,6-Diamidino-2-phenylindole; FLS: Fibroblast-Like Synoviocytes; NR: non-responders; ns: non-significant; R: responders; SP: sulfapyridine.

Fig. 6

Primary FLS from PsA patients are more sensitive to IL-36 stimulation (A) PsA (n=6 individual patients) and RA (n=7 individual patients) FLS were stimulated with increasing doses of rhIL-36α (0.1–100 ng/ml) for 48 h. IL-8 concentration in the supernatants was determined by ELISA; the percentage of increase compared with supernatants of untreated cells is shown. *P < 0.05 as assessed by Kruskal–Wallis with Dunn’s post-test. (B) IL-8 was measured in the supernatants of PsA (n=3) and RA (n=3) FLS stimulated with rhIL-36α for 48 h with or without pre-treatment (1 h) with 100 ng/ml of rhIL-36RA. The percentage of increase compared with supernatants of untreated cells (left panel) and the percentage of decrease compared with cells stimulated with IL-36α alone (right panel) are shown. *P < 0.05 as assessed by Kruskal–Wallis with Dunn’s post-test (left panel) or the Mann–Whitney U test (right panel). (C) Protein expression of total and phosphorylated NF-κB in PsA- and RA-FLS stimulated with 100 ng/ml of rhIL-36α for 15, 30 or 60 min. Actin is used as loading control. A representative blot of three independent experiments is shown. (D) Quantification of western blot signal for phosphorylated NF-κB using ImageJ software. Signals were normalized to the Actin signal and the fold increase is calculated relative to the untreated condition. Results are presented as mean (s.e.m). *P < 0.05 as assessed by Mann–Whitney U test for each timepoint. (E) IL-1RL2 (gene encoding IL-36Rrp2) expression was assessed in PsA- and RA-FLS by RT-quantitative PCR. FLS: Fibroblast-Like Synoviocytes; NF-κB: nuclear factor κB.