Distinct expression of interleukin (IL)-36α, β and γ, their antagonist IL-36Ra and IL-38 in psoriasis, rheumatoid arthritis and Crohn's disease

Abstract

Interleukin (IL)-36α, IL-36β and IL-36γ are expressed highly in skin and are involved in the pathogenesis of psoriasis, while the antagonists IL-36Ra or IL-38, another potential IL-36 inhibitor, limit uncontrolled inflammation. The expression and role of IL-36 cytokines in rheumatoid arthritis (RA) and Crohn's disease (CD) is currently debated. Here, we observed that during imiquimod-induced mouse skin inflammation and in human psoriasis, expression of IL-36α, γ and IL-36Ra, but not IL-36β and IL-38 mRNA, was induced and correlated with IL-1β and T helper type 17 (Th17) cytokines (IL-17A, IL-22, IL-23, CCL20). In mice with collagen-induced arthritis and in the synovium of patients with RA, IL-36α, β, γ, IL-36Ra and IL-38 were all elevated and correlated with IL-1β, CCL3, CCL4 and macrophage colony-stimulating factor (M-CSF), but not with Th17 cytokines. In the colon of mice with dextran sulphate sodium-induced colitis and in patients with CD, only IL-36α, γ and IL-38 were induced at relatively low levels and correlated with IL-1β and IL-17A. We suggest that only a minor subgroup of patients with RA (17-29%) or CD (25%) had an elevated IL-36 agonists/antagonists ratio, versus 93% of patients with psoriasis. By immunohistochemistry, IL-36 cytokines were produced by various cell types in skin, synovium and colonic mucosa such as keratinocytes, CD68⁺ macrophages, dendritic/Langerhans cells and CD79α⁺ plasma cells. In primary cultures of monocytes or inflammatory macrophages (M1), IL-36β and IL-36Ra were produced constitutively, but IL-36α, γ and IL-38 were produced after lipopolysaccharide stimulation. These distinct expression profiles may help to explain why only subgroups of RA and CD patients have a potentially elevated IL-36 agonists/antagonists ratio.

Keywords: Crohn's disease; IL-36; psoriasis; rheumatoid arthritis.

Figure 1

Expression of interleukin (IL)−36 cytokines in mouse models of inflammation. Expression of IL‐36α, β and γ, IL‐36Ra and IL‐38 mRNA in mouse skin during imiquimod (IMQ)‐induced inflammation (n = 3 mice per time‐point, upper panels), in hind paws of mice with collagen‐induced arthritis (CIA) (n = 4–5 mice per time‐point, middle panels) or in the distal colon of mice with dextran sulphate sodium (DSS)‐induced colitis (n = 7 mice, lower panels) at early, peak, stabilized (stab) and/or resolution (res) phases of inflammation (see Materials and methods), as assessed by reverse transcription–quantitative polymerase chain reaction (RT–qPCR). Control groups (CT) comprised non‐induced mice at the different time‐points. The results represent cytokine mRNA expression relative to hypoxanthine–guanine phosphoribosyltransferase (HPRT) and are shown as individual values and mean. The dotted line indicates the cytokine mRNA expression level observed in mouse skin at the peak of inflammation. (a) P < 0·05 compared to CT group, as assessed by Mann–Whitney U‐test.

Figure 2

Expression of interleukin (IL)‐36 cytokines in patients with psoriasis, rheumatoid arthritis (RA) and Crohn's disease (CD). (a) Expression of IL‐36α, β and γ, IL‐36Ra and IL‐38 mRNA in human healthy skin (CT, n = 14) and psoriasis samples (PSO, n = 15) (upper panels), in synovial tissue of patients with OA (n = 6) or RA (n = 17) (middle panels) and in colonic biopsies of patients with CD from normal, non‐inflamed (CT, n = 15) or inflamed (CD, n = 16) areas (lower panels). The results represent cytokine mRNA expression relative to hypoxanthine–guanine phosphoribosyltransferase (HPRT) and are shown as individual values and mean. The different P‐values are indicated, as assessed by unpaired Student's t‐test, as well as the mean fold increase in expression over the CT group. (b) Cytokine protein levels, as assessed by enzyme‐linked immunosorbent assay (ELISA) in synovial fluid of RA patients (n = 30) versus osteoarthritis (OA) patients (n = 29). The results are shown as individual values and mean. The different P‐values are indicated, as assessed by unpaired Student's t‐test. (c) Induction of cytokine mRNA expression in PSO, RA or CD (CRO) is presented in individual patients as fold increase over the mean value calculated in the CT or OA groups. Agonists are presented using the indicated yellow colour‐code, antagonists with the green colour‐code. The dotted areas identify patients with an elevated agonists/antagonists ratio. (d) Cluster of genes with positive statistically significant correlations (P < 0·05, as assessed by Pearson's bivariate correlation analysis) at the mRNA and/or protein expression level in patients with PSO, RA or CD are presented in grey. In white are presented genes whose expression does not correlate with the grey ones; n.d. = not done. See Tables 1–4 (Supporting information, File S1) for expression of these genes in PSO, RA and CD patients.

Figure 3

Immunohistochemical analysis of interleukin (IL)‐36 cytokines in psoriasis. (a) Sections from human healthy skin (n = 3 donors, upper panels) or from patients with psoriasis (n = 3, lower panels) were stained for IL‐36α, β or γ. Representative images are shown. Enlarged images 1 and 2 correspond to the respective boxed areas. Scale bar = 50 μm. (b) Serial sections from psoriatic skin were stained for CD79α (pan‐B lymphocytes), CD55 (pan‐fibroblasts) or CD68 (macrophages, dendritic and Langerhans cells). Enlarged images 1 and 2 correspond to the respective boxed areas. Scale bar = 50 μm. The box presents the symbols used to show representative cell types.

Figure 4

Immunohistochemical analysis of interleukin (IL)‐36 cytokines in rheumatoid arthritis (RA). (a) Sections of human synovial tissue from patients with OA (n = 3, upper panels) or from patients with RA (n = 7, lower panels) were stained for IL‐36α, β or γ. Representative images are shown. Enlarged images correspond to the respective boxed areas. Scale bar = 50 μm. (b) Semiquantitative analysis of the immunohistochemistry (IHC) stainings. The results represent individual values and mean. *P < 0·05 compared to CT osteoarthritis (OA) group, as assessed by Mann–Whitney U‐test. (c) Serial sections from RA synovium were stained for CD79α (pan‐B lymphocytes), CD55 (pan‐fibroblasts), CD68 (pan‐macrophages and dendritic cells) or with an isotype control antibody (IsoCT). Enlarged images correspond to the respective boxed areas. Scale bar = 50 μm. The box presents the symbols used to show representative cell types. (d) Co‐localization of IL‐36α with CD68 (pan‐macrophages and dendritic cells) or with CD79α (pan‐B lymphocytes) in the synovial tissue of RA patients as assessed by confocal microscopy. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). Representative images are shown. Examples of IL‐36α⁺CD68⁺ (yellow arrow) and IL‐36α⁺CD79α⁺ (pink arrow) cells are shown in the merged image. Scale bar = 50 μm.

Figure 5

Immunohistochemical analysis of interleukin (IL)‐36 cytokines in Crohn's disease (CD). (a) Sections from human colonic biopsies of patients with CD from normal, non‐inflamed (unaffected, n = 4 donors, upper panels) or inflamed (CD, n = 4, lower panels) areas were stained for IL‐36α, β or γ. Representative images (mucosa) are shown. Enlarged images correspond to the respective boxed areas. Scale bar = 50 μm. (b) Serial sections from inflamed CD colon were stained for CD79α (pan‐B lymphocytes), CD55 (pan‐fibroblasts) or CD68 (macrophages and dendritic cells). Scale bar = 50 μm. The box presents the symbols used to show representative cell types.

Figure 6

Expression of interleukin (IL)‐36 cytokines in cultured cells. (a) Primary cultures of normal human epidermal keratinocytes from two different healthy donors, synovial fibroblasts from three different rheumatoid arthritis (RA) patients [rheumatoid arthritis synovial fibroblasts (RASF)] or Caco2 enterocytes were stimulated for 24 h with polyI:C (25 μg/ml) or a combination of five cytokines [M5: IL‐17A, IL‐22, oncostatin M, IL‐1β, tumour necrosis factor (TNF)‐α, all at 10 ng/ml]. CD14⁺ monocytes (mono), M1 macrophages, M2 macrophages, monocyte‐derived dendritic cells (moDC) and osteoclasts (Oc) were prepared from three different healthy blood donors and stimulated for 8 h with lipopolysaccharide (LPS) (100 ng/ml). Expression of IL‐36α, β, γ, IL‐36Ra and IL‐38 mRNA was assessed by reverse transcription–quantitative polymerase chain reaction (RT–qPCR). The results represent cytokine mRNA expression relative to hypoxanthine–guanine phosphoribosyltransferase (HPRT), shown as mean ± standard error of the mean (s.e.m.). *P < 0·05 compared to unstimulated cultures (CT), as assessed by Mann–Whitney U‐test. (b) Summary of the cell sources and different expression profiles of IL‐36 cytokines in psoriasis, RA and CD. Macro = macrophage; DC = dendritic cells; Kera = keratinocyte; Endoth = endothelial cell; B = plasma cell; Fibro = fibroblast; Entero = enterocyte. Other cytokines, which correlate with IL‐36 cytokines are also indicated (+).