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. 2013 Mar 1;15(2):R38.
doi: 10.1186/ar4192.

The severity of experimental arthritis is independent of IL-36 receptor signaling

Céline Lamacchia, Gaby Palmer, Emiliana Rodriguez, Praxedis Martin, Solenne Vigne, Christian A Seemayer, Dominique Talabot-Ayer, Jennifer E Towne, Cem Gabay

The severity of experimental arthritis is independent of IL-36 receptor signaling

Céline Lamacchia et al. Arthritis Res Ther. .

Abstract

Introduction: Interleukin (IL)-36 refers to three related IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, that bind to the IL-36 receptor (IL-36R). IL-36 exerts proinflammatory effects in skin and lung and stimulates T cell responses. In the present study, we examined the expression and function of IL-36R and its ligands in experimental arthritis.

Methods: Collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and K/BxN serum transfer-induced arthritis were induced according to standard protocols. Messenger RNA levels for IL-36R and its ligands in the joints of mice with CIA were determined by RT-qPCR. Mice with CIA were injected with a blocking monoclonal anti-IL-36R, a blocking anti-IL-1RI, or their isotype-matched control antibodies at the time of arthritis onset. Anti-IL-36R or control antibodies were also injected at the time of AIA induction. Finally, IL-36R-deficient mice were examined in AIA and serum transfer-induced arthritis. The development and severity of arthritis were assessed by clinical and histological scoring.

Results: IL-36R, IL-36Ra and IL-36γ mRNA were detected in the joints of mice with CIA, but their levels did not correlate with arthritis severity. As opposed to anti-IL-1RI antibody treatment, the injection of an anti-IL-36R antibody was devoid of effect on the development and severity of CIA. The severity of joint inflammation and structural damage in AIA was also unaltered by anti-IL-36R antibody treatment. Finally, the severity of AIA and K/BxN serum transfer-induced arthritis was similar in IL-36R-deficient and wild-type mice.

Conclusions: The development and severity of experimental arthritis are independent of IL-36R signaling.

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Figures

Figure 1
Figure 1
Analysis of IL-36R, IL-36γ and IL-36Ra mRNA expression in normal and inflamed joints during collagen-induced arthritis (CIA). IL-36 receptor (IL-36 R) (A), IL-36γ (B), IL-36R antagonist (IL-36Ra) (C), IL-1RI (D), IL-1β (E) and IL-1Ra (F) mRNAs levels were determined by RT-qPCR in normal (clinical score = 0) or inflamed (low clinical scores: 0.5 to 1.5; high clinical scores: 2.0 to 3.0) ankles of type II collagen (CII)-immunized DBA-1 mice in the first nine days after the appearance of clinical symptoms (early arthritis). (A-F) mRNA expression was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Each data point represents a single mouse; horizontal lines show the mean. *P < 0.05 versus score 0, as assessed by analysis of variance, followed by unpaired two-tailed Student's t-test.
Figure 2
Figure 2
Efficacy of a blocking monoclonal anti-IL-36 receptor (R) antibody in vivo. BALB/c mice (n = 5/group) were pre-treated intranasally (i.n.) with a rat IgG2a anti-mouse IL-36R antibody (M616; 50 μg/mouse; squares), an isotype control antibody (M10; 50 μg/mouse; triangles) or PBS (circles) 2 h prior to i.n. challenge with recombinant IL-36γ (1 μg/mouse) on days 0, 1 and 2. A fourth group of mice, used as negative control, only received i.n. injection of PBS (inverted triangles). On day 2, bronchoalveolar lavage fluids recovered from mice 4 h after the last injection were used to assess (A) total leucocyte and neutrophil counts and (B) chemokine (CCL20, CCL11 and CCL24) protein levels by DuoSet ELISA. (A-B) Results are shown as individual values for each mouse (symbols) and mean values (lines). ***P < 0.001 versus isotype control-treated mice, as assessed by unpaired two-tailed Student's t-test.
Figure 3
Figure 3
Treatment with a monoclonal anti-IL-36 receptor (R) antibody does not modify the course of collagen-induced arthritis (CIA). CII-immunized mice (n = 10/group) were treated with anti-IL36R (M616, squares, white columns), anti-IL-1RI (M147, triangles, dashed columns) or isotype-matched control antibodies (4G8 (circles, grey columns) for M616, 4D2 (triangles, black columns) for M147), as described in Materials and methods. Results show the incidence of arthritis (A), the clinical severity of articular inflammation (B) and the number of arthritic paws (C) on days 21 to 41. The systemic inflammatory response (D) was assessed by measuring circulating IL-6 levels on day 41 after the first immunization. Joint sections from all mice were evaluated on day 41 for histological scores (E) and histological features (F). All sections were scored for inflammation, cartilage erosion and neutrophil infiltration. Images are representative of H&E- or toluidine blue-stained sections of knee joints for each group (original magnification × 10). (G) Levels of CXCL-1 (ng/mL) were determined by ELISA in ankle extracts of each mouse on day 41 and normalized by the total protein concentration (mg/mL). (B, C, E) Values are the mean ± standard error of the mean. ***P < 0.001, anti-IL-1RI versus isotype control 4D2; &P < 0.05 and &&P < 0.01, anti-IL-1RI versus anti-IL-36R, as assessed by Kruskal-Wallis test (B and C) or by analysis of variance (ANOVA), followed by unpaired two-tailed Student's t-test (E). (D, G) Results are shown as individual values for each mouse (symbols) and mean values (lines) **P < 0.01, anti-IL-1RI versus isotype control 4D2, as assessed by ANOVA, followed by unpaired two-tailed Student's t-test.
Figure 4
Figure 4
The course of antigen-induced arthritis (AIA) is not modified by treatment with a monoclonal anti-IL-36 receptor (R) antibody or in IL-36R-deficient mice. Time course of knee joint inflammation is shown in anti-IL-36R antibody-treated mice (A, upper panel) and in IL-36R knockout (KO) mice (B, left panel) with AIA. Joint inflammation was measured by external gamma counting of 99mtechnetium (Tc) accumulation on days 1, 3 and 7 after mBSA injection into the left knee. Results are expressed as the ratio of 99mTc uptake in the left arthritic knee over the right non-inflamed knee. For each time point, the mean ± standard error of the mean (SEM) of the ratios is shown for anti-IL-36R antibody (n = 7; triangles), 9B5 (rat IgG2a anti-human CD44) isotype-matched control antibody-treated mice (n = 7; gray circles), or PBS-treated mice (n = 11; open circles), wild-type (WT) C57BL/6J mice (n = 9; black line) and IL-36R-/- mice (n = 7; dashed line). (A, lower panels; B, right panel) Histological scores for synovial inflammation and cartilage degradation 8 and 14 days after intra-articular mBSA injection. Results shown represent the mean ± SEM for anti-IL-36R antibody (n = 5 on day 8, n = 6 on day 14, black columns), 9B5 isotype control (n = 3 on day 8, n = 4 on day 14, hatched columns), PBS-treated mice (n = 3 on day 8, n = 4 on day 14, open columns) (A, lower panels), WT C57BL/6J mice (n = 9 on day 8, white columns) and IL-36R-/- mice (n = 7 on day 8, black columns) (B, right panel).
Figure 5
Figure 5
Incidence and severity of K/BxN serum transfer-induced arthritis are not reduced in IL-36 receptor (R) knockout (KO) mice. Incidence of arthritis (A), arthritis severity scores (B) and the number of affected paws (C) are shown for WT (n = 10, black line) and IL-36R KO (n = 7, dashed line) mice. (A and C) Results are shown as the mean ± standard error of the mean.
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