Amelioration of human lupus-like phenotypes in MRL/lpr mice by overexpression of interleukin 27 receptor alpha (WSX-1)

Abstract

Objective: In the present work, we investigate the role of interleukin (IL)27/IL27 receptor alpha (Ralpha) (WSX-1) in the development of autoimmune disorders in the MRL/lpr mouse, which is considered as an experimental model of systemic lupus erythaematosus (SLE) in humans.

Methods: We generated two strains of WSX-1 transgenic mice in the MRL/lpr background with different expression levels of WSX-1, and investigated the effect of WSX-1 overexpression on survival, glomerulonephritis and immunological properties.

Results: In comparison with wild type (WT) MRL/lpr and transgenic (Tg) low (TgL) mice, Tg high (TgH) mice exhibited a prolonged lifespan and no apparent development of autoimmune nephritis. Production of anti-dsDNA antibody and total IgG and IgG2a were significantly lower in TgH mice than those of TgL and WT mice. The expressed amounts of interferon (IFN)gamma and IL4 mRNA by CD4+ T cells from Tg mice decreased in a dose-dependent fashion. CD4+ splenic lymphocytes in TgH mice were more subject to the IL27-mediated suppression of cytokine production. In vitro stimulation of CD4+ T cells by IL27 resulted in over phosphorylation of STAT3 in TgH cells than in WT cells.

Conclusion: WSX-1 overexpression in the MRL/lpr background rendered the autoimmune prone mice protected from the development of autoimmune diseases. Our results suggest that IL27 signalling may be a therapeutic target against autoimmune diseases, including human SLE.

Figure 1. Prolonged survival of MRL/lpr mice with high expression of WSX-1. A. The cumulative survivals of wild type (WT), transgenic low (TgL) and high (TgH) mice were monitored weekly (n = 20 per group). *p<0.05 by the Kaplan–Meier method. There was no difference in the survival rate between WT and TgL mice. B. Western blot analysis of the transgenic expression of WSX-1 in CD4⁺ T cells from WT mice and two Tg-positive MRL/lpr mouse lines, TgL and TgH. The lysates of purified splenic CD4⁺ T cells from 16-week-old WT, TgL and TgH mice were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition and transferred to membranes. Expression levels for β-actin protein was visualised for each sample as a loading control. The transgene products demonstrate robust, moderate and faint expression of WSX-1 in TgH, TgL and WT mice T cells, respectively.

Figure 2. Amelioration of glomerulonephritis and decreased Ig deposition in transgenic high (TgH) mice. The kidneys were fixed in 10% formalin for 24 h at 4°C. Paraffin sections (4 µm) were stained either with H&E, periodic acid Schiff stain, or periodic acid-methenamine silver (PAM). Microscopic examination of the kidney glomerulus of a 24-week-old wild type (WT) mouse (A), transgenic low (TgL) mouse (B) and TgH mouse (C) (periodic acid Schiff staining; original magnification, ×400). IgG (D, E and F), IgG1 (G, H and I) and IgG2a (J, K and L) deposits in the glomeruli of a 24-week-old WT, TgL and TgH mice were visualised using immunofluorescent anti-Ig staining (×400) and quantitative analysed by image software. WT: IgG (83.8 (7.25)), IgG1 (68.4 (6.4)) and IgG2a (76.8 (4.9)), TgL: IgG (81.0 (8.48)), IgG1 (71.4 (7.3)) and IgG2a (73.0 (4.6)), TgH:IgG (25.2 (9.65)), IgG1 (22.8 (6.45)), IgG2a (19.6 (3.2)). These representative data obtained from 30 glomerular cross sections per kidney (six mice per group) with similar staining patterns. M. The severity of glomerulonephritis was evaluated by the score of glomerular proliferative activity. The scores of glomerular proliferative activity of 24-week-old WT, TgL and TgH mice were 2.38 (0.356), 1.92 (0.564) and 0.74 (0.378), respectively.

Figure 3. Decreased autoreactive immune responses in transgenic high (TgH) mice. A. Sera collected from 16-week-old wild type (WT), transgenic low (TgL) and TgH mice were examined for anti-nuclear antibodies (ANA) as described in Materials and methods. The same sera in (A) were measured for anti-dsDNA antibodies (B) or for Ig levels (C). D. The expression levels of interferon (IFN)γ and interleukin (IL)4 mRNA in CD4⁺ T cells and those of IL12 and IL10 mRNA in B220⁺CD3⁺ cell-depleted splenocytes were examined by quantitative PCR. Data shown are mean (SD) of 10 mice per group. *p<0.05 by unpaired Student t test.

Figure 4. Decrease in CD3⁺B220⁺CD4^–CD8^– T cell and upregulated expression of activation markers on transgenic high (TgH) CD4⁺ T cells. A. Spleens were removed from 16-week-old wild type (WT) and TgH mice, and single cell suspensions were stained for the expression of surface markers, followed by flow cytometry. Numbers are the percentage of CD3⁺B220⁺CD4^–CD8^– T cells, CD8⁺ T cells and CD4⁺ T cells against total cell populations. B. Surface expression levels of CD69, CD25, CD62L and CD44 were analysed by flow cytometry. Numbers are the percentage of the total CD4⁺ T cells.

Figure 5. IL27 suppression of cytokine production by activated CD4⁺ T cells. A. CD4⁺ T cells from wildtype (WT) and transgenic high (TgH) mice were stimulated with plate-bound anti-CD3 antibody plus soluble anti-CD28 antibody (1 μg/ml) in the absence or presence of interleukin (IL)27 for 24 h. Culture supernatants were measured for the production of respective cytokines. Data shown are mean (SD) of 10 mice per group. *p<0.05 by unpaired Student t test. B. CD4⁺ T cells from WT and TgH mice were stimulated as in Materials and methods. Phosphorylation of signal transducer and activator of transcription (STAT)1 or STAT3 in whole cell lysates in WT and TgH mice was analysed with anti-phosphotyrosine (α-pY) -STAT1 or anti-pY-STAT3 antibodies, respectively. The filter was stripped and re-probed with anti-STAT1 or anti-STAT3 antibodies to ensure the same amounts of samples were loaded. Experiments were repeated three times with similar results.