Remission of systemic lupus erythematosus disease activity with regulatory cytokine interleukin (IL)-35 in Murphy Roths Large (MRL)/lpr mice

Abstract

The immunological mechanisms mediated by regulatory cytokine interleukin (IL)-35 are unclear in systemic lupus erythematosus (SLE). We investigated the frequency of CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) regulatory T (Treg ) and IL-10(+) regulatory B (Breg ) cells and related immunoregulatory mechanisms in a female Murphy Roths Large (MRL)/lpr mouse model of spontaneous lupus-like disease, with or without IL-35 treatment. A remission of histopathology characteristics of lupus flare and nephritis was observed in the MRL/lpr mice upon IL-35 treatment. Accordingly, IL-35 and IL-35 receptor subunits (gp130 and IL-12Rβ2) and cytokines of MRL/lpr and BALB/c mice (normal controls) were measured. The increased anti-inflammatory cytokines and decreased proinflammatory cytokines were possibly associated with the restoration of Treg and Breg frequency in MRL/lpr mice with IL-35 treatment, compared to phosphate-buffered saline (PBS) treatment. mRNA expressions of Treg -related FoxP3, IL-35 subunit (p35 and EBI3) and soluble IL-35 receptor subunit (gp130 and IL12Rβ2) in splenic cells were up-regulated significantly in IL-35-treated mice. Compared with the PBS treatment group, IL-35-treated MRL/lpr mice showed an up-regulation of Treg -related genes and the activation of IL-35-related intracellular Janus kinase/signal transducer and activator of transcription signal pathways, thereby indicating the immunoregulatory role of IL-35 in SLE. These in vivo findings may provide a biochemical basis for further investigation of the regulatory mechanisms of IL-35 for the treatment of autoimmune-mediated inflammation.

Keywords: cytokines; interleukin-35; regulatory B cells; regulatory T cells; systemic lupus erythematosus.

Figure 1

Clinical characteristics of Murphy Roths Large (MRL)/lpr mice with interleukin (IL)-35 or phosphate-buffered saline (PBS) injection. The results of each score system of the signs: (a) proteinuria, (b) leucocytuira, (c) lupus flare, (d) glomerulonephritis, (e) interstitial nephritis and (f) vessels infiltration are presented as bar charts with the arithmetic mean plus standard error of the mean (s.e.m.). *P < 0·05, **P < 0·01, ***P < 0·001, PBS treatment versus IL-35 treatment; ^†P < 0·05, ^††P < 0·01, ^†††P < 0·001, BALB/c control mice versus PBS-treated MRL/lpr mice; ^‡P < 0·05, ^‡‡‡P < 0·001, BALB/c control mice versus IL-35-treated MRL/lpr mice (n = 5 in each group).

Figure 2

Plasma concentrations of anti-nuclear antibody and anti-double-stranded DNA antibody in Murphy Roths Large (MRL)/lpr and BALB/c mice treated with interleukin (IL)-35 or phosphate-buffered saline (PBS) injection. Concentrations of plasma (a) anti-nuclear antibody (ANA) and (b) anti-ds-DNA antibodies are presented as bar charts with the arithmetic mean plus standard error of the mean (s.e.m.). *P < 0·05, ***P < 0·001, PBS versus IL-35 treatment; ^††P < 0·01, ^†††P < 0·001, BALB/c control mice versus PBS-treated MRL/lpr mice; ^‡P < 0·05, BALB/c control mice versus IL-35-treated MRL/lpr mice (n = 5 in each group).

Figure 3

RT² profiler polymerase chain reaction (PCR) array of mouse T helper (Th) cell differentiation. The results of epigenetically regulated gene expressions of Th1 cells, Th2 cells, inducible regulatory T cells (iT_reg) and natural (n)T_reg, Th17 cells and conventional T cells versus T_reg cells in (a) phosphate-buffered saline (PBS)-treated Murphy Roths Large (MRL)/lpr versus BALB/c mice (in ratio) and (b) interleukin (IL)-35 treated versus PBS-treated MRL/lpr mice (as ratio) are grouped according to the instruction of the classification of kit and presented as box-and-whisker plots with the median [interquartile range (IQR)]. Mann–Whitney U-test was used to assess the differences of mRNA expression among different epigenetically regulated genes of Th cells. *P < 0·05, **P < 0·01.

Figure 4

The differences of mRNA expression of p35, EBI3, gp130, IL-12Rβ2 and forkhead box protein 3 (FoxP3) of splenic cells between interleukin (IL)-35 and phosphate-buffered saline (PBS) treatment. Total RNA extracted from splenic cells from Murphy Roths Large (MRL)/lpr and BALB/c mice was reverse-transcribed and analysed by quantitative polymerase chain reaction (qPCR). Results were presented as Bar charts showing the arithmetic mean plus standard error of the mean (s.e.m.). *P < 0·05, PBS versus IL-35 treatment; ^††P < 0·01, BALB/c control mice versus PBS-treated MRL/lpr mice; ^‡P < 0·05, ^‡‡P < 0·01, ^‡‡‡P < 0·001, BALB/c control mice versus IL-35-treated MRL/lpr mice (n = 5 in each group).

Figure 5

The differences of mRNA expression of p35, EBI3, gp130, IL-12Rβ2 and forkhead box protein 3 (FoxP3) of thymic cells between interleukin (IL)-35 and phosphate-buffered saline (PBS) treatment. Total RNA extracted from thymic cells from Murphy Roths Large (MRL)/lpr and BALB/c mice was reverse-transcribed and analysed by quantitative polymerase chain reaction (qPCR). Results were presented as bar charts showing the arithmetic mean plus standard error of the mean (s.e.m.). Differences among each group of mice in the same treatment were compared by Kruskal–Wallis analysis of variance (anova), followed by Dunn’s post-test. ^†P < 0·05, BALB/c control mice versus PBS-treated MRL/lpr mice; ^‡P < 0·05, BALB/c control mice versus IL-35-treated MRL/lpr mice (n = 5 in each group).

Figure 6

Plasma concentrations of interleukin (IL)-35 and soluble IL-35R component gp130 and IL-12Rβ2 in Murphy Roths Large (MRL)/lpr and BALB/c mice with IL-35 or phosphate-buffered saline (PBS) treatment. Results of the concentrations of plasma (a) IL-35, (b) gp130 and (c) IL-12Rβ2 are presented as bar charts with the arithmetic mean plus standard error of the mean (s.e.m.). *P < 0·05, **P < 0·01, ***P < 0·001, PBS versus IL-35 treatment; ^†††P < 0·001, BALB/c control mice versus PBS-treated MRL/lpr mice; ^‡‡‡P < 0·001, BALB/c control mice versus IL-35-treated MRL/lpr mice (n = 5 in each group).

Figure 7

Characterization of % CD4⁺CD25⁺forkhead box protein 3 (FoxP3)⁺ regulatory T cells (T_reg), the ratios of CD4⁺CD25⁺FoxP3⁺ T_reg %/CD4⁺CD25⁻ effector T cell % and % IL-10⁺ regulatory B cells (B_reg) in Murphy Roths Large (MRL)/lpr and BALB/c mice treated with IL-35 or phosphate-buffered saline (PBS). Bar charts showed the arithmetic mean plus standard error of the mean (s.e.m.) of the frequency of CD4⁺CD25⁺FoxP3⁺ T_regs in (a) splenic, (b) thymic and (c) peripheral blood cells; and the ratios of CD4⁺CD25⁺FoxP3⁺ T_reg %/CD4⁺CD25⁻ effector T cell % in (d) splenic, (e) thymic and (f) peripheral blood cells; and the frequency of IL-10⁺ B_reg cells in (g) splenic, (h) thymic and (i) peripheral blood cells. *P < 0·05, ***P < 0·001, IL-35 versus PBS treatment; ^†P < 0·05, ^††P < 0·01, ^†††P < 0·001. BALB/c control mice versus PBS-treated MRL/lpr mice; ^‡P < 0·05, ^‡‡P < 0·01, ^‡‡‡P < 0·001, BALB/c control mice versus IL-35-treated MRL/lpr mice (n = 5 in each group).

Figure 8

Expression of gp130 and IL-12Rβ2 on splenic CD4⁺ T helper (Th) cell surface in Murphy Roths Large (MRL)/lpr and BALB/c mice with interleukin (IL)-35 or phosphate-buffered saline (PBS) treatment. Isolated splenic cells were stained with immunofluorescent antibodies and analysed by flow cytometry. Cell surface expression of gp130 and IL-12Rβ2 on CD4⁺ Th cells was presented as bar charts with the arithmetic mean plus standard error of the mean (s.e.m.). *P < 0·05, **P < 0·01, ***P < 0·001, IL-35 versus phosphate-buffered saline (PBS) treatment; ^†P < 0·05, ^††P < 0·01, BALB/c control mice versus PBS-treated MRL/lpr mice; ^‡P < 0·05, BALB/c control mice versus IL-35-treated MRL/lpr mice (n = 5 in each group).

Figure 9

Plasma concentrations of cytokines in Murphy Roths Large (MRL)/lpr and BALB/c mice with interleukin (IL)-35 or phosphate-buffered saline (PBS) treatment. Bar charts are shown with the arithmetic mean values of the plasma levels of (a) interferon (IFN)-γ, (b) IL-12p70, (c) IL-21, (d) IL-2, (e) IL-27, (f) IL-10, (g) tumour necrosis factor (TNF)-α, (h) IL-6 and (i) IL-17A.*P < 0·05, **P < 0·01, IL-35 versus PBS treatment; ^†P < 0·05, ^††P < 0·01, ^†††P < 0·001, BALB/c control mice versus PBS-treated MRL/lpr mice; ^‡P < 0·05, BALB/c control mice versus IL-35-treated MRL/lpr mice (n = 5 in each group).