IL-27 Regulated CD4+IL-10+ T Cells in Experimental Sjögren Syndrome

Abstract

Interleukin 27 (IL-27) plays diverse immune regulatory roles in autoimmune disorders and promotes the generation of IL-10-producing CD4⁺ T cells characterized by producing the immunosuppressive cytokine IL-10. However, whether IL-27 participates in pathological progress of Sjögren syndrome (SS) through regulating CD4⁺IL-10⁺ T cells remains unknown. Here we aimed to explore the potential role of IL-27 and CD4⁺IL-10⁺ T cells in the pathogenesis of SS. The IL-27 gene knockout non-obese diabetic (Il-27^-/-NOD) mice were generated and injected with exogenous IL-27. Exogenous injection of IL-27 and neutralization of IL-27 with anti-IL-27 antibody in NOD mice were performed. The histopathologic changes in submandibular glands, lacrimal glands and lung, salivary flow rate, and percentages of CD4⁺IL-10⁺ T cells were determined. And, ovalbumin-immunized C57L/B6 mice were injected with IL-27 to detect the percentage of CD4⁺IL-10⁺ T cells. In vitro, splenic naive T cells from C57L/B6 mice were cultured with IL-27 for 4 days to induce the differentiation of CD4⁺IL-10⁺ T cells. In addition, IL-27, IL-10, and CD4⁺IL-10⁺ T cells were determined in health control and SS patients. The results showed that Il-27^-/-NOD mice had more severe disease and lower level of CD4⁺IL-10⁺ T cells than control mice. And IL-27 promoted the generation and differentiation of CD4⁺IL-10⁺ T cells in vivo and in vitro significantly. In agreement with the findings in the SS-like mice, patients with SS showed lower levels of IL-27, IL-10, and CD4⁺IL-10⁺ T cells. Our findings indicated that IL-27 deficiency aggravated SS by regulating CD4⁺IL-10⁺ T cells. Targeting IL-27 and CD4⁺IL-10⁺ T cells may be a novel therapy for patients with SS.

Keywords: CD4+IL-10+ T cells; Sjögren syndrome; immunosuppression; interleukin-10; interleukin-27.

Figure 1

Interleukin 27 gene deficiency aggravated SS in NOD mice. (A) The physical appearance manifestation of rash in 12-week-old female NOD and Il-27^−/−NOD mice. (B–D) Histological analysis, SGs, LGs, and lung from representative NOD and Il-27^−/−NOD mice stained with hematoxylin and eosin to assess inflammation (top, magnification ×100; bottom, magnification ×400), n = 5.

Figure 2

Lower levels of salivary flow rate and CD4⁺IL-10⁺ T cells in Il-27^−/−NOD mice. (A) Histological analysis, SG from representative 12-week-old female NOD and Il-27^−/−NOD and IL-27–treated Il-27^−/−NOD mice stained with hematoxylin and eosin to assess inflammation (top, magnification ×100; bottom, magnification ×400). (B) The lymphocyte infiltration in SG of mice were evaluated for histological scores. (C) Salivary flow rate, (D) representative flow cytometry results, and (E) the percentage and (F) absolute number of splenic CD4⁺IL-10⁺ T cells in NOD and Il-27^−/−NOD and IL-27–treated Il-27^−/−NOD mice. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, n = 5.

Figure 3

Interleukin 27 suppressed the inflammation in NOD mice. (A,C) Histological analysis, SG from representative mice stained with hematoxylin and eosin to assess inflammation (magnification ×100). Arrows indicate lymphocytes infiltrating focus. (B,D) Salivary flow rate of 12-week-old female NOD mice. (E,G) Representative flow cytometry results and (F,H) the percentage of splenic CD4⁺IL-10⁺ T cells in NOD mice. Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, n = 5.

Figure 4

CD4⁺IL-10⁺ T cells generation and differentiation were promoted by IL-27. (A) IL-27 treatment schedule. (B) Representative flow cytometry results and (C) the percentage of splenic CD4⁺IL-10⁺ T cells in IL-27–treated or control OVA-immunized 9-week-old female C57BL/6 mice. (D) Representative flow cytometry results, and (E) the percentage of differentiated CD4⁺IL-10⁺ T cells. Error bars indicate SEM. *p < 0.05, **p < 0.01, n = 5.

Figure 5

Interleukin 27 was related to decreased CD4⁺IL-10⁺ T cells in SS patients. (A,B) Plasma levels of IL-27 and IL-10 in HCs (n = 14) and SS patients (n = 14) were detected by ELISA. (C) Representative flow cytometry results and (D) the percentage of CD4⁺IL-10⁺ T cells in HCs (n = 20) and SS patients (n = 17). Sjögren syndrome patients were divided into antibodies-positive (+) and antibodies-negative (–) two groups. The correlations of CD4⁺IL-10⁺ T cells with (E) anti-SSA antibodies, (F) anti-SSB antibodies, and (G) ANAs were analyzed in SS patients. Error bars indicate SEM. *p < 0.05, **p < 0.01.