Anti-inflammatory roles of interleukin-35 in the pathogenesis of Japanese cedar pollinosis

Abstract

Background: Interleukin (IL)-35 has been recently identified as an anti-inflammatory cytokine in allergic inflammation. However, its biological role in the pathogenesis of allergic rhinitis has not been fully elucidated.

Objective: The purpose of this study was to investigate the anti-inflammatory activity of IL-35 in the pathogenesis of allergic rhinitis in patients with Japanese cedar pollinosis (JCP).

Methods: Peripheral blood mononuclear cells were collected from healthy controls and JCP patients during the off-season for pollen. Peripheral blood mononuclear cells were incubated with Cry j 1, a major allergen of Japanese cedar pollen and production of IL-5, IL-13, and IL-35 were measured by enzyme-linked immunosorbent assay. Th2 (CD4⁺ST2⁺) cells and group 2 innate lymphoid cells were isolated from peripheral blood mononuclear cells of JCP patients, and the inhibitory effects of IL-35 on cell differentiation, proliferation and mRNA expression of IL-5, IL-13, and GATA3 were examined. B cells were also isolated and the effects of IL-35 on total IgE production were examined.

Results: Cry j 1-induced production of IL-5 and IL-13 was significantly increased in peripheral blood mononuclear cells from JCP patients, whereas Cry j 1-induced IL-35 production was significantly decreased compared with healthy controls. IL-35 significantly inhibited Th2 cell differentiation, group 2 innate lymphoid cell proliferation, and mRNA expression of IL-5, IL-13, and GATA3 in Th2 cells and group 2 innate lymphoid cells. IL-35 also inhibited IgE production from B cells.

Conclusion: IL-35 is an important anti-inflammatory cytokine in JCP, and its biological roles include the downregulation of Th2 cells and group 2 innate lymphoid cells, and the inhibition of IgE production from B cells. These findings demonstrate that IL-35 may have the potential to exert anti-allergic effects for the treatment of allergic rhinitis.

Keywords: B cell; Group 2 innate lymphoid cell; IL-35; Japanese cedar pollinosis; Th2 cell.

Fig. 1. Cry j 1-induced production of interleukin (IL)-5, IL-13, and IL-35 from peripheral blood mononuclears cells (PBMCs). PBMCs were incubated with Cry j 1, a major allergen of Japanese cedar pollen for 5 days. Concentrations of IL-5, IL-13, and IL-35 in cell-free supernatants were measured by enzyme-linked immunosorbent assay. ^*p < 0.05, by the Mann-Whitney U test. HC (n = 15), JCP patients (n = 17). HC, healthy controls; JCP, Japanese cedar pollinosis.

Fig. 2. (A, B) Effects of interleukin (IL)-35 on IL-33-induced cell differentiation and mRNA expression of IL5, IL-13, and GATA3 in Th2 cells. Isolated CD4⁺T cells were cultured with IL-33 (100 ng/mL) in the presence or absence of IL-35 (10–1,000 ng/mL) for 5 days and the ratio of Th2 (CD4⁺ST2⁺) cells in total CD4⁺T cells was examined using flow cytometry. Sorted Th2 cells (C) were cultured with recombinant human IL-33 (100 ng/mL) in the presence or absence of recombinant human IL-35 (10–1,000 ng/mL) for 6 hours, and mRNA expression of IL-5, IL-13, and GATA3 was measured by reverse transcription-polymerase chain reaction (D). ^*p < 0.05, and ^*p < 0.01, by the Mann-Whitney U test. (n = 7).

Fig. 3. Effects of interleukin (IL)-35 on cell proliferation and mRNA expression of IL-5, IL-13, and GATA3 in group 2 innate lymphoid cells (ILC2s). (A) Sorted ILC2s were incubated with IL-2 (50 ng/mL) in the presence or absence of IL-35 (100 ng/mL) for 2 weeks. (B) The numbers of viable ILC2s were counted using a cell counting kit-8, and are shown as optical density. (C) Proliferated ILC2s (1× 10⁴ cells/ mL) were stimulated with IL-33 (100 ng/mL) in the presence or absence of IL-35 (100 ng/mL) for 6 hours, and mRNA expression of IL-5, IL-13, and GATA3 were measured by reverse transcription-polymerase chain reaction. ^*p < 0.05, by the Mann-Whitney U test (n = 6).

Fig. 4. Effects of interleukin (IL)-35 on IgE production from B cells. Isolated B cells were cultured with CD40L (100 ng/mL) alone or with IL-4 (100 ng/mL) or IL-4 plus IL-21 (100 ng/mL) in the presence or absence of IL-35 (100 ng/mL) for 72 hours. (A and B) Total IgE concentration in cell-free supernatants was measured by enzyme-linked immunosorbent assay. ^*p < 0.05, and ^*p < 0.01, by the Mann-Whitney U test. (n = 6).