IL-10 promotes Th17 cell differentiation by enhancing STAT1-dependent IL-6 production via IgE-stimulated mast cells

Abstract

Mast cells (MCs) are tissue-resident cells of hematopoietic origin that play an important role in host's defense mechanism against nematodes. However, excessive activation of these cells contributes to the development of certain allergic diseases. Immunoglobin E (IgE) is one of the well-known molecules that activate MCs. Even in the absence of specific antigens, the binding of highly cytokinergic IgE to FcεRI on MCs prolongs their survival and induces cytokine production without enhancing their degranulation. In the present study, we examined the effects of the members of the interleukin-10 (IL-10) family of cytokines on IgE-mediated MCs functions. The receptors including Il10r1, Il10r2, and Il20r2, but not Il20r1, Il22r1 or Il28r1, were constitutively expressed in mouse bone marrow cell-derived cultured MCs (BMCMCs), suggesting that IL-10 may influence MCs function. Indeed, we found that only IL-10 could influence upon BMCMCs function; IL-10 enhanced prolongation of survival, promoted IL-6 and/or IL-13 production dependently of STAT1 and STAT3, and suppressed tumor necrosis factor production independently of STAT1 and STAT3 on IgE-stimulated BMCMCs. Moreover, the IL-10-mediated enhancement of IL-6 production by IgE-stimulated BMCMCs promotes Th17 cell expansion. These results suggest that IL-10 has a dual role as an anti-inflammatory and pro-inflammatory cytokine in MCs functions.

Keywords: IL-10; Mast cells; STAT1; STAT3; Th17 cells.

Fig. 1

Expression of IL-10R family members in BMCMCs. Expression levels of Il10r1, Il10r2, Il20r1, Il20r2, Il22r1, and/or Il28r1 mRNA in BMCMCs (n = 3, derived from distinct mice) in the presence or absence of IgE (SPE-7) (A) and NIH3T3 (B) determined by qPCR. HMC-1 mRNA was used as a negative control (A). Data show the mean ± SEM and the representative result of two independent experiments. *p < 0.001, HMC-1 vs. BMCMCs, and ^†p< 0.05, medium vs. SPE-7. NS not significant.

Fig. 2

IL-10 affects survival and cytokine production but not degranulation in IgE-stimulated BMCMCs. BMCMCs (n = 5–10 derived from distinct mice) were stimulated with or without IgE (SPE-7) in the presence or absence of 1–100 ng/mL IL-10 and 100 ng/mL IL-19, IL-20, IL-22, IL-24, IL-26, IL-28 A, IL-28B, or IL-29 for 1 h (A), 3 days (B), and 24 h (C). (A) The levels of β-hexosaminidase release. P/I = PMA + Ionomycin, “-” = no cytokine. (B) Cell survival assessed in PI- and annexin V-negative cells by FACS analysis. *p < 0.05, no cytokine (-) vs. SPE-7. (C) Levels of IL-6, IL-13, and TNF in culture supernatants, as determined by ELISA. *p < 0.05, vs. no cytokine “0”. Data show the mean ± SEM and the representative result of two independent experiments.

Fig. 3

Effects of IL-10 on the production of IL-6, but not on TNF, by BMCMCs depend on STAT1 and STAT3. BMCMCs (A) or 0.5 µM cucurbitacin I-treated BMCMCs (B,C) (n = 5–10 derived from distinct mice) were stimulated with or without IgE (SPE-7) in the presence or absence of 1–100 ng/mL IL-10 for 24 h. The levels of IL-6 and TNF in the culture supernatants were determined using ELISA. (A) The cultivation using Stat1^+/+ and Stat1^−/− BMCMCs. *p < 0.05, Stat1^+/+ BMCMCs vs. Stat1^−/− BMCMCs. (B) Culture of cucurbitacin-I-treated wild-type BMCMCs. *p < 0.05, vehicle-treated BMCMCs vs. cucurbitacin I-treated BMCMCs. (C) The cultivation using cucurbitacin I-treated Stat1^−/− BMCMCs. Data show the mean ± SEM and the representative result of two independent experiments.

Fig. 4

IL-10 promotes Th17 cell differentiation by enhancing STAT1-dependent IL-6 production in IgE-stimulated mast cells. Naïve CD4⁺ T cells were co-cultured with wild-type and Il6^−/− BMCMCs (n = 4–5 derived from distinct mice) or with Stat1^+/+ and Stat1^−/− BMCMCs (n = 4–5 derived from distinct mice) in the presence of anti-mouse CD3 mAb and IgE (SPE-7), with or without 100 ng/mL rmIL-10, for 4 days. The cells were then stimulated with PMA + ionomycin in the presence of monensin for 6 h. Intracellular IL-17 and Foxp3 expression levels were determined by FACS analysis. (A) Representative FACS results. (B) Data show the mean ± SEM from (A), and the representative results of two independent experiments. *p < 0.05, medium vs. rmIL-10, and ^†p < 0.05, wild-type BMCMCs vs. Il6^−/− BMCMCs.