IL-27 Expression and Responsiveness in Human Uterine Epithelial Cells and Fibroblasts In Vitro and the Role of Estradiol

Abstract

Interleukin (IL)-27 is a pleiotropic cytokine that regulates multiple aspects of innate and adaptive immunity, but whose role in immune protection of the female reproductive tract is unknown. Although not constitutively expressed by human uterine epithelial cells and fibroblasts in culture, IL-27 secretion was upregulated after treatment with the viral ligand poly (I:C) in a type I interferon (IFN)-dependent manner, with higher levels measured in fibroblasts than epithelial cells. Estradiol increased poly (I:C)-induced IL-27 production by fibroblasts, but not epithelial cells. While both cell types expressed the IL-27 receptor, only fibroblasts responded to recombinant IL-27 with increased expression of the antiviral genes, APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) and MxA, and the tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO). Estradiol inhibited IL-27-mediated induction of IDO in fibroblasts through estrogen receptor alpha, but had no effect on APOBEC3G. IL-27 pretreatment also potentiated poly (I:C) upregulation of the antiviral genes, OAS2 and APOBEC3G, in fibroblasts. Thus, IL-27 is part of the antiviral response by uterine cells against potential pathogens. The effect of estradiol on IL-27 production and sensitivity by fibroblasts demonstrates a selective hormone action on individual cell types in the uterus and suggests that IL-27 may have differential effects during the menstrual cycle.

Keywords: IL-27; epithelial cells; estradiol; poly (I:C); stromal fibroblasts; uterus.

FIG. 1.

Poly (I:C) induces IL-27 mRNA expression and secretion in uterine epithelial cells (A, B) and stromal fibroblasts (C, D). Confluent monolayers of epithelial cells and fibroblasts were treated with 25 μg/mL of poly (I:C). mRNA (A, C) and secretions (B, D) were recovered after 24 and 48 h, respectively, and levels of IL-27 measured by RT-PCR and ELISA (n = 4–5). For type I interferon (IFN) receptor complex (IFNAR) blockade (E, F), both epithelial cells and fibroblasts were pretreated with αIFNAR2 or IgG control (ISO) for 1 h before poly (I:C) treatment for the following 24 h. αIFNAR2 was maintained in cell culture media throughout the experiment (n = 3). mRNA expression is normalized to levels of β-actin. *P < 0.05; **P < 0.01; ***P < 0.001.

FIG. 2.

Estradiol upregulates poly (I:C)-induced secretion of IL-27 by uterine stromal fibroblasts. Confluent monolayers of uterine epithelial cells (A), uterine fibroblasts (B), endocervical fibroblasts (C), and ectocervical fibroblasts (D) were pretreated with estradiol (E₂) at 5 × 10⁻⁸ M for 48 h before washout and subsequent treatment with E₂ and poly (I:C) (25 μg/mL) for 48 h. IL-27 levels were measured in cell secretions by ELISA (n = 4–5). *P < 0.05.

FIG. 3.

IL-27 receptor expression in uterine epithelial cells and stromal fibroblasts. mRNA was recovered from untreated confluent monolayers of epithelial cells and fibroblasts, and levels of IL-27Rα and gp130 were determined by RT-PCR. Expression levels of IL-27Rα and gp130 are both normalized to levels of β-actin in each cell type.

FIG. 4.

IL-27 reduces IL-27Rα and induces IDO and A3G expression in uterine stromal fibroblasts. Confluent monolayers of fibroblasts were treated with recombinant human IL-27 for 24 h before mRNA recovery and analysis for IL-27Rα (A), gp130 (B), IDO (C), APOBEC3G (A3G) (D), MxA (E), OAS2 (F), and TLR3 (G) by RT-PCR. *P < 0.05. APOBEC3G, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; IDO, indoleamine 2,3-dioxygenase.

FIG. 5.

Estradiol inhibits IL-27-induced IDO expression through ERα in uterine stromal fibroblasts. Confluent monolayers of matched epithelial cells (A, B) and fibroblasts (C, D) were pretreated with estradiol (E₂) at 5 × 10⁻⁸ M for 48 h before washout and subsequent treatment with E₂ or IL-27 (100 ng/mL) for 24 h, after which mRNA expression levels for APOBEC3G (A3G) and IDO were measured by RT-PCR (n = 4–5). For ER blockade, Rx was added to confluent monolayers of fibroblasts (E) at 5 × 10⁻⁶ M for 1 h before addition of E₂ at 5 × 10⁻⁸ M for a further 48 h, followed by washout and treatment with E₂ or IL-27 (100 ng/mL) for a further 24 h. Rx was maintained in the media at 5 × 10⁻⁶ M for the entire duration of the experiment (n = 3). *P < 0.05. ER, estrogen receptor; Rx, Raloxifene.

FIG. 6.

IL-27 potentiates the upregulation of OAS2 and APOBEC3G (A3G) in uterine stromal fibroblasts. Confluent monolayers of fibroblasts were pretreated with IL-27 (1 and 100 ng/mL) for 48 h before washout and subsequent treatment with poly (I:C) (25 μg/mL) for 24 h, after which mRNA expression levels for OAS2 (A) and A3G (B) were measured by RT-PCR (n = 4). ***P < 0.001.