Calcitriol inhibits TNF-α-induced inflammatory cytokines in human trophoblasts

Abstract

Elevated placental proinflammatory cytokine release is associated with miscarriage, preterm labor and preeclampsia. Specifically, tumor necrosis factor-α (TNF-α)-induced cytokines may threaten pregnancy outcome. Since trophoblasts produce calcitriol, a hormone with strong immunosuppressive properties, we assessed the effects of this secosteroid on inflammatory cytokines induced in trophoblasts by challenge with TNF-α. The effects of calcitriol on synthesis of mRNAs encoding interleukin-6 (IL-6), interferon-γ (IFN-γ), and TNF-α were measured by real time RT-PCR. Secreted cytokines were quantified by ELISA. The effects of TNF-α on CYP24A1, chorionic gonadotropin (hCG), 3β-hydroxysteroid dehydrogenase (HSD3B1) and P₄₅₀-aromatase (CYP19) mRNA expression were also studied. TNF-α stimulated IL-6, IFN-γ and its own expression more than 3-fold over controls (P < 0.05). Calcitriol inhibited the expression profile of inflammatory cytokine genes in a dose–response manner (P < 0.05). This effect was prevented by addition of the vitamin D receptor antagonist TEI-9647. TNF-α also significantly inhibited expression of hCG, HSD3B1 and CYP19 genes, and stimulated CYP24A1 gene expression. These data show that calcitriol prevents TNF-α induction of inflammatory cytokines through a process likely to be mediated by the vitamin D receptor. We conclude that TNF-α inhibits placental hormone synthesis and stimulates calcitriol catabolism by regulating enzymes involved in these processes.

Introduction

Placental production of calcitriol results from the conversion of 25-hydroxyvitamin D₃ (25-OHD₃) into 1,25-dihydroxyvitamin D₃ (calcitriol) by the activity of cytochrome P₄₅₀ 25-OHD₃-1-alpha-hydroxylase (CYP27B1) (Dusso et al., 2005). Recently, research has been conducted in order to investigate the role of locally generated calcitriol as a modulator of fetal–placental development and function. Previous studies from our laboratory have demonstrated that calcitriol regulates the secretion of various placental hormones (Barrera et al., 2007, Barrera et al., 2008). The placenta expresses a wide spectrum of cytokines (Bennett et al., 1996), and current data support the concept that increased production of proinflammatory (Th1) cytokines such as interferon-γ (IFN-γ), interleukin-6 (IL-6) and TNF-α is potentially harmful for pregnancy outcome, while predominance of anti-inflammatory (Th2) cytokines is thought to be beneficial for pregnancy success (Darmochwal-Kolarz et al., 1999, Peltier, 2003). In models for human diseases, systemic administration of calcitriol elicits strong immunosuppressive effects, attenuating various Th1-triggered diseases (Szodoray et al., 2008). Indeed, the ability of the secosteroid to shift T cell responses from Th1 to Th2 has been demonstrated (Daniel et al., 2008). Furthermore, it has been demonstrated that calcitriol-treated first trimester NK decidual cells synthesize less cytokines than controls (Evans et al., 2006).

Preeclampsia is considered a placenta-dependent disorder with local and systemic abnormalities and has been associated with increased release of Th1 cytokines such as TNF-α (Vince et al., 1995, Saito et al., 1999a). Other factors known to induce placental TNF-α are hypoxia and infections, which also cause maternal immune activation. Since TNF-α is one of the key mediators of the inflammatory response, we used it to induce an inflammatory condition in cultured trophoblasts in order to study the effect of calcitriol upon TNF-α-induced Th1 cytokine gene expression and release. Additionally, considering that previous studies have shown adverse effects of TNF-α on placental functions (Leisser et al., 2006, Kilani et al., 2007), we investigated the effects of this cytokine upon placental hormonogenesis by studying the regulatory actions of TNF-α upon genes encoding hormone chorionic gonadotropin (hCG) and the enzymes P₄₅₀-aromatase (CYP19) and 3β-hydroxysteroid dehydrogenase (HSD3B1), implicated in the production of estradiol and progesterone, respectively.

The regulation of the calcitriol-degrading enzyme 1,25-dihydroxyvitamin D₃ 24-hydroxylase (CYP24A1) by TNF-α was also examined in view of ample evidence showing that serum calcitriol and placental production of this hormone are decreased in preeclampsia (Halhali et al., 2000, Diaz et al., 2002, Halhali et al., 2007), while serum TNF-α and placental production of this cytokine are both augmented in this disease (Wang and Walsh, 1996, Laskowska et al., 2006).