IL-27 affects helper T cell responses via regulation of PGE2 production by macrophages

https://doi.org/10.1016/j.bbrc.2014.07.096Get rights and content

Highlights

  • IL-27 suppressed COX-2 expression and PGE2 secretion by LPS-stimulated macrophages.

  • PGs secreted by IL-27R-deficient macrophages lead to enhanced Th1/17 differentiation.

  • STAT1 downstream of IL-27 receptor was responsible for the suppression of COX-2.

  • IL-27 affects Th1/17 differentiation in an indirect way via regulating PG secretion.

Abstract

IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. The immunosuppressive effect of IL-27 largely depends on induction of IL-10-producing Tr1 cells. To date, however, effects of IL-27 on regulation of immune responses via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrow-derived macrophages (BMDMs) from WSX-1 (IL-27Rα)-deficient mice and found enhanced IFN-γ and IL-17A secretion by CD4+ T cells as compared with that of control BMDMs. We then found that PGE2 production and COX-2 expression by BMDMs from WSX-1-deficient mice was increased compared to control macrophages in response to LPS. The enhanced production of IFN-γ and IL-17A was abolished by EP2 and EP4 antagonists, demonstrating PGE2 was responsible for enhanced cytokine production. Murine WSX-1-expressing Raw264.7 cells (mWSX-1-Raw264.7) showed phosphorylation of both STAT1 and STAT3 in response to IL-27 and produced less amounts of PGE2 and COX-2 compared to parental RAW264.7 cells. STAT1 knockdown in parental RAW264.7 cells and STAT1-deficiency in BMDMs showed higher COX-2 expression than their respective control cells. Collectively, our result indicated that IL-27/WSX-1 regulated PGE2 secretion via STAT1–COX-2 pathway in macrophages and affected helper T cell response in a PGE2-mediated fashion.

Introduction

IL-27 is a novel heterodimeric cytokine consisting of EBI-3 and p28 which are structurally related to the IL-12/IL-23 subunits p40 and p35/p19, respectively [1]. IL-27 is produced by activated antigen presenting cells (APCs) including dendritic cells (DCs) and macrophages [2], [3], [4]. IL-27 signals through its heterodimeric receptor consisting of WSX-1 and gp130 [2], [3], [4]. Initial studies demonstrated that IL-27 plays a role in Th1 induction and enhances proliferation of naïve CD4+ T cells [1], [2], [3], [4]. Recent studies, however, have demonstrated the anti-inflammatory function of IL-27 by generating Tr1 cells [5]. In addition to its regulation of T cell function, IL-27 also regulates APCs in an autocrine manner [4].

Prostaglandins (PGs) are a group of biologically active lipid mediators that are derived from arachidonic acid, and mediate a variety of functions. While cyclooxygenase (COX)-1 constitutively exists and mediates PG production in various tissues/organs, COX-2 is induced in immune cells, such as macrophages, by stimulation including growth factors, and mediates PG production in inflammatory venues [6]. Among various PGs produced, PGE2 has a dominant role in inflammation/immune response [7], [8]. Interestingly, recent reports have demonstrated that PGE2 promotes Th1 differentiation and Th17 expansion in vitro via its receptors, EP2 and EP4 [9], [10]. Regulation of PGs, especially PGE2, may thus be a potent target of anti-inflammation strategies.

To date, functions of IL-27 on lipid mediators are poorly understood. In this study, we firstly demonstrated that lack of IL-27 signaling resulted in enhanced production of PGE2 by bone marrow-derived macrophages (BMDMs), which led to increased production of IL-17A and IFN-γ by CD4+ T cells. We also demonstrated that IL-27 negatively regulated COX-2 expression in a STAT1-dependent way. These findings clearly showed that IL-27 regulates PGE2 production by macrophages and “indirectly” regulates Th1/17 differentiation. IL-27 modulation of PGE2 production is a novel mechanism by which IL-27 shows its anti-inflammatory/immunosuppressive function.

Section snippets

Mice

Female WSX-1−/− mice were generated as described previously [11]. Female STAT1−/− mice were provided by Dr. Yoshimura (Keio University, Japan). C57BL/6J mice as a control were purchased from Japan CLEA, Inc. Mice were maintained under specific pathogen-free conditions and used between 8 and 14 weeks of age. All experiments were approved by the Animal Care and Use Committee at Nippon Boehringer Ingelheim Co., Ltd. or Saga University.

Reagents

Recombinant murine (rm) M-CSF was purchased from eBioscience.

WSX-1-deficient BMDM conditioned medium promoted IFN-γ and IL-17A secretion by CD4+ T cells

LPS stimulation of BMDMs induced expression of IL-27 subunits, EBI-3 (peak at around 12 h after stimulation) and p28 (peak as early as 3–6 h after stimulation) (Supplemental Fig. 1A). LPS stimulation also induced expression of WSX-1 (data not shown). LPS stimulation of macrophages not only induced IL-27R expression but also primed the cells responsive to IL-27 stimulation by STAT1 activation. IL-27 stimulation of LPS-pre-treated BMDMs, but not untreated BMDMs, induced phosphorylation of both

Discussion

Here we firstly reported that IL-27 negatively regulated PGE2 production from murine macrophages and it resulted in the modulation of IFN-γ and IL-17A production from CD4+ T cells in vitro.

In the present study, CM from WSX-1-deficient BMDMs after LPS stimulation induced higher amount of IFN-γ and IL-17A from CD4+ T cells (Fig. 1). WSX-1-deficient BMDMs produced higher level of PGE2 and cytokines such as IL-6 and IL-12p40, than control BMDMs, (Figs. 2A and 3). Inhibition of PGE2 production by

Acknowledgments

This study was supported in part by grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan (H.Y. and H.H.). The authors are grateful to Drs. Akihiko Yoshimura and Christopher Hunter for helpful discussion and Ms. Furukawa for secretarial work.

References (20)

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