Progesterone inhibits Toll-like receptor 4-mediated innate immune response in macrophages by suppressing NF-κB activation and enhancing SOCS1 expression
Introduction
Progesterone, a required steroid hormone in mammalian, is produced by the granulosa cells and corpus luteum of the ovary [1]. In addition to its essential role in establishment and maintenance of pregnancy [2], a number of studies have demonstrated that progesterone has immune suppressive properties [3], [4], [5]. Many studies have demonstrated that progesterone may affect the incidence of some autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA) [6]. Progesterone also has been shown to have direct effects on T lymphocytes at concentrations commensurate with pregnancy [3]. Even at the physiological concentrations, progesterone can suppress LPS-stimulated production of the proinflammatory cytokines TNF-α and IL-1β [4]. However, the mechanisms of how progesterone affects innate and adaptive immune response need to be fully investigated.
Toll-like receptors (TLRs) are important pattern-recognition receptors (PRRs) in innate immune responses. After recognizing microbial conserved pathogen-associated molecule patterns such as lipopolysaccharide (LPS), viral RNA and CpG oligodeoxynucleotide (ODN) motifs, TLRs activate immune cells to produce proinflammatory cytokines and type I interferon (IFN-α/β) to initiate innate immune responses. With the exception of TLR3, TLRs activate immune cells by means of the adaptor protein MyD88 through a cascade of the kinases involving downstream mitogen-activated protein kinase (MAPK) and transcription factor NF-κB to induce the production of inflammatory cytokines [7], [8]. However, it remains far from understanding how progesterone modulates TLR signaling.
Considering the activation of TLRs signaling may contribute to abortion induced by microbial infection when pregnant [9], [10], and progesterone may inhibit several signal pathways triggered by TLRs [11], [12], in this study, we investigated whether progesterone at pregnancy level could inhibit TLR-mediated proinflammatory cytokine production, and if so, what is the underlying mechanisms. Concentrations of progesterone (10−7 M) correspond to peripheral blood levels during pregnancy [13]. We demonstrated that progesterone can inhibit TLR4-induced IL-6, NO production through suppression of NF-κB activation and enhancement of SOCS1 expression in macrophages.
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Cell line and culture
The murine macrophage cell line RAW264.7 was obtained from American Type Culture Collection. RAW264.7 cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated fetal calf serum (Invitrogen), 2 mM l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified incubator of 5% CO2.
Reagents, antibodies and plasmids
LPS (Escherichia coli, O26:B6) was purchased from Sigma (St Louis, MO). Phosphorothioate-modified CpG ODN was synthesized by Sybersyn Co. (Sybersyn, Beijing, China). The sequence was
Progesterone pretreatment inhibits LPS and CpG ODN-induced IL-6 production in macrophages
TLR4 and TLR9 are reported to play pivotal roles in recognizing bacterial LPS and unmethylated CpG ODN, respectively. TLRs-mediated intracellular signaling subsequently induces activation of NF-κB and the secretion of proinflammatory cytokines [17], [18]. We investigated the effects of 17β-Estradiol (E2) and progesterone (P) pretreatment on cytokine production in RAW264.7 cells induced by subsequent stimulation with TLR agonist. The cells were pretreated with 17β-Estradiol (E2) or progesterone
Discussion
In the present study, we demonstrate the immunomodulatory effects of 17β-Estradiol and progesterone on the macrophages, by showing that progesterone regulates TLR agonists activated TLR signaling in macrophages. Pretreatment of macrophages with progesterone, not 17β-Estradiol, inhibited LPS and CpG ODN-induced IL-6 and NO production. The results suggested that progesterone and estrogen differentially regulate LPS and CpG ODN-activated TLR signaling in macrophages.
In addition to the inhibitory
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (30300187), Zhejiang Provincial Natural Science Foundation of China (Y206036) and Zhejiang Provincial Health Bureau Science Foundation of China (2006A080).
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