miR-155 mediates suppressive effect of progesterone on TLR3, TLR4-triggered immune response
Highlights
► Progesterone treatment down regulates LPS- and poly(I:C)-induced miR-155 expression in macrophages. ► Progesterone pretreatment inhibits LPS-induced IL-6 as well as LPS- and poly(I:C) induced IFN-β production via decreasing the activity of miR-155. ► Progesterone suppresses TLRs-triggered immune response by regulating miR-155, and the decreased miR-155 contributes to inhibit TLR-induced IL-6 and IFN-β via increased SOCS1 expression.
Introduction
Progesterone is a sex hormone produced by the granulosa cells and corpus luteum of the ovary. In addition to its essential role in establishment and maintenance of pregnancy [1], it has been demonstrated that progesterone has immune suppressive properties [2], [3], [4], progesterone may affect the incidence of some autoimmune diseases [5]. Moreover, at concentrations commensurate with pregnancy, progesterone also has been shown to have direct inhibitory effects on T lymphocytes [2]. Even at the physiological concentrations, progesterone can suppress LPS-induced production of the proinflammatory cytokines TNF-α and IL-1β [3].
Toll-like receptors (TLRs) play an important role in innate immune responses against bacterial and viral pathogens [6]. TLR-triggered macrophages can produce proinflammatory cytokines and type I interferon (IFNα/β) to initiate innate immune responses through MyD88-dependent or MyD88-independent signaling pathway. Among TLRs, TLR4 signals through either MyD88 or TRIF, while TLR3 only utilizes TRIF. The MyD88 pathway involves the early phase of NF-κB activation, which leads the production of inflammatory cytokines. The TRIF pathway activates interferon(IFN)-regulatory factor (IRF3) and is involved in the late phase of NF-κB activation, both of which lead to the production of IFN-β and the expression of IFN-inducible genes [7]. Considering the activation of TLRs signaling may contribute to abortion induced by microbial infection when pregnant [8], [9], and that progesterone may inhibit several signal pathways triggered by TLRs, we have investigated whether progesterone at pregnancy level could inhibit TLR-mediated proinflammatory cytokine production and its underlying mechanisms in our previous studies. Our results have shown that progesterone treatment inhibits TLR4-mediated innate immune response in macrophages by suppressing NF-κB activation and enhancing SOCS1 expression [10]. In this study, we sought to examine whether modulation of microRNAs (miRNAs) may play a role in the inhibitory effect of progesterone on signaling by TLRs.
miRNAs are small (18–25 nucleotide long), noncoding RNAs that suppress gene expression at the posttranscriptional level, resulting in either translation inhibition or mRNA degradation [11]. Up to now, over 1000 miRNAs have been identified and the prediction that each miRNA may recognize several hundred target sequences; the current challenge is to identify these targets and understand how miRNAs are regulated within the cells. It is particularly important to consider the mounting evidences demonstrating their contributions to diseases and their roles in cellular mechanisms such as differentiation, metabolism, and immunity [12]. miRNAs such as miR-146, miR-155 and miR-21 were shown to be ubiquitously induced in response to TLRs signaling in monocytes [12], [13], [14], [15], [16]. Direct roles of miRNAs in innate immune response were initiated by a report that identified miR-146 as a negative feedback regulator in TLR signaling by targeting IL-1R-associated kinase (IARK)1 and TNF receptor-associated factor (TRAF) 6 [13]. Numerous targets also have been identified for miR-155 such as c-Maf, SHIP1 and SOCS1 [16], [17], [18]. Mice that are deficient in miR-155 have defects in B cell differentiation and possessing severe deficiencies in immune responses when exposed to pathogens [16], [19], [20].
Considering the potential roles of miRNAs in LPS-induced cytokine production after progesterone treatment, we hypothesized that miR-146, miR-155 and miR-21 could play a role in progesterone-mediated TLR signaling regulation. Concentrations of progesterone (10−7 M) correspond to peripheral blood levels during pregnancy as we previously reported [10]. Here we report for the first time that progesterone treatment regulates LPS- and poly(I:C)-induced miR-155 expression in macrophages by inhibiting NF-κB activation, and that regulated miR-155 by progesterone results in decreased IL-6 and IFN-β production in TLR activated macrophages by enhancing SOCS1 expression.
Section snippets
Cell culture and transfection
The murine macrophage cell line RAW264.7 was obtained from American Type Culture Collection and cultured as described previously [10]. For cell transfection, 1 × 105 cells were seeded into 24-well plates and incubated overnight and then transfected with RNAs using INTERFERin (Polyplus transfection) according to the manufacturer's instructions. During progesterone treatment and LPS stimulation, cells were cultured in serum-free DMEM medium.
Reagents and antibodies
LPS (Escherichia coli, O26:B6) and poly(I:C)
Progesterone treatment inhibited LPS-induced IL-6 production in macrophages
Our previous report has shown that progesterone inhibits LPS-induced secretion of the proinflammatory cytokine IL-6 in macrophages [10]. In the present study, we detected the mRNA and protein production of LPS-induced IL-6 in progesterone-pretreated macrophages at different time points. As shown in Fig. 1A, LPS-induced IL-6 mRNA expression increased after 1 h, peaked around 6 h and declined at 9 h. After 1 h of stimulation with LPS, we observed the IL-6 mRNA level in progesterone-treated cells was
Discussion
The severity of host response in some diseases differs between gender, and this dimorphism has been attributed to the immunomodulating effects of reproductive steroid hormones such as progesterone and estrogen. Progesterone, the principal hormone secreted by the corpus luteum, is also believed to be necessary for the maintenance of pregnancy in humans and animals. Progesterone plays an important role in protecting conceptus from maternal immune attack. Disrupted progesterone synthesis or
Conflicts of interest
The authors have no financial conflicts of interest.
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (30971505, 31170860), Zhejiang Provincial Natural Science Foundation of China (Y206036).
References (28)
- et al.
Inhibitory effects of progesterone differ in dendritic cells from female and male rodents
Gend Med
(2008) - et al.
Regulation of innate and adaptive immunity by the female sex hormones oestradiol and progesterone
FEMS Immunol Med Microbiol
(2003) - et al.
Pattern recognition receptors and inflammation
Cell
(2010) - et al.
Toll-like receptor 3 agonist induces impairment of uterine vascular remodeling and fetal losses in CBA × DBA/2 mice
J Reprod Immunol
(2007) - et al.
Progesterone inhibits Toll-like receptor 4-mediated innate immune response in macrophages by suppressing NF-κB activation and enhancing SOCS1 expression
Immune Lett
(2009) MicroRNAs: genomics, biogenesis, mechanism, and function
Cell
(2004)- et al.
Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein
Immunity
(2009) - et al.
Bonmassar E. miR-155 gene: a typical multifunctional microRNA
Biochim Biophys Acta
(2009) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt cycle method
Methods
(2001) - et al.
Reproductive functions of progesterone receptors
Recent Prog Horm Res
(2002)