Exclusive Temporal Stimulation of IL-10 Expression in LPS-Stimulated Mouse Macrophages by cAMP Inducers and Type I Interferons.

Orna Ernst,Yair Glick,Yifat Glucksam-Galnoy,Muhammad Athamna,Doron Gerber,Bibek Bhatta,Iris Ben-Dror,Tsaffrir Zor

doi:10.3389/fimmu.2019.01788

Abstract

Expression of the key anti-inflammatory cytokine IL-10 in lipopolysaccharide (LPS)-stimulated macrophages is mediated by a delayed autocrine/paracrine loop of type I interferons (IFN) to ensure timely attenuation of inflammation. We have previously shown that cAMP synergizes with early IL-10 expression by LPS, but is unable to amplify the late type I IFN-dependent activity. We now examined the mechanism of this synergistic transcription in mouse macrophages at the promoter level, and explored the crosstalk between type I IFN signaling and cAMP, using the β-adrenergic receptor agonist, isoproterenol, as a cAMP inducer. We show that silencing of the type I IFN receptor enables isoproterenol to synergize with LPS also at the late phase, implying that autocrine type I IFN activity hinders synergistic augmentation of LPS-stimulated IL-10 expression by cAMP at the late phase. Furthermore, IL-10 expression in LPS-stimulated macrophages is exclusively stimulated by either IFNα or isoproterenol. We identified a set of two proximate and inter-dependent cAMP response element (CRE) sites that cooperatively regulate early IL-10 transcription in response to isoproterenol-stimulated CREB and that further synergize with a constitutive Sp1 site. At the late phase, up-regulation of Sp1 activity by LPS-stimulated type I IFN is correlated with loss of function of the CRE sites, suggesting a mechanism for the loss of synergism when LPS-stimulated macrophages switch to type I IFN-dependent IL-10 expression. This report delineates the molecular mechanism of cAMP-accelerated IL-10 transcription in LPS-stimulated murine macrophages that can limit inflammation at its onset.

Highlights

The TLR4 ligand, LPS, stimulates macrophages to produce and secrete multiple pro-inflammatory mediators [1]
We recently demonstrated that elevated intra-cellular cAMP synergizes with LPS at IL-10 expression and secretion in LPSstimulated RAW264.7 macrophages only at the early (3 h), but not late (24 h), phase [11]
We further showed that the loss of cAMP effect at the late phase was specific to IL-10 expression, while general cAMP-dependent transcriptional activity was retained [11]

Summary

Introduction

The TLR4 ligand, LPS, stimulates macrophages to produce and secrete multiple pro-inflammatory mediators [1]. Expression of the anti-inflammatory cytokine IL-10 peaks with a delay that is due to the essential involvement of LPS-stimulated type I interferons (IFN) that act in an autocrine and paracrine manner [2,3,4,5,6,7]. In LPS-stimulated RAW264.7 macrophages, IL-10 Promoter Activation by cAMP there is an approximately 10 h time gap between the TNFα and IL-10 expression peaks [8]. Synergistic IL-10 expression has been demonstrated in macrophages stimulated by a cAMP inducer and agonists of other TLRs [9, 10]. We further demonstrated that the enhancement of LPS-stimulated IL-10 expression by cAMP and by autocrine type I IFN is temporally distinct [11]. In the current study we explored the mechanism of IL-10 expression temporal regulation at the promoter level

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Conclusion