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. 2019 Apr 17:2019:3451461.
doi: 10.1155/2019/3451461. eCollection 2019.

The cAMP Pathway Amplifies Early MyD88-Dependent and Type I Interferon-Independent LPS-Induced Interleukin-10 Expression in Mouse Macrophages

Orna Ernst  1   2 , Yifat Glucksam-Galnoy  1 , Muhammad Athamna  1   3 , Iris Ben-Dror  1 , Hadar Ben-Arosh  1 , Galit Levy-Rimler  1 , Iain D C Fraser  2 , Tsaffrir Zor  1
Affiliations

The cAMP Pathway Amplifies Early MyD88-Dependent and Type I Interferon-Independent LPS-Induced Interleukin-10 Expression in Mouse Macrophages

Orna Ernst et al. Mediators Inflamm. .

Abstract

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine, secreted by macrophages and other immune cells to attenuate inflammation. Autocrine type I interferons (IFNs) largely mediate the delayed expression of IL-10 by LPS-stimulated macrophages. We have previously shown that IL-10 is synergistically expressed in macrophages following a costimulus of a TLR agonist and cAMP. We now show that the cAMP pathway directly upregulates IL-10 transcription and plays an important permissive and synergistic role in early, but not late, LPS-stimulated IL-10 mRNA and protein expression in mouse macrophages and in a mouse septic shock model. Our results suggest that the loss of synergism is not due to desensitization of the cAMP inducing signal, and it is not mediated by a positive crosstalk between the cAMP and type I IFN pathways. First, cAMP elevation in LPS-treated cells decreased the secretion of type I IFN. Second, autocrine/paracrine type I IFNs induce IL-10 promoter reporter activity only additively, but not synergistically, with the cAMP pathway. IL-10 promoter reporter activity was synergistically induced by cAMP elevation in macrophages stimulated by an agonist of either TLR4, TLR2/6, or TLR7, receptors which signal via MyD88, but not by an agonist of TLR3 which signals independently of MyD88. Moreover, MyD88 knockout largely reduced the synergistic IL-10 expression, indicating that MyD88 is required for the synergism displayed by LPS with cAMP. This report delineates the temporal regulation of early cAMP-accelerated vs. late type I IFN-dependent IL-10 transcription in LPS-stimulated murine macrophages that can limit inflammation at its onset.

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Figures

Figure 1
Figure 1
The cAMP pathway synergistically stimulates early, but not late, LPS-induced IL-10 transcription. (a–d) Early synergism at the IL-10 promoter reporter and IL-10 secretion levels. RAW264.7 macrophages, transfected with a full mouse IL-10 promoter reporter construct, were incubated with LPS (10 ng/ml) and/or isoproterenol (Iso, 1 μM) for 3 h (a, b) or for up to 24 h (c, d). (b, d) The medium was collected, and the secretion of endogenous IL-10 was measured by ELISA. Data represent the mean ± SD (n = 3). (a, c) Luciferase reporter data expressed as the mean ± SD (n = 3) of values normalized against Renilla luciferase activity. (a, b) ∗ p < 0.003 and ∗∗ p < 0.0005 relative to resting cells. (c) p < 0.05 relative to resting cells for all treatments except for LPS at 3 h and Iso at 24 h. (d) Values of isoproterenol treatment were indistinguishable from control values. p < 0.05 relative to resting cells for LPS and for LPS+Iso at all time points. (c, d) p < 0.05 for LPS+Iso relative to LPS only at 3 h and 8 h. The experiments shown in (a–d) were carried out 45, 48, 14, and 12 times, respectively, with similar results. (e) Decay rate of the reporter. RAW264.7 macrophages, transfected with a full mouse IL-10 promoter reporter construct, were incubated with LPS (10 ng/ml) and isoproterenol (Iso, 1 μM) for 3 h, the stimuli-containing medium was removed, and decay of the reporter was measured at the indicated time points along the dashed line. Luciferase reporter data expressed as the mean ± SD (n = 3) of values normalized against Renilla luciferase activity, relative to unstimulated control cells. p < 0.05 relative to resting cells at all time points. The experiment was carried out twice. (f) Early synergism at the IL-10 mRNA level. RAW264.7 macrophages were stimulated with LPS (10 ng/ml) and/or isoproterenol (Iso, 1 μM) or the PDE4 inhibitor rolipram (20 μM) for the indicated time. Total RNA was isolated from the cells, and IL-10 mRNA levels were assessed by real-time PCR. The intensity of IL-10 mRNA in unstimulated cells, normalized by HPRT mRNA, was set to 1. Data represent the mean ± SD (n = 3). p < 0.05 relative to resting cells for Iso at 1 h only and for all other treatments at all time points. p < 0.05 for LPS relative to LPS+Iso or LPS+rolipram only at 1-4 h. The experiment was carried out twice.
Figure 2
Figure 2
Early synergistic elevation of LPS-stimulated IL-10 expression by the cAMP pathway in BMDM and in vivo is diminished with time. (a) BMDM from C57BL/6 mice were incubated with LPS (10 ng/ml) and/or isoproterenol (Iso, 1 μM) for up to 24 h. The medium was collected, and secretion of endogenous IL-10 was measured by ELISA. Data represent the mean ± SD (n = 6). Values of isoproterenol treatment were indistinguishable from control values. p < 0.05 for LPS+Iso relative to either LPS or control and for LPS relative to control, at all time points. (b) BALB/c mice were IP-injected with the cAMP inducer PCERA-1 (1 mg/kg) or with vehicle 30 min prior to IP administration of LPS (5 mg/kg). Blood was collected at the indicated time points. IL-10 and TNFα serum levels were measured by ELISA. Data expressed as the mean ± SEM (n = 6 for t = 1-1.5 h, n = 3 for other time points). p < 0.05 for LPS+PCERA-1 relative to LPS only at 1-2 h in the IL-10 assay (b, left) and at 1-3.5 h in the TNFα assay (b, right). Cytokine levels were undetected following administration of PCERA-1 alone or vehicle. The experiments were carried out twice.
Figure 3
Figure 3
The insensitivity of late-phase IL-10 induction to isoproterenol does not result from partial receptor desensitization. (a, b) RAW264.7 macrophages were transfected with a luciferase reporter regulated by 4 repeats of a consensus CRE sequence. Luciferase reporter data expressed as the mean ± SD (n = 3) of values normalized against Renilla luciferase activity, relative to unstimulated control cells. (a) The cells were preincubated for 21 h with either LPS (10 ng/ml) or vehicle, washed and further incubated for the following 3 h with isoproterenol (Iso, 1 μM) and/or LPS (10 ng/ml). (b) The cells were treated for 3 h or 24 h with isoproterenol (Iso, 1 μM) or with the PDE4 inhibitor rolipram (20 μM). ∗ p = 0.0002. (c) RAW264.7 macrophages were incubated for the indicated time with LPS (10 ng/ml) in the presence or absence of the PDE4 inhibitor rolipram (20 μM). IL-10 and TNFα secretion was measured by ELISA. Data expressed as the mean ± SD (n = 6). p < 0.05 for LPS+rolipram relative to LPS alone at all time points except for IL-10 at 24 h. The experiments were carried out twice (a, c) or three times (b) with similar results.
Figure 4
Figure 4
Isoproterenol inhibits LPS-stimulated type I IFN secretion. RAW264.7 macrophages were incubated with LPS (10 ng/ml)±isoproterenol (Iso, 1 μM) for 1 h. The LPS-containing medium was then removed, and the conditioned medium was collected following 3 h without further stimulus. (a) IL-10 secretion from the macrophages was measured by ELISA. (b) Type I IFN level in the media was measured by a luciferase reporter ISRE-L929 cell line assay. Data expressed as the mean ± SD (n = 3) of values normalized against Renilla luciferase activity and divided by 10,000. ∗ p < 0.003 for cells costimulated with LPS+isoproterenol compared to cells stimulated with LPS alone. The experiment was carried out 5 times with similar results.
Figure 5
Figure 5
Isoproterenol synergistically amplifies only direct LPS-induced IL-10 transcription at the early phase, but not late, type I IFN-dependent LPS activity. (a, b) RAW264.7 macrophages were transfected with a full mouse IL-10 promoter reporter construct. (a) Left panel: nontransfected RAW264.7 macrophages were incubated for 21 h with either LPS (10 ng/ml) or vehicle, and the conditioned medium (CM) was collected. Then, the IL-10 promoter reporter cells were incubated for 3 h with either CM from LPS-treated cells in the presence or absence of isoproterenol (Iso, 1 μM) or with CM from vehicle-treated cells together with isoproterenol. The LPS antagonist polymyxin B (50 μM) was added to the CM in all treatments. Right panel: synergism between isoproterenol and LPS (10 ng/ml) is shown for comparison. (b) The IL-10 promoter reporter cells were incubated for 8 h with isoproterenol (Iso, 1 μM) and/or either mouse IFNα (2000 units/ml, left panel) or LPS (10 ng/ml, right panel). (a, b) Luciferase reporter data expressed as the mean ± SD (n = 3) of values normalized against Renilla luciferase activity, relative to unstimulated control cells. ∗ p < 0.01, ∗∗ p < 0.03 for cells monostimulated relative to cells costimulated. The experiments were carried out twice with similar results.
Figure 6
Figure 6
MyD88 is required for synergistic IL-10 expression by LPS and cAMP. (a) RAW264.7 macrophages were transfected with a full mouse IL-10 promoter reporter construct. The cells were treated for 3 h with a combination of isoproterenol (Iso, 1 μM) and the PDE4 inhibitor rolipram (20 μM), in the presence or absence of a TLR agonist, either LPS (10 ng/ml; TLR4), Pam2Cys (0.1 ng/ml; TLR2/6), poly(I:C) (50 μg/ml; TLR3), or imiquimod (50 μM; TLR7). Luciferase reporter data expressed as the mean ± SD (n = 3) of values normalized against Renilla luciferase activity. ∗ p < 0.0001 compared to control cells stimulated with isoproterenol+rolipram. Reporter activity following administration of any TLR agonist alone (without the cAMP inducers) was indistinguishable from control values. (b) BMDM from WT (C57BL/6) and corresponding MyD88 knockout mice were incubated for 3 h with LPS (10 ng/ml) and/or isoproterenol (Iso, 1 μM). The medium was collected, and secretion of endogenous IL-10 was measured by ELISA. Data represent the mean ± SD (n = 6). ∗ p < 0.01 compared to WT. The experiments were carried out five (a) or three times (b) with similar results.
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