Resolvin D1 receptor stereoselectivity and regulation of inflammation and proresolving microRNAs - PubMed (original) (raw)

Resolvin D1 receptor stereoselectivity and regulation of inflammation and proresolving microRNAs

Sriram Krishnamoorthy et al. Am J Pathol. 2012 May.

Abstract

Resolution of acute inflammation is an active process that involves the biosynthesis of specialized proresolving lipid mediators. Among them, resolvin D1 (RvD1) actions are mediated by two G protein-coupled receptors (GPCRs), ALX/FPR2 and GPR32, that also regulate specific microRNAs (miRNAs) and their target genes in novel resolution circuits. We report the ligand selectivity of RvD1 activation of ALX/FPR2 and GPR32. In addition to RvD1, its aspirin-triggered epimer and RvD1 analogs each dose dependently and effectively activated ALX/FPR2 and GPR32 in GPCR-overexpressing β-arrestin systems using luminescence and electric cell-substrate impedance sensing. To corroborate these findings in vivo, neutrophil infiltration in self-limited peritonitis was reduced in human ALX/FPR2-overexpressing transgenic mice that was further limited to 50% by RvD1 treatment with as little as 10 ng of RvD1 per mouse. Analysis of miRNA expression revealed that RvD1 administration significantly up-regulated miR-208a and miR-219 in exudates isolated from ALX/FPR2 transgenic mice compared with littermates. Overexpression of miR-208a in human macrophages up-regulated IL-10. In comparison, in ALX/FPR2 knockout mice, RvD1 neither significantly reduced leukocyte infiltration in zymosan-induced peritonitis nor regulated miR-208a and IL-10 in these mice. Together, these results demonstrate the selectivity of RvD1 interactions with receptors ALX/FPR2 and GPR32. Moreover, they establish a new molecular circuit that is operative in the resolution of acute inflammation activated by the proresolving mediator RvD1 involving specific GPCRs and miRNAs.

Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

RvD1, AT-RvD1, and their stable analogs directly act on hALX/FPR2. Ligand-receptor interaction was monitored using the HEK-ALX/FPR2 β-arrestin system (see Materials and Methods). Dose-response activation curves were obtained for RvD1 and DHA (A), RvD1-ME (B), 17(R/S)-methyl RvD1-ME (C), and AT-RvD1 (D). Results are expressed as mean ± SEM (n = 3 to 4). RLU, relative luminescence units.

Figure 2

Figure 2

RvD1, AT-RvD1, and their stable analogs directly act on hGPR32. Ligand-receptor interactions were determined using the GPR32 β-arrestin system as in Figure 1. Dose-response activation curves are shown for RvD1 and DHA (A), RvD1-ME (B), 17(R/S)-methyl RvD1-ME (C), and AT-RvD1 (D). Results are expressed as mean ± SEM (n = 4 to 6). RLU, relative luminescence units.

Figure 3

Figure 3

RvD1, Rv analogs, and compound 43 produce hGPR32-dependent changes in impedance. Ligand-receptor–dependent changes in impedance were continuously recorded with real-time monitoring across cell monolayers using an ECIS system with hGPR32-expressing β-arrestin cells. Results are tracings obtained from incubations with CHO-GPR32 β-arrestin cells plus RvD1 (1, 10, and 100 nmol/L) (A); compound 43 (1, 10, and 100 nmol/L) (B); 100 nmol/L RvD1, RvD1-ME, and 17(R/S)-methyl RvD1-ME (C); and 100 nmol/L RvD1 directly compared with native DHA (D). Each tracing is representative of n = 3. Insets in A and B show dose-dependent changes in cell impedance determined at 5 minutes of incubation of GPR32-overexpressing cells with RvD1 or compound 43 or vehicle alone. Results are expressed as mean ± SEM (n = 3).

Figure 4

Figure 4

RvD1 reduction in PMN infiltration is enhanced in hALX/FPR2-overexpressing transgenic (Tg) mice peritonitis. hALX/FPR2-Tg mice and WT littermates were injected with zymosan (1 mg per mouse i.p.), with or without RvD1-ME at 10 ng per mouse i.v. Inflammatory exudates were collected at 24 hours and total exudate leukocytes were determined (see Materials and Methods): total leukocytes (left panel) and PMNs (middle panel). The right panel shows numbers of total leukocytes and resident macrophages in control mice. Results are expressed as mean ± SEM (n = 3 mice). *P < 0.05, WT, zymosan versus zymosan + RvD1; †P = 0.05, zymosan, WT versus ALX/FPR2-Tg; ‡P < 0.01, zymosan + RvD1 WT versus ALX/FPR2-Tg.

Figure 5

Figure 5

RvD1 stimulates specific proresolving miRNAs in hALX/FPR2 transgenic (Tg) mice. miRNA fractions were isolated from peritonitis exudate samples of WT littermates and ALX/FPR2-Tg mice challenged with zymosan (1 mg per mouse) compared with mice given RvD1 (10 ng per mouse with zymosan). Real-time PCR analyses of indicated miRNAs were performed and results were analyzed by the 2−ΔCT method. Fold change in expression levels in RvD1-treated mice compared with zymosan alone were calculated from 2−ΔCT values. Results are expressed as mean ± SEM (n = 3 in each group) fold change in levels of miR-208a (A), miR-219 (B), and miR-21, miR-146b, and miR-302d (C). *P < 0.05 for RvD1-treated versus zymosan-treated WT or Tg mice; †P < 0.05 for zymosan + RvD1–treated Tg mice versus zymosan + RvD1–treated WT mice. D: miR-208a and miR-219 expression levels determined by real-time PCR in peritoneal cells from untreated mice.

Figure 6

Figure 6

Regulation of acute inflammation and miRNA by RvD1 in ALX/FPR2 knockout mice. Number of total leukocytes (A) and PMNs (B) in peritoneal lavage fluid from WT or ALX/FPR2−/− mice injected with zymosan (1 mg per mouse, i.p.) with or without RvD1-ME (10 ng per mouse, i.v.). Lavage fluid samples were collected 24 hours after initiation of peritonitis, and cells were stained with anti-Ly-6G and F4/80 antibodies. Results are expressed as mean ± SEM (n = 6 mice per group). *P < 0.05 versus zymosan-treated group. Expression of miR-208a (C) and miR-219 (D) in exudate cells from WT or ALX/FPR2−/− mice 24 hours after injection of zymosan plus RvD1-ME (10 ng per mouse, i.v.) or zymosan plus saline. Relative expression of miRNAs was determined using real-time PCR. Results are expressed as mean ± SEM of fold changes in expression (n = 6 mice per group). **P < 0.001 versus zymosan-treated group.

Figure 7

Figure 7

Regulation of IL-10 levels by RvD1 in peritonitis and by miR-208a in human macrophages. A: IL-10 levels were determined in mALX/FPR2−/− mice (n = 3 to 6). *P < 0.05 versus mice receiving zymosan alone. B: IL-10 levels were determined in human macrophages transiently overexpressing miR-208a or control vector (n = 3). *P < 0.05 versus control vector–transfected macrophages.

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