MicroRNAs in resolution of acute inflammation: identification of novel resolvin D1-miRNA circuits

Abstract

Mechanisms controlling resolution of acute inflammation are of wide interest. Here, we investigated microRNAs (miRNAs) in self-limited acute inflammatory exudates and their regulation by resolvin D1 (RvD1). Using real-time PCR analysis, we found in resolving exudates that miR-21, miR-146b, miR-208a, miR-203, miR-142, miR-302d, and miR-219 were selectively regulated (P<0.05) in self-limited murine peritonitis. RvD1 (300 ng/mouse or 15 μg kg(-1)) reduced zymosan-elicited neutrophil infiltration into the peritoneum 25-50% and shortened the resolution interval (R(i)) by ∼4 h. In peritonitis at 12 h, RvD1 up-regulated miR-21, miR-146b, and miR-219 and down-regulated miR-208a in vivo. In human macrophages overexpressing recombinant RvD1 receptors ALX/FPR2 or GPR32, these same miRNAs were significantly regulated (P<0.05) by RvD1 at concentrations as low as 10 nM, recapitulating the in vivo circuit. In addition, RvD1-miRNAs identified herein target cytokines and proteins involved in the immune system, e.g., miR-146b targeted NF-κB signaling, and miR-219 targeted 5-lipoxygenase and reduced leukotriene production. RvD1 also reduced nuclear translocation of NF-κB and SMAD and down-regulated phospho-IκB. Taken together, these results indicate that resolvin-regulated specific miRNAs target genes involved in resolution and establish a novel resolution circuit involving RvD1 receptor-dependent regulation of specific miRNAs.

Figure 1.

Strategy for identifying RvD1-regulated miRNAs involved in resolution. Scheme of temporal and differential miRNA expression and analyses of resolving exudates.

Figure 2.

Strategy for identifying RvD1-regulated miRNAs involved in resolution. A) Self-limited acute inflammation (i.e., murine peritonitis) was used to obtain exudates (see Fig. 1). For temporal analysis, lavages were collected at 4, 12, 24, and 48 h after zymosan A injection. For resolution indices of self-limited peritonitis, peritoneal lavages were collected from zymosan A (1 mg/mouse, i.p.)-challenged mice (n=3–16 mice/time point). Total leukocytes (left panel), PMN (triangles) and monocytes (circles) (middle panel), and elicited MΦs (nablas; right panel) were determined (see Materials and Methods). Results are means ± se. Dotted lines indicate time points; PMN number values were used to determine resolution indices as in ref. ; i.e., T_max, time of maximum PMN infiltration (Ψ_max); T₅₀, time to achieve 50% reduction in PMN number (Ψ₅₀) from Ψ_max; R_i, resolution interval (T₅₀ − T_max; time interval between T_max and T₅₀). Right panel, inset shows the rate of accumulation of 17- and 14-hydroxy-docosahexaenoic acid (HDHA) markers of D-series resolvin (solid circles) and maresin (open squares) pathway activation, respectively, in resolving exudates replotted here for comparison (50). B) Representative flow cytometry dot plots of Ly-6G and CD11b expression in exudate cells.

Figure 3.

miRNA array from resolving exudates. A) Peritoneal leukocytes were collected at 4, 12, and 24 h after zymosan A (1 mg/mouse, i.p.) injection, and miRNA fractions were isolated. Heat map cluster represents relative expression of ∼300 target mouse miRNAs from resolving exudates (n=3 mice/time point) determined with real-time PCR array after normalization with housekeeping small RNAs (see Materials and Methods). Dendrogram was generated to cluster miRNAs according to their relative expression with the Manhattan distance and Ward's method using StatMiner Software. High-resolution full-size heat map is included in Supplemental Fig. S1. Relative expression intensities are indicated in a green-red color code based on ΔC_t values. B) Scatterplot of relative expression of ∼300 miRNAs analyzed in exudate cells at 4 and 12 h peritonitis. Red and green lines in the scatterplot indicate 2.5- and 0.4-fold changes in expression between 12 and 4 h, which were used for defining candidate miRNA (red and green diamonds). Black line represents no changes. C) Time course of miRNA relative expression in resolving exudates. miRNAs were selected among those that displayed fold changes >2.5 or <0.4 at 12 h of zymosan-induced peritonitis compared to 4 h. Relative expression was assessed with real-time PCR array (see Materials and Methods). Results are expressed as means of 3 mice.

Figure 4.

Real-time PCR analysis of miRNAs reveals their specific temporal regulation in resolving exudates. Kinetics of relative expression for indicated miRNAs was determined from exudates collected at different time intervals after zymosan A injection using real-time PCR after normalization (see Materials and Methods). Results are expressed as means ± se (n=3–6 mice) *P < 0.05 vs. 4 h.

Figure 5.

Resolvin D1 controls acute inflammation, accelerates resolution, and selectively regulates miRNA expression in exudates. A, B) Male FVB mice were administered zymosan alone (1 mg/mouse, i.p.; A, open circles) or zymosan plus RvD1 (300 ng/mouse, i.p.; A, shaded circles); exudates were collected at indicated time points to determine total leukocytes (A) and PMN and monocytes/MΦs (B) using flow cytometry, as described in Materials and Methods. C) Resolution indices. Solid circles and open triangles (left panel) indicate each of the parameters used to define the resolution indices (right panel) in zymosan (left panel, gray lines) or zymosan plus RvD1 (black lines) group, as described by Bannenberg et al. (4). Arrow denotes time of zymosan and RvD1 addition. Results are expressed as means ± se (n=3–16 mice). *P < 0.05 vs. zymosan. Inset: RvD1 structure. D) Scatterplot and temporal profile of miRNA expression from exudates collected 12 h after injection of zymosan alone or zymosan plus RvD1 (300 ng/mouse, i.p.). Red and green lines in scatterplot represent boundaries of 2.5- and 0.4-fold changes by RvD1 compared to zymosan-treated mice, which were used as cutoff values to define RvD1-regulated miRNA. Results are means of 3 mice.

Figure 6.

Resolvin D1 temporally regulates miRNA expression in resolving exudates: real-time PCR. miRNA fractions were extracted from peritoneal exudates at 4, 12, 24, and 48 h after injection of zymosan alone or zymosan plus RvD1 and quantified using real-time PCR after normalization (see Materials and Methods). Dotted lines indicate no changes in miRNA expression. Shaded bars indicate statistically significant expression levels. Results are expressed as means ± se (n=3–6 mice). *P < 0.05 vs. zymosan alone at each time point.

Figure 7.

Resolvin D1 regulates miRNA expression via recombinant receptors ALX/FPR2 and GPR32. Regulation of miRNA levels by RvD1 in human MΦs transfected with ALX/FPR2 or GPR32. Relative expression of indicated miRNAs from MΦs incubated with RvD1 (10.0 nM, 6 h, 37°C) compared to vehicle alone was determined using real-time PCR analysis. Results are expressed as means ± se (n=3–5 healthy subjects). Results for miR-208 are representative of 5 healthy subjects. Bottom right panel: receptor expression levels by PCR in ALX/FPR2- and GPR32-overexpressing MΦs from a representative healthy subject. *P < 0.05 vs. vehicle alone; *P < 0.05 vs. MΦ-mock transfected cells.

Figure 8.

RvD1-regulated miRNAs target genes with specific roles in inflammation and resolution. Human MΦs were transfected with plasmids containing hsa-miR-146b, as described in Materials and Methods. Figure shows the expression level of indicated miRNA (top left) and down-regulated target mRNAs in MΦ-miR compared to MΦ-mock transfected cells determined using real-time PCR 48 h after transfection. Results are expressed as means ± se (n=3–4 healthy subjects). Result for IL-8 mRNA level is representative of 4 healthy subjects. *P < 0.05 vs. MΦ-mock transfected cells. C8A, complement component 8, polypeptide α; CAMP, cathelicidin antimicrobial peptide; CHUK, conserved helix-loop-helix ubiquitous kinase (IKK-α); CRP, C reactive protein; DMBT1, deleted in malignant brain tumors 1; IFN, interferon; IL, interleukin; IL1F, IL-1 family member; IL1R1, IL-1 receptor 1; IL1RAP, IL-1 receptor accessory protein; IL1RAPL2, IL-1 receptor accessory protein-like 2; IL1RL2, IL-1 receptor-like 2; LALBA, lactalbumin α; LPB, lipopolysaccharide binding protein; LTF, lactotransferrin; NOS2, nitric oxide synthase 2 (iNOS); PGLYRP, peptidoglycan recognition protein; PTAFR, platelet-activating factor receptor; S100A12, S100 calcium-binding protein A12; SFTPD, surfactant protein D; TLR, Toll-like receptor; TRAF, tumor necrosis factor receptor-associated factor.

Figure 9.

RvD1-regulated miRNAs target genes with specific roles in inflammation and resolution. Human MΦs were transfected with plasmids containing miR-208a (A) or miR-219 (B), as described in Materials and Methods. Each panel shows the expression levels of indicated miRNAs (left) and down-regulated target mRNAs in MΦ-miR compared to MΦ-mock transfected cells determined using real-time PCR 48 h after transfection. Results are expressed as means ± se (n=3–4 healthy subjects). *P < 0.05 vs. MΦ-mock transfected cells. 5-LO, arachidonate 5-lipoxygenase (ALOX5); CD40L, CD40 ligand; PDCD4, programmed cell death 4; PLCG2, phospholipase Cγ2; TBXA2R, thromboxane A₂ receptor; TNF-RII, tumor necrosis factor-α receptor 2.

Figure 10.

IPA networks connecting target genes of RvD1-regulated miRNAs. Genes that were significantly down-regulated in MΦ-miR are indicated in red within each network.

Figure 11.

RvD1 regulates miRNA target genes in self-limited murine acute peritonitis. Messenger RNA fractions were isolated from peritoneal exudates 4, 12, 24, and 48 h after injection of zymosan with or without RvD1. Top panels: relative expression levels of IKK, PDCD4, and 5-LO measured using real-time PCR and normalized to GAPDH levels. Bottom panel: IL-10 protein levels determined by ELISA and normalized to total protein. Results are expressed as means ± se of fold change above zymosan alone (n=3–9 mice). *P < 0.05 vs. zymosan alone. 5-LO, arachidonate 5-lipoxygenase (ALOX5); IKK, IκB kinase [conserved helix-loop-helix ubiquitous kinase (CHUK)]; IL-10, interleukin 10; PDCD4, programmed cell death 4.

Figure 12.

miR-219 regulates 5-LO protein levels and LTB₄ generation. A) Western blot analyses show 5-LO protein levels in MΦ-vector and MΦ-miR-219, determined 72 h after transfection. Results are expressed as means ± se of relative intensities of immunoreactive bands of target proteins quantitated and normalized to actin, used as loading control (n=3 healthy subjects). *P < 0.05 vs. MΦ-vector. Inset: representative immunoblot radiograph visualized using chemiluminescence. B) Quantitation of LTB₄ and PGE₂ levels using LC-MS/MS of MΦ-vector and MΦ-miR-219 cells incubated with 5 × 10⁶ zymosan particles/10⁶ MΦs (2 h, 37°C). Representative chromatogram from n = 3 healthy subjects is shown. Inset: mean ± se percentage reduction in LTB₄ levels in miR-219 transfected MΦs and MS/MS spectrum of LTB₄ (n=3 healthy subjects). *P < 0.05 vs. MΦ-vector. C) RvD1 regulates activation of transcription factors in human monocytes. Peripheral blood monocytes (see Materials and Methods) were incubated with RvD1 (10 nM) or vehicle alone for 90 min at 37°C. Activation of indicated transcription factors was assessed using a competitive ELISA kit. Heat map depicts fold change in nuclear transcription factor levels by RvD1 compared to vehicle alone (heat map generated from mean of n=4 healthy subjects). See Supplemental Data for list of TF abbreviations. D) Left panel: percentage reduction by RvD1 in nuclear translocation of NF-κB and SMAD in human monocytes. Results are expressed as means ± se (n=4 healthy subjects). *P < 0.05 vs. vehicle alone). Right panel: RvD1 regulation of phospho-IκB levels in human monocytes. Peripheral blood monocytes were incubated with 10 nM RvD1 or vehicle for 15 min, followed by 1 ng TNF-α or vehicle for 90 min at 37°C. Phospho-IκB levels were measured. Results are from 4 healthy subjects.