Induction of IL-4Rα-dependent microRNAs identifies PI3K/Akt signaling as essential for IL-4-driven murine macrophage proliferation in vivo - PubMed (original) (raw)
Induction of IL-4Rα-dependent microRNAs identifies PI3K/Akt signaling as essential for IL-4-driven murine macrophage proliferation in vivo
Dominik Rückerl et al. Blood. 2012.
Abstract
Macrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4-receptor/PI3K/Akt-signaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4-driven MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα-induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4-driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting MΦ expansion and alternative activation during type 2 inflammatory settings.
Figures
Figure 1
Validation of differential expression of miRNAs in AAMΦ in vivo and in vitro. (A) Peritoneal MΦ were isolated from Thio-injected BALB/c mice or from _B malayi_–infected BALB/c (WT) or IL-4Rα−/− (−/−) mice and expression of the indicated miRNAs assessed by qRT-PCR. Each data point shown reflects data from individual mice. (B) Chi3l3 expression in the cells isolated in panel A. (C) Thio-elicited, adherence-purified MΦ were incubated with rIL-4 (IL-4), LPS/IFNγ (L/I), or without stimulus (−) for 16 hours and analyzed as in panel A. (D) Chi3l3 expression in the cells isolated in panel C. One of 3 separate experiments shown. *P < .05; **P < .01; ***P < .001; n.s. indicates not significant.
Figure 2
Kinetics of miR-378-3p induction during in vitro alternative activation. Thio-elicited, adherence-purified MΦ were incubated with rIL-4 (black squares), LPS and rIFNγ (gray triangles), or with medium alone (open squares) for the indicated time and analyzed for miRNA and mRNA expression. Statistics indicate differences between rIL-4–treated samples and media controls. **P < .01; ***P < .001. Each data point represents mean and SEM of 3 individual animals. One of 2 separate experiments shown.
Figure 3
miR-378-3p targets the IL-4R/PI3K/Akt-signaling pathway. (A) Schematic depiction of the IL-4R–signaling cascade highlighting putative targets for miR-378-3p. Yellow indicates predicted by TargetScan; red: differentially expressed genes in pre- and anti-miR-378-3p–transfected fibroblasts. (B) Analysis of rIL-4–elicited STAT-6 phosphorylation by intracellular FACS staining. Histogram of STAT-6 (pY641) expression in Thio-MΦ preincubated with rIL-4 (orange line) or medium (red line) for 24 hours before restimulation with rIL-4 (open histograms) or medium (filled gray histograms) for 5 minutes. Timeline of pSTAT-6 (pY641) expression in rIL-4 (orange squares) or medium (red circles) pretreated, rIL-4–stimulated (colored symbols) or unstimulated (gray symbols) cells. Data show median fluorescence intensity of 6 individual animals ±SEM. For unstimulated controls, cells were pooled from several animals. Asterisks indicate statistical differences between rIL-4–preincubated and freshly stimulated MΦ. ***P < .001; **P < .01. (C) Luciferase-assay using the 3′UTR of wild-type Akt-1 (Akt-1) or constructs with mutated seed-region binding sites for both predicted miR-378-3p binding sites (mut) or without insert (empty). Data indicate cotransfection with a miR-378-3p mimic (black bars) or a scrambled control (open bars). Data are pooled from 5 separate experiments and depicted as relative luminescence compared with no-RNA controls (gray bars). Bars not connected by the same letters are statistically significantly different. (D) Representative Western blot analysis of RAW264.7 cells 12 hours after transfection with a mimic (pre-378) or inhibitor (anti-378) of miR-378-3p or appropriate negative controls (pre- and anti-neg). Samples were separated on 4%-12% Bis-Tris gels and stained for Akt-1 and β-actin at the same time. (E) Densitometric analysis of the samples analyzed in panel D. Data are representative of 4 separate experiments. Columns not connected by the same letters are statistically significantly different.
Figure 4
Akt-inhibition modulates alternative activation of MΦ in vitro. Thio-MΦ were stimulated with rIL-4 (+) or medium (−) after preincubation with the indicated concentration of triciribine for 1 hour and analyzed for expression of alternative activation markers by qRT-PCR. Data are representative of 3-4 animals per group. Error bars indicate SEM. One of 4 experiments shown. Asterisks indicate statistical differences compared with IL-4–treated samples. ***P < .001, **P < .01.
Figure 5
Time course of miR-378-3p expression and markers of proliferation after B malayi implantation. (A) Gene expression of miR-378-3p and Akt1 in peritoneal MΦ isolated from B malayi implanted C57BL/6 mice at the indicated time points after implantation. N indicates naive animals. Data points represent individual animals or separate pools of animals. (B) FACS analysis of Ki67 expression and BrdU incorporation in the MΦ analyzed in panel A. Data from the same experiment shown in supplemental Figure 4 in Jenkins et al. One experiment of 1 shown. ***P < .001; **P < .01; *P < .05.
Figure 6
Akt inhibition and miR-378-3p overexpression negatively regulate MΦ proliferation. (A) BALB/c mice were injected intraperitoneally with a single dose of triciribine (1 mg/kg) or vehicle control 1 hour before injection of IL-4c or PBS. After 24 hours cells were analyzed for proliferation and alternative activation by FACS analysis. Each data point is representative of an individual animal. Pooled data from 3 separate experiments shown. (B) Conversion of alamarBlue by RAW264.7 cells 24 and 48 hours after transfection with a miR-378-3p mimic (black squares) or inhibitor (black circles) or the appropriate negative controls (open symbols). Data representative of 3 separate experiments. (C) FACS-analysis of Vybrant CFDA SE dilution in the cells analyzed in panel A. (D) Gene expression of miR-378-3p target genes and genes associated with cell proliferation in RAW264.7 cells 48 hours after transfection with a miR-378-3p mimic (black bars) or the appropriate negative control (open bars). ***P < .001; **P < .01; *P < .05; n.s. indicates not significant.
Figure 7
Induction of miR-378-3p is specific to IL-4–mediated signaling in MΦ. (A) Thio-elicited, adherence-purified MΦ were incubated with rIL-4 (black squares), rM-CSF (gray circles), or with medium alone (open squares) for the indicated time and analyzed for miRNA and mRNA expression. Statistics indicate differences between rM-CSF–treated samples and rIL-4 controls. ***P < .001. Each data point represents mean and SEM of 6 individual animals. Results from a single experiment shown. (B) CD11b+F4/80+ (MΦ), CD19+F4/80− (B cells), CD4+CD11b− lymphocytes (CD4+) and CD8+CD11b− lymphocytes (CD8+) were FACS-sorted from IL-4c injected (filled bars) or PBS-injected control animals (open bars) and subjected to qRT-PCR. Data are depicted as fold change above PBS controls. Statistics indicate differences between IL-4c–treated samples and PBS-controls. ***P < .001; *P < .05. Bars depict mean and SEM of 10 individual animals per group pooled from 2 independent experiments, except for the CD8+ data, which is from 1 representative experiment. The second experiment is not shown because Ccna2 in the PBS controls was not detectable resulting in a fold change of infinity for the IL-4c treatment.
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