Enhancement of macrophage inflammatory responses by CCL2 is correlated with increased miR-9 expression and downregulation of the ERK1/2 phosphatase Dusp6

Abstract

Macrophage polarization plays a central role in both protective immunity and immunopathology. While the role of cytokines in driving macrophage polarization is well characterized, less is understood about the role of chemokines. The purpose of this study was to determine if CC chemokine 2 (CCL2/MCP1) could influence macrophage polarization in response to subsequent activation with cytokines and microbial products. Treatment of bone marrow-derived macrophages with CCL2 alone did not result in increased expression of either classical or alternatively-activated macrophage genes as compared to standard skewing cytokines or Toll-like receptor agonists. However, subsequent stimulation of CCL2 pre-treated macrophages with classical activation stimuli resulted in enhanced expression of genes associated with classical activation. This enhancement correlated with increased phosphorylation of ERK1/2 kinases, a decrease in expression of the ERK phosphatase Dusp6 and enhanced expression of miR-9. These results indicate that CCL2 supports the classical activation of macrophages, with miR-9 mediated down-regulation of Dusp6 and enhanced ERK-mediated signal transduction possibly mediating this enhanced pro-inflammatory gene expression.

Keywords: Chemokines; Gene regulation; Macrophages; Micro-RNA; Signal transduction.

FIGURE 1

Gene expression by BMDMs following stimulation with CCL2. A&B) Cells were treated with the indicated stimuli for 6 hours, and mRNA expression of A) Tnfa and B) Retnla was determined by qPCR. CCL2=100 ng/ml, IL-4=10ng/ml, IFNγ=10 U/ml. (*) = p <0.05 vs. Control. C–E) BMDMs were treated with the indicated concentrations of CCL2 and mRNA expression was analyzed by qPCR. Results are representative of two independent experiments. For (C) and (D), (*) = p<0.05 vs. all groups at the specific timepoint; For (E), (*) = p<0.05 vs. 0 and 10 ng/ml CCL2.

FIGURE 2

Regulation of the CCL2/CCR2 axis by extracellular signals. BMDMs were treated with the indicated stimuli for 6 hours, and mRNA expression of A&C) Ccl2 and B&D) Ccr2 was determined by qPCR. CCL2=100 ng/ml, IL-4=10ng/ml, IFNγ=10 U/ml, LPS=100ng/ml. Results are representative of three independent experiments. (*) = p<0.05 vs. control.

FIGURE 3

Effects of CCL2 stimulation on BMDM morphology in vitro. BMDMs were either A) left unstimulated, or treated with B) 100 ng/ml CCL2, C) 10 U/ml IFNγ + 100 ng/ml LPS or D) 10 ng/ml IL-4 for 24 hours in Lab-Tek 8-well chambered glass slides, with 2 replicate wells for each culture condition. Following incubation, cells were fixed with methanol and stained with Diff-Quik. A minimum of five distinct fields were observed in each replicate well via light microscopy. Representative images were captured using CellSens software. Images are representative of two independent experiments.

FIGURE 4

Enhancement of classical activation gene expression in CCL2 pre-treated BMDMs. Following maturation in L-cell conditioned media, BMDMs were replated in either minimal media or media supplemented with 100 ng/ml CCL2. A&B) Following pre-treatment with CCL2 for 18 hours, BMDMs were treated with the indicated cytokines/TLR ligands (IFNγ=10 U/ml, LPS=−100ng/ml, IL−4=10 ng/ml); 6 hours post-stimulation, mRNA was isolated for analysis by qPCR. Results are representative of four independent experiments. C) BMDMs were supplemented for 18 hours with varying concentrations of CCL2, followed by IFNγ/LPS for an additional 6 hours. mRNA was then isolated for analysis by qPCR. Fold expression levels were generated compared to untreated and unstimulated BMDMs that were cultured alongside the stimulated cells for the indicated time periods. Results are representative of three independent experiments. D) BMDMs were supplemented for 18 hours with varying concentrations of CCL2, followed by IFNγ/LPS for 24 hours. Supernatants from cultured BMDMs were then analyzed by multiplex bead assay (Luminex). Results are representative of two independent experiments. (*) = p<0.05 vs Untreated (A&B) or 0 ng/ml CCL2 (C&D).

FIGURE 5

Expression of activation markers on CCL2 pre-treated BMDMs. Following maturation in L-cell conditioned media, BMDMs were replated in either minimal media or media supplemented with 100 ng/ml CCL2. The following day, cells were treated with 10 U/ml IFNγ + 100 ng/ml LPS for 24 hours, and cells were fixed and stained for flow cytometry. Expression of A) IFNγR, B) TLR4, C) CCR2 and D) CCR7 in PE-fluorescence was observed on F4/80+ BMDMs. Long dashed line: no PE antibody, Solid line: Untreated, Short dashed line: CCL2 pre-treated BMDMs. Results are representative of two independent experiments.

FIGURE 6

Enhancement of ERK 1/2 signaling in CCL2 pre-treated BMDMs. A) Untreated or CCL2 pre-treated BMDMs were stimulated with 10 U/ml IFNγ + 100 ng/ml LPS for the indicated timepoints, cells were lysed and ERK1/2 phosphorylation was analyzed by SDS-PAGE electrophoresis and Western Blot. Results are representative of three independent experiments. B) Untreated or CCL2 pre-treated BMDMs were stimulated with IFNγ/LPS for 60 minutes, cells were lysed and ERK1/2 phosphorylation was quantitatively analyzed via Luminex bead assay. Results are representative of two independent experiments. (*) = p<0.05 vs. Wild-type. C) Untreated or CCL2 pre-treated BMDMs were restimulated with IFNγ/LPS for 6 hours, with additional samples treated with either vehicle control (DMSO) or the ERK1/2 inhibitor GDC-0994 (50 nM). Expression of Nos2 mRNA was analyzed via qPCR. (**) = p<0.01 vs. Untreated.

FIGURE 7

Regulation of DUSP6 and miR-9 expression in BMDMs by CCL2. A) Following maturation in L-cell conditioned media, BMDMs were treated with the indicated concentrations of CCL2 for 18 hours, and expression of Dusp6 mRNA was analyzed via qPCR. Results are representative of two independent experiments. B) BMDMs were treated with the indicated concentrations of CCL2 for 18 hours, and DUSP6 protein expression was analyzed via SDS-PAGE electrophoresis and Western Blot. Results are representative of two independent experiments. C) Schematic representation of the DUSP6 3′ UTR and putative matching seed regions for miR-9 and miR-145, adapted from an in silico study performed using TargetScanMouse6.2 (www.targetscan.org). D) Untreated or CCL2 pre-treated BMDMs were stimulated with the indicated cytokines/TLR ligands for 6 hours, and miR-9 expression was analyzed by miR-specific cDNA synthesis followed by qPCR. Results are representative of two independent experiments. (*) = p<0.05 vs. unstimulated.