MEK1/2 Inhibition Promotes Macrophage Reparative Properties

Abstract

Macrophages have important functional roles in regulating the timely promotion and resolution of inflammation. While many of the intracellular signaling pathways involved in the pro-inflammatory responses of macrophages are well characterized, the components that regulate macrophage reparative properties are less well understood. We identified the MEK1/2 pathway as a key regulator of macrophage reparative properties. Pharmacological inhibition of the MEK1/2 pathway (MEKi) significantly increased expression of IL-4/IL-13 (M2) responsive genes in murine bone marrow-derived and alveolar macrophages. Deletion of the MEK1 gene using LysM^Cre+/+MEK1^fl/fl macrophages as an alternate approach yielded similar results. MEKi enhanced STAT6 phosphorylation, and MEKi induced changes in M2 polarization were dependent on STAT6. In addition, MEKi-treatment significantly increased both murine and human macrophage efferocytosis of apoptotic cells (AC) independent of macrophage polarization and STAT6. These phenotypes were associated with increased gene and protein expression of Mertk, Tyro3, and Abca1, three proteins that promote macrophage efferocytosis. We also studied the effects of MEKi on in vivo macrophage efferocytosis and polarization. MEKi treated mice had increased efferocytosis of apoptotic PMNs instilled into the peritoneum. Furthermore, administration of MEKi after LPS-induced lung injury led to improved recovery of weight, fewer neutrophils in the alveolar compartment, and greater macrophage M2 polarization. Collectively, these results show that MEK1/2 inhibition is capable of promoting reparative properties of both murine and human macrophages. These studies suggest that the MEK1/2 pathway may be a therapeutic target to promote the resolution of inflammation via modulation of macrophage functions.

Introduction

Macrophage responses to infection, injury, and other inflammatory stimuli are shaped by the complex milieu of signals from the surrounding environment. Macrophage plasticity allows these cells to adopt different polarized phenotypes that may evolve over time based on cell origin and environmental stimuli. The functional dichotomy of M(IFN-γ) or M(LPS) (M1) and M(IL-4/IL-13) (M2) polarization has been investigated as opposite ends on a spectrum of activation states regulating the function of macrophages (1). LPS-stimulated macrophages release pro-inflammatory cytokines, including IL-1β, IL-12, and TNF-α, and are effective at killing bacteria; (2, 3) whereas IL-4/IL-13 stimulated cells down-regulate inflammatory programming and up-regulate genes involved in wound repair (4, 5). Under this paradigm, a transition from an M1 to M2 phenotype facilitates a reparative phenotype that promotes resolution of inflammation (6, 7), and there are both human and murine studies indicating that this transition occurs in vivo (8, 9).

The cellular signaling networks regulating macrophage responses to M1 stimuli such as LPS or IFN-γ have been characterized in detail, and there are numerous other pathways that direct macrophages to develop distinct phenotypic and functional states (1, 10, 11). In contrast, a complete understanding of the signaling pathways that regulate macrophage M2 polarization and reparative properties is lacking. Because of this, manipulation of macrophage cell signaling targets as a therapeutic strategy to promote the resolution of inflammation via harnessing the reparative properties of macrophages remains limited.

The mitogen-activated protein kinases MEK1 (Map2k1) and MEK2 (Map2k2) participate in intracellular signaling networks and exert control on downstream effector molecules, ERK1 and ERK2 via MEK1/2 dependent serine and tyrosine phosphorylation (12). MEK1 and MEK2 share 80% amino acid identity, suggesting that they may be functionally redundant. In certain cases, deletion of both MEK1 and MEK2 is required for phenotypes to emerge (13). However, Mek2^−/− mice are phenotypically normal, whereas MEK1 deletion is embryonic lethal suggesting that MAPK cascade signaling is dependent on select isoforms in specific settings (14). Recognition that altered activation of proteins in the RAS-RAF-MEK-ERK1/2 pathway occurs in many human cancers has led to the development of inhibitor compounds targeting MEK1 and MEK2 to disrupt oncogenic pathway over-activation (15-17). More recently, immune-related targets of MEK pathways have been described. For example, inhibition of the MEK1/2 pathway in macrophages has been shown to regulate LPS responses (18, 19), and mice treated with MEK1/2 inhibitors within 6 hours of cecal ligation had reduced inflammation and multi-organ dysfunction (20). However, to our knowledge, the role of MEK1/2 in regulating of IL-4/IL-13 polarization has not been characterized.

In this current study, we characterized the effect of MEK1/2 pathway inhibition on macrophage phenotypes during resting states and IL-4/IL-13 polarization. The MEK1/2 inhibitor (MEKi) PD0325901 significantly increased expression of murine macrophage IL-4/IL-13 responsive genes including Retnla, Ym1, Ccl17, and Tgfb1, and membrane proteins, CD71 and CD206. This up-regulation occurred by a mechanism that included increased STAT6 pathway activity, as measured by increased STAT6-phosphorylation that coincided with decreased levels of SOCS1 and SOCS3. In addition, MEKi-treatment significantly increased both murine and human macrophage efferocytosis of apoptotic cells (AC) independent of macrophage polarization. MEKi-treatment induced increased murine macrophage gene expression and cell surface localization of Mertk, Tyro3, and Abca1, three proteins that promote macrophage efferocytosis of AC. We also found that the in vivo clearance of apoptotic neutrophils was enhanced by MEKi treatment. Furthermore, inhibition of MEK1/2 after induction of LPS-induced lung injury was associated with earlier recovery of weights, reduced lung neutrophils, and increased macrophage M2 polarization. To our knowledge, these studies are the first to evaluate the effects of a MEKi on macrophage reparative function. These studies have broad relevance, as the regulation of macrophage polarization and efferocytosis using MEKi has important therapeutic implications for multiple diseases such as lung infection and inflammation, atherosclerosis, and cancer.

Methods

Mice

C57Bl/6, Balb/c, and Stat6^−/− mice on a Balb/c background (a gift of Steven F. Ziegler, Ph.D.) were used for these studies. Mice were matched for age (8-12 weeks) and gender. LysM^Cre+/+MEK1^fl/fl mice were generated by crossing MEK1^fl/fl mice with LysM^Cre+/+ mice (Jackson Laboratories, Bar Harbor, ME) (13, 14). Genotyping was performed using PCR probe sets as described (13). Animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington.

qPCR Primers, Antibodies, and Inhibitors

Validated TaqMan FAM primer probes for the murine genes Retnla Mm00445109, Hprt Mm01545399, Ym1 Mm00657889, Tgfb1 Mm00441724 Ccl17 Mm01244826, Arg1 Mm00475988, Mertk Mm00434920, Abca1 Mm00442646, PU.1 Mm00488140, IL10 Mm00439614 and human genes Hprt1 Hs02800695, Tgm2 Hs00190278, Tgfb1 HS00998133, Mrc1 HS00267207, and Ccl17 HS00171074 were purchased from Life Technologies (Carlsbad, CA). Antibodies to phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (cat. no. 9101), p44/42 MAPK (Erk1/2) (137F5) (cat. no. 4695), Stat6 (cat. no. 9362), SOCS1 (A156) (cat. no. 3950), SOCS3 (cat. no. 2923), MEK1 (D2R10) (cat. no. 12671), β-Actin (D6A8) (cat. no. 8457), and anti-rabbit IgG HRP-linked (cat. no. 7074) were from Cell Signaling Technology (Danvers, MA). Anti-Stat6 (phospho Y641) (ab54461) was from Abcam (Cambridge, MA). Mouse RELMα antibody (cat. no. AF1523) was from R&D Systems (Minneapolis, MN). Antibodies used for flow cytometry included: Abca1 Dyelight-488 conjugate (Pierce, PA5-22908) and rabbit IgG isotype control FITC conjugate (Novus, NBP1-43957), anti-mouse antibodies to CD11c PE-conjugate (clone N418), CD206-FITC (clone C068C2), FITC conjugate Rat IgG2a κ (clone RTK2758), CD11b PE-Cy7 (cloneM1/70), CD45 APC-Cy7 (clone 30-F11), Ly6G-FITC (clone 1A8), and CD71 PercP Cy 5.5 (clone R17217) from Biolegend (San Diego, CA), anti-mouse Mertk PE conjugate (clone DS5MMER), efluor450 F4/80 (clone BM8), anti-CD14 PE-conjugate (clone 61D3), and anti-mouse CD71-PE (clone R17217), and CD11c APC (clone N418) from eBioscience (San Diego, CA), anti-mDtk PE conjugate (FAB759P, R&D), anti-mAxl PE-conjugate (FAB8541P, R&D), anti-mouse Siglec F PE (clone E50-2440), and PE conjugated isotype controls Rat IgG2a κ (cat. no. 553930) and Rat IgG1 (cat. no. 553925) from BD Pharmigen (San Jose, CA). Mouse Fc block, purified CD16/CD32 clone 93 was from eBioscience. Human Fc block, purified CD16/CD32 was from BD. PD0325901, PD98059, and U0126 were obtained from InvivoGen (San Diego, CA) or Sigma (St. Louis, MO).

Isolation and Culture of Murine Bone Marrow-Derived Macrophages (BMDM)

Bone marrow cells were isolated from femurs and tibias of C57Bl/6, Balb/c, Stat6^−/−, and LysM^Cre+/+MEK1^fl/fl mice, and cultured in Mac medium as described (21). BMDM were re-plated on day 7 after harvest and allowed to adhere overnight at 37°C. Recombinant murine IL-4 (10 ng/ml) and IL-13 (10 ng/ml; Life Technologies) in Mac medium were added to cells. PD0325901 was added at 0.5 μM while U0126 and PD98059 were added at 10 μM each. Equal amounts of DMSO were added as necessary for carrier controls.

Isolation and Culture of Murine Alveolar Macrophages (AM)

Naïve C57Bl/6 mice were euthanized by Beuthanasia-D and underwent three serial bronchoalveolar lavages (BAL) with 0.9 ml BAL buffer (PBS + 5 mM EDTA). BAL cells were plated in Mac medium, and AM allowed to adhere to tissue culture plates for 1 hour at 37°C. Wells were rinsed twice with warm PBS and Mac medium with IL-4+IL-13 was added back with either DMSO (carrier) or PD0325901 for 48 hours.

Isolation and Culture of Human Macrophages and Neutrophils

The University of Washington Institutional Review Board approved a protocol to acquire venous blood from healthy and consenting adult donors. Blood was collected in BD Vacutainer collection tubes with EDTA (Thermo Fisher Scientific). Neutrophils and peripheral blood mononuclear cells (PBMC) were isolated from whole blood by dextran sedimentation removal of erythrocytes, followed by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) as previously described (22). To further purify monocytes from total PBMCs, Dynabeads Untouched Human Monocytes Kit (Invitrogen, Carlsbad, CA) was used following the recommended protocol. This isolation protocol routinely resulted in recovery of high purity (>90%) of CD14+ monocytes from total PBMCs. Monocytes were cultured in RPMI-1640 supplemented with 5% Pen/Strep, Sodium Bicarbonate, and 100 ng/ml recombinant human M-CSF (PeproTech, Rocky Hill, NJ) (MDM medium) to generate monocyte derived macrophages (human MDM). Monocytes were seeded into 12-well tissue culture plates at a density of 4.5 ×10⁵ cells/well in 2 ml total volume and incubated at 37°C. On day 5 post-isolation, an additional 0.25 ml of MDM medium was added to each well. Cultures were monitored for adherence and confluency and used for experiments between days 7-10.

Protein Lysates and Western Blotting

To collect protein, macrophage cultures were rinsed twice with cold PBS and lysed in 200 μl cold RIPA lysis buffer containing EDTA with addition of complete Mini EDTA-free protease inhibitor and PhosSTOP phosphatase inhibitor tablets (Roche, Mannheim, Germany). Protein concentrations were determined by BCA assay (Pierce, Rockford, IL) and 5-10 μg of total protein for each sample were prepared in Life Technologies Bolt LDS sample buffer and Reducing Agent by heating at 70°C for 10 minutes. Samples were loaded into Bolt 4-12% gradient gels for SDS-PAGE. Proteins were transferred onto 0.45 μm PVDF (NEF1002, Perkin Elmer, Inc. Shelton, CT). Blots were developed with Supersignal West Femto Maximum Sensitivity Substrate (Pierce) and imaged using Ultra Quant Molecular Imaging and Analysis Software for an Omega Ultra-Lum gel imaging system. Minimal adjustments to brightness/contrast were equally applied to whole gel images. Blots were stripped with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) and re-probed for loading controls. Densitometry analyses were performed using ImageJ software.

Quantitative real-time PCR

To measure gene expression, total RNA was first isolated using a NucleoSpin RNA isolation kit (Clontech Laboratories, Inc., Mountain View, CA). RNA concentrations were determined with a NanoDrop1000 (Thermo Fisher Scientific) and diluted so equal concentrations (up to 1.5μg) of total RNA was used as the template for cDNA synthesis with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Equal amounts of cDNA template were used in qPCR reactions with verified primer probes and the SensiMix II Probe Hi-Rox kit (Bioline, Taunton, MA). Reactions were measured on an ABI HT7900 fast real-time PCR machine. Data analysis was performed as previously described (23). Briefly, duplicate replicates were measured to obtain the average Ct for a sample. The ΔCt was the difference between the average Ct for a specific cDNA and control Hprt. The ΔΔCt was the average ΔCt at a given time point minus the average ΔCt of a control sample. The data are expressed as relative quantification (RQ), which is the fold change and is calculated as 2^−ΔΔCt.

Efferocytosis Assay and Flow Cytometry

Human PMN or Jurkat cells were either left unlabeled or labeled with CellTrace CFSE (Invitrogen) following the manufacturer's protocols. To induce apoptosis, PMN were incubated 20-24 h in either Mac or MDM medium at a concentration of 5 × 10⁶ cells/ml in Falcon tubes covered with breath-easy membrane (USA Scientific, Ocala, FL). Cultures were subjected to gentle tumbling at room temperature. Jurkat cell apoptosis was induced by stimulation with soluble Fas ligand (sCD95L;Sigma) for 6 h. Macrophages were either left resting or stimulated with IL-4/IL-13 (BMDM and BAL AM) or IL-4 (MDM) and either carrier (DMSO) or PD0325901 for indicated lengths of time. Adherent macrophages were rinsed with PBS, and unlabeled and CFSE+ PMN (5:1 PMN:macrophage) or Jurkat cells (2:1 Jurkat:macrophage) were added to macrophage cultures for 2 h at 37°C. After washing away non-adherent cells, macrophages were incubated with 1mM EDTA in PBS and lifted with a cell scraper. Collected macrophages were washed, incubated with Fc block, and stained for F4/80, CD11b, and CD14. Flow cytometry was performed on a Canto RUO (BD Biosciences) and analyzed using FlowJo software (Treestar, Ashland, OR). To examine surface staining of Mertk, Tyro3, Axl, and Abca1, BMDM were stimulated with IL-4/IL-13 for 48 h with either carrier or MEKi. Samples were collected as described above. Technical triplicates for each sample were processed for each experiment. The normalized ΔMFI for each was determined by subtracting the average MFI of isotype control samples from either carrier or MEKi from the individual MFI of each sample.

Peritoneal Macrophage Efferocytosis

C57Bl/6 mice received MEKi PD0325901 (IP; 20 μg/kg in 10% DMSO) or vehicle control (IP; 10% DMSO) in sterile PBS. Human PMNs were aged overnight while tumbling at room temperature to induce apoptosis. Apoptotic PMN were pelleted, resuspended in sterile PBS, and 5 × 10⁶ cells in 200 μl PBS injected into the peritoneal cavity. After 2 hours mice were sacrificed by CO₂ asphyxiation and the peritoneal cavity was subjected to lavage with cold PBS. 50,000 total lavage cells were used to for cytospin preparations that were then stained with Diff-Quick (Siemens, Neward, DE). An individual blinded to the identity of the samples quantified the percent of macrophages with an ingested cell by counting a minimum of 200 macrophages in random fields of view from each cytospin.

Experimental LPS-Induced Acute Lung Injury

C57Bl/6 mice were anesthesitized using isoflurane and positioned for oropharyngeal E. coli LPS instillation (1.5 μg/g in 50 μl sterile PBS). Mice were monitored daily for weight change and activity. Groups of mice received MEKi PD0325901 (IP; 20 μg/kg in 10% DMSO) or vehicle control (IP; 10% DMSO) on days 1 and 3 after LPS delivery. Mice were sacrificed at days 2 and 4 after LPS challenge and subjected to three serial bronchoalveolar lavages with 0.9 ml PBS + 5 mM EDTA. Total BAL cells were enumerated on a Cellometer Auto 2000 (Nexcelom Bioscience LLC, Lawrence, MA) and staining by Diff-Quick was performed on cytospin preparations. BAL macrophages were isolated by adherence to tissue culture plates for 1 hour with non-adherent cells removed by washing with PBS. BAL macrophage RNA was isolated and used as the template for cDNA that was then used in qPCR to determine relative gene expression. Surface CD71 levels were measured by multicolor flow cytometry on BAL macrophages that were identified as CD45⁺, Ly6G⁻, CD11c⁺, SigF⁺ cells.

Statistics

Statistical analyses were performed using GraphPad Prism 6 software. Samples were analyzed by Student's t test or by ANOVA for multiple comparisons, as appropriate. Significance was considered as P<0.05.

Results

MEK1/2 pathway inhibition augments M2 gene expression

Regulation of macrophage reparative function is an area of potential therapeutic importance requiring characterization of the intracellular signaling pathways driving these functions. In order to identify novel pathways modulating macrophage M2 programming, we screened several compounds for their role in M2 gene expression (not shown) and chose to further investigate the MEK1/2 pathway as a novel regulator of macrophage reparative function. This pathway was constitutively active, as measured by phosphorylation of downstream ERK1/2 (pERK1/2) in both murine bone marrow-derived macrophages (murine BMDM) (Fig. 1A) and human monocyte derived macrophages (human MDM) in culture (Fig. 1B), and it remained active after M2 polarization with IL-4/IL-13.

FIGURE 1.

BMDM M2 gene expression is increased during IL-4/IL-13 polarization by the MEK1/2 inhibitor PD0325901. (A) One of three representative experiments showing constitutive expression of pERK1/2 in resting (M0) and IL-4/IL-13 treated conditions in murine BMDM. There was early reduction of pERK1/2 at 15-60 minutes post-stimulation with MEKi (PD0325901). (B) One of three representative experiments showing constitutive expression of pERK1/2 in resting (M0) and IL-4 treated human MDM. Western blots of protein lysates from 0 and 48 hours show decreased pERK1/2 after PD0325901 treatment. (C) M0 and IL-4/IL-13-treated BMDM exposed to carrier control or MEKi over 48 h were processed for qRT-PCR to determine relative quantification (RQ) of Retnla, Ym1, Ccl17, Tgfb1, and Arg1 mRNA normalized to time-matched M0 + carrier control samples. Treatment with MEKi led to a significant increase in IL-4/IL-13 dependent gene expression of Retnla, Ym1, Ccl17, Tgfb1, but not Arg1. Data from 3-4 biological replicates (mean±SEM). (D) At 48 h, IL-4/IL-13 treated cells exposed to carrier control or MEKi were processed for surface staining of CD71 and CD206. Change in mean fluorescent intensity (ΔMFI) was determined by subtracting the MFI of the isotype control samples from that of each antibody. MEKi treatment led to increased surface expression of both CD71 and CD206. Data mean ± SD are from triplicate samples from one representative experiment of two. (E) LPS-treated BMDM were treated with IL-4/IL-13 + carrier or PD0325901. At 48 h, MEKi treatment led to increased expression of Retnla, Ym1, and Tgfb1. Data are mean ± SD from 6 independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

To study the role of MEK1/2 in M2 polarization, we exposed resting (M0) or IL-4/IL-13-stimulated (M2) murine BMDM to the MEK1/2 inhibitor (MEKi) PD0325901 or carrier (DMSO) control. We confirmed significant reduction of pERK1/2 in MEKi-treated murine and human cells (Fig 1A,B). We analyzed the effect of MEK1/2 inhibition on M2 gene expression at 4-48 h post-IL-4/IL-13 stimulation. We found that MEKi significantly increased murine BMDM expression of a number of M2 genes, including Retnla, Ym1, Ccl17, and Tgfb1, in a time-dependent fashion with over a 200-fold increase of Retnla in MEKi compared to carrier IL-4/IL-13 stimulated cells (Fig 1C). Surface expression of M2 markers, CD71 and CD206, were also increased by MEKi (Fig 1D). Although most M2 genes were upregulated by MEKi, Arg1 did not change (Fig 1C). This MEKi-dependent phenotype was also observed during murine BMDM IL-4 polarization (Supplemental Fig. 1) and largely absent upon treatment of resting cells. Therefore, the significant MEKi-dependent modulation of M2 gene expression required the presence of IL-4 or IL-4/IL-13.

Since we have shown that macrophage repolarization from M1 to M2 occurs in vivo (23), we investigated whether MEKi could promote greater M2 phenotypes after LPS-stimulation. Murine BMDMs were M1 polarized with LPS 24 h prior to stimulation with IL-4/IL-13 in the presence of MEKi or carrier control. Consistent with our previous findings, the MEKi-treatment significantly increased Retnla, Ym1, and Tgfb1 mRNA compared to carrier-treated samples (Fig. 1E). Therefore, the MEKi-dependent increase of M2 gene expression occurred regardless of the initial polarized state of the macrophage.

To confirm that MEK1/2 pathway inhibition and not off-target effects of PD0325901-treatment were responsible for the increased M2 gene expression, we first tested two additional MEK1/2 pathway inhibitors, U0126 and PD98059, which had variable effects on increasing Retnla expression (Fig. 2A). At the doses tested, we found variable suppressive effects on the MEK1/2 pathway as determined by loss of pERK1/2 at 48 h. The second generation MEKi, PD0325901, had the greatest suppressive effect (63% reduction), followed by U0126 (25% reduction), and no significant inhibition of pERK1/2 by PD98059 at 48 h (Fig. 2B-C). These changes in pERK1/2 correlated with effects on Retnla gene (Fig. 2A) and protein (Fig. 2B,D) expression. PD0325901 treatment resulted in a greater increase in Retnla than U0126 treatment; and PD98059 treatment did not alter Retnla expression.

FIGURE 2.

Efficacy of MEK1/2 pathway inhibition results in differential enhancement of BMDM IL-4/IL-13 polarization. BMDM were stimulated with IL-4/IL-13 with the addition of media (control), DMSO (+ carrier), PD0325901, U0126, or PD98059 for 48 hours. Cells were processed for RNA and protein. (A) Relative quantification (RQ) of Retnla gene expression normalized to M0 showing the greatest effect of PD0325901 (mean ± SD of 4 biological replicates). (B) BMDM protein lysates were probed for ERK1/2 phosphorylation (pERK1/2), ERK1/2, Relmα, and β-actin and quantified using densitometry. (C) Relative quantification of the ratio of pERK1/2:ERK1/2 expressed as percent of IL-4/IL-13 + carrier controls showing the greatest effect of PD0325901. (D) Ratio of Relmα:β-Actin also demonstrating the greatest effect of PD0325901. (E) BMDM protein lysates from LysM^Cre+/+MEK1^fl/fl BMDM (LysM^Cre+/+) compared to LysM^Cre−/−MEK1^fl/fl controls (LysM^Cre−/−) demonstrating a reduction in MEK1 protein from LysM^Cre+/+ cells. Lane 1: M0 + Carrier, Lane 2: IL-4/IL-13 + Carrier, Lane 3: IL-4/IL-13 + MEKi. (F) Relative quantification of Retnla gene expression from IL-4/IL-13 treated LysM^Cre+/+ and LysM^Cre−/− BMDM normalized to M0 (mean ± SD of triplicate samples). Data are from one of two representative experiments. (G) BAL alveolar macrophages were IL-4/IL-13 polarized with the addition of either DMSO (+ carrier) or PD0325901 (+MEKi) ex vivo for 48 hours. qRT-PCR was used to measure the relative quantification (RQ) of Retnla, Ym1, Tgfb1, and Ccl17. Data are ± SD of the fold increase in PD0325901 compared to carrier. (n=7 biological replicates for each group collected from 2 independent experiments.) Statistical comparisons are versus carrier-treated samples or as indicated. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

As an alternate approach to pharmacological inhibition of the MEK1/2 pathway, we created a genetic knock-out of MEK1 in murine BMDM by breeding MEK1^fl/fl to LysM^Cre mice. This resulted in a substantial decrease of MEK1 protein in murine BMDM derived from LysM^Cre+/+MEK1^fl/fl compared to LysM^Cre−/−MEK1^fl/fl mice (45.7 ± 4.2% of LysM^Cre−/− levels) (Fig. 2E). After exposure to IL-4/IL-13 for 24 hours, Retnla expression was significantly increased in MEK1-deficient murine BMDM compared to control (Fig. 2F). Collectively, these data demonstrate that inhibition of the MEK1/2 pathway during murine BMDM stimulation with IL-4/IL-13 results in a marked increase in gene expression and protein production of several macrophage IL-4/IL-13 responsive genes in MEKi compared to carrier-treated samples. Data with MEK1-deficient murine BMDMs suggest that loss of MEK1 alone is sufficient to promote increased IL-4/IL-13 polarization. Together, these data strongly support a role of the MEK1/2 pathway in regulating macrophage IL-4/IL-13 responses.

To determine if MEKi treatment had similar effects on different macrophage populations, we isolated murine alveolar macrophages (AM) from bronchoalveolar lavage. AM were IL-4/IL-13 polarized ex vivo with either carrier or PD0325901 for 48 h. Corresponding to the murine BMDM phenotype, expression of Retnla, Ym1, Tgfb1, and Ccl17 were significantly increased in MEKi compared to carrier-treated controls (Fig. 2G). Thus, MEK1/2 pathway inhibition induced similar changes in IL-4/IL-13 responsive gene expression in macrophages from different tissue sources.

MEKi treatment enhances STAT6 pathway activation during IL-4/IL-13 stimulation

Macrophages require the transcription factor STAT6 for increased expression of many IL-4/IL-13 regulated genes (24). Based on our M2 gene expression results, we hypothesized that the MEK1/2 pathway negatively regulates STAT6 activation. We examined phosphorylation of STAT6 (pSTAT6) in M2-polarized cells treated with MEKi or carrier control as a measure of STAT6 pathway activation. At 24 and 48 h, MEKi-treatment increased pSTAT6 levels compared to carrier-treated controls (Fig 3A, B). Because pSTAT6 is negatively regulated by SOCS1 and SOCS3 (25), we also measured these protein levels from the same samples. MEKi-treatment significantly decreased SOCS3 at 24 and 48 hours and SOCS1 at 48 hours compared to carrier-treated controls (Fig 3A, B). As these data implicate MEK1/2 pathways in maintaining SOCS1 and SOCS3 levels, and thereby reducing pSTAT6, we asked if STAT6 alone is responsible for the MEKi-dependent effects on M2 gene expression. Using BMDM from wild-type (WT) and Stat6^−/− mice, we evaluated the effect of the MEKi or carrier control on M2 gene expression. As expected, Stat6^−/− murine BMDM failed to respond to IL-4/IL-13, with no increase in M2 genes Retnla or Ym1 (Fig 3C). In the presence of the MEKi, the upregulation of M2 genes and protein were significantly reduced in Stat6^−/− BMDM; demonstrating a dependency on STAT6 (Fig 3C,D). However, there was a small increase in Retnla mRNA in MEKi treated Stat6^−/− cells suggesting additional pathway(s) may be working in concert with STAT6. This small increase in Retnla mRNA in MEKi-treated compared to carrier-treated conditions was also observed in resting wild-type murine BMDM (Fig. 1), although the magnitude of change is very small compared to IL-4/IL-13 stimulating conditions. Despite this small change, Relmα protein levels (Retnla protein product) were only increased in WT cells treated with MEKi and not Stat6^−/− cells (Fig 3D). Previous studies have shown that the transcription factor PU.1 increases Retnla and Ym1 expression after IL-4 stimulation (26). Here, we show that MEKi-treatment significantly increased PU.1 expression at 12-48 hours after stimulation (Fig 3E), thereby representing another potential mechanism by which MEKi may function to increase M2 gene expression. Collectively, these results show that MEK1/2 pathway activity dampens the STAT6-dependent response to IL-4/IL-13 stimulation.

FIGURE 3.

MEKi increases BMDM STAT6 pathway activation during IL-4/IL-13 stimulation. (A) BMDM protein lysates collected at 1, 4, 24, and 48 hours after stimulation of M0 + Carrier and IL-4/IL-13 + Carrier or + MEKi. Blots were probed for pSTAT6, STAT6, SOCS1, SOCS3, and β-Actin one representative experiment of n=4-6. (B) Densitometry quantitation of the ratio of pSTAT6/STAT6, SOCS1/Actin, and SOCS3/Actin normalized to carrier-treated samples demonstrating increased pSTAT6 and reduced SOCS1 and SOCS3 proteins in MEKi treated samples. (C, D) BMDM from wild-type (WT) or Stat6^−/− Balb/c mice were stimulated with IL-4/IL-13 with the addition of DMSO (+ Carrier) or 0.5 μM PD0325901 (+MEKi). (C) At 48 h, Retnla, and Ym1 mRNA expression was measured and expressed as fold change (RQ) relative to respective M0 conditions. There was marked reduction in both Retnla and Ym1 in Stat6−/− compared to WT cells. Data are from 3-5 biological replicates and show the mean for each sample comparing matched carrier and inhibitor treated samples. (D) Protein lysates were collected at serial time points after stimulation. Relmα, STAT6 and β-Actin were detected by western blot. There was no detectable STAT6 or Relmα proteins in Stat6−/− cells compared to that of WT. Blots are from one representative experiment of three. (E) BMDM were stimulated with IL-4/ IL-13 with the addition of carrier or MEKi. RNA was collected over 48 h to determine the relative quantification (RQ) of PU.1 normalized to time-matched M0 conditions. MEKi treatment led to increases in PU.1 mRNA starting at 12 h (mean of 3-4 independent experiments). *P<0.05, **P <0.01.

MEK1/2 inhibition increases macrophage efferocytosis of apoptotic cells

A critical reparative function of macrophages is their ability to clear apoptotic cells (AC) in the process referred to as efferocytosis. We sought to directly test the hypothesis that MEKi-treatment would increase macrophage efferocytosis of AC. Efferocytosis was evaluated by FACS quantification of macrophage uptake of CFSE-labeled apoptotic human neutrophils (human PMN). The method of human PMN aging routinely resulted in a high percentage of apoptosis (~70% Annexin-V-positive, data not shown). Resting (M0) or M2 murine BMDMs were treated with carrier control or MEKi for 48 hours prior to incubation with apoptotic CFSE-labeled human PMNs. Compared to carrier-treatment, MEKi-treatment significantly increased M2 polarized murine BMDM efferocytosis of apoptotic human PMNs (Fig. 4A). In addition, we observed a similar MEKi-dependent increase in efferocytosis using M2 polarized murine alveolar macrophages (Fig. 4B). Surprisingly, we observed that the MEKi phenotype did not require M2 polarization such that MEKi treatment of M0 macrophages alone resulted in increased AC efferocytosis (Fig 4C). Further experiments determined that the MEKi increase in AC efferocytosis was not limited to human PMN as the target AC, as use of apoptotic human Jurkat cells in the same assay resulted in similar findings (data not shown and Fig. 4D). The MEKi-dependent increase in efferocytosis was time dependent, as the phenotype occurred with 24 h but not 6 h of MEKi treatment (Fig. 4E). In addition, we tested whether MEKi would increase AC efferocytosis of both resting or IL-4 polarized (M2) human MDM. Consistent with results from murine macrophages, the MEKi increase in efferocytosis was also observed in human MDM independent of macrophage polarization (Fig. 4E), as MEKi-treatment increased efferocytosis of resting (M0) (Fig. 4F, representative plots) and M2 (Fig. 4G, representative plots) human MDM by 1.53-fold ± 0.31 SD and 1.60 ± 0.28 SD, respectively, compared to carrier controls. Together, these data indicate that MEKi treatment increases efferocytosis of AC by resting or M2 polarized macrophages from different tissue sources and species of origin.

FIGURE 4.

MEKi increases macrophage efferocytosis of apoptotic cells. Murine BMDM (A) or alveolar macrophage (AM) (B) were stimulated with IL-4/IL-13 or BMDM cultured as M0 (C) with the addition of DMSO (+ Carrier) or 0.5 μM PD0325901 (+MEKi) for 48 hours. Unlabeled and CFSE-labeled, apoptotic human neutrophils (PMN) were added to BMDM cultures. Efferocytosis was quantified as the percentage of CSFE⁺ macrophages using FACS. In all MEKi treated samples, efferocytosis was significantly enhanced. Data are mean percent BMDM CFSE⁺ (A,C) (n=3) or (B) AM CFSE⁺ (n=6 each) from paired carrier and MEKi-treated samples. (D) M0 BMDM treated with either carrier or MEKi for 6 or 24 hours were incubated with Jurkat as AC target, and efferocytosis evaluated by flow cytometry. MEKi increased efferocytosis in M0 cells at 24 h but not at 6 h. Data are the mean ± SD from triplicate samples from one representative experiment of three. (E) Apoptotic PMNs were added to human MDM (M0 or IL-4 treated) with either carrier or MEKi. Efferocytosis was enhanced by MEKi across all conditions. Data are mean ± SD of the fold increase in the percentage of MDM CFSE⁺ from 4 different donors. (F,G) Representative contour plots from a single human donor showing CD14⁺ MDM after efferocytosis of either unlabeled PMN (+ Unl PMN) or CFSE-labeled PMN (+ CFSE PMN) from (F) resting (M0) or (G) IL-4 (M2) stimulated with either carrier or MEKi. *P<0.05, **P<0.01, ***P<0.001.

MEKi-treatment increases macrophage Mertk, Tyro3, and Abca1 independent of IL-4/IL-13 polarization and STAT6

As the increase in AC efferocytosis occurred after 24 hour MEKi treatment, we hypothesized that changes in efferocytosis-related genes and proteins, such as the TAM family member Mertk or Abca1 (27), would also be observed within this time period. We found increased mRNA of both Mertk and Abca1 in resting and M2 BMDM and murine AM treated with MEKi compared to their carrier-treated controls (Fig. 5A-C). Additionally, the MEKi-dependent increase in Mertk and Abca1 was independent of Stat6 (Fig. 5B), further demonstrating that the MEKi effects on efferocytosis occur independent of M2 polarization. We next examined surface expression of all three TAM receptors (Mertk, Tyro3, and Axl) and Abca1 on murine BMDMs exposed to MEKi or carrier control (Fig. 5D-E). While there was a modest increase in the percentage of murine BMDM Mertk positive after MEKi treatment, we did not observe significant differences in the percentage of murine BMDM positive for Axl, Tyro3, or Abca1 (data not shown). However, there was a significant increase in the mean fluorescent intensity (MFI) of Mertk, Abca1, and Tyro3, but not Axl, in MEKi treated compared to carrier-treated controls. Collectively, these data show that MEKi treatment increases mRNA and protein surface localization of macrophage proteins that facilitate efferocytosis.

FIGURE 5.

MEKi increases macrophage MertK, Tyro3, and Abca1 independent of polarization and STAT6. (A) BMDM from WT mice were left resting (M0) or polarized with IL-4 /IL-13 for 24 hours with the addition of either DMSO (+ Carrier) or 0.5 uM PD0325901 (+MEKi). (B) BMDM from Wild-type (WT) or Stat6^−/− mice or (C) murine alveolar macrophages were IL-4/IL-13 polarized for 48 hours with the addition of either vehicle or MEKi (PD0325901). RNA was collected to determine relative expression (RQ) of Mertk and Abca1 compared to M0 + carrier control. Mertk and Abca1 were increased in MEKi-treated M0 and M2 polarized WT and Stat6−/− BMDM, and in WT alveolar macrophages. Data are mean ± SD of 3-7 biological replicates. (D) Representative contour plots showing IL-4/IL-13 BMDMs polarized for 48 hours with the addition of either carrier or MEKi. Gates were set based on Mertk isotype control staining to evaluate the percent of macrophages that are Mertk⁺. (E,F) Quantitation of FACS data showing a MEKi-dependent increase in % BMDM Mertk⁺, ΔMFI of Mertk⁺, Abca1 and Tyro3. Data are ± SEM of 2-3 biological replicates. *P<0.05, **P <0.01, ***P <0.001.

MEKi treatment of mice promotes reparative macrophage function

We used two in vivo models to test the ability of the MEKi to alter macrophage efferocytosis and M2 polarization. To explore if MEKi can increase macrophage efferocytosis of apoptotic PMNs in vivo, we pretreated mice with MEKi or carrier control 24 h prior to peritoneal instillation of apoptotic human neutrophils. At 2 h post-instillation, % efferocytosis was quantified from cytospins of cells recovered from peritoneal lavage (Fig. 6A). We found ~2 fold increase in % efferocytosis in MEKi treated mice, a similar fold change to that seen in our in vitro assays (Fig 4).

FIGURE 6.

MEKi promotes in vivo macrophage efferocytosis and M2 polarization. (A) Mice received either carrier or MEKi 24 h prior to intraperitoneal delivery of apoptotic neutrophils. Additional control mice did not receive PMNs. Mice were subjected to peritoneal lavage and cytospin preparations were made from recovered cells and stained with Diff-Quick. The percent of macrophages with ingested cells was quantified and data from three independent experiments are shown. MEKi treatment led to a significant increased in efferocytosis. (B-E) In a separate in vivo model of lung injury, mice received oropharyngeal delivery of LPS on day 0 and MEKi on days 1 and 3 post-LPS. Mice were monitored for weight change (B) and euthanized on days 2 and 4 for assessment of BAL cell counts and differential (C,D). On day 4, alveolar macrophages were isolated for assessment of M2 gene and protein expression (E). (B) Initial weight loss was similar between MEKi and carrier control groups, and MEKi treated mice had faster recovery of their weights starting at day 2. Day 2 (n=26-27/condition), Days 3-4 (n=16/condition). (C,D) On days 2 and 4, there were fewer BAL total cells due to reduced numbers of neutrophils in MEKi treated mice. Macrophage numbers were similar in both groups. (n=5-6 mice/group; 2-3 experimental replicates). (E) On day 4, alveolar macrophages from MEKi treated mice had greater expression of Ym1 and Ccl17 mRNA (normalized to carrier control) and greater expression of CD71 as measured by FACS. (n=10-12/group). *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001.

We also used an experimental LPS-induced model of lung injury to examine the effects of MEKi on macrophage polarization. In this model, LPS was instilled into the lung, and MEKi or carrier control was administered starting at 24 h (D1) post-injury (Fig. 6B-E). All LPS-treated mice had similar weight loss at D1 post-LPS. On D2, the MEKi treated mice had improved activity (not shown) and faster recovery of weight (Fig 6B). On D2 and D4, MEKi treated mice had fewer neutrophils in the alveolar space and similar macrophage numbers (Fig. 6 C-D). On D4, there was increased M2 polarization of alveolar macrophages in MEKi treated mice as demonstrated by greater M2 gene expression (Ccl17 and Ym1) and surface expression of the M2 marker, CD71 (Fig. 6E). These findings indicate a beneficial effect of MEKi treatment after the induction of lung injury with greater M2 polarization of alveolar macrophages.

Discussion

In our studies, we demonstrated a novel role for the MEK1/2 pathway as a negative regulator of macrophage M2 polarization and clearance of apoptotic cells. Pharmacologic inhibition of this pathway demonstrated significant augmentation of both M2 gene and protein expression and a marked enhancement of macrophage efferocytosis. Importantly, we also demonstrated enhanced M2 polarization in macrophages from LysM^Cre+/+MEK1^fl/fl mice, confirming a critical role of MEK1 in regulating macrophage polarization.

By using Stat6^−/− mice, we confirmed dependency of MEKi-dependent changes in M2 gene and protein expression on this transcription factor. We examined STAT6 and phospho-STAT6 levels in MEKi mice compared to controls, and found that early phospho-STAT6 levels were unaffected by MEKi but later levels were increased in the presence of MEKi. Since SOCS1 and SOCS3 are important inhibitors of STAT6, we examined expression of these proteins (28). We found reduced levels of both SOCS1 and SOCS3 in MEKi treated mice, suggesting that MEKi functions via dampening inhibitors to STAT6 pathway activation. We also found MEKi-dependent increases in other STAT6-dependent transcription factors such as PU.1, which has been shown to regulate Ym-1 and Retnla (26). These finding suggesting more complex regulation of transcription factors by MEKi and may be one explanation as to why many (Ym1, Ccl17, Retnla, Tgfb1) but not all (Arg1, Il10) M2 gene markers were enhanced by MEKi.

The clearance of apoptotic neutrophils by macrophages is another important process for tissue homeostasis and the resolution of inflammation. Macrophage receptors, including the Tyro family of proteins facilitate ingestion of AC and induction of signals to dampen macrophage pro-inflammatory responses (29). Defects in macrophage efferocytosis may contribute to prolonged and unresolved inflammation, and there is evidence that macrophages from COPD patients and cigarette smokers have marked defects in efferocytosis (30). In addition, macrophage efferocytosis is thought to contribute to the regulation atherosclerosis, such that increased macrophage efferocytosis may decrease atherosclerotic plaque size and promote resolution of local inflammation (31, 32). Therefore, the therapeutic potential to stimulate or enhance macrophage efferocytosis of AC has broad implications for the treatment of many human diseases (33).

Here, we directly tested the hypothesis that MEKi promotes macrophage efferocytosis. Our data demonstrate that MEK1/2 inhibition results in a robust increase in both murine and human macrophage efferocytosis of AC. We found that effects on efferocytosis were independent of IL-4/IL-13 and Stat6. MEKi significant increased both gene and protein expression of Mertk, Tyro3, and Abca1, three proteins that promote macrophage efferocytosis. Furthermore, regulation of these genes and proteins by MEKi were STAT6 independent. Importantly, we found that mice treated with MEKi had increased efferocytosis of apoptotic PMNs suggesting a novel therapeutic approach to alter macrophage function to improve health.

We previously showed that alveolar macrophages change polarized states in a murine model of lung injury, going from a pro-inflammatory (M1) to reparative (M2) phenotype (34). Altered activation states may be critical determinants in tempering lung injury and promoting resolution. To test if MEKi can modulate M2 repolarization in vivo, we induced lung injury with LPS and delivered the MEKi after lung injury induction. We found that MEKi-treated mice had faster recovery of weight and activity, reduced neutrophil numbers in the lung, and greater macrophage M2 polarization as defined by gene expression of Ym1, Ccl17, and surface expression of CD71 compared to carrier-treated mice. We did not find altered pro-inflammatory gene expression by the alveolar macrophage (not shown), although others have reported that MEK1/2 inhibitors reduce pro-inflammatory macrophage gene expression in vitro (results we have also observed; data not shown). Lack of this finding in our in vivo studies may reflect the timing of MEKi delivery (after the peak of pro-inflammatory cytokine induction) or the timing of our assessment of macrophage gene expression. Importantly, in our studies, mice received the inhibitor 24 h after LPS challenge which may be a more realistic application of therapies to modulate lung injury.

Overall, we identified a novel pathway regulating macrophage M2 polarization and efferocytosis, features that contribute to macrophage reparative function. Others have also shown beneficial effects of MEKi on inflammation (20, 35, 36). However, our study demonstrates novel effects of MEKi on macrophage reparative functions that can be modulated in vivo. Application of these findings and strategies to modulate macrophage biology could have broad impact across diseases.

Abbreviations

BMDM

murine bone marrow-derived macrophages

MDM

human monocyte-derived macrophage

PMN

human polymorphonuclear leukocyte

MEKi

MEK1/2 inhibitor

BAL

bronchoalveolar lavage