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. 2022 Feb 22;19(4):540–553. doi: 10.1038/s41423-022-00841-w

MEF2C promotes M1 macrophage polarization and Th1 responses

Xibao Zhao 1,#, Qianqian Di 1,#, Han Liu 2,#, Jiazheng Quan 1, Jing Ling 3, Zizhao Zhao 3, Yue Xiao 1, Han Wu 1, Zherui Wu 1, Wengang Song 4, Huazhang An 4,✉, Weilin Chen 1,✉
PMCID: PMC8975968  PMID: 35194174

Abstract

The polarization of macrophages to the M1 or M2 phenotype has a pivotal role in inflammation and host defense; however, the underlying molecular mechanism remains unclear. Here, we show that myocyte enhancer factor 2 C (MEF2C) is essential for regulating M1 macrophage polarization in response to infection and inflammation. Global gene expression analysis demonstrated that MEF2C deficiency in macrophages downregulated the expression of M1 phenotypic markers and upregulated the expression of M2 phenotypic markers. MEF2C significantly promoted the expression of interleukin-12 p35 subunit (Il12a) and interleukin-12 p40 subunit (Il12b). Myeloid-specific Mef2c-knockout mice showed reduced IL-12 production and impaired Th1 responses, which led to susceptibility to Listeria monocytogenes infection and protected against DSS-induced IBD in vivo. Mechanistically, we showed that MEF2C directly activated the transcription of Il12a and Il12b. These findings reveal a new function of MEF2C in macrophage polarization and Th1 responses and identify MEF2C as a potential target for therapeutic intervention in inflammatory and autoimmune diseases.

Keywords: Myocyte enhancer factor 2C, Macrophage polarization, Interleukin-12, T helper type 1 response, Inflammation

Subject terms: Infection, Interleukins

Introduction

Macrophages are critical players in immune responses, such as inflammation, immunoregulation and tissue remodeling [1–4]. These cells are essential for the initiation and resolution of pathogen- or damage-induced inflammation [2]. Macrophages are also capable of phagocytosis and antigen presentation, providing these cells with a powerful ability to regulate innate and adaptive immunity and resist pathogen infection [5–7].

Currently, there are at least four definitions of macrophage activation, including terms such as M1 and M2, alternative and classic activation, regulatory macrophages, and subdivisions originating from parent terms [8]. However, the diversity of terminology and inconsistent use of markers to describe macrophage activation might impede research. To avoid confusion, we classified classically activated macrophages (M1) and alternatively activated macrophages (M2) in our research. These populations can be phenotypically polarized by surrounding microenvironmental stimuli to activate specific functional programs [9, 10]. M1 macrophages can be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) or interferon-γ (IFN-γ) following stimulation with bacterial products such as lipopolysaccharide (LPS), leading to the production of high levels of proinflammatory cytokines, such as interleukin 12 (IL-12) and IL-23, which promote proinflammatory responses. In contrast, M2 macrophages are induced by M-CSF, IL-4, and IL-13. M2 macrophages express high levels of M2-associated phenotypic markers, such as IL-10 and arginase 1 (Arg1), and drive immune regulation and tissue remodeling [11–13]. As a critical feature of the innate immune system, macrophage polarization is a dynamic process that is tightly regulated. Dysregulation of this process can allow bacterial and helminth infection [14, 15], as well as conditions such as inflammation [16], cancer [17, 18], metabolic disorders and insulin resistance [19]. Fibroblast growth factor 2 (FGF2) affects macrophage polarization and is a critical regulator of immunity in the tumor microenvironment [20]. Although macrophage polarization has been intensively studied, the molecular mechanism underlying macrophage polarization remains largely unknown.

IL-12 is a heterodimeric cytokine composed of p35 and p40 subunits (Il12a and Il12b), and the p40 subunit is shared by IL-23 [21]. IL-12 plays a crucial role in the regulation of innate and adaptive immune responses during infection and inflammation [22, 23]. Previous studies have demonstrated that IL-12 is a phenotypic marker of M1 macrophages [11, 24]. IL-12-deficient macrophages tend to differentiate into M2 macrophages [25]. IL-12 promotes CD4+ T cell differentiation into T helper 1 (Th1) cells and the production of interferon-γ [21]. Studies have shown that M1 macrophage polarization can lead to a potent Th1 response and form a link between innate resistance and adaptive immunity [11, 21]. Th1 cells have important immune functions, and abnormal activation can cause inflammation and autoimmune diseases, such as inflammatory bowel disease and collagen-induced arthritis [26, 27].

Myocyte-specific enhancer factor 2c (Mef2c) belongs to the MADS-box transcription factor family and contains highly conserved MADS and MEF2 domains in the N-terminus. These domains are essential for DNA binding, dimerization and interactions with other factors [28]. The carboxy-terminal segment is much less conserved and confers transcriptional activation properties [29]. MEF2 is a critical transcriptional switch in the adult body that links metabolism and immunity [30]. Deletion of MEF2A recapitulated PGE2 treatment and abolished type I interferon induction after exposure to innate immune stimuli [31]. Stochastic activation of the hippocampal CREB-pCREB-miR466f-3p-MEF2A axis modulates individual variations in spatial learning and memory capability in inbred mice [32]. MEF2C can activate or enhance gene expression by binding to the promoter region via a consensus A/T-rich DNA sequence known as the MEF2 site [33]. MEF2C is essential for the development and differentiation of bone, neuronal cells, and cardiac and skeletal muscle [34, 35]. In addition, MEF2C also has important functions in myeloid leukemia [36, 37], IFN-I-dependent inflammatory responses [38], and B cell proliferation [39]. Interestingly, MEF2C binding sites were the most highly enriched motifs in a ChIP-seq search for de novo enhancer sequences in M. tuberculosis-infected macrophages [40]. This finding indicated that MEF2C plays an important role in macrophage responses to infection and requires further study. Here, we demonstrated that MEF2C promoted M1 macrophage polarization by regulating the expression of macrophage lineage-specific cytokines. Using chromatin immunoprecipitation (ChIP) and double luciferase reporter assays, we revealed that MEF2C was directly involved in the transcriptional regulation of the Il12a and Il12b subunits. Furthermore, we used myeloid-specific Mef2c-knockout (Mef2cflox/flox Lyz2-cre) mice to demonstrate that Mef2c deficiency reduced IL-12 production and impaired Th1 responses, leading to susceptibility to Listeria monocytogenes infection and resistance to DSS-induced IBD in vivo. These findings reveal a new function of Mef2c in macrophage polarization and Th1 responses, suggesting that Mef2c-mediated IL-12 transcriptional regulation is crucial for the phenotypic polarization of macrophages and related immune-associated diseases.

Materials and methods

Mice

Mef2cfl/fl mice were purchased from the Jackson Laboratory (Stock No: 025556). Briefly, Mef2cfl/fl mice possess loxP sites flanking the second coding exon of the Mef2c gene. When crossed with mice that express tissue-specific Cre recombinase, exon 2 of the Mef2c gene is deleted in the Cre-expressing tissues of the resulting offspring.

Lyz2-cre mice were purchased from Nanjing Biomedical Research Institute of Nanjing University. Mef2cfl/fl mice were crossed with Lyz2-cre mice to obtain Mef2cfl/fl Lyz2-cre mice. Murine genotypes were determined by PCR analysis of tail-snip DNA using the following primers: Mef2c lox forward: 5ʹ-TTCAGGTGACCTCATTTGAACC-3ʹ; Mef2c lox reverse: 5ʹ-GGAGCCATTGCTCATAAGAAAG-3ʹ; Lyz2-cre forward: 5ʹ-GAACGCACTGATTTCGACCA-3ʹ; and Lyz2-cre reverse: 5ʹ-GCTAACCAGCGTTTTCGTTC-3ʹ.

Mice were maintained and bred in pathogen-free conditions. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approval was granted by the Institutional Animal Care and Use Committee of Shenzhen University, Shenzhen, China.

Cell culture

M1 and M2 macrophages were obtained from mouse bone marrow cells that were cultured for 5 days in RPMI-1640 medium supplemented with GM-CSF (20 ng/ml, #AF-315-03, Peprotech) or M-CSF (20 ng/ml, # AF-315-02, Peprotech). HEK 293 T and THP1 cells were obtained from the American Type Culture Collection and cultured in DMEM containing 10% fetal bovine serum. All cell cultures were maintained in the appropriate media at 37 °C with 5% CO2 and 95% humidity.

Plasmids and reagents

Plasmids expressing Flag-tagged full-length Mef2c and luciferase reporter plasmids containing wild-type or mutant promoters of Il12a and Il12b were constructed using standard molecular biology techniques. Antibodies against iNOS (# 13120) and Arg1 (# 93668) were purchased from Cell Signaling Technology. Antibodies against MEF2C (# 10056-1-AP), IFN-γ (# 15365-1-AP), F4/80 (# 28463-1-AP), CD86 (# 13395-1-AP), CD206 (# 18704-1-AP) and β-actin (# 60008) were purchased from Proteintech Group. For flow cytometry, anti-CD11c-APC (# 117309), anti-CD206-PE (# 141705), anti-CD4-FITC (# 100501), anti-IFN-γ-AF647 (# 505816), anti-IL-4-APC (# 504105), anti-CD25-AF700 (# 102024), anti-IL-12-PE/Cy7 (# 505209), anti-F4/80-BV421 (# 123131), anti-CD11b-FITC (# 101206) and 7-AAD (# 420403) were purchased from BioLegend. LPS (# L3024) was purchased from Sigma–Aldrich. Animal-free recombinant murine IL-4 (# AF-214-14) was purchased from Peprotech.

Reactive oxygen species assay

Intracellular ROS levels were measured using a Reactive Oxygen Species Assay Kit (# S0033S, Beyotime Biotechnology) according to the manufacturer’s protocol.

Chromatin immunoprecipitation (ChIP) analysis

Chromatin immunoprecipitation assays were performed using EZ-ChIP Chromatin Immunoprecipitation Kits (# 17-371, Millipore) according to the manufacturer’s protocol with the following primers: Il12a (IP: MEF2C), 5ʹ-ACTGTTAGGGCTAATAGGCAAAC-3ʹ and 5ʹ-CCTGATGGTTTTACTTTCCTCTG-3ʹ; Il12b (IP: MEF2C), 5ʹ-GTTAGCCATTGCCGCCTCTATTC-3ʹ and 5ʹ-CGTCGAAATCCCAGGTTAAGAG-3ʹ; Il12a (IP: PU.1), 5ʹ-GGACTGTGTCTGGTGGCCAAG-3ʹ and 5ʹ- GAAGCTCATTTAAGGGAGCTGTTCC-3ʹ; Il12b (IP: PU.1 and NF-κB), 5ʹ-GATGCACTCAGGGAGGCAAGG-3ʹ and 5ʹ-CTGACTAGTCTCAATTGCAACACTG-3ʹ; Il12a (IP: IRF8), 5ʹ-CCTATCTTCTTCCTGTTTCTACTGCCAC-3ʹ and 5ʹ-GTGCTGAGGGCTGATTTCAACG-3ʹ; Il12b (IP: IRF8), 5ʹ-CCCAGAATGTTTTGACACTAGTTTTCAG-3ʹ and 5ʹ-GCTCCTGGTGCTTATATACTCTACTCC-3ʹ; and Il12a (IP: NF-κB), 5ʹ-CACCCAGTGAGGCTGTCTTGG-3ʹ and 5ʹ-GAGTTCGAAGCTTGCAATGACAAGC-3ʹ.

Dual-luciferase reporter assays

HEK 293 T cells or Raw 264.7 cells (2 × 104 cells per well) were seeded in 96-well plates and then cotransfected with Mef2c-expressing plasmids (100 ng per well), the indicated Il12a or Il12b luciferase reporter plasmids (80 ng per well) and the Renilla luciferase construct pRL-TK (Promega, for normalization of the transfection efficiency, 10 ng per well). After 24 h, with or without LPS stimulation, the cells were harvested, and dual-luciferase assays were performed using a dual-specific luciferase assay kit (# E1960, Promega) according to the manufacturer’s protocols.

Quantitative PCR (qPCR) and ELISA

Total RNA was extracted from cells using RNAiso Plus (#9109, Takara) according to the manufacturer’s instructions. Gene expression was determined by Hieff qPCR SYBR Green Master Mix (no ROX) (#11201ES03, Yeasen), and gene expression was normalized to β-actin using the ΔΔCt method. The following gene-specific primers were used: mIl12a forward, 5ʹ-CTGTGCCTTGGTAGCATCTA-3ʹ and reverse, 5ʹ-TTTCACTCTGTAAGGGTCTG-3ʹ; mIl12b forward, 5ʹ- AGGTGCGTTCCTCGTAGAGA-3ʹ and reverse, 5ʹ-AAAGCCAACCAAGCAGAAGA-3ʹ; mIl23a forward, 5ʹ-GCACCTGCTTGACTCTGAC-3ʹ and reverse, 5ʹ-CGCTGCCACTGCTGACTA-3ʹ; mIl10 forward, 5ʹ-GCTGCGGACTGCCTTCA-3ʹ and reverse, 5ʹ-TGCATTAAGGAGTCGGTTAGCA-3ʹ; mArg1 forward, 5ʹ-CAGAAGAATGGAAGAGTCAG-3ʹ and reverse, 5ʹ-CAGATATGCAGGGAGTCACC-3ʹ; mChil3 forward, 5ʹ-GGATGGCTACACTGGAGAAA-3ʹ and reverse, 5ʹ-AGAAGGGTCACTCAGGATAA-3ʹ; mIfng forward, 5ʹ-GATGCATTCATGAGTATTGCCAAGT-3ʹ and reverse, 5ʹ-GTGGACCACTCGGATGAGCTC-3ʹ; mTbet forward, 5ʹ-CAACAACCCCTTTGCCAAAG-3ʹ and reverse, 5ʹ-TCCCCCAAGCAGTTGACAGT-3ʹ; mIl4 forward, 5ʹ-AGATCACGGCATTTTGAACG-3ʹ and reverse, 5ʹ-TTTGGCACATCCATCTCCG-3ʹ; mGata3 forward, 5ʹ-AGAACCGGCCCCTTATGAA-3ʹ and reverse, 5ʹ-AGTTCGCGCAGGATGTCC-3ʹ; mFoxp3 forward, 5ʹ-GGCGAAAGTGGCAGAGAGGTAT-3ʹ and reverse, 5ʹ-AAGACCCCAGTGGCAGCAGAA-3ʹ; mIl1b forward, 5ʹ-AACTGTTCCTGAACTCAACTGT-3ʹ and reverse, 5ʹ-GAGATTTGAAGCTGGATGCTCT-3ʹ; mIl6 forward, 5ʹ- TAGTCCTTCCTACCCCAATTTCC-3ʹ and reverse, 5ʹ-TTGGTCCTTAGCCACTCCTTC-3ʹ; mTnf forward, 5ʹ-AAGCCTGTAGCCCACGTCGTA-3ʹ and reverse, 5ʹ-GGCACCACTAGTTGGTTGTCTTTG-3ʹ; mNos2 forward, 5ʹ-CAGCACAGGAAATGTTTCAGC-3ʹ and reverse, 5ʹ-TAGCCAGCGTACCGGATGA-3ʹ; mCox2 forward, 5ʹ-CCCTGAAGCCGTACACATCA-3ʹ and reverse, 5ʹ-TGTCACTGTAGAGGGCTTTCAATT-3ʹ; mMef2c forward, 5ʹ-AGAAGGCTTATGAGCTGAGCG-3ʹ and reverse, 5ʹ-ACTCGGTGTACTTGAGCAACAC-3ʹ; mActb forward, 5ʹ- AGTGTGACGTTGACATCCGT-3ʹ and reverse, 5ʹ- GCAGCTCAGTAACAGTCCGC-3ʹ; hIL12B forward, 5ʹ-TGCCCATTGAGGTCATGGTG-3ʹ and reverse, 5ʹ- CTTGGGTGGGTCAGGTTTGA-3ʹ; hIL23A forward, 5ʹ-CTCAGGGACAACAGTCAGTTC-3ʹ and reverse, 5ʹ-ACAGGGCTATCAGGGAGCA-3ʹ; hIL10 forward, 5ʹ-TCAAGGCGCATGTGAACTCC-3ʹ and reverse, 5ʹ-GATGTCAAACTCACTCATGGCT-3ʹ; hARG1 forward, 5ʹ-TGGACAGACTAGGAATTGGCA-3ʹ and reverse, 5ʹ-CCAGTCCGTCAACATCAAAACT-3ʹ; and hACTB forward, 5ʹ-TGGAGAAAATCTGGCACCACACC-3ʹ and reverse, 5ʹ-GATGGGCACAGTGTGGGTGACCC-3ʹ.

ELISA kits (Invitrogen) were used to determine the concentrations of IL-1β (# 88-7013-22), IL-6 (# 88-7064-22), TNF-α (# 88-7346-22), IL-12p70 (# 88-7121-22), and IL-10 (# 88-7105-22) in culture supernatants or serum according to the manufacturer’s protocols.

Phagocytosis and bacteria-killing assays

Phagocytosis and bacterial killing were assessed using a gentamicin protection assay. In short, Listeria monocytogenes-infected macrophages (MOI = 0.1 or 1) were cultured in medium without antibiotics for 30 min at 37 °C. Subsequently, gentamicin (50 μg/mL) was added, and the cells were incubated for an additional 30 min at 37 °C. Then, the cells were lysed in water for 10 min at 0, 1, 2, 4, and 6 h after the addition of fresh medium without gentamicin. Serial dilutions were plated on BHI plates containing chloramphenicol, and colonies were counted to determine CFUs.

Plasmid transfection and RNA interference assay

Plasmids or siRNA oligos were transfected into cells using jetPRIME (# 114-15, PolyPlus) according to the manufacturer’s protocol. At 24 h or 48 h after transfection, the cells were harvested or stimulated with LPS (100 ng/ml) and analyzed by qPCR. The siRNA sequences were as follows: Control siRNA: 5ʹ-UUCUCCGAACGUGUCACGUUU-3ʹ; mMef2a siRNA: 5ʹ-GUGGCAGUCUUGGAAUGAATT-3ʹ; mMef2c siRNA: 5ʹ-CCCACCUGGCAGCAAGAACAC-3ʹ; mMef2d siRNA: 5ʹ-GAUCCAGCGAAUCACUGAUTT-3ʹ; and hMEF2C siRNA: 5ʹ-CACCUGGUAACUUGAACAATT-3ʹ.

RNA sequencing

RNA-seq and data analysis were conducted by Seqhealth Technology Co., Ltd. (Wuhan, China). In brief, total RNA was extracted from LPS-stimulated BMDMs using TRIzol reagent, and DNA was then digested using DNase I. Next, 2 μg of total RNA was used for stranded RNA sequencing library preparation using a KCTM Stranded mRNA Library Prep Kit for Illumina® (Catalog No. DR08402, Wuhan Seqhealth Co., Ltd. China) according to the manufacturer’s instructions. PCR products corresponding to 200–500 bps were enriched, quantified and sequenced on a NovaSeq 6000 sequencer (Illumina) with a PE150 model.

Raw sequencing data were first filtered by Trimmomatic (version 0.36). Low-quality reads were discarded, and reads that were contaminated with adaptor sequences were trimmed. Clean data were mapped to the mouse reference genome using STRA software (version 2.5.3a) with default parameters. Differentially expressed genes between groups were identified using the edgeR package (version 3.12.1). A P value cutoff of 0.05 and a fold-change cutoff of 1.5 were used to determine the statistical significance of differences in gene expression. Gene ontology (GO) analysis of differentially expressed genes was performed using KOBAS software (version: 2.1.1), and a P value cutoff of 0.05 was used to determine statistically significant enrichment.

Listeria monocytogenes infection assay

Listeria monocytogenes bacteria were cultured in BHI broth overnight at 37 °C, centrifuged (5000 × g for 5 min at 4 °C) and resuspended in PBS. Age- and sex-matched Mef2cfl/fl and Mef2cfl/fl Lyz2-cre mice (aged 8–10 weeks) were intravenously injected with Listeria monocytogenes (2 × 105 CFU/mouse). Three days later, serum was collected for ELISA. The mice were euthanized, and the liver and spleen were collected to determine the bacterial load. The livers from Listeria monocytogenes-infected mice were fixed and stained with HE and then examined for histological changes under a light microscope. To analyze CD4+ T cell responses, the spleens of Listeria monocytogenes-infected mice were isolated, and single-cell suspensions were prepared for qPCR analysis of Ifng, Tbet, Il4, Gata3 and Foxp3 expression and flow cytometric analysis of IFN-γ, IL-4 and CD25 expression in CD4 T cells. For cytokine staining, the cells were pretreated with PMA (50 ng/ml, #S7791, Selleck), brefeldin A (3.0 ug/ml, #S7046, Selleck) and ionomycin (1 mg/ml, #SQ23377, MedChemExpress) for 5 h at 37 °C before flow cytometric analysis.

Induction of experimental colitis

Age- and sex-matched groups of littermates (aged 8–10 weeks) were administered 3% (w/v) DSS (#160110, MP Biomedical) in drinking water for 7 days, followed by a 2-day recovery period with normal drinking water before sacrifice. The colons were cut into small pieces in 500 μl of PBS and then centrifuged at 400 × g for 5 min. The supernatants were used to analyze IL-1β, IL-6, TNF-α and IL-12p70 production in colon tissue by ELISA, and the cell pellets were used for qPCR analysis of Il1b, Il6, Tnf, Il12a, Il12b and Cox2 mRNA expression in the colon tissue from Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice.

Isolation of colonic lamina propria mononuclear cells (LPMCs)

Experimental colitis was induced in Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice with DSS. Freshly isolated colon tissues were opened longitudinally and washed with sterile HBSS. The colons were cut into small pieces (<5 mm) and washed three times with HBSS. The tissues were then incubated in HBSS (with 5 mM EDTA and 1 mM DTT, but without Ca2+ and Mg2+) for 20 min at 37 °C in a 200 rpm shaking incubator. After the suspended cells were removed, the colon pieces were collected in 50 ml tubes containing warmed digestion solution (containing 0.5 mg/ml DNase I and 0.5 mg/ml collagenase D in PBS). All colon pieces were incubated in digestion solution for 45 min at 37 °C in a 200 rpm shaking incubator. A Percoll gradient was used to isolate LPMCs from cell suspensions after adequate digestion. IFN-γ, IL-4 and CD25 expression in CD4+ T cells and CD11c, CD206 and IL-12 expression in macrophages were analyzed by flow cytometry. For cytokine staining, the cells were pretreated with PMA (50 ng/ml, #S7791, Selleck), brefeldin A (3.0 ug/ml, #S7046, Selleck) and ionomycin (1 mg/ml, #SQ23377, MedChemExpress) for 5 h at 37 °C before flow cytometric analysis.

Statistical analyses

The data are presented as the means ± SD of one of three independent experiments, and the data were analyzed by GraphPad Prism. To analyze the differences between two groups, a two-tailed Student’s t test was used. Mouse survival was analyzed using the Kaplan–Meier method. P < 0.05 was considered to indicate statistical significance.

Results

Mef2c is involved in macrophage polarization

Macrophages are a heterogeneous population of immune cells that change their phenotype and physiology in response to cytokines and microbial signals. M1 macrophages and M2 macrophages have different patterns of cytokine expression. M1 macrophages are engaged in inflammatory, microbicidal and tumoricidal activities and are characterized as having an IL-12highIL-10low phenotype, whereas M2 macrophages are involved in the resolution of inflammation. To investigate whether Mef2c is involved in macrophage polarization, we crossed Mef2cflox/flox mice, which possess loxP sites flanking the second coding exon of the Mef2c gene (Fig. S1a), with Lyz2-cre mice to obtain Mef2cflox/flox Lyz2-cre mice in which Mef2c is ablated (Fig. S1b and c). Global gene expression analysis indicated that Mef2c deficiency affected the expression of a large number of genes (Fig. S2a). Among these differentially expressed genes (DEGs), M1 macrophage markers, such as Il12b, Nos2 and Il12a, were downregulated, whereas the expression of the M2 macrophage markers Arg1 and Chil3 was upregulated (Fig. 1a and b). Gene ontology (GO) analysis indicated that Mef2c positively regulated IL-12 production (Fig. S2b).

Fig. 1.

Fig. 1

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Mef2c is involved in macrophage polarization. a Mef2cflox/flox and Mef2cflox/flox Lyz2-cre macrophages were stimulated with LPS (100 ng/ml) for 2 h. Total RNA was extracted, and RNA-seq analysis was performed. Heatmaps of the selected gene panels are shown. b Volcano plot of the differentially expressed genes (downregulated and upregulated genes are shown in blue and red, respectively)

To further investigate the involvement of Mef2c in macrophage polarization, we used GM-CSF and M-CSF to induce M1 and M2 macrophage differentiation, respectively. We also cultured M1 macrophages with M-CSF to induce M1-to-M2 polarization and M2 macrophages with GM-CSF to induce M2-to-M1 polarization (Fig. 2a). We then used qPCR to analyze the mRNA expression of Il12a, Il12b, Il23a, and Il10 in the cells. The changes in the expression of the cytokines demonstrated that the culture conditions successfully induced macrophage phenotypic polarization with plasticity (Fig. 2b–e). To explore the possible function of Mef2c in the inflammatory process and macrophage polarization, we analyzed the level of MEF2C protein in M1 and M2 macrophages. Immunoblotting showed that MEF2C protein levels were higher in bone marrow-derived macrophages (BMDMs) differentiated with GM-CSF (M1) than in those differentiated with M-CSF (M2) (Fig. 2f). Treating M1 macrophages with M-CSF resulted in relatively lower levels of MEF2C protein in the converted M2 macrophages (M1-to-M2), whereas treating M2 macrophages with GM-CSF resulted in relatively higher levels of MEF2C protein in the converted M1 macrophages (M2-to-M1) (Fig. 2g). Collectively, these data suggest that Mef2c is involved in macrophage polarization.

Fig. 2.

Fig. 2

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High expression of MEF2C in M1 macrophages. a Schematic diagram of the strategy used to induce M1 or M2 and M1-to-M2 or M2-to-M1 polarization. Bone marrow-derived cells were differentiated for 5 days into M1 macrophages with GM-CSF (20 ng/ml) and into M2 macrophages with M-CSF (20 ng/ml). On Day 5, GM-CSF-induced M1 macrophages were induced with M-CSF (20 ng/ml)-containing medium for 24 h to undergo M1-to-M2 polarization, and on Day 5, M-CSF-induced M2 macrophages were induced with GM-CSF (20 ng/ml)-containing medium for 24 h to undergo M2-to-M1 polarization. b–e qPCR analysis of Il12a (b), Il12b (c), Il23a (d) and Il10 (e) expression in macrophages following M1, M2, M1-to-M2 or M2-to-M1 polarization. f Immunoblot analysis of MEF2C expression in monocytes collected on Day 0 (mono) or macrophages following M1 or M2 polarization. g Immunoblot analysis of MEF2C expression in macrophages following M1-to-M2 or M2-to-M1 polarization. The data are representative of three independent experiments. The data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Mef2c influences the expression of macrophage polarization lineage-specific genes

We next investigated the ability of Mef2c to regulate the expression of M1 and M2 macrophage phenotypic markers. Since our results showed that MEF2C protein levels were higher in M1 macrophages than in M2 macrophages, we analyzed the effects of knocking down Mef2c in M1 macrophages and overexpressing Mef2c in M2 macrophages. qPCR analysis showed that Mef2c knockdown in M1 macrophages inhibited LPS-induced expression of the M1 phenotypic markers Il12a (Fig. 3a), Il12b (Fig. 3b) and Il23a (Fig. 3c) but increased LPS-induced expression of the M2 macrophage phenotypic markers Il10 (Fig. 3d), Arg1 (Fig. 3e) and Chil3 (Fig. 3f). To further confirm these results, we knocked down MEF2C in THP1 cells and verified that the specific siRNA could efficiently inhibit MEF2C expression (Fig. S3a). The results showed that MEF2C knockdown decreased IL12B (Fig. S3b) and IL23A (Fig. S3c) expression but increased IL10 (Fig. S3d) and ARG1 (Fig. S3e) expression in LPS-stimulated THP1 cells. In addition, we found that Mef2c did not affect NF-κB or MAPK pathway activation in macrophages (Fig. S3f).

Fig. 3.

Fig. 3

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Mef2c influences the expression of macrophage polarization-specific genes. a–f Bone marrow-derived cells were differentiated for 5 days into M1 macrophages by stimulation with GM-CSF (20 ng/ml). M1 macrophages were then transfected with control or Mef2c siRNA for 48 h and then stimulated with LPS (100 ng/ml) for the indicated times. qPCR analysis of Il12a (a), Il12b (b), Il23a (c), Il10 (d), Arg1 (e), and Chil3 (f) mRNA expression. g–l Bone marrow-derived cells were differentiated for 5 days into M2 macrophages by stimulation with M-CSF (20 ng/ml). M2 macrophages were transfected with vector or Mef2c expression plasmid for 48 h and then stimulated with LPS (100 ng/ml) for the indicated times. qPCR analysis of Il12a (g), Il12b (h), Il23a (i), Il10 (j), Arg1 (k), and Chil3 (l) mRNA expression. The data are representative of three independent experiments. The data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

In addition, our results demonstrated that Mef2c overexpression in M2 macrophages increased the expression of the M1 macrophage phenotypic markers Il12a (Fig. 3g), Il12b (Fig. 3h) and Il23a (Fig. 3i) but decreased the expression of the M2 macrophage phenotypic markers Il10 (Fig. 3j), Arg1 (Fig. 3k) and Chil3 (Fig. 3l). Mef2a and Mef2d also belong to the MADS-box transcription factor family, and we next investigated whether Mef2a and Mef2d could regulate Il12a, Il12b and Il10 expression. We first designed Mef2a- and Mef2d-specific siRNAs and found that these siRNAs efficiently inhibited Mef2a (Fig. S4a) and Mef2d (Fig. S4e) expression in macrophages. The qPCR results showed that Mef2a knockdown increased Il12a (Fig. S4b), Il12b (Fig. S4c) and Il10 (Fig. S4d) expression in macrophages. Moreover, Mef2d knockdown promoted LPS-induced Il12a (Fig. S4f), Il12b (Fig. S4g) and Il10 (Fig. S4h) expression in macrophages. These results suggested that Mef2c plays a different role in regulating inflammatory gene expression than Mef2a or Mef2d. Taken together, these results indicate that Mef2c affects the expression of macrophage polarization lineage-specific genes by promoting the expression of M1 macrophage markers and inhibiting the expression of M2 macrophage markers.

Mef2c deficiency impairs M1 macrophage polarization

We next used myeloid-specific Mef2c knockout (Mef2cflox/flox Lyz2-cre) mice to further investigate the role of Mef2c in macrophage polarization in vitro and in vivo. In accordance with the knockdown experiments, qPCR analysis showed that Mef2c deficiency suppressed the expression of the LPS-induced M1 macrophage markers Il12a (Fig. 4a), Il12b (Fig. 4b) and Il23a (Fig. 4c) and promoted the expression of the LPS-induced M2 macrophage markers Il10 (Fig. 4d), Arg1 (Fig. 4e) and Chil3 (Fig. 4f). Immunoblot analysis also indicated that Mef2c knockout decreased LPS-induced iNOS expression (Fig. 4g) and increased IL-4-induced ARG1 expression (Fig. 4h). We monitored macrophage phenotype by flow cytometric analysis of CD11c and CD206 as phenotypic markers of M1 and M2 macrophages, respectively. We found that CD11c expression was downregulated in Mef2c-deficient macrophages, whereas CD206 was upregulated (Fig. 4i), suggesting that Mef2c deficiency impairs M1 macrophage polarization.

Fig. 4.

Fig. 4

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Mef2c deficiency impairs macrophage polarization. qPCR analysis of Il12a (a), Il12b (b), Il23a (c), Il10 (d), Arg1 (e), and Chil3 (f) mRNA expression in Mef2cflox/flox and Mef2cflox/flox Lyz2-cre peritoneal macrophages stimulated with LPS (100 ng/ml) for the indicated times. g Immunoblot analysis of iNOS expression in Mef2cflox/flox and Mef2cflox/flox Lyz2-cre peritoneal macrophages stimulated with LPS (100 ng/ml) for the indicated times. h Immunoblot analysis of ARG1 expression in Mef2cflox/flox and Mef2cflox/flox Lyz2-cre peritoneal macrophages stimulated with IL-4 (10 ng/ml) for the indicated times. i Flow cytometric analysis of CD11c and CD206 expression by Mef2cflox/flox and Mef2cflox/flox Lyz2-cre peritoneal macrophages stimulated with LPS (100 ng/ml) or IL-4 (10 ng/ml) for 16 h. j qPCR analysis of Il12a, Il12b, Il10, and Arg1 mRNA expression by peritoneal macrophages collected from Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice 2 h after intraperitoneal injection of LPS (5 mg/kg). The data are representative of three independent experiments. The data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Subsequently, we investigated the effects of Mef2c deficiency on macrophage polarization in vivo. Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice received an intraperitoneal injection of LPS, and peritoneal macrophages were collected 2 h later. qPCR analysis showed that Mef2c deficiency suppressed the expression of the M1 macrophage markers Il12a and Il12b in vivo but promoted the expression of the M2 macrophage markers Il10 and Arg1 (Fig. 4j). Previous studies have shown that IL-10 signaling is important for inflammatory gene expression [41]. To exclude the effect of IL-10 overproduction on inflammatory gene expression in Mef2c-deficient mice/macrophages, we used an IL-10R antibody to block IL-10 signaling. The results showed that blocking IL-10 signaling could significantly promote the expression of the inflammatory genes Il12a, Il12b and Il23a in Mef2cflox/flox and Mef2cflox/flox Lyz2-cre macrophages both in vitro (Fig. S5a) and in vivo (Fig. S5b). However, Mef2cflox/flox macrophages still expressed higher levels of the LPS-induced inflammatory genes Il12a, Il12b and Il23a than Mef2cflox/flox Lyz2-cre macrophages both in vitro (new Fig. S5a) and in vivo (new Fig. S5b), indicating that Mef2c regulated inflammatory gene expression independent of IL-10 signaling. These results suggest that Mef2c deficiency impairs M1 macrophage polarization both in vitro and in vivo.

Mef2c-deficient mice show increased susceptibility to Listeria monocytogenes infection

Polarization influences the phenotype and function of macrophages. To investigate the effect of Mef2c deficiency on the antibacterial innate immune response in vivo, we challenged Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice with Listeria monocytogenes. The ELISA results indicated that IL-12 levels in the serum of Mef2cflox/flox Lyz2-cre mice were significantly lower than those of Mef2cflox/flox mice (Fig. 5a). We also observed that the levels of the inflammatory cytokines IL-6 (Fig. 5b) and TNF-α (Fig. 5c) in the serum of Mef2cflox/flox Lyz2-cre mice were lower than those of Mef2cflox/flox mice. In contrast, IL-10 levels in the serum of Mef2cflox/flox Lyz2-cre mice were higher than those of Mef2cflox/flox mice (Fig. 5d). These results revealed that the inflammatory response to Listeria monocytogenes infection was impaired in Mef2c-deficient mice. To evaluate the ability of Mef2c to regulate Listeria monocytogenes clearance in vivo, we determined the bacterial burden in the spleen and liver in Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice after Listeria monocytogenes infection. The colony formation assays showed that the bacterial burden in the spleen and liver was significantly increased in Mef2c-deficient mice, indicating that Mef2c-deficient mice had a lower ability to resist Listeria monocytogenes infection than wild-type control mice (Figs. 5e and f). However, a significant difference in the bacterial burden in the livers of Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice was observed only on Day 5 after Listeria monocytogenes infection, suggesting that Mef2c affects adaptive immunity by regulating macrophage polarization and cytokine production. In addition, we observed that the pathological abscesses induced in the spleen and liver by Listeria monocytogenes infection were more serious in Mef2cflox/flox Lyz2-cre mice than in Mef2cflox/flox mice (Fig. 5g). The formation of microabscesses is a histological hallmark of Listeria monocytogenes-infected livers. Hematoxylin and eosin (HE) staining revealed more microabscesses in Mef2cflox/flox Lyz2-cre mice than in Mef2cflox/flox mice (Fig. 5h). More importantly, Mef2cflox/flox Lyz2-cre mice showed lower resistance to Listeria monocytogenes infection in terms of overall survival than Mef2cflox/flox mice (Fig. 5i). These results suggest that Mef2c deficiency increases the susceptibility of Mef2cflox/flox Lyz2-cre mice to Listeria monocytogenes infection.

Fig. 5.

Fig. 5

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Mef2c deficiency reduces the host response to Listeria monocytogenes infection. ELISA analysis of IL-12p70 (a), IL-6 (b), TNF-α (c), and IL-10 (d) concentrations in the serum of Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice 3 days after intravenous injection of Listeria monocytogenes (2 × 105 CFU/mouse). Listeria monocytogenes burdens in the spleens (e) and livers (f) of Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice on Days 0, 1, 3 5, and 7 after intravenous injection of Listeria monocytogenes (2 × 105 CFU/mouse). Liver abscesses in Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice were evaluated 3 days after intravenous injection of Listeria monocytogenes (2 × 105 CFU/mouse) by light microscopy (g) and HE staining (h); scale bar = 200 μm. i Survival of Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice administered Listeria monocytogenes (5 × 105 CFU/mouse) by intravenous injection (n = 20 per group). j Pathogen burden in peritoneal macrophages isolated from Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice infected with Listeria monocytogenes (MOI = 0.1) for the indicated times. k Flow cytometric analysis of ROS production by peritoneal macrophages isolated from Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice and stimulated with LPS (100 ng/ml) for the indicated times. l qPCR analysis of Ifng and Tbet expression in spleen cells obtained from Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice 3 days after intravenous injection of Listeria monocytogenes (2 × 105 CFU/mouse) and cultured for 48 h with gentamicin in the presence of anti-CD3 and anti-CD28 antibodies. m Flow cytometric analysis of IFN-γ expression in CD4+ T cells obtained from the spleens of Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice 3 days after intravenous injection of Listeria monocytogenes (2 × 105 CFU/mouse). n Naïve CD4+ T cells were sorted from lymph nodes obtained from wild-type mice and cultured in the presence of anti-CD3, anti-CD28, and anti-IL-4 antibodies and supernatant from LPS-stimulated wild-type or Mef2c-deficient macrophages prior to the induction of Th1 cell differentiation in vitro. The proportion of CD4+ IFN-γ+ T cells was then analyzed by flow cytometry. The data are representative of three independent experiments. The data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

To further explore the mechanism of Mef2c in the host response against Listeria monocytogenes infection, we evaluated Listeria monocytogenes killing ability and ROS production in macrophages. Mef2c deficiency damaged the capacity of macrophages to kill bacteria but did not affect the engulfment of Listeria monocytogenes (Fig. 5j and Fig. S6a). Furthermore, we observed that Mef2c-deficient macrophages produced less ROS than their wild-type counterparts (Fig. 5k). M1 macrophage polarization promoted the acquisition of antigen-presenting functions and IL-12 production, leading to efficient induction of Th1 responses. The qPCR results showed that splenocytes from Listeria monocytogenes-infected Mef2cflox/flox Lyz2-cre mice expressed less Ifng and Tbet mRNA, which are markers of a Th1 response, than those from Mef2cflox/flox mice (Fig. 5l), whereas there was no difference in the mRNA expression levels of the Th2 markers Il4 and Gata3 (Fig. S6b) or the Treg marker Foxp3 (Fig. S6c). We also used flow cytometry to analyze CD4+ T cell differentiation in mice after Listeria monocytogenes infection. The frequency of CD4+ IFN-γ+ T cells among the splenocytes of Mef2cflox/flox Lyz2-cre mice was significantly lower than that among the splenocytes of Mef2cflox/flox mice, indicating an impaired Th1 response against Listeria monocytogenes infection in Mef2cflox/flox Lyz2-cre mice (Fig. 5m). However, Mef2c deficiency in macrophages had no effect on Th2 (Fig. S6d) or Treg (Fig. S6e) differentiation. We also evaluated the potential of culture supernatants derived from wild-type or Mef2c-deficient macrophages to affect the differentiation of T cells in vitro. Flow cytometric analysis revealed a higher frequency of CD4+ IFN-γ+ T cells in the presence of wild-type macrophage culture supernatant than in the presence of Mef2c-deficient macrophage culture supernatant (Fig. 5n). These results demonstrate that Mef2c deficiency increases susceptibility to Listeria monocytogenes infection by suppressing M1 macrophage polarization and subsequently initiating Th1 adaptive immune responses.

Mef2c deficiency attenuates experimental murine colitis in vivo

Inflammatory bowel disease is closely related to the abnormal activation of macrophages and Th1 cells. We further investigated the effects of Mef2c deficiency on DSS-induced acute colitis in mice. As shown in Fig. 6a, colitis-induced body weight loss was significantly alleviated in Mef2cflox/flox Lyz2-cre mice compared with Mef2cflox/flox mice. Furthermore, the disease activity index (diarrhea and rectal bleeding) was obviously reduced in Mef2cflox/flox Lyz2-cre mice compared with Mef2cflox/flox mice (Fig. 6b). Similarly, colon length was significantly increased in Mef2cflox/flox Lyz2-cre mice compared with Mef2cflox/flox mice (Fig. 6c). Less inflammatory cell infiltration and only slight epithelial damage were observed in the colons of Mef2cflox/flox Lyz2-cre mice after colitis induction than in those of Mef2cflox/flox mice (Fig. 6d). qPCR analysis showed lower levels of proinflammatory cytokines and mediators (such as Il1b, Il6, Tnf, Nos2, Il12a, Il12b, and Cox2) in the colons of Mef2cflox/flox Lyz2-cre mice after colitis induction than in the colons of control Mef2cflox/flox mice (Fig. 6e). ELISA also showed significantly lower levels of IL-1β, IL-6, TNF-α, and IL-12 production in the colons of Mef2cflox/flox Lyz2-cre mice after colitis induction than in the colons of Mef2cflox/flox mice (Fig. 6f). These results indicated that Mef2c deficiency attenuated DSS-induced acute colitis in mice in vivo.

Fig. 6.

Fig. 6

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Mef2cflox/flox Lyz2-cre mice show attenuated experimental colitis in vivo. Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice were administered drinking water with or without 3% DSS for 7 days to induce acute colitis, followed by a 2-day recovery period with normal drinking water. Body weight changes (a) and DAI scores (b) in DSS-induced experimental colitis mice. c Gross morphology of colons from Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice and colon lengths. d HE-stained images of colon sections. e qPCR analysis of Il1b, Il6, Tnf, Il12a, Il12b, and Cox2 mRNA expression in the colons of Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice. f ELISA analysis of IL-1β, IL-6, TNF-α, and IL-12p70 production in the colons of Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice. The data are representative of three independent experiments. The data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Immunofluorescence analysis showed that compared with that in Mef2cflox/flox mice, CD86 expression was decreased in F4/80+ macrophages in colon tissue of Mef2cflox/flox Lyz2-cre mice (Fig. 7a), while CD206 expression was increased (Fig. 7b), suggesting that Mef2c deficiency inhibited macrophage M1 polarization during DSS-induced colitis. We used flow cytometry to analyze macrophage polarization by measuring the expression of CD11c and CD206 as phenotypic markers of M1 and M2 macrophages, respectively. The results showed that CD11c expression was downregulated in the colons of Mef2cflox/flox Lyz2-cre mice after colitis induction compared with that in the colons of Mef2cflox/flox mice (Fig. 7c), while CD206 expression was upregulated (Fig. 7d). Furthermore, flow cytometric analysis showed fewer IL-12+ macrophages in the colons of Mef2cflox/flox Lyz2-cre mice after colitis induction than in the colons of Mef2cflox/flox mice (Fig. 7e). We also used flow cytometry to analyze IFN-γ production by CD4+ T cells in the colon tissues of mice. The frequency of CD4+ IFN-γ+ T cells in the colon tissues of Mef2cflox/flox Lyz2-cre mice was significantly lower than that in the colons of Mef2cflox/flox mice, indicating an impaired Th1 response in Mef2cflox/flox Lyz2-cre mice in vivo (Fig. 7f). These results were confirmed by immunohistochemical analysis showing significantly reduced levels of IFN-γ expression in the colons of Mef2cflox/flox Lyz2-cre mice after colitis induction (Fig. 7g), thus providing further evidence that Mef2c deficiency impairs the Th1 response in the colons of Mef2cflox/flox Lyz2-cre mice. However, conditional Mef2c deficiency had no effect on Th2 (Fig. S7a) or Treg (Fig. S7b) differentiation. These data indicate that Mef2c deficiency suppresses the generation of proinflammatory macrophages and impairs Th1 responses in mice with experimental colitis.

Fig. 7.

Fig. 7

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Mef2c deficiency decreases proinflammatory macrophages and Th1 responses in experimental colitis. Mef2cflox/flox and Mef2cflox/flox Lyz2-cre mice were administered drinking water with or without 3% DSS for 7 days to induce acute colitis, followed by a 2-day recovery period with normal drinking water. a Immunofluorescence analysis of F4/80 and CD86 expression in the colon tissue of DSS-induced experimental colitis mice. b Immunofluorescence analysis of F4/80 and CD206 expression in the colon tissue of DSS-induced experimental colitis mice. Flow cytometric analysis of CD11c (c), CD206 (d) and (e) IL12B expression in macrophages from the colon tissue of DSS-induced experimental colitis mice. f Flow cytometric analysis of IFN-γ expression in CD4 T cells from the colon tissue of DSS-induced experimental colitis mice. g Immunohistochemical analysis of IFN-γ expression in the colon tissue of DSS-induced experimental colitis mice. The data are representative of three independent experiments. The data represent the mean ± SD. *P < 0.05, ***P < 0.001

MEF2C directly regulates the transcription of Il12a and Il12b

To investigate the molecular mechanism by which Mef2c regulates macrophage polarization, we performed double luciferase reporter gene assays on macrophages transfected with the Il12a and Il12b promoter luciferase reporter plasmids. Mef2c upregulated the luciferase reporter activity of the Il12a (Fig. 8a) and Il12b (Fig. 8b) promoters. These results were confirmed in ChIP assays showing that MEF2C binds directly to the promoters of Il12a and Il12b (Fig. 8c, d). We then used the JASPAR database (http://jaspar.genereg.net/) to predict MEF2C binding sites and constructed mutant Il12a and Il12b promoter luciferase reporter plasmids by deleting the predicted MEF2C binding sites. Double luciferase reporter gene assays showed that the expression of the mutant forms of the Il12a (Fig. 8e) and Il12b (Fig. 8f) promoters was not regulated by Mef2c. Since the binding of MEF2C to the Il12a and Il12b promoters is dynamic, to further investigate the mechanism of the direct regulation of Il12a and Il12b expression by Mef2c after LPS exposure, we transfected wild-type and mutant Il12a or Il12b promoter luciferase reporter plasmids into Raw 264.7 cells and then stimulated the cells with LPS. The results revealed that Mef2c could increase wild-type Il12a or Il12b but not mutant Il12a or Il12b luciferase reporter gene expression after LPS stimulation (Fig. 8g and h). To confirm these results, we transfected wild-type and mutant Il12a or Il12b luciferase reporter plasmids into HEK 293 T cells with or without the Myd88 plasmid and obtained similar results (Fig. S8a and b). These data verified that Mef2c could directly regulate Il12a and Il12b expression after LPS exposure. Studies have shown that many transcription factors can regulate IL-12 production, such as PU.1, IRF8 and NK-κB [42–44]. Next, we investigated whether Mef2c deficiency affected the binding of other transcription factors to the Il12a and Il12b promoters. The ChIP assay showed that Mef2c deficiency suppressed PU.1 binding to the Il12a (Fig. S8c) and Il12b (Fig. S8d) promoters and induced IRF8 binding to the Il12a (Fig. S8e) and Il12b (Fig. S8f) promoters but had no effect on NF-κB (Fig. S8g and h). The results suggested that Il12a and Il12b expression might be involved in the regulation of multiple transcription factors. Taken together, these results indicate that MEF2C affects macrophage polarization through direct transcriptional regulation of Il12a and Il12b.

Fig. 8.

Fig. 8

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MEF2C directly regulates the transcription of Il12a and Il12b. Dual-luciferase reporter assays were performed 24 h after the cotransfection of HEK 293 T cells with Mef2c plasmids and Il12a (a) and Il12b (b) luciferase reporter plasmids. ChIP assays were used to analyze MEF2C recruitment to the Il12a (c) and Il12b (d) promoters in peritoneal macrophages stimulated with or without LPS. Schematic diagrams of Il12a (e) and Il12b (f) mutant luciferase reporter plasmids (left). Dual-luciferase reporter assays were performed 24 h after the cotransfection of HEK 293 T cells with Mef2c plasmids and Il12a (e) and Il12b (f) mutant luciferase reporter plasmids. Raw 264.7 cells were transfected with Mef2c plasmids or wild-type and mutant Il12a (g) and Il12b (h) luciferase reporter plasmids for 24 h and then stimulated with LPS (100 ng/ml). Dual-luciferase reporter assays were performed. The data are representative of three independent experiments and represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

In the present study, we uncovered a previously unappreciated role for the transcription factor Mef2c, which is involved in promoting M1 macrophage polarization and regulating the Th1 response. These results suggest that Mef2c plays an important role in innate and adaptive immunity. Macrophage plasticity allows for efficient changes in phenotype and physiology in response to cytokines and microbial signals [45]. M-CSF and GM-CSF are involved in the regulation of macrophage heterogeneity. Based on differences in phenotype and function, macrophages can be divided into M1 (classically activated) or M2 (alternatively activated) phenotypes [6]. Immunoblot analysis revealed that M1 macrophages expressed higher levels of Mef2c protein than M2 macrophages. Moreover, we demonstrated the plasticity of Mef2c expression in macrophages during the conversion of the M1 and M2 phenotypes. These findings suggest that Mef2c plays an important role in regulating macrophage polarization.

M1 and M2 macrophages differ in their patterns of cytokine expression. M1 macrophages produce large amounts of proinflammatory cytokines, which are involved in resistance to pathogens, whereas M2 macrophages produce the anti-inflammatory mediators responsible for tissue repair and tumor progression [46]. qPCR analyses showed that Mef2c knockdown or knockout in macrophages inhibited LPS-induced expression of M1 macrophage markers (such as Il12a, Il12b, and Il23a), while the expression of M2 macrophage markers (such as Il10, Arg1 and Chil3) was increased. Similarly, flow cytometric analysis demonstrated that CD11c (M1 macrophage marker) expression was downregulated in Mef2c-deficient macrophages, while CD206 (M2 macrophage marker) expression was upregulated, indicating that Mef2c influences M1 macrophage polarization. However, we did not find significant changes in the expression of Il1b, Il6, or Tnf in Mef2c-knockdown or -knockout macrophages compared to wild-type macrophages in vitro. These proinflammatory cytokines are commonly regarded as markers of M1 macrophages [6]. Although Mef2c has been reported to suppress NF-κB activation [47], our findings showed that Mef2c had no obvious effect on the NF-κB signaling pathway. It is possible that Mef2c has distinct functions in different cells and regulates macrophage polarization through an NF-κB-independent pathway. However, Mef2cflox/flox Lyz2-cre mice produced less IL-1β, IL-6 and TNF-α in response to Listeria monocytogenes compared to their wild-type counterparts, as determined by ELISA. Moreover, using a DSS-induced experimental colitis model, we showed that IL-1β, IL-6 and TNF-α production in the colons of Mef2cflox/flox Lyz2-cre mice was significantly decreased after colitis induction. These results suggest that although Mef2c does not directly affect the production of some proinflammatory cytokines, it influences macrophage phenotypes and functions, thus changing the microenvironment of the tissue in vivo and regulating the production of related cytokines.

In addition, M1 and M2 macrophages show distinctive glucose and amino acid metabolism. M1 macrophages exhibit a shift toward the glycolytic pathway, whereas oxidative phosphorylation is the dominant pathway in M2 macrophages [48, 49]. It can be suggested that this shift in macrophage metabolism improves the ability if cells to regulate responses to pathogen infection and inflammation. In addition, nitric oxide (NO), an inflammatory signaling molecule, plays an important role in inflammatory and antibacterial responses. In M1 macrophages, iNOS catalyzes the conversion of L-arginine to reactive nitrogen intermediates (RNIs), which have strong antimicrobial activity. In contrast, M2 macrophages have increased expression of ARG1, which catalyzes the conversion of L-arginine into ornithine and polyamines [50, 51]. Immunoblot analysis showed that iNOS expression was decreased in Mef2c-deficient macrophages, whereas ARG1 expression was increased. These findings suggest that Mef2c influences M1 macrophage polarization, and further studies of the mechanism are required to fully elucidate the biology of Mef2c.

Previous studies have shown that M1 macrophages facilitate inflammatory responses and resist pathogen infection [6]. Based on the ability of Mef2c to promote M1 macrophage polarization, we investigated the effects of LPS challenge or Listeria monocytogenes infection on Mef2cflox/flox Lyz2-cre mice. We found that Mef2c deficiency was associated with reduced production of proinflammatory cytokines under these conditions compared to those in wild-type mice. We also found that mice with myeloid-specific Mef2c knockout were more susceptible to Listeria monocytogenes infection than wild-type mice based on the higher bacterial loads in the spleen and liver and lower survival rates after Listeria monocytogenes infection.

ROS are essential for pathogen clearance [52] and play an important role in promoting the polarization of macrophages to a proinflammatory phenotype [53]. Our data showed that macrophages from Mef2cflox/flox Lyz2-cre mice produced less ROS and exhibited a reduced capacity to kill bacteria than those from wild-type mice, which is consistent with our observation that Mef2c-deficient mice had increased susceptibility to Listeria monocytogenes infection. Furthermore, the impaired Th1 response in Mef2cflox/flox Lyz2-cre mice after Listeria monocytogenes infection also accounts for the increased susceptibility of these mice to infection. While appropriate inflammatory responses can facilitate the clearance of pathogens, an excessive response leads to uncontrolled inflammation and the exacerbation of disease [54]. Using the DSS-induced experimental colitis model, we showed that the inflammatory response was impaired in Mef2cflox/flox Lyz2-cre mice, which had less severe colonic tissue damage than wild-type mice, and that these changes were associated with a decrease in M1 macrophages and a reduced Th1 response in the colon. Consequently, we conclude that Mef2c deficiency in macrophages results in impaired polarization toward the M1 phenotype.

To further clarify the mechanism by which Mef2c functions as a transcription factor to regulate macrophage polarization, we used the JASPAR database (http://jaspar.genereg.net/) to predict MEF2C binding sites in the promoter sequences of genes encoding macrophage lineage-specific cytokines. This analysis predicted the presence of an A/T-rich DNA consensus sequence in the promoters of the genes encoding Il12a and Il12b. Chromatin immunoprecipitation (ChIP) and double luciferase reporter assays showed that MEF2C bound to the promoters of the genes encoding the Il12a and Il12b subunits to increase their transcription. Previous studies have shown that macrophage polarization is regulated by many transcription factors, such as IRF5 and KLF4 [11, 55]. IRF5 directly activates Il12a, Il12b and Il23a transcription but represses Il10 transcription. MEF2C also directly activates Il12a and Il12b transcription. Furthermore, our global gene expression analysis revealed a slight upregulation in KLF4 expression (approximately 1.35-fold), which could promote M2 macrophage polarization by interacting with STAT6. Therefore, it can be hypothesized that Mef2c coordinates with these transcription factors to influence the expression of macrophage lineage-specific genes.

In summary, our study demonstrated that Mef2c directly activated the transcription of Il12a and Il12b and then promoted M1 macrophage polarization and Th1 responses, resulting in resistance to Listeria monocytogenes infection and susceptibility to DSS-induced experimental colitis. In addition, we showed that Mef2c regulated the ability of macrophages to kill bacteria and produce ROS, but the detailed mechanism still needs further investigation. These findings provide new insights into the mechanism of macrophage polarization and reveals that Mef2c is a potential therapeutic target for inflammatory and autoimmune diseases.

Supplementary information

41423_2022_841_MOESM1_ESM.docx (3.5MB, docx)

MEF2C promotes M1 macrophage polarization and Th1 response

Acknowledgements

This work was supported by grants from the National Key Research and Development Program of China (2016YFA0502201) awarded to Prof. Huazhang An; the National Natural Science Foundation of China (Nos. U1801283, 31870908), the Guangdong Provincial Science and Technology Program (No. 2019B030301009) and the SZU Top Ranking Project (No. 86000000210) awarded to Prof. Weilin Chen; the National Natural Science Foundation of China (No. 81771711) awarded to Prof. Wengang Song; and the Guangdong Provincial Science and Technology Program (No. 2019A1515110086) awarded to Xibao Zhao. We thank Jessica Kate Tamanini (Scientific Editor, Shenzhen University School of Medicine) for editing the manuscript.

Author contributions

XZ, HA, and WC participated in the research design. XZ, QD, HL, and JQ conducted the experiments. JL, ZZ, WS, QD, YX, HW, and ZW contributed analytic tools or new reagents. XZ, WC, and HA performed the data analysis. XZ, WC, and HA contributed to the writing of the manuscript.

Competing interests

The authors declare no competing interests.

Footnotes

These authors contributed equally: Xibao Zhao, Qianqian Di, Han Liu.

Contributor Information

Huazhang An, Email: anhz@immunol.org.

Weilin Chen, Email: cwl@szu.edu.cn.

Supplementary information

The online version contains supplementary material available at 10.1038/s41423-022-00841-w.

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MEF2C promotes M1 macrophage polarization and Th1 response

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