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  • Open Access

Different responses to oxidized low-density lipoproteins in human polarized macrophages

  • 1,
  • 2Email author,
  • 1Email author,
  • 1,
  • 2,
  • 2 and
  • 1
Lipids in Health and Disease201110:1

https://doi.org/10.1186/1476-511X-10-1

  • Received: 28 November 2010
  • Accepted: 4 January 2011
  • Published:

Abstract

Background

Oxidized low-density lipoprotein (oxLDL) uptake by macrophages plays an important role in foam cell formation. It has been suggested the presence of heterogeneous subsets of macrophage, such as M1 and M2, in human atherosclerotic lesions. To evaluate which types of macrophages contribute to atherogenesis, we performed cDNA microarray analysis to determine oxLDL-induced transcriptional alterations of each subset of macrophages.

Results

Human monocyte-derived macrophages were polarized toward the M1 or M2 subset, followed by treatment with oxLDL. Then gene expression levels during oxLDL treatment in each subset of macrophages were evaluated by cDNA microarray analysis and quantitative real-time RT-PCR. In terms of high-ranking upregulated genes and functional ontologies, the alterations during oxLDL treatment in M2 macrophages were similar to those in nonpolarized macrophages (M0). Molecular network analysis showed that most of the molecules in the oxLDL-induced highest scoring molecular network of M1 macrophages were directly or indirectly related to transforming growth factor (TGF)-β1. Hierarchical cluster analysis revealed commonly upregulated genes in all subset of macrophages, some of which contained antioxidant response elements (ARE) in their promoter regions. A cluster of genes that were specifically upregulated in M1 macrophages included those encoding molecules related to nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) signaling pathway. Quantitative real-time RT-PCR showed that the gene expression of interleukin (IL)-8 after oxLDL treatment in M2 macrophages was markedly lower than those in M0 and M1 cells. HMOX1 gene expression levels were almost the same in all 3 subsets of macrophages even after oxLDL treatment.

Conclusions

The present study demonstrated transcriptional alterations in polarized macrophages during oxLDL treatment. The data suggested that oxLDL uptake may affect TGF-β1- and NF-κB-mediated functions of M1 macrophages, but not those of M0 or M2 macrophages. It is likely that M1 macrophages characteristically respond to oxLDL.

Keywords

  • Mannose Receptor
  • Foam Cell Formation
  • Antioxidant Response Element
  • cDNA Microarray Analysis
  • Ingenuity Pathway Analysis Software

Background

Atherosclerosis is a major cause of cardiovascular disease, which is one of the leading morbidities worldwide [1]. Atherosclerosis has been suggested to be merely a lipid-storage disease; however, it is now recognized as an inflammatory condition of the vessel wall characterized by infiltration of macrophages and T cells [2]. Monocytes are recruited into the arterial intima and differentiate into macrophages. They take up oxidized low-density lipoprotein (oxLDL) via scavenger receptors, and then become foam cells that play a crucial role in the initiation of atherosclerotic lesions [3]. Foam cells have been shown to affect many atherogenic events, including recruitment of monocytes and neutrophils by producing chemokines, such as monocyte chemoattractant protein (MCP)-1 [4] and interleukin (IL)-8 [5], formation of necrotic cores in atherosclerotic plaques [3], and production of matrix metalloproteases (MMPs), which degrade the extracellular matrix comprising the fibrous cap of plaque [6]. Therefore, macrophages immunologically interact with surrounding inflammatory cells during the process of differentiation into foam cells in atherogenic processes.

Over the past several decades, a number of studies have demonstrated that macrophages do not represent a homogenous cell population. Stein et al. described an alternative subset of macrophages induced by IL-4, characterized by high mannose receptor (MR) expression [7]. Since then, it has been demonstrated that monocyte-derived macrophages can be polarized into two subsets in vitro. One subset consists of classically activated macrophages (M1 macrophages) polarized with lipopolysaccharide (LPS) and interferon (IFN)-γ, which are characterized by CD86 expression and production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, IL-1, and IL-6. The other subset consists of alternatively activated macrophages (M2 macrophages) polarized with Th2 cytokines, such as IL-4 and/or IL-13, which are characterized by MR expression [8].

Recently, Bouhlel et al. confirmed the presence of M2 macrophages within human atherosclerotic lesions by identifying the expression of M2 markers, including IL-10 and MR in human carotid plaques [9]. They also reported that macrophages expressing M2 markers show a different distribution from foam cells. These results suggested the presence of heterogeneous subsets of macrophages in human atherosclerotic lesions. However, it remains unclear which type of macrophages differentiate into foam cells or how they contribute to atherogenesis.

The present study was performed to elucidate the contributions of M1 and M2 macrophages to atherogenesis during differentiation into foam cells. Martinez et al. investigated the polarization of human monocytes toward M1 or M2 macrophages using cDNA microarray analysis, and found distinct sets of genes specifically upregulated in either subset of macrophages [10]. Cho et al. also examined the transcriptional differences in human monocyte-derived macrophages during oxLDL uptake by cDNA microarray analysis [11]. However, there have been no previous studies of the whole transcriptional alterations in human M1 or M2 macrophages during oxLDL uptake. To investigate the roles of these macrophage subsets during differentiation into foam cells, we examined the transcriptional alterations of M1 or M2 macrophages during oxLDL treatment.

Methods

Materials

Lymphoprep was purchased from AXIS-SHILD (Rodelokka, Oslo, Norway). Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM/F12) was obtained from Invitrogen (Carlsbad, CA), and RPMI-1640 culture medium (endotoxin-free) was from Sigma-Aldrich (St. Louis, MO). Recombinant human macrophage-colony stimulating factor (M-CSF), IFN-γ, and IL-4 were obtained from R&D Systems (Minneapolis, MN). LPS from Escherichia coli (serotype O111:B4) was obtained from List Biological Laboratories Inc. (Campbell, CA). OxLDL (endotoxin level < 0.5 EU/ml), which was prepared with 3.5 μM CuSO4 in PBS at 37°C for 24 h, was purchased from Biomedical Technologies (Stoughton, MA). The average level of thiobarbituric acid-reactive substances (TBARS) in this study was 76.23 ± 7.89 nmol malondialdehyde equivalents/mg LDL protein (mean ± SD). Anti-CD14 antibody, anti-CD86 antibody, and anti-MR antibody were obtained from eBioscience (San Diego, CA). All procedures were performed under endotoxin-free conditions.

Cells

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers with informed consent from buffy coats by density-gradient centrifugation using Lymphoprep. The purity of monocytes was > 95% as determined by flow cytometric analysis using anti-CD14 antibody (data not shown). The monocytes were suspended in DMEM/F12, and plated onto tissue culture dishes at a density of 1 × 106 cells/cm2 for 2 h at 37°C. The adherent cells were differentiated into macrophages by incubation with 100 ng/mL M-CSF in RPMI-1640 medium supplemented with 20% fetal calf serum (FCS) for 7 days (these cells were defined as M0 macrophages). Macrophage polarization was performed as described by Martinez et al. with slight modifications [10]. To obtain M1 or M2 macrophages, M0 macrophages were further incubated with 10 pg/mL LPS plus 20 ng/mL IFN-γ or 20 ng/mL IL-4 in RPMI-1640 with 5% FCS for 18 h, respectively. After polarization, media were removed, and each subset of macrophages was incubated for a further 6 h in the presence or absence of 100 μg/mL oxLDL. The study was approved by the Ethical Committee of Juntendo University.

Flow cytometric analysis

The M1 or M2 polarized macrophages were washed with PBS. After washing, cells were stained with PE-Cy5- or FITC-conjugated antibodies or with corresponding isotype controls for 20 min at 4°C. Then, flow cytometry was performed to determine the expression of cell surface antigens using FACSCalibur (BD Biosciences, Franklin Lakes, NJ), as described previously [12]. Data were analyzed using Cell Quest software (BD Biosciences).

Quantitative real-time RT-PCR

Total RNA was extracted and purified from macrophages using an RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was synthesized from 50 ng/μL of total RNA using an ExScript RT-PCR Kit (Takara-Bio, Shiga, Japan). Primers were selected using Perfect Real-Time Primer Support System provided by Takara. Real-time RT-PCR was performed using SYBR Premix Ex Taq (Takara-Bio) and an ABI 7900HT Sequence Detector System (Applied Biosystems, Foster City, CA). The amplification program included an initial denaturation step at 95°C for 10 s, 40 cycles of denaturation at 95°C for 10 s, and annealing and extension at 60°C for 30 s. After amplification, dissociation curves were acquired to determine the specificity of PCR products. The relative cDNA concentrations were established using a standard curve plotted with sequential tenfold dilutions of cDNA synthesized from QPCR Human Reference Total RNA (Stratagene, La Jolla, CA). The data were normalized relative to peptidylprolyl isomerase A (PPIA) as an internal control.

cDNA microarray analysis

cDNA synthesis and aminoallyl labeling of RNA were performed using an amino-allyl RNA amplification kit (Sigma-Aldrich) according to the manufacturer's instructions. The Cy3- or Cy5-labeled aminoallyl RNA was concentrated using Microcon YM-30 (Millipore, Bedford, MA), mixed with hybridization buffer supplied with the kit, and denatured at 95°C for 2 min. The hybridization mixture was applied onto a "3D-Gene" human oligo chip 25 k (Toray Industries, Tokyo, Japan), and incubated according to the manufacturer's instructions. After washing and drying the DNA chip slides, the fluorescent signals were quantified by ScanArray Lite (PerkinElmer Life Sciences, Boston, MA) and analyzed using ScanArray Express software. After subtraction of the mean background level, the fluorescence intensity was normalized relative to the mean sample intensity in each chip. Any given gene was analyzed if its normalized intensity was more than 2-4. We defined genes showing a change in expression of > 2-fold during oxLDL treatment as significantly up- or downregulated (log2 ratios were greater than +1 or less than -1).

Ingenuity pathway analysis

Ingenuity Pathway Analysis (IPA) software (version 8.7; Ingenuity Systems, Redwood, CA) was utilized to determine the possible biological pathways and intermolecular networks between candidate genes. A detailed description of IPA software can be found on the Ingenuity Systems website http://www.ingenuity.com/. The significantly up- or downregulated genes were overlaid onto a global molecular network developed from information contained in the Ingenuity Knowledge Base.

Functional gene ontology analysis identified the biological functions that were most significant to molecules in the network. The network molecules associated with biological functions in the Knowledge Base were considered for the analysis. Right-tailed Fisher's exact test was used to calculate the P-values determining the probability that each biological function assigned to that network was due to chance alone. IPA generates significant biological networks that are particularly enriched with the genes of interest, called "focus genes." It calculates a network score that takes into account the number of focus genes and the size of the networks, indicating the likelihood of focus genes in a network being found together by chance. The higher the score, the lower is the probability of finding the observed Network Eligible Molecules in a given network by chance. Network analysis produces a graphical representation of the molecular relationships between the identified genes. Molecules are represented as nodes, and the biological relationship between two nodes is represented as a line. All relationships are supported by at least 1 reference from the literature, from a textbook, or from canonical information stored in the Knowledge Base.

Statistical analysis

The data were expressed as the means ± SD and were analyzed for significant differences by one-way or two-way analysis of variance (ANOVA) and, Bonferroni's post hoc test using GraphPad Prism (version 5.00; GraphPad Software, La Jolla, CA).

Results

Characteristics of M1 or M2 polarized macrophages

Human monocyte-derived macrophages cultured for 7 days in the presence of M-CSF can be polarized toward M1 macrophages by further treatment with 100 ng/mL LPS plus 20 ng/mL IFN-γ for 18 h [10]. However, under our experimental conditions, almost all cells were damaged by such high a concentration of LPS, as demonstrated by trypan blue staining (data not shown). Therefore, we differentiated M-CSF-treated monocytes into M1 macrophages by incubation with 10 pg/mL LPS plus 20 ng/mL IFN-γ. We confirmed the polarized cells as M1 and M2 macrophages by quantitative real-time RT-PCR and flow cytometric analysis (Figure 1). Consistent with the previous report of Martinez et al. [10], M1 macrophages showed higher levels of proinflammatory cytokine mRNA expression, such as TNF-α, IL-1β, and IL-6, than M0 or M2 macrophages. In contrast, M2 macrophages showed markedly elevated expression of MRC1, which encodes MR (Figure 1A). The level of CD86 expression on M1 macrophages was higher than that on M2 macrophages, while MR was expressed on M2 macrophages but not on M1 macrophages (Figure 1B).
Figure 1
Figure 1

Expression of M1 and M2 macrophage polarized markers in human monocyte-derived macrophages. A. TNFα, IL1β, and IL6 gene expression as markers of M1, and MRC1 gene expression as a marker of M2 were analyzed by RT-PCR. Each panel shows data from one of 3 representative experiments. B. Surface expression of CD86 (M1 marker) and mannose receptor (MR) (M2 marker) were analyzed by flow cytometry. The level of CD86 expression was higher in M1 than in M2 macrophages, while the expression level of MR was higher in M2 than in M1 macrophages.

Transcriptional profile

After confirming that the 2 subsets of macrophages were properly polarized to M1 or M2 macrophages, cDNA microarray analysis was performed to investigate the alterations during oxLDL treatment. Of the 25392 probe sets on "3D-Gene" human oligo chip 25 k, we eliminated 1125 probe sets as controls and backgrounds. Any given gene was eliminated if its normalized intensity was less than 2-4. We also eliminated non-altered genes that showed changes in expression level of less than 2-fold during oxLDL treatment. Finally, we identified 2025, 2265, and 2249 genes that were significantly up- or downregulated in M0, M1, and M2 macrophages, respectively (Figure 2). Among these genes, 1526, 1819, and 1880 genes were upregulated in M0, M1, and M2 macrophages by oxLDL treatment, respectively (All transcriptional profiles are shown in Additional file 1). Table 1 shows the top 30 genes that were most markedly upregulated by oxLDL. IL8, TRIM16, and ADM were commonly upregulated in all subsets of macrophages. Twenty-eight genes in the top 30 upregulated genes in M2 macrophages (93% of the top 30 genes) were also upregulated in M0, while 15 genes in the top 30 upregulated genes in M2 macrophages were upregulated in M1 cells.
Figure 2
Figure 2

Genes showing significantly altered expression in oxLDL-treated macrophages on cDNA microarray analysis. The logarithmically transformed intensities of all the genes in oxLDL-treated macrophages were plotted against those in non-treated macrophages (Left panels). Genes were eliminated if the normalized intensity was less than 2-4, or if the change in alteration during oxLDL treatment was less than 2-fold (Right panels). Finally, 2025, 2265, and 2249 genes were identified as showing significantly regulated expression in M0, M1, and M2 macrophages, respectively.

Table 1

Genes upregulated by oxLDL in polarized macrophages

The top 30 genes upregulated by oxLDL in M0

Gene Symbol

Ref Seq ID

M0

M1

M2

NCBI official full name

IL8

NM_000584

6.200

3.200

5.177

interleukin 8

CCL1

NM_002981

6.028

0.937

-

chemokine (C-C motif) ligand 1

SERPINB2

NM_002575

5.485

-

-

serpin peptidase inhibitor, clade B (ovalbumin), member 2

IL1B

NM_000576

5.162

2.700

2.684

interleukin 1, beta

PTGS2

NM_000963

4.985

-

-

prostaglandin-endoperoxide synthase 2

TM4SF1

NM_014220

4.836

-

1.238

transmembrane 4 L six family member 1

CCL19

NM_006274

4.834

0.368

-

chemokine (C-C motif) ligand 19

POPDC3

NM_022361

4.557

1.491

-

popeye domain containing 3

INHBA

NM_002192

4.476

2.335

-

inhibin, beta A

CKB

NM_001823

4.378

0.920

2.107

creatine kinase, brain

IFIT1

NM_001001887

4.152

0.717

5.734

interferon-induced protein with tetratricopeptide repeats 1

CCL5

NM_002985

4.075

-0.218

1.616

chemokine (C-C motif) ligand 5

IFIT5

NM_012420

4.039

-

-

interferon-induced protein with tetratricopeptide repeats 5

TNFAIP6

NM_007115

4.033

0.019

-

tumor necrosis factor, alpha-induced protein 6

TRIM16

NM_006470

3.990

2.992

3.453

tripartite motif-containing 16

ADM

NM_001124

3.973

4.807

3.202

adrenomedullin

CCL4L2

NM_207007

3.905

1.321

0.448

chemokine (C-C motif) ligand 4-like 2

ISG20

NM_002201

3.839

0.952

4.442

interferon stimulated exonuclease gene 20kDa

EREG

NM_001432

3.726

2.793

-

epiregulin

CSF2

NM_000758

3.644

1.605

0.724

colony stimulating factor 2 (granulocyte-macrophage)

RHOF

NM_019034

3.638

-

3.020

ras homolog gene family, member F (in filopodia)

GSTM3

NM_000849

3.632

2.635

2.543

glutathione S-transferase mu 3 (brain)

AKR1C3

NM_003739

3.497

2.490

3.432

aldo-keto reductase family 1, member C3

CYP7B1

NM_004820

3.482

-

-

cytochrome P450, family 7, subfamily B, polypeptide 1

CCL4

NM_002984

3.466

0.742

-0.123

chemokine (C-C motif) ligand 4

CCL24

NM_002991

3.433

0.413

-

chemokine (C-C motif) ligand 24

NT5E

NM_002526

3.406

-

-

5'-nucleotidase, ecto (CD73)

IFI44L

NM_006820

3.325

-0.074

4.474

interferon-induced protein 44-like

COX11

NM_004375

3.306

1.946

1.111

COX11 cytochrome c oxidase assembly homolog (yeast)

NOL12

NM_024313

3.281

0.937

1.313

nucleolar protein 12

The top 30 genes upregulated by oxLDL in M1

Gene Symbol

Ref Seq ID

M0

M1

M2

NCBI official full name

ADM

NM_001124

3.973

4.807

3.202

adrenomedullin

S1RT5

NM_012241

3.039

3.486

2.137

sirtuin 5

CXCR5

NM_032966

1.986

3.437

1.815

chemokine (C-X-C motif) receptor 5

KLHL21

NM_014851

2.380

3.324

-

kelch-like 21 (Drosophila)

IL8

NM_000584

6.200

3.200

5.177

interleukin 8

DPYSL3

NM_001387

3.064

3.192

3.497

dihydropyrimidinase-like 3

RAB43

NM_198490

1.264

3.003

3.324

RAB43, member RAS oncogene familyprovided

TRIM16

NM_006470

3.990

2.992

3.453

tripartite motif-containing 16

C1orf66

NM_015997

0.382

2.970

1.953

chromosome 1 open reading frame 66

PIP4K2A

NM_005028

2.325

2.967

3.127

phosphatidylinositol-5-phosphate 4-kinase, type II, alpha

AGAP3

NM_031946

1.289

2.945

1.091

ArfGAP with GTPase domain, ankyrin repeat and PH domain 3

INPP4A

NM_001566

0.129

2.872

1.872

inositol polyphosphate-4-phosphatase, type I, 107kDa

DAGLA

NM_006133

1.545

2.834

2.807

diacylglycerol lipase, alpha

EREG

NM_001432

3.726

2.793

-

epiregulin

AGAP11

NM_133447

0.971

2.791

2.318

ankyrin repeat and GTPase domain Arf GTPase activating protein 11

ADAMTS10

NM_030957

0.683

2.773

0.952

ADAM metallopeptidase with thrombospondin type 1 motif, 10

KCP

NM_199349

1.269

2.746

1.861

kielin/chordin-like protein

ZFAT

NM_020863

1.758

2.743

2.002

zinc finger and AT hook domain containing

LZTR1

NM_006767

1.092

2.714

3.134

leucine-zipper-like transcription regulator 1

MON1B

NM_014940

0.234

2.712

-0.754

MON1 homolog B (yeast)

IL1B

NM_000576

5.162

2.700

2.684

interleukin 1, beta

STAM

NM_003473

-0.222

2.697

-0.150

signal transducing adaptor molecule (SH3 domain and ITAM motif) 1

MAFG

NM_032711

2.775

2.684

2.997

v-maf musculoaponeurotic fibrosarcoma oncogene homologG (avian)

OSGIN1

NM_182981

2.427

2.672

2.002

oxidative stress induced growth inhibitor 1

ZNF673

NM_017776

0.606

2.669

-

zinc finger family member 673

IREB2

NM_004136

-

2.648

-

iron-responsive element binding protein 2

GSTM3

NM_000849

3.632

2.635

2.543

glutathione S-transferase mu 3 (brain)

UBR2

NM_015255

2.160

2.619

2.371

ubiquitin protein ligase E3 component n-recognin 2

SFT2D3

NM_032740

1.146

2.577

1.609

SFT2 domain containing 3

FAM70A

NM_017938

-

2.577

2.614

family with sequence similarity 70, member A

The top 30 genes upregulated by oxLDL in M2

Gene Symbol

Ref Seq ID

M0

M1

M2

NCBI official full name

IFIT2

NM_001547

2.559

-

5.893

interferon-induced protein with tetratricopeptide repeats 2

IFIT1

NM_001001887

4.152

0.717

5.734

interferon-induced protein with tetratricopeptide repeats 1

IL8

NM_000584

6.200

3.200

5.177

interleukin 8

MX2

NM_002463

2.482

0.562

4.552

myxovirus (influenza virus) resistance 2 (mouse)

IFI44L

NM_006820

3.325

-0.074

4.474

interferon-induced protein 44-like

ISG20

NM_002201

3.839

0.952

4.442

interferon stimulated exonuclease gene 20kDa

IDO1

NM_002164

-

0.623

4.335

indoleamine 2,3-dioxygenase 1

PLSCR1

NM_021105

2.002

-0.179

4.119

phospholipid scramblase 1

IFIT3

NM_001549

2.306

-0.483

3.943

interferon-induced protein with tetratricopeptide repeats 3

SGPP2

NM_152386

3.229

-

3.626

sphingosine-1-phosphate phosphatase 2

OR9I1

NM_001005211

2.923

1.683

3.574

olfactory receptor, family 9, subfamily I, member 1

VNN1

NM_004666

0.568

0.896

3.569

vanin 1

DPYSL3

NM_001387

3.064

3.192

3.497

dihydropyrimidinase-like 3

TRIM16

NM_006470

3.990

2.992

3.453

tripartite motif-containing 16

EPSTI1

NM_033255

2.678

-0.642

3.439

epithelial stromal interaction 1 (breast)

AKR1C3

NM_003739

3.497

2.490

3.432

aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II)

RAB43

NM_198490

1.264

3.003

3.324

RAB43, member RAS oncogene family

APOL6

NM_030641

1.925

0.723

3.291

apolipoprotein L, 6

USP18

NM_017414

1.686

0.052

3.288

ubiquitin specific peptidase 18

ADM

NM_001124

3.973

4.807

3.202

adrenomedullin

RSAD2

NM_080657

1.858

0.083

3.194

radical S-adenosyl methionine domain containing 2

LZTR1

NM_006767

1.092

2.714

3.134

leucine-zipper-like transcription regulator 1

PIP4K2A

NM_005028

2.325

2.967

3.127

phosphatidylinositol-5-phosphate 4-kinase, type II, alpha

CSF2RA

NM_172247

1.683

2.109

3.115

colony stimulating factor 2 receptor, alpha, low-affinity (granulocyte-macrophage)

TNFRSF9

NM_001561

2.899

1.702

3.100

tumor necrosis factor receptor superfamily, member 9

ARHGAP44

NM_014859

1.136

1.919

3.053

Rho GTPase activating protein 44

RHOF

NM_019034

3.638

-

3.020

ras homolog gene family, member F (in filopodia)

CWF19L1

NM_018294

1.271

2.098

3.005

CWF19-like 1, cell cycle control (S. pombe)

MAFG

NM_032711

2.775

2.684

2.997

v-maf musculoaponeurotic fibrosarcoma oncogene homolog G (avian)

TNFRSF4

NM_003327

1.815

1.376

2.974

tumor necrosis factor receptor superfamily, member 4

Non-polarized (M0) and polarized M1 and M2 macrophages were treated with or without oxLDL for 6 h. The changes in gene expression were analyzed by cDNA microarray analysis as described in the Materials and methods section. The top 30 upregulated genes in each subset of macrophages are listed. The values denote fold changes (log2 ratio) of normalized intensities during oxLDL treatment. Letters in boldface indicate genes that were also upregulated by more than 2-fold in the two other subsets. "-" indicates genes that did not show significantly altered expression. Twenty-eight of the top 30 upregulated genes in M2 macrophages (93% of the top 30 genes) were also upregulated in M0, while 15 genes in the top 30 upregulated genes in M2 macrophages were upregulated in M1 cells.

Functional gene ontology

To identify oxLDL treatment-related biological functions of polarized macrophages, bioinformatics aspects of differentially expressed genes during oxLDL treatment were further analyzed using IPA software. The 1566, 1738, and 1749 genes of M0, M1, and M2 macrophages were identified by IPA software as functionally intentional genes, and categorized into 65, 84, and 80 groups according to functional gene ontology, respectively. Figure 3 shows the top 10 functional ontology categories which contain the molecules altered by oxLDL treatment. Eight of the top 10 ontology categories of M0 macrophages were also found in the top 10 of M2 macrophages, whereas only 3 ontology categories of M1 macrophages were found in the top 10 of M2.
Figure 3
Figure 3

Gene ontology analysis. The genes showing significantly upregulated expression in oxLDL-treated macrophages were functionally categorized into groups according to gene ontology. The top 10 functional ontology categories in each subset of macrophages (M0, M1, and M2) are shown in order of P-value. Right-tailed Fisher's exact test was used to calculate the P-value determining the probability that each biological function assigned to that network was due to chance alone. Eight of the top 10 ontology categories were found in both M0 and M2. *Common in M0 and M1. **Common in M0 and M2. *** Common in M0, M1, and M2.

Molecular network analysis

We performed molecular network analysis using IPA software to elucidate the molecular relationships when each subset of macrophages was treated with oxLDL. The top 5 highly scoring networks of each subset are shown in Table 2. Among these networks, the highest scoring network was found in M1 macrophages as Network #1, including molecules related to "carbohydrate metabolism," "DNA replication, recombination and repair," and "embryonic development." Interestingly, most of the molecules in network #1 were related to transforming growth factor (TGF)-β1 directly or indirectly (Figure 4).
Table 2

Molecular network analysis using IPA

Top 5 Networks in M0

ID

Molecules in Network

Score

Focus Molecules

Top Functions

#1

BARX2, CD226, CRABP1, CTH, Cytokeratin, FOLR2, FOS, GCLM, GCNT1, HHEX, HTATIP2, IGDCC3, KCNJ2, KRT8, KRT18, KRT20, KRT6A, LPCAT3, MAF, MAFB, MAFF, MAFG, MT1A, musculoaponeurotic fibrosarcoma oncogene, NFE2L3, NRF1, PAX6, PLAGL1, PVR, S100P, TGFBI, TH1 Cytokine, TLE1, TMSB4, TMSB4X

33

31

Cellular Assembly and Organization,

Cellular Function and Maintenance,

Hair and Skin Development and Function

#2

Alpha tubulin, APEX2, ARRDC3, CALB2, Calbindin, CSF1, CXORF21, DHCR24, DUB, EHMT2, FAM105A, FAM107A, GSC, HDAC6, HOOK2, LMO2, LRRC58, MIR124, NTRK3, oxidoreductase, POU5F1, PPP1R12C, REST, RYK, STAU1, TRPM2, TSPAN14, Tubulin, USP12, USP28, USP34, USP41, USP48, USP49, ZBED3

28

29

Auditory and Vestibular System Development and Function,

Genetic Disorder,

Metabolic Disease

#3

ANAPC1, ANAPC2, ANAPC5, APC, ASNS, BIRC3, CD3EAP, CDC20, CHD2, CKAP2, Cpt, CPT2, Cyclin A, DTYMK, E3 RING, EIF5, GLCE, GPC1, HEXIM1, HGF, IMPDH2, Integrin alpha V beta 3, ISG20, JMJD1C, MYL12A, NAMPT, POLR1A, SLC40A1, SPATC1, SPDEF, TMEM158, TOPBP1, UBR2, Vegf, VHL

28

29

Protein Degradation, Protein Synthesis,

Cardiovascular System Development and Function

#4

ALDH2, ASXL1, C14ORF1, CBX4, CCDC106, CDK5RAP2, Cyclooxygenase, DIO3, DUSP8, FHL1, JDP2, LSM2, MIP1, MYBPC3, P38 MAPK, PCGF2, PHYHIP, PLEKHN1, PORCN, SEMA7A, SENP2, Stat1 dimer, STK36, SUFU, Tnf receptor, TNFRSF9, TNFRSF11A, TNFSF9, TRAF, TRAF1, TRAF5, TRAF2-TRAF5, TRAIP, TREM1, WNT4

27

28

Cellular Assembly and Organization,

Cellular Function and Maintenance,

Skeletal and Muscular System Development and Function

#5

ANKRD29, APEX1, BAG5, CKB, CKMT1B, CNOT3, CNOT6, CNOT8, Creatine Kinase, DNAJA4, DNAJB6, DNAJC15, DNMBP, Fibrinogen, Hsp70, Hsp90, Hsp22/Hsp40/Hsp90, HSPA2, HSPA1A, ICAM1, IFT52, IP6K2, MIR1, Nos, Pka catalytic subunit, PLSCR1, SH3KBP1, SLC25A30, SPRY2, SRXN1, TIMP3, TNKS1BP1, TNPO2, TRIM2, XPNPEP3

26

27

Cellular Assembly and Organization,

Cellular Development,

Cellular Growth and Proliferation

Top 5 Networks in M1

ID

Molecules in Network

Score

Focus Molecules

Top Functions

#1

ABCD1, AQP8, C13ORF15, CALML4, CCNE2, CDC7, DBF4, FXYD6, GALM, GIN1, GYG1, GYG2, GYS1, H1FX, HNMT, HYAL2, LPCAT3, ORC2L, ORC5L, ORC6L, OVOL1, PDLIM5, PHGDH, PSPH, RBM19, RBMS3, SBNO2, SEMA7A, SLC23A2, SLC25A14, SLC35A1, TBC1D1, TGFB1, TRIM7, UGDH

41

35

Carbohydrate Metabolism,

DNA Replication, Recombination, and Repair,

Embryonic Development

#2

ATAD3B, CAPZB, CHTF18, CPD, CPT2, FXC1, FXYD1, GAMT, GPC1, GTF3C2, GTF3C4, HMG CoA synthase, HMGCS1, HNRNPA0, IQGAP3, IREB2, LMNB2, LSM2, LSM6, LSM7, MINA, Mir125b (mouse), MYC, Ndpk, NME2, NME6, NME7, PDS5B, RAD21, RANBP6, REC8, RFX2, RNGTT, RRM2B, TOR2A

34

32

Cellular Assembly and Organization,

Genetic Disorder,

Metabolic Disease

#3

ACTN4, APEX2, ATRX, DNA-directed DNA polymerase, Erm, HOOK2, ICAM1, IGSF8, LAS1L, LIG3, LIG4, LRSAM1, MARCH3, MYOZ1, NEIL1, NOM1, PNKP, POLE2, POLG2, POLM, RNF2, RNF6, RNF10, RNF25, RNF166, RNF185, SLC9A1, TRIM37, TRP, TRPC4, UBE2D2, UBE2E3, UBE2 H (includes EG:7328), VIL1, ZNRF1

34

32

DNA Replication, Recombination, and Repair,

Gene Expression,

Cell-mediated Immune Response

#4

ARL15, BRD1, BTBD2, C14ORF1, C1ORF103, CCDC106, CCDC90B, CD3EAP, CDKN3, DNA-directed RNA polymerase, EGLN1, EHMT2, FEZ1, FXR1, GTF3A, histone-lysine N-methyltransferase, KBTBD7, KIF11, KIF2C, KLK10, LMO2, MELK, POLR1A, POLR3 D, POLR3E, POLR3F, RIT1, SETDB1, S TAB2, THAP8 (includes EG:199745), TLE1, TMSB4X, UNC119, Vegf, ZNF24 (includes EG:7572)

30

32

Cell Cycle,

Cellular Assembly and Organization,

DNA Replication, Recombination, and Repair

#5

ABCC1, ABCD3, ACIN1, ATP5B, ATPase, CRISPLD2 (includes EG:83716), DTX1, DUSP1, E2F1, FABP1, FAM177A1, GRAP, IL34, Immunoglobulin, INHBA, INHBC, LIF, MAFF, MARK4, MIR124, MYH9, NAA15, NOTCH1, POMC, RDH10, RSF1, RYK, SKIV2L, SLC22A18AS, S TAB1, SYNGR2, Trypsin, WRN (includes EG:7486), WWP1, ZNF790

30

31

Cellular Development,

Nervous System Development and Function,

Hematological System Development and Function

Top 5 Networks in M2

ID

Molecules in Network

Score

Focus Molecules

Top Functions

#1

AKR1C3, ARL15, C1ORF103, CETN3, DCP1A, DCTPP1, DDX6, DDX20, DNA-directed RNA polymerase, DUB, ETV3, FILIP1L, FXYD6, GANAB, GSTM3 (includes EG:2947), GTF3C4, GXYLT1, JMJD1C, NUP155, POLR1A, POLR1E, POLR3F, PRMT1, RHOF, RIF1, SDCCAG8, SNRPA1, TARS, TRAF6, UNC119, USP20, USP41, USP45, VHL, ZFP161

36

33

Infection Mechanism,

Infectious Disease,

Cell Cycle

#2

ATN1, BANP, C8ORF4, CBFA2T2, CLK2, CLOCK, CRY1, DBP, EIF2B1, Fascin, H1FX, HIST1H2AE (includes EG:3012), Histone H1, HNRNPL, Importin beta, KHDRBS3, KIAA0913, LBR, LTBP4, LTC4 S, MAST2, OGG1, Pkc(s), RNF19A, SAFB, SDCBP2, SLC20A2, SRPK1, STK36, SUFU, TIMELESS, TM4SF1, TPM2, XPC, ZNF652

32

31

Behavior,

Nervous System Development and Function,

Lipid Metabolism

#3

ATF7IP, ATPase, BACE1, CAPN3, CASP2, CASP7, CASP10, Caspase, CTRL, ENTPD2, hydrolase, ICAM1, IDE, ITM2C, KIAA1632, KYNU, LGMN, MAGED1, MEOX2, NAIP, NANP, NGFR, NUP160, PAPPA, peptidase, PLEKHF2 (includes EG:79666), RECQL5, RTN3, SENP5 (includes EG:205564), SPPL2B, SPTBN1, THTPA, UBAC2, UBR5, XAF1

32

31

Protein Degradation,

Protein Synthesis,

Post-Translational Modification

#4

API5, ARRDC3, ASPH, Calbindin, CKB, CNNM4, COX11, COX15, COX10 (includes EG:1352), CPT1B, Creatine Kinase, Cytochrome c oxidase, ENO3, FICD, FOXP1, GPRASP2, HTR2B, HTT, JAKMIP1, KLF16, NDUFS1, NEFH, PHB (includes EG:5245), PPP1R16B, RCOR2, REST, RGS14, RNF34, SASH1, SCN4B, SNN, STRN, Thymidine Kinase, VAPA, WAC

32

31

Gene Expression,

Neurological Disease,

Cellular Compromise

#5

ALDH7A1, BPI, Cbp, Ciap, CLIP2, CST7, DBT, DUSP2, G0S2, GFPT2, KLF3, KRT19, lymphotoxin-alpha1-beta2, MICA, NFkB (complex), NKIRAS1, PELI3, POU2F2, RBCK1, REL/RELA/RELB, RNF216, S100P, S100Z, SH3RF1, SIAH2, SLC2A6, SNIP1, SUMO4, TNIP2, TRIM32, TRIM69, TXNRD1, WTAP, ZMYND11, ZNF71

30

30

Carbohydrate Metabolism,

Lipid Metabolism,

Small Molecule Biochemistry

Molecular network analysis was performed using IPA software. The top 5 groups of molecules in the network are listed in order of network score, which IPA calculates as shown below. We focused on the highest scoring network #1 in M1 macrophages, which had a score of 41 and included 35 molecules matched with the Ingenuity Knowledge Base.

Network Score = -log10(P-value)

Figure 4
Figure 4

Molecular network analysis of the highest scoring network in Table 2. The molecular network in network #1 of M1 macrophages shown in Table 2 and corresponding data of M0 and M2 macrophages are shown. Nearly all the molecules in M1 were related to TGF-β1 directly or indirectly. Molecules are represented as nodes, and the biological relationships between pairs of nodes are represented as lines. The intensity of the node color indicates the degree of upregulation (red) or downregulation (blue). The numbers below the nodes denote fold changes (log2 ratio) of normalized intensities during oxLDL treatment. Nodes are displayed using various shapes representing the functional class of the gene product. Lines are displayed with various labels describing the nature of the relationship between the nodes; i.e., A for Activation, E for Expression, LO for Localization, PD for Protein - DNA binding, PP for Protein - Protein binding, RB for Regulation of Binding, and T for Transcription.

Hierarchical cluster analysis

Hierarchical cluster analysis allows us to visually comprehend differential patterns over multiple microarray datasets. To analyze the hierarchical clusters over subsets of macrophages, we constructed a heat map where red and green indicated up- and downregulation, respectively (Figure 5). We employed 3196 genes, expression levels of which were significantly altered by oxLDL treatment in at least one subset of macrophages. A total of 251 genes were commonly identified as upregulated genes in all subsets of macrophages, including TRIM16, HMOX1, TXNRD1, GCLM, and DUSP1, all of which contain an antioxidant response element (ARE) in their promoter regions, which serves as a binding site for nuclear factor erythroid 2-related factor 2 (Nrf2). Hierarchical cluster analysis identified 3 clusters the genes of which were upregulated in one subset but not in the other subsets (Figure 5). Cluster A included 17 annotated genes that were upregulated in M0, but not in the other subsets (Table 3). The genes in cluster A belonged to ontology categories including "cell-mediated immune response," "cellular movement," "hematological system development and function," and "immune cell trafficking." There were 72 annotated genes in cluster B, which were specifically upregulated in M1. These 72 genes were related to "gene expression" and "cellular development." They included NFKB2, encoding nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), and PIK3R4, encoding phosphoinositide-3-kinase (PI3K), both of which are molecules related to the NF-κB signaling pathway. In cluster C, 28 annotated genes were identified as specifically upregulated in M2. These genes were associated with "carbohydrate metabolism," "lipid metabolism," and "small molecule biochemistry."
Figure 5
Figure 5

Heat map constructed by hierarchical cluster analysis. Red and green in the heat maps indicate up- and downregulation during oxLDL treatment, respectively. The left map includes 3196 genes expression levels of which were significantly altered by oxLDL treatment in at least one subset of macrophages. Genes in three clusters, denoted as A to C, were specifically upregulated only in one subset. There were 17, 72, and 28 annotated genes in cluster A, B, and C, respectively (right map).

Table 3

Genes included in each cluster

Cluster A.

Gene Symbol

Ref Seq ID

M0

M1

M2

NCBI official full name

CHI3L2

NM_004000

1.170712

0.008113

-0.453072

chitinase 3-like 2

PARP8

NM_024615

1.018767

0.147437

-0.206628

poly (ADP-ribose) polymerase family, member 8

MAP4K4

NM_145686

1.115230

-0.302140

-0.763189

mitogen-activated protein kinase kinase kinase kinase 4

CD44

NM_001001391

1.312520

-0.201936

-0.048693

CD44 molecule

CYP27B1

NM_000785

1.111109

-0.055705

-0.015432

cytochrome P450, family 27, subfamily B, polypeptide 1

SLC1A2

NM_004171

1.466166

-0.088519

0.225126

solute carrier family 1 (glial high affinity glutamate transporter), member 2

CKMT1

NM_020990

1.436353

0.264923

0.338353

creatine kinase, mitochondrial 1B

MYOD1

NM_002478

1.665245

0.211625

0.330158

myogenic differentiation 1

CD40

NM_001250

1.319905

-0.606033

0.379319

CD40 molecule, TNF receptor superfamily member 5

PRKAR2B

NM_002736

1.349370

-0.472458

0.347752

protein kinase, cAMP-dependent, regulatory, type II, beta

KIAA0913

NM_015037

1.244477

-0.318545

0.447287

KIAA0913

SNX10

NM_013322

1.138265

-0.351416

0.635676

sorting nexin 10

HHIP

NM_022475

1.177555

-0.537186

0.506197

hedgehog interacting protein

BIRC3

NM_001165

1.499122

-0.583781

-0.065715

baculoviral IAP repeat-containing 3

ZBTB7C

NM_001039360

1.293925

-1.001009

0.404020

zinc finger and BTB domain containing 7C

PACRGL

NM_145048

2.026548

-0.460488

0.187722

PARK2 co-regulated-like

CCL23

NM_005064

2.471357

-0.192407

-0.036793

chemokine (C-C motif) ligand 23

Cluster B.

Gene Symbol

Ref Seq ID

M0

M1

M2

NCBI official full name

NDN

NM_002487

-0.169667

1.457695

-0.617320

necdin homolog (mouse)

FAM129C

NM_173544

-0.110208

1.321559

-0.841954

family with sequence similarity 129, member C

NME6

NM_005793

-0.315804

1.345342

-0.759802

non-metastatic cells 6, protein expressed in (nucleoside-diphosphate kinase)

TMEM99

NM_145274

-0.390527

1.497197

-0.676125

transmembrane protein 99

ATP2B1

NM_001682

-0.371182

1.535878

-0.824423

ATPase, Ca++ transporting, plasma membrane 1

KIF1A

NM_004321

-0.362399

1.333531

-0.928733

kinesin family member 1A

TMEM188

NM_153261

-0.375073

1.157188

-0.955238

transmembrane protein 188

PLEKHG4

NM_015432

-0.414580

1.052587

-0.706013

pleckstrin homology domain containing, family G (with RhoGef domain) member 4

NOXA1

NM_006647

-0.322207

1.015456

-0.592170

NADPH oxidase activator 1

CUX1

NM_001913

-0.236206

1.147118

-0.551592

cut-like homeobox 1

SBNO2

NM_014963

-0.294540

1.271977

-0.630553

strawberry notch homolog 2 (Drosophila)

TIGD5

NM_032862

-0.376362

1.224225

-0.526717

tigger transposable element derived 5

CTSL2

NM_001333

-0.428772

1.153800

-0.359408

cathepsin L2

ZNF24

NM_006965

-0.385988

1.069071

-0.360632

zinc finger protein 24

STK32C

NM_173575

-0.634840

1.124350

-0.544619

serine/threonine kinase 32C

NUDT17

NM_001012758

-0.524910

1.111636

-0.519731

nudix (nucleoside diphosphate linked moiety X)-type motif 17

CALML4

NM_033429

-0.433422

1.089113

-0.492229

calmodulin-like 4

C7orf33

NM_145304

-0.435642

1.106760

-0.512504

chromosome 7 open reading frame 33

FBXO22

NM_012170

-0.203452

1.309369

-0.481216

F-box protein 22

LINGO1

NM_032808

-0.174931

1.312372

-0.445152

leucine rich repeat and Ig domain containing 1

ZMYND19

NM_138462

-0.243665

1.351306

-0.517691

zinc finger, MYND-type containing 19

CCR1

NM_001295

-0.165232

1.419892

-0.480175

chemokine (C-C motif) receptor 1

ATRX

NM_000489

-0.498685

1.285463

-0.371568

alpha thalassemia/mental retardation syndrome X-linked

FRMD4B

NM_015123

-0.477381

1.252296

-0.287931

FERM domain containing 4B

CEP164

NM_014956

-0.419638

1.269277

-0.283263

centrosomal protein 164kDa

LANCL2

NM_018697

-0.393507

1.324253

-0.242555

LanC lantibiotic synthetase component C-like 2 (bacterial)

UBA5

NM_024818

-0.478021

1.390785

-0.479541

ubiquitin-like modifier activating enzyme 5

GGCX

NM_000821

-0.380201

1.330677

-0.476370

gamma-glutamyl carboxylase

FOXN3

NM_005197

-0.772837

1.196028

-0.268613

forkhead box N3

TP53BP1

NM_005657

-0.797822

1.249895

-0.460222

tumor protein p53 binding protein 1

ORC6

NM_014321

-0.633454

1.305326

-0.322922

origin recognition complex, subunit 6

ST7

NM_018412

-0.678132

1.309238

-0.290689

suppression of tumorigenicity 7

OGFOD1

NM_018233

0.061541

1.413933

-0.438702

2-oxoglutarate and iron-dependent oxygenase domain containing 1

UBE2B

NM_003337

0.146284

1.431435

-0.295974

ubiquitin-conjugating enzyme E2B (RAD6 homolog)

GALNTL4

NM_198516

0.178950

1.470650

-0.270045

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-like 4

HSPA14

NM_016299

0.185892

1.419392

-0.264205

heat shock 70kDa protein 14

PEX13

NM_002618

0.137507

1.527385

-0.274508

peroxisomal biogenesis factor 13

ATP2B2

NM_001001331

0.084953

1.689567

-0.394052

ATPase, Ca++ transporting, plasma membrane 2

TAAR2

NM_014626

0.168770

1.695874

-0.439255

trace amine associated receptor 2

HUS1

NM_004507

0.098273

1.666199

-0.226370

HUS1 checkpoint homolog (S. pombe)

RIC8B

NM_018157

0.214093

1.650594

-0.307883

resistance to inhibitors of cholinesterase 8 homolog B (C. elegans)

DNAJC17

NM_018163

0.172153

1.482705

-0.492935

DnaJ (Hsp40) homolog, subfamily C, member 17

TMEM130

NM_152913

0.206368

1.399311

-0.480833

transmembrane protein 130

AP2A2

NM_012305

0.285524

1.391232

-0.458091

adaptor-related protein complex 2, alpha 2 subunit

TTTY13

NR_001537

0.381981

1.560081

-0.451429

testis-specific transcript, Y-linked 13 (non-protein coding)

AKT1S1

NM_032375

0.385837

1.517956

-0.371995

AKT1 substrate 1 (proline-rich)

WNT3

NM_030753

0.009319

1.560600

-0.801987

wingless-type MMTV integration site family, member 3

ZNF443

NM_005815

0.154517

1.457293

-0.742146

zinc finger protein 443

TFCP2L1

NM_014553

0.207710

1.525949

-0.955561

transcription factor CP2-like 1

KIAA0355

NM_014686

0.278629

1.724807

-0.576470

KIAA0355

ASAP2

NM_003887

0.524513

1.733462

-0.459752

ArfGAP with SH3 domain, ankyrin repeat and PH domain 2

C14orf49

NM_152592

0.578042

1.624871

-0.365254

chromosome 14 open reading frame 49

NFKB2

NM_002502

0.652839

1.882528

-0.343188

nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100)

ZNF586

NM_017652

0.328381

1.848104

-0.284595

zinc finger protein 586

RNMTL1

NM_018146

0.277170

1.645924

-0.133286

RNA methyltransferase like 1

FOSL2

NM_005253

0.386974

1.569486

-0.229451

FOS-like antigen 2

SATB1

NM_002971

0.542138

1.252296

-0.749110

SATB homeobox 1

ASB6

NM_177999

0.666548

1.536652

-0.584820

ankyrin repeat and SOCS box-containing 6

SLC5A3

NM_006933

0.682674

1.388148

-0.424733

solute carrier family 5 (sodium/myo-inositol cotransporter), member 3

C8orf84

NM_153225

0.637255

1.709652

-0.981619

chromosome 8 open reading frame 84

MON1B

NM_014940

0.234404

2.712191

-0.753618

MON1 homolog B (yeast)

PIGO

NM_152850

0.134053

2.574539

-0.004491

phosphatidylinositol glycan anchor biosynthesis, class O

STAM

NM_003473

-0.221822

2.697280

-0.150394

signal transducing adaptor molecule (SH3 domain and ITAM motif) 1

CCDC45

NM_138363

-0.046055

2.118025

-0.312274

coiled-coil domain containing 45

GYS1

NM_002103

-0.699205

1.685994

-0.629003

glycogen synthase 1 (muscle)

RENBP

NM_002910

-0.403657

1.957579

-0.580833

renin binding protein

BTD

NM_000060

-0.348646

1.647239

-0.685758

biotinidase

FAM172A

NM_032042

-0.409833

1.717425

-0.665763

family with sequence similarity 172, member A

PIK3R4

NM_014602

-0.188899

2.325311

-0.729078

phosphoinositide-3-kinase, regulatory subunit 4

RSAD1

NM_018346

-0.251002

2.260460

-0.748812

radical S-adenosyl methionine domain containing 1

ZNF626

NM_145297

-0.252970

1.842391

-0.910235

zinc finger protein 626

CTH

NM_001902

-1.060839

2.365953

-1.038817

cystathionase (cystathionine gamma-lyase)

Cluster C.

Gene Symbol

Ref Seq ID

M0

M1

M2

NCBI official full name

MXD3

NM_031300

-0.800223

0.116059

1.984245

MAX dimerization protein 3

CD14

NM_000591

-1.193454

-0.266058

2.053786

CD14 molecule

BARX1

NM_021570

-1.650218

0.236670

1.728783

BARX homeobox 1

ZNF331

NM_018555

-1.729498

-0.028187

1.786943

zinc finger protein 331

GPR54

NM_032551

-0.085064

-0.929503

1.669680

KISS1 receptor

ANP32A

NM_006305

-0.155917

-0.929503

1.827632

acidic (leucine-rich) nuclear phosphoprotein 32 family, member A

C19orf54

NM_198476

-0.205921

-0.803972

1.634746

chromosome 19 open reading frame 54

G0S2

NM_015714

-0.367904

-0.858834

2.131860

G0/G1switch 2

SPATA3

NM_139073

-0.301818

-0.432004

1.766064

spermatogenesis associated 3

FOXA2

NM_153675

-0.306075

-0.533883

1.983144

forkhead box A2

PSORS1C2

NM_014069

-0.423428

-0.713119

1.449567

psoriasis susceptibility 1 candidate 2

SNN

NM_003498

-0.492216

-1.239138

1.185086

stannin

CDKN2B

NM_078487

-0.404103

-0.847300

1.024029

cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)

ZNF257

NM_033468

-0.916132

-1.388935

1.189490

zinc finger protein 257

ZBTB4

NM_020899

-0.296438

-1.533325

0.937391

zinc finger and BTB domain containing 4

CASP2

NM_032983

-1.527530

-0.751505

1.026536

caspase 2, apoptosis-related cysteine peptidase

TCTEX1D1

NM_152665

-1.661006

-0.348442

1.280638

Tctex1 domain containing 1

FILIP1L

NM_014890

-0.967885

-0.212315

1.018932

filamin A interacting protein 1-like

USP52

NM_014871

-0.885665

-0.310932

1.097774

PAN2 poly(A) specific ribonuclease subunit homolog (S. cerevisiae)

SETD8

NM_020382

-1.020324

-0.599401

0.697655

SET domain containing (lysine methyltransferase) 8

C13orf31

NM_153218

-1.442594

-0.330041

0.688789

chromosome 13 open reading frame 31

POLD1

NM_002691

-1.311086

-0.314006

0.812769

polymerase (DNA directed), delta 1, catalytic subunit 125kDa

DNMT3A

NM_022552

-1.207327

-0.289399

0.489584

DNA (cytosine-5-)-methyltransferase 3 alpha

TGFBI

NM_000358

-1.324217

-0.324120

0.308245

transforming growth factor, beta-induced, 68kDa

ANP32C

NM_012403

-1.493138

-0.380761

0.494101

acidic (leucine-rich) nuclear phosphoprotein 32 family, member C

ITGA2B

NM_000419

-1.109218

0.018385

1.026536

integrin, alpha 2b (platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41)

RGS3

NM_130795

-1.022299

-0.038087

0.656812

regulator of G-protein signaling 3provided

MARCH1

NM_017923

-1.374411

0.233896

0.729622

membrane-associated ring finger (C3HC4) 1provided

The top functional ontology categories

 

Cluster A.

P -value

   Cell-mediated Immune Response

9.46E-06-2.65E-02

   Cellular Movement

9.46E-06-3.76E-02

   Hematological System Development and Function

9.46E-06-3.57E-02

   Immune Cell Trafficking

9.46E-06-2.49E-02

   Cardiovascular Disease

2.64E-05-3.76E-02

Cluster B.

P -value

   Cell Cycle

1.31E-04-3.28E-02

   Cell Cycle

1.31E-04-3.28E-02

   DNA Replication, Recombination, and Repair

1.31E-04-4.51E-02

   Gene Expression

2.16E-03-3.35E-02

   Cellular Development

3.51E-03-3.64E-02

   Nervous System Development and Function

3.51E-03-4.7E-02

Cluster C.

P -value

   Carbohydrate Metabolism

1.12E-04-4.09E-02

   Lipid Metabolism

1.12E-04-4.09E-02

   Small Molecule Biochemistry

1.12E-04-4.09E-02

   Cell Death

3.76E-04-4.81E-02

   Nervous System Development and Function

7.79E-04-3.33E-02

Three clusters were extracted from the heat map shown in Figure 5. There were 17, 72, and 28 annotated genes in clusters A, B, and C, respectively. The top functional ontology categories and the corresponding P-values in each subset are shown. NFKB2 and PIK3R4 were found in cluster B. The values denote fold changes (log2 ratio) of normalized intensities during oxLDL treatment.

Quantitative real-time RT-PCR analysis

All data from cDNA microarray analysis in the present study were comparisons between cells with and without oxLDL treatment. To compare gene expression levels among subsets of macrophages, we performed quantitative real-time RT-PCR for two genes: IL8 listed in the top 30 genes that were commonly upregulated in all subsets of macrophages (Table 1) and HMOX1 encoding heme oxygenase (HO)-1 as a representative of genes containing an ARE.

Consistent with previous reports, the expression levels of IL8 were higher in non-stimulated M1 macrophages than in M2 [10], and oxLDL treatment induced higher levels of IL8 expression in M0 macrophages [13, 14] (Figure 6A). Moreover, the expression level of IL8 was significantly upregulated by oxLDL treatment in M1 macrophages, whereas its expression level after oxLDL treatment in M2 was markedly lower than those in M0 and M1 macrophages (P < 0.05). It has been known for several decades that oxLDL treatment increases, while IL-4 treatment decreases IL-8 production in human monocyte-derived macrophages [13, 15]. However, a recent report of microarray analysis indicated that oxLDL treatment induced no changes in human monocyte-derived macrophages [11]. This may have been due to various factors, such as individual variations or duration of oxLDL treatment [16].
Figure 6
Figure 6

Quantitative real-time RT-PCR. A. IL8 mRNA expression levels. The expression level of IL8 was significantly upregulated by oxLDL treatment in M1 macrophages (P < 0.05). IL8 expression level after oxLDL treatment in M2 was markedly lower than those in M0 and M1 macrophages (P < 0.05). B. HMOX1 mRNA expression levels. OxLDL treatment significantly enhanced HMOX1 gene expression by 7.6-fold (**P < 0.0005), 5.8-fold (*P < 0.05), and 5.9-fold (*P < 0.05) in M0, M1, and M2 macrophages, respectively. HMOX1 gene expression levels were almost the same in all 3 subsets of macrophages even after oxLDL treatment. Each bar shows the mean ± SD of 3 experiments.

HO-1 is expressed in vascular endothelial cells and macrophages in the early stages of atherosclerotic lesions and in foam cells in the advanced stages, and is known for its antiinflammatory actions [17, 18]. HMOX1 is known to be upregulated by oxidized phospholipids in murine macrophages polarized toward M1 or M2 [19]. Treatment with oxLDL yielded markedly higher levels of HMOX1 expression in all subsets of macrophages: i.e., 7.6-fold (P < 0.0005), 5.8-fold (P < 0.05), and 5.9-fold (P < 0.05) changes in M0, M1, and M2 macrophages compared to corresponding non-treated controls, respectively. HMOX1 gene expression levels were almost the same in all 3 subsets of macrophages even after oxLDL treatment (Figure 6B).

Discussion

In the present study, we demonstrated transcriptional alterations during oxLDL treatment, which has been suggested to be a model of the early stages of foam cell formation, in human polarized macrophages. Our study demonstrated that: 1) 93% of the top 30 genes upregulated by oxLDL treatment in M2 macrophages were also upregulated in M0; 2) the top 10 functional ontology categories in M2 macrophages were similar to those in M0; 3) almost all of the molecules in the highest scoring molecular network of M1 were related either directly or indirectly to TGF-β1; 4) there were commonly upregulated genes in all subset of macrophages, some of which contained ARE in their promoter regions; 5) hierarchical cluster analysis revealed a cluster specifically upregulated in M1, including genes encoding molecules related to the NF-κB signaling pathway; 6) in quantitative real-time RT-PCR, the level of IL8 gene expression after oxLDL treatment in M2 macrophages was markedly lower than those in M0 and M1 macrophages; and 7) HMOX1 gene expression levels were almost the same in all 3 subsets of macrophages even after oxLDL treatment.

The top genes expression of which was upregulated by oxLDL treatment in M2 but not M1 macrophages were highly correlated with the genes that were upregulated in M0 (Table 1). Moreover, the top altered ontology categories during oxLDL treatment in M2 macrophages were more similar to those of M0 than M1 (Figure 3). It has been reported that M-CSF-induced macrophages (M0 macrophages in the present study) have a similar transcriptional profile to M2 macrophages [10]. The transcriptional alteration during oxLDL treatment in M2 macrophages was also relatively similar to that in M0 macrophages but not M1 cells (Table 1 and Figure 3).

The data of hierarchical cluster analysis are shown in Figure 5. Commonly upregulated genes in all subsets of macrophages included some ARE-containing genes; e.g., TRIM16, HMOX1, TXNRD1, GCLM, and DUSP1. TRIM16, HMOX1, TXNRD1, GCLM, and DUSP1 encode tripartite motif-containing 16 (TRIM16), HO-1, thioredoxin reductase (Txnrd) 1, glutamate-cysteine ligase, modifier subunit (GCLM), and dual specificity phosphatase (DUSP) 1, respectively. These genes were upregulated during oxLDL treatment in all subsets of macrophages in the present study. ARE is a binding site for the transcription factor Nrf2 [20]. The Nrf2-ARE pathway plays a crucial role in protection against oxidative stress [21]. On exposure to oxidative stress, Nrf2 translocates to the nucleus, binds to the ARE [22], and activates the genes, including TRIM16, HMOX1, TXNRD1, GCLM, and DUSP1. These data were consistent with the recent report that oxidized phospholipids upregulated expression of ARE-containing genes in murine bone marrow-derived macrophages [19].

TGF-β has been suggested to have antiinflammatory properties [23], and it thought to be produced by alternatively activated macrophages [24]. Activation of M1 macrophages might be altered by M2-derived TGF-β. As TGF-β downregulates scavenger receptors, such as scavenger receptor type A (SR-A) I/II and CD36 [25], and upregulates ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1 [26], TGF-β is also thought to have protective effects against the development of atherosclerosis. However, the contribution of TGF-β to the development of atherosclerosis is more complicated, taking account of clinical data. It is controversial whether TGF-β levels in blood from patients are positively or negatively correlated with cardiovascular disease [27]. In molecular network analysis, the molecules in the highest scoring network of M1 macrophages, but not M0 or M2, were related directly or indirectly to TGF-β1 (Figure 4). However, no molecules in the known TGF-β signal transduction pathway, including TGF-β receptors (TβRs) and SMADs, were altered by oxLDL treatment in this study. The results of cDNA microarray analysis (Figure 4) and real-time RT-PCR analysis (data not shown) indicated that oxLDL treatment slightly induced TGF-β1 gene expression in M1 macrophages. TGF-β generally plays an important role in maintaining normal vessel wall conditions, including the expression of contractile proteins in vascular smooth muscle cells (VSMCs) [28]. Under atherogenic conditions, however, TGF-β reduces extracellular matrix production from VSMCs and enhances leukocytes recruitment to atherosclerotic plaques, resulting in plaque rupture. Our results suggest that TGF-β-related molecules were affected by oxLDL stimulation, and that TGF-β promoted proinflammatory activities in M1 macrophages as in VSMCs. These findings suggest that oxLDL regulates the functions of M1 macrophages through an as yet unknown TGF-β-mediated cascade. It is therefore necessary to elucidate the detailed TGF-β-related functions regulated by oxLDL stimulation in various cells.

NF-κB is present in an inactive form bound to an inhibitor protein (I-κB) in the cytoplasm. On stimulation, NF-κB is released from I-κB, is translocated to the nucleus, and binds to the promoter DNA, followed by production of many types of inflammatory cytokine [29, 30]. The NF-κB signaling pathway is known to be activated by oxLDL in a CD36-dependent manner [31]. Interestingly, cluster B included genes related to the NF-κB signaling pathway, such as NF-κB and PI3K (Figure 5 Table 3). The results of molecular network analysis indicated that oxLDL treatment induced upregulation of the growth factor receptor-mediated NF-κB signaling pathway in M1 but not M0 or M2 macrophages, while I-κB was upregulated in M0 and M2 but not M1 (Additional file 2). Thus, it seems that oxLDL stimulated the NF-κB signaling pathway specifically in M1.

There have been some reports partially conflicting with this study [10, 11], probably due to differences in experimental conditions, such as oxLDL concentrations, TBARS levels, or duration of treatment. Further studies are required to determine whether M1 macrophages contribute to foam cell formation. In this study, we primarily measured mRNA levels, and all samples were obtained from healthy volunteers. Measurements of protein levels and data derived from atherosclerotic subjects should be included in the next study.

Conclusions

The present study demonstrated the effects of oxLDL on transcriptional alterations in polarized macrophages. The data suggested that oxLDL uptake may affect TGF-β1- and NF-κB-mediated functions of M1 macrophages, but not M0 or M2 macrophages. It is likely that M1 macrophages characteristically respond to oxLDL. Further studies are required to evaluate the roles of TGF-β1- and NF-κB-mediated macrophage functions in the early stages of foam cell formation.

Declarations

Acknowledgements and Funding

This study was supported in part by the "High-Tech Research Center" Project for Private Universities: matching fund subsidy, by a Grant-in-Aide (S0991013) for the Foundation of Strategic Research Projects in Private Universities from Ministry of Education, Culture, Sport, Science, and Technology, Japan, and by the Mizutani Foundation for Glycoscience (to K. I.).

Authors’ Affiliations

(1)
Department of Cardiovascular Medicine, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
(2)
Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1, Tomioka, Urayasu City, Chiba 278-0021, Japan

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© Hirose et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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