gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

CXCL4 induces a unique transcriptome in monocyte-derived macrophages

Meeting Contribution

  • corresponding author C. A. Gleissner - University of Heidelberg, Germany; La Jolla Institute for Allergy & Immunology, La Jolla/California, U.S.A.
  • I. Shaked - University of Heidelberg, Germany
  • K. M. Little - University of Heidelberg, Germany
  • K. Ley - University of Heidelberg, Germany

Deutsche Gesellschaft für Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Düsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf15

DOI: 10.3205/10dgaf15, URN: urn:nbn:de:0183-10dgaf150

Published: March 23, 2011

© 2011 Gleissner et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

In atherosclerotic arteries, blood monocytes differentiate to macrophages in the presence of growth factors like macrophage colony-stimulation factor (MCSF) and chemokines like platelet factor 4 (CXCL4). To compare the gene expression signature of CXCL4-induced macrophages with MCSF-induced macrophages or macrophages polarized with IFN-γ/LPS (M1) or IL-4 (M2), we cultured primary human peripheral blood monocytes for six days. mRNA expression was measured by Affymetrix gene chips and differences were analyzed by Local Pooled Error test, Profile of Complex Functionality and Gene Set Enrichment Analysis. 375 genes were differentially expressed between MCSF- and CXCL4-induced macrophages, 206 of them overexpressed in CXCL4 macrophages coding for genes implicated in the inflammatory/immune response, antigen processing/presentation, and lipid metabolism. CXCL4-induced macrophages overexpressed some M1 and M2 genes and the corresponding cytokines at the protein level, however, their transcriptome clustered with neither M1 nor M2 transcriptomes. They almost completely lost the ability to phagocytose zymosan beads. Genes linked to atherosclerosis were not consistently up- or downregulated. Scavenger receptors showed lower and cholesterol efflux transporters higher expression in CXCL4- than MCSF-induced macrophages, resulting in lower LDL content. We conclude that CXCL4 induces a unique macrophage transcriptome distinct from known macrophage types, defining a new macrophage differentiation that we propose to call M4.


Introduction

During atherogenesis blood monocytes are thought to enter the arterial wall and differentiate into macrophages, which sustain an inflammatory milieu and promote plaque formation.

As demonstrated by in vitro and in vivo data, macrophages present in chronically inflamed tissues may assume different phenotypes. The best defined polarization types are M1 and M2 [1]. According to the “classical” paradigm, M1 macrophages can be obtained through activation by interferon-γ (IFNγ), tumor necrosis factor-α (TNF-α), or lipopolysaccharide (LPS)), whereas the “alternative” M2 macrophages can be induced through activation by interleukin-4 (IL-4), IL-10, or IL-13. M1 and M2 are probably not the only macrophage phenotypes present in vivo.

In atherosclerosis, there is evidence for the presence of several different macrophage phenotypes within atherosclerotic plaques, some with features of M1 and M2 [2]. In addition, other differentiation types like CD14-CD68+ and CD14+CD68- macrophages have been identified in coronary artery lesions (10). In vivo, differentiation of macrophages toward different phenotypes has been associated with certain drugs, growth factors, and other mediators. M2 differentiation is induced by PPARγ agonists, whereas MCSF preferentially induces CD14+CD68+ macrophages [3] and hemoglobin-haptoglobin promotes differentiation toward CD163highHLA-DRlow macrophages [4].

Macrophage colony-stimulating factor (MCSF) and platelet factor-4 (CXCL4) are known to promote differentiation of monocytes into macrophages in vitro. Knock-out mice lacking MCSF (CSF1) are protected from atherogenesis [5]. CXCL4 inhibits monocyte apoptosis and promotes macrophage differentiation, too [6]. It is released from platelets upon activation in micromolar concentrations. In vivo, presence of CXCL4 within atherosclerotic lesions has been shown to correlate with clinical parameters [7]. Eliminating the PF4 gene coding for CXCL4 by homologous recombination has been shown to reduce lesion formation in a mouse model of atherosclerosis [8].

While the transcriptomes of MCSF-induced macrophages and their M1 or M2 polarizations have been extensively studied [1], the published data on the phenotype of CXCL4-induced macrophages is scarce. Furthermore, it remains unclear whether the CXCL4 macrophage can be attributed to any of the known polarization patterns.


Methods

Monocyte-derived macrophages: Human peripheral blood was obtained with approval from the institutional review board from healthy volunteer donors. Briefly, monocytes were isolated from human peripheral blood by gradient centrifugation and subsequent negative bead isolation. Monocytes were cultured for six days in the presence of 100 ng/mL MCSF or 1 µmol/L CXCL4.

Affymetrix gene chip experiments: For each condition RNA was isolated and hybridized to a separate gene array.

ELISA and cytokine bead arrays: Protein concentration of selected cytokines was measured in cell culture supernatants using ELISA or cytokine bead arrays.

Statistics: For statistical analysis, the open source statistical software package R (http://www.r-project.org/) was used including the Local Pooled Error (LPE) test for differential expression discovery between two conditions [9]. To assess functional networks regulated in each macrophage type, profiled complex functionality was analyzed employing the ProfCom software [9]. Gene set enrichment was analyzed using an open access software for gene set enrichment analysis (GSEA) (28) to assess potential similarities between the CXCL4-induced gene expression profile and the known M1 and M2 signatures. The latter were extracted from gene expression data of monocyte-macrophage differentiation and polarization published by Mantovani et al. [1] (GEO data set 2430). Hierarchical clustering was used to determine the level of similarity between the three normalized groups [9]. All genes were included in the analysis.


Results

After six days in culture both MCSF- as well as CXCL4-induced macrophages (which we suggest to call M0 and M4, respectively) displayed a morphology characteristic of macrophages. When comparing the overall gene expression signature of M0 and M4 macrophages, the two gene expression patterns were found to be similar and highly correlated (r=0.934, P<0.0001, see Figure 1 [Fig. 1]). Out of 26,051 probe sets expressed above the detection limit in at least one macrophage sample, 460 annotated probe sets were significantly up- or downregulated with a FDR<0.05 corresponding to a total number of 375 regulated genes. Two hundred six of these genes displayed higher and 169 lower expression levels in M4 macrophages as compared to M0 macrophage.

Based on the genes found to be differentially expressed between M0 and M4 macrophages by LPE test, we sought to identify functional processes as defined by gene ontology that were transcriptionally overrepresented in M4 macrophages. Applying profiling of complex functionality (ProfCom) analysis, we found a number of biological processes that were associated with genes expressed in M0 or M4 macrophages. Most prominently, both M0 and M4 macrophage overexpressed genes related to the inflammatory and the immune response. In M4 macrophages, CCL18 and TNFSF10 (TRAIL) were overexpressed, whereas in M0 macrophages AIF1 (allograft inflammatory factor), ALOX5 (arachidonate 5-lipoxygenase), and IL1RN (interleukin-1 receptor antagonist) were found in the inflammation and immune response gene sets (in all cases P<0.05).

Genes involved in antigen processing and presentation were significantly overrepresented in M4 macrophages (P<0.05), including HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DQA1 (coding for MHC class II), as well as the costimulatory surface molecule CD86. By contrast, genes overexpressed in MCSF-induced macrophages were more likely to be implicated in chemotaxis (represented by the chemokines CCL3,CCL7, and the chemokine receptor CCR1, P<0.05) or cell adhesion (as indicated by the integrin genes ITGAV, ITGA6, ITGB8B, and the COL6A gene coding for the extracellular matrix component collagen 6A, P<0.05).

M0 macrophages display a gene expression pattern very similar to that of M2 macrophages, whereas the gene signature of M1 macrophages is distinct. We therefore sought to assess whether the M4 macrophage transcriptome is comparable to either of these polarization types. When looking at selected genes characteristic for M1 or M2 polarization, it became clear that a large number of polarization marker genes were not differentially expressed between M0 and M4 macrophages. On the other hand, a small number of marker genes displayed significant differential expression between M0 and M4 macrophages, however, there was no clear pattern for preferential expression of M1 or M2 markers in either of the macrophage types. Measuring protein levels of cytokines released into cell culture supernatants largely confirmed this pattern with IL-6, TNF (both M1), CCL18, and CCL22 (both M2) levels being higher in M4 macrophages, and IL-10 (M2) levels higher in M0 macrophages. No differences were seen for the levels of IL-1β, IL-8, and IL-12p70.

To generate larger gene sets for M1 or M2 polarization, we compared gene array data sets for M1 and M2 polarized macrophages published by Mantovani et al. [1]. Genes with a FDR<0.05 as determined by LPE testing between M1 and M2 were included in the gene sets. Using these gene sets, we performed gene set enrichment analysis (GSEA) for M1 and M2 genes. This demonstrated no significant overexpression of either of the gene sets in M0 or M4 macrophages and establishes that M4 macrophages are neither M1 nor M2 but represent a distinct phenotype.

To corroborate this hypothesis, we employed hierarchical clustering, now including all genes of the M1, M2, and M4 macrophage expression data. This analysis confirmed that the M4 transcriptome significantly differs from M0, M1 or M2 macrophages and represents a unique macrophage phenotype. In fact, M1 and M2 are more similar to each other than to M4.

To understand the potential relevance of CXCL4-induced macrophages in atherogenesis, we further investigated the list of differentially expressed genes. As indicated by gene ontology analysis, these included several chemokines, matrix metalloproteases and two members of the cathepsin family, but also some genes implicated in lipid metabolism and foam cell formation. Most of these gene groups did not display a consistent pattern, indicating that both pro-atherogenic and anti-atherogenic genes were expressed in M4 macrophages. Thus, while MMP7 showed higher expression in M4 than M0 macrophages, MMP8 and MMP12 expression were higher in M0 macrophages.


Discussion

This paper reports the first comprehensive analysis of the transcriptome of monocyte-derived macrophages induced by the chemokine platelet factor-4 (CXCL4). Our study demonstrates that (1) CXCL4 induces a macrophage phenotype that is distinct from that induced by MCSF, (2) the CXCL4-induced transcriptome shares similarities with, but is also distinct from each of the classical M1 and M2 phenotypes, and (3) the transcriptome of CXCL4-induced macrophages is not clearly pro- or anti-atherogenic. Based on its unique properties, we suggest calling the macrophage polarization induced by CXCL4 ‘M4 macrophages’.

Our knowledge about heterogeneity of polarized macrophages has increased significantly over the past several years. Thus, in addition to the “classical” M1 macrophages (characterized by high expression of pro-inflammatory cytokines, iNOS expression and production of reactive oxygen species) and M2 macrophages (expressing high levels of mannose receptor, dectin-1 and arginase), a number of M2 subsets has been characterized [1]. These include M2 macrophages activated by IL-4 or IL-13 (now termed M2a), macrophages activated by immune complexes (termed M2b), and macrophages polarized with glucocorticoids or IL-10 (M2c). The MCSF-induced transcriptome as well as the corresponding M1 and M2 (more specifically M2a) transcriptomes have been studied by Mantovani et al. using Affymetrix gene chips [1]. These experiments showed a close similarity between MCSF-induced and M2a macrophages. Furthermore, they demonstrated differences between M1 and M2a in genes involved in metabolic activities as well as genes coding for chemokines.

Similar to MCSF, the platelet chemokine CXCL4 has been demonstrated to prevent monocyte apoptosis and promote macrophage differentiation from human peripheral blood monocytes [6]. Surprisingly, the phenotype of these CXCL4-induced macrophages has not been studied in detail. Our data suggest that CXCL4 induces a macrophage phenotype that shares similarities with both M1 and M2 macrophages. Thus, some M1- as well as M2-related genes are overexpressed in M4 macrophages as compared to MCSF-induced macrophages. This finding was confirmed for a number of cytokines on the protein level. Most importantly, an unbiased analysis using different approaches like gene set enrichment, modified principal component, and hierarchical clustering analysis all confirmed the uniqueness of the CXCL4-induced macrophage transcriptome.

Platelets as well as monocytes and monocyte-derived macrophages are present within atherosclerotic lesions, and it is now clear that both contribute to lesion formation [10]. The platelet chemokine CXCL4 is known to promote atherosclerosis as demonstrated in CXCL4-deficient PF4-/- mice. On the Apoe-/- background, the PF4-/- mice showed about 60% reduction of lesion size in the aorta [8]. One way by which CXCL4 may contribute to atherogenesis is by promoting macrophage differentiation from monocytes present in the arterial wall. It had been speculated that CXCL4 may induce a macrophage polarization favorable for the development of atherosclerotic lesions. Our in vitro data suggest that CXCL4 alone is not sufficient to promote atherosclerosis, because compared to MCSF-induced M0 macrophages CXCL4-induced M4 macrophages express a number of atherosclerosis-related genes at higher and others at lower levels.

In vivo, entire platelets with their granule contents are present in atherosclerotic lesions and not isolated CXCL4. Thus, CXCL4 may synergize with other platelet elements to induce a pro-atherosclerotic macrophage phenotype that is believed to be lacking in PF4-/- mice. In fact, it has been demonstrated in Apoe-/- mice that pharmacological inhibition of heterodimerization of CXCL4 with CCL5, which is also released from activated platelets, resulted in significant reduction of lesion formation [11]. We recently showed that CXCL4-induced macrophage lack expression of the hemoglobin scavenger receptor CD163 and that in human atherosclerotic lesions expression of CXCL4 and CD163 are inversely correlated [12]. This supports the notion that the M4 macrophage phenotype can actually be identified within human atherosclerotic lesions and may have pathophysiological relevance in atherosclerosis.

In summary, our data provide new insight into the process of macrophage differentiation. By comparing the transcriptome of MCSF- and CXCL4-induced macrophages in vitro, we identify M4 macrophages and provide novel starting points for further atherosclerosis- and other disease-related research.


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