Gene Expression Profiling of the Extracellular Matrix Signature in Macrophages of Different Activation Status: Relevance for Skin Wound Healing

Abstract

The extracellular matrix (ECM) provides structural support for tissue architecture and is a major effector of cell behavior during skin repair and inflammation. Macrophages are involved in all stages of skin repair but only limited knowledge exists about macrophage-specific expression and regulation of ECM components. In this study, we used transcriptome profiling and bioinformatic analysis to define the unique expression of ECM-associated genes in cultured macrophages. Characterization of the matrisome revealed that most genes were constitutively expressed and that several genes were uniquely regulated upon interferon gamma (IFNγ) and dexamethasone stimulation. Among those core matrisome and matrisome-associated components transforming growth factor beta (TGFβ)-induced, matrix metalloproteinase 9 (MMP9), elastin microfibril interfacer (EMILIN)-1, netrin-1 and gliomedin were also present within the wound bed at time points that are characterized by profound macrophage infiltration. Hence, macrophages are a source of ECM components in vitro as well as during skin wound healing, and identification of these matrisome components is a first step to understand the role and therapeutic value of ECM components in macrophages and during wound healing.

Keywords: extracellular matrix; gene expression profiling; matrisome; pro- and anti-inflammatory macrophage; skin wound healing.

Figure 1

Transcriptome analysis of the matrisome of primary human macrophages. (a) Non-averaged hierarchical clustered intensity plot (distant metrics–euclidean, linkage rule–ward’s) of core matrisome and matrisome-associated genes in macrophages is shown. Clusters of intermediate (green), low (blue) and high (red) expression are highlighted. The normalized intensity values of the individual replicates are shown. The highest expressed gene within the core matrisome TGFBI (transforming growth factor beta induced) and the matrisome-associated MMP9 (matrix metalloproteinase 9) are indicated. (b) The proportion of entities within the core matrisome or matrisome-associated cluster are shown in a Venn diagram. The numbers and percentages of regulated genes found in subcategories are listed.

Figure 2

Identification of highly expressed core matrisome and matrisome-associated genes in primary human macrophages. The most highly expressed genes of the core matrisome (a), cell–matrix interaction-mediating integrin (b) and matrisome-associated genes (c) are listed according to intermediate (light red) and high (red) expression. Normalized expression values are given and the complete set of genes with respective expression values is given in the Table S1.

Figure 3

Regulation of core matrisome and matrisome-associated genes after interferon gamma (IFNγ) and dexamethasone (Dexa) stimulation. (a,b) The proportion of entities among the regulated genes in IFNγ- (a) or dexamethasone-primed macrophages (b) that are found in the core matrisome or matrisome-associated data set are shown in a Venn diagram. The numbers and percentages of genes in the subcategories are listed. (c,d) Expression intensity plots for IFNγ- (c) or dexamethasone-primed macrophages (d) are shown. Regulated genes within the core matrisome and the matrisome-associated cluster are highlighted in black. (fold change ≥ 2, p < 0.05, false discovery rate (FDR) correction by Benjamini-Hochberg, medium versus IFNγ or medium versus dexamethasone).

Figure 4

Cluster analysis of regulated matrisome genes in macrophages stimulated with IFNγ or dexamethasone. (a–d) Clustered genes of the core matrisome (a,b) and matrisome-associated cluster (c,d) regulated in monocyte-derived macrophages (MDMs) after IFNγ (a,c) or dexamethasone stimulation (b,d, Dexa) are displayed. Non-averaged hierarchical clustered intensity plot (distant metrics–euclidean, linkage rule–ward’s) of differentially expressed genes in cells cultured in normal medium (M) and medium containing IFNγ (I) or dexamethasone (D) is shown and the fold change (FC) is given.

Figure 5

Validation of matrisome proteins in the in vivo wound bed in a murine wound healing model. (a) In wounds that were harvested three days post wounding the macrophage-specific F4/80 as well as transforming growth factor beta induced (TGFBI), MMP9 and EMILIN-1 were detected by immunofluorescence analysis within the wound (a’) or the intact skin (a´´). (b) TGFBI, MMP9, EMILIN-1, netrin-1 and gliomedin abundance (arrowhead) was analyzed by immunoblotting in lysates of intact skin (skin) or wounds three (D3) and five days post wounding (D5). Actin was used as loading control. As EMILIN-1 and gliomedin were tested on a single blot, the same actin control is included in both panels. Molecular weights of Thermo Scientific™ PageRuler™ Plus Prestained 10–250 kDa Protein Ladder bands are given. *, unspecific band; scale bars (a), 1000 µm; (a´, a´´), 200 µm.

Figure 6

Potential wound healing mechanisms involving the macrophage-derived extracellular matrix (ECM). (a) Macrophages could produce collagen IV, VI and laminins to provide their own substrate and/or bridging proteins for their adhesion to basement memranes. (b) Macrophages may promote the formation of a microfibrillar network within the wound by producing the beaded filament forming collagen VI. Collagen XI may be utilized by macrophages to interact with fibrillar collagens and support their organization while macrophage-derived netrin-1 may provide guidance signals to organize the wound. Macrophages may secrete fibulin-5 and EMILINs to act as a scaffold protein that organize and link elastic fibres. (c) Macrophage-derived gliomedin that binds to perlecan might participate in basement membrane formation/stabilization and influence keratinocytes to regulate epithelial wound closure. By producing testican-1, which is a target of TGF-β and induces epithelial-to-mesenchymal transition (EMT), macrophages may support epithelial wound closure. (d) Macrophage-derived testican-1 may also regulate metalloproteinase activity to promote the degradation of provisional wound matrix supporting vascularization, while fibulin-5 synthetized by macrophages may regulate angiogenesis by modulating VEGF signaling. (e) Macrophages may express proteases (A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS2), MMP9, MMP1) and their inhibitors (TIMP1–3) to generate bioactive ECM fragments (matrikines) and may also be a source of ECM molecules that harbor these bioactive fragments (collagen VI, laminins, versican).