Cell plasticity in wound healing: paracrine factors of M1/ M2 polarized macrophages influence the phenotypical state of dermal fibroblasts

Abstract

Background: Macrophages and fibroblasts are two major players in tissue repair and fibrosis. Despite the relevance of macrophages and fibroblasts in tissue homeostasis, remarkably little is known whether macrophages are able to influence the properties of fibroblasts. Here we investigated the role of paracrine factors secreted by classically activated (M1) and alternatively activated (M2) human macrophages on human dermal fibroblasts (HDFs).

Results: HDFs stimulated with paracrine factors from M1 macrophages showed a 10 to > 100-fold increase in the expression of the inflammatory cytokines IL6, CCL2 and CCL7 and the matrix metalloproteinases MMP1 and MMP3. This indicates that factors produced by M1 macrophages induce a fibroblast phenotype with pro-inflammatory and extracellular matrix (ECM) degrading properties. HDFs stimulated with paracrine factors secreted by M2 macrophages displayed an increased proliferation rate. Interestingly, the M1-activated pro-inflammatory fibroblasts downregulated, after exposure to paracrine factors produced by M2 macrophages or non-conditioned media, the inflammatory markers as well as MMPs and upregulated their collagen production.

Conclusions: Paracrine factors of M1 or M2 polarized macrophages induced different phenotypes of HDFs and the HDF phenotypes can in turn be reversed, pointing to a high dynamic plasticity of fibroblasts in the different phases of tissue repair.

Figure 1

Characterization of macrophages after M1 or M2 polarization. After stimulation with LPS/IFNG, M1 macrophages showed a dendritic morphology while IL4/IL13 (M2) stimulated and unstimulated macrophages showed a rounded and/or spindle-shaped morphology (A). The three primary macrophages subsets showed, compared to reference gene YWHAZ, a high expression of CD68. In M1 polarized macrophages the CD68 gene expression is downregulated while the expression of CD14 is upregulated compared to M2 or unstimulated macrophages (B). LPS/IFNG-stimulated (M1) macrophages showed upregulated gene expression of IL1B, IL6, CCL2 and CD40 (C). IL4/IL13-stimulated (M2) macrophages upregulated the gene expression of CLEC10A, MRC1 and tended to upregulate CCL18. IL1R2 showed a high expression in M2 and unstimulated macrophages and was downregulated in M1 polarized macrophages (D). At protein level, more CCL2 was observed in conditioned medium from M1 macrophages. CCL18 protein secretion showed, like CCL2, values that correlated with gene expression (E). * p < 0.05, Difference between LPS/IFNG and IL4/IL13 stimulated macrophages, ** p < 0.01, *** p < 0.001. # p < 0.05, Difference between LPS/IFNG stimulated and unstimulated macrophages, ### p < 0.001. ^^ p < 0.01, Difference between IL4/IL13 stimulated and unstimulated macrophages. Data were analyzed using one-way ANOVA followed by Tukey’s post-test. Gene expression analysis n = 4, protein secretion n = 3.

Figure 2

Morphology of HDFs stimulated with CM of M1 polarized, M2 polarized, or unstimulated macrophages. HDFs stimulated with CM of M1 polarized macrophages; 24 h (A), 72 h (D), and 144 h (G). After 24 h, most of the fibroblasts showed a spindle-shaped morphology although some rounded fibroblasts were seen. After 72 h, the fibroblasts adopted a rounded morphology that was most prominent after 144 h. HDFs stimulated with CM of M2 polarized macrophages; 24 h (B), 72 h (E) and 144 h (H). After 24 h the cells showed a spindle-like morphology, which was changed into an elongated spindle-like morphology after 72 h. HDFs stimulated with CM from unstimulated macrophages 24 h (C), 72 h (F), and 144 h (I). The fibroblasts showed a spindle-like morphology after 24 h, which was not changed in time.

Figure 3

CM from M1 macrophages induces a pro-inflammatory HDF. HDFs upregulated the gene expression of pro-inflammatory genes CCL2, IL6 and CCL7 after stimulation with CM of M1 polarized macrophages compared to M2 polarized and unstimulated macrophages (A). HDFs stimulated with CM of M1 macrophages secreted significantly more CCL2, IL6 and CCL7 after 24 h and 48 h, whereas secretion levels of these proteins by fibroblasts stimulated with CM of M2 macrophages or unstimulated macrophages were below the detection limit (B). ** p < 0.01, Difference between HDFs stimulated with CM of M1 polarized and CM of M2 polarized macrophages, *** p < 0.001. # p < 0.05, Difference between HDFs stimulated with CM of M1 polarized and CM of unstimulated macrophages, ## p < 0.01, ### p < 0.001. Gene expression analysis data were analyzed using two-way ANOVA followed by Bonferroni’s post-test, n = 4. Protein secretion data were analyzed using one-way ANOVA followed by Tukey’s post-test n = 3.

Figure 4

CM from M1 macrophages induces dermal fibroblasts with extracellular matrix degradation properties. HDFs upregulated the gene expression of MMP1, MMP2, MMP3, MMP14 and TIMP1 after stimulation with CM of M1 polarized macrophages compared to M2 polarized and unstimulated macrophages (A). HDFs stimulated with CM of M1 macrophages secreted significantly more MMP1, MMP2 and MMP3 protein compared to fibroblasts stimulated with CM of M2 macrophages or unstimulated macrophages (B). HDFs stimulated with M1 CM showed a higher net proteolytic activity compared to HDFs stimulated with CM of M2 and unstimulated macrophages (C). ** p < 0.01, Difference between HDFs stimulated with CM of M1 polarized and CM of M2 polarized macrophages, *** p < 0.001. ## p < 0.01, Difference between HDFs stimulated with CM of M1 polarized and CM of unstimulated macrophages, ### p < 0.001. Gene expression analysis data were analyzed using two-way ANOVA followed by Bonferroni’s post-test, n = 4. MMP secretion was analyzed using one-way ANOVA followed by Tukey’s post-test, n = 3. Proteolytic activity was analyzed using one-way ANOVA followed by Tukey’s post-test, n = 4.

Figure 5

CM of M1 polarized, M2 polarized or unstimulated macrophages do not induce myofibroblast differentiation of HDFs. HDFs stimulated with CM of unstimulated macrophages showed, compared to fibroblasts stimulated with CM of M1 polarized macrophages, an upregulated gene expression of ACTA2 after 48 h, 72 h and 144 h. HDFs stimulated with CM of M2 polarized macrophages showed a higher gene expression of ACTA2 compared to HDFs stimulated with CM of M1 macrophages after 144 h (A). No differences were observed in TAGLN gene expression between the three conditions in all time points (A). No differences in ACTA2 protein expression were seen between the three conditions after 144 h. TGFB1 stimulated fibroblasts were used as positive control (B). Fibroblasts stimulated with CM of M1 macrophages contract the collagen 10% more than fibroblasts stimulated with CM of M2 and unstimulated fibroblasts. Fibroblasts stimulated with TGFB1 contract the collagen 50% more compared to the other stimulations (C). * p < 0.05, Difference between HDFs stimulated with CM of M1 polarized and CM of M2 polarized macrophages, *** p < 0.001. # p < 0.05, Difference between HDFs stimulated with CM of M1 polarized and CM of unstimulated macrophages, ## p < 0.01, ### p < 0.001. ^ p < 0.05, Difference between HDFs stimulated with CM of M2 polarized and CM of unstimulated macrophages. ≅≅≅ p < 0.001, Difference between HDFs stimulated with TGFB1 and CM of M1 polarized macrophages. §§§ p < 0.001, Difference between HDFs stimulated with TGFB1 and CM of M2 polarized macrophages. ❖❖❖ p < 0.001, Difference between HDFs stimulated with TGFB1 and CM of unstimulated polarized macrophages. Gene expression analysis data were analyzed using two-way ANOVA followed by Bonferroni’s post-test, n = 4. ACTA2 protein expression was shown in red and nuclei in blue (DAPI), original magnification 200×. Collagen gel contraction analysis data were analyzed using one-way ANOVA followed by Tukey’s post-test, n = 3.

Figure 6

Proliferation of HDFs is induced by CM of M2 macrophages. After 72 h, no differences in cell numbers were seen between HDFs stimulated with CM of M1 polarized, M2 polarized or unstimulated macrophages. After 144 h, stimulation with CM of M2 macrophages increased the dermal fibroblast cell number, exclusively (A). This is due to proliferation of the HDFs as shown by MKI67 protein staining (B). More MKI67 positive nuclei (green) were seen in fibroblasts stimulated with CM of M2 macrophages compared to fibroblasts stimulated with CM of M1 and unstimulated macrophages after 144 h (C). *** p < 0.001, Difference between HDFs stimulated with CM of M1 polarized and CM of M2 polarized macrophages. # p < 0.05, Difference between HDFs stimulated with CM of M2 polarized and CM of unstimulated macrophages. ^ p < 0.05, Difference between 72 h and 144 h after stimulation of HDFs with CM of M2 polarized macrophages. Cell numbers and MKI67 positive nuclei were analyzed using two-way ANOVA followed by Bonferroni’s post-test n = 3. MKI67 protein expression was shown in green, nuclei in blue (DAPI) and original magnification was 200×.

Figure 7

Influence of CM of M1 polarized, M2 polarized or unstimulated macrophages on extracellular matrix deposition by fibroblasts. After 144 h, HDFs stimulated with CM of M1 macrophages showed reduced COL1A1 and COL3A1 gene expression levels compared to stimulation with CM of M2 and unstimulated macrophages (A). No differences in COL1A1 deposition by fibroblasts were seen after the three different stimulations after 72 h. However, less collagen type I protein deposition was seen in fibroblasts stimulated with CM of M1 macrophages compared to stimulation with CM of unstimulated macrophages after 144 h (B). ** p < 0.01, Difference between HDFs stimulated with CM of M1 polarized and CM of M2 polarized macrophages. ## p < 0.01, Difference between HDFs stimulated with CM of M1 polarized and CM of unstimulated macrophages. Gene expression analysis data were analyzed using two-way ANOVA followed by Bonferroni’s post-test, n = 4. COL1A1 protein expression was shown in red, nuclei in blue (DAPI) and original magnification was 200×.

Figure 8

Fibroblasts stimulated with CM of M1 macrophages followed by stimulation with CM of M2 macrophage or non-CM (switch). When stimulation of HDFs with CM of M1 macrophages is followed by CM of M2 macrophages or non-CM (switch), the pro-inflammatory genes CCL2 and IL6 were completely downregulated and showed the same gene expression as fibroblasts stimulated with only CM of M2 macrophages after 72 h and 144 h (A). MMP1 gene expression after the CM switch was downregulated at 144 h, whereas TIMP1 expression remained similar (B). COL1A1 gene expression was upregulated after the CM switch compared to fibroblasts stimulated with CM of M1 macrophages at 144 h. This gene expression level was similar to fibroblasts stimulated with CM of M2 macrophages (C). After the switch no differences were seen in COL3A1 gene expression compared fibroblasts stimulated with CM of M1 or CM of M2 macrophages (C). * p < 0.05, Difference between HDFs stimulated with CM of M1 polarized and CM of M2 polarized macrophages, ** p < 0.01, *** p < 0.001. §§ p < 0.01, Difference between HDFs stimulated with CM of M1 polarized and the switch to non-CM, §§§ p < 0.001. # p < 0.05, Difference between HDFs stimulated with CM of M1 polarized and the CM switch, ### p < 0.001. ≅≅ p < 0.01, Difference between HDFs stimulated with CM of M2 polarized and the switch to non-CM, ≅≅≅ p < 0.001. ^ p < 0.05, Difference between HDFs stimulated with CM of M2 polarized and the CM switch. Gene expression analysis data were analyzed using two-way ANOVA followed by Bonferroni’s post-test, n = 4.