3D in vitro M2 macrophage model to mimic modulation of tissue repair

Abstract

Distinct anti-inflammatory macrophage (M2) subtypes, namely M2a and M2c, are reported to modulate the tissue repair process tightly and chronologically by modulating fibroblast differentiation state and functions. To establish a well-defined three-dimensional (3D) cell culture model to mimic the tissue repair process, we utilized THP-1 human monocytic cells and a 3D collagen matrix as a biomimetic tissue model. THP-1 cells were differentiated into macrophages, and activated using IL-4/IL-13 (M_IL-4/IL-13) and IL-10 (M_IL-10). Both activated macrophages were characterized by both their cell surface marker expression and cytokine secretion profile. Our cell characterization suggested that M_IL-4/IL-13 and M_IL-10 demonstrate M2a- and M2c-like subtypes, respectively. To mimic the initial and resolution phases during the tissue repair, both activated macrophages were co-cultured with fibroblasts and myofibroblasts. We showed that M_IL-4/IL-13 were able to promote matrix synthesis and remodeling by induction of myofibroblast differentiation via transforming growth factor beta-1 (TGF-β1). On the contrary, M_IL-10 demonstrated the ability to resolve the tissue repair process by dedifferentiation of myofibroblast via IL-10 secretion. Overall, our study demonstrated the importance and the exact roles of M2a and M2c-like macrophage subtypes in coordinating tissue repair in a biomimetic model. The established model can be applied for high-throughput platforms for improving tissue healing and anti-fibrotic drugs testing, as well as other biomedical studies.

Fig. 1. Macrophage differentiation and activation in 3D collagen matrices.

a Schematic workflow for macrophage differentiation and activation towards M_IL-4/IL-13 and M_IL-10 in 3D collagen matrix. b Representative images of M_IL-4/IL-13 and M_IL-10 were gathered using bright-field microscopy. A quantitative morphological analysis, namely c cell area and d cell aspect ratio, was conducted using an image analysis toolbox. Data are presented as a violin plot. At least 100 cells from three independent experiments were analyzed.

Fig. 2. Quantitative analysis of cell surface markers of M_IL-4/IL-13 and M_IL-10.

Cell surface markers were analyzed by immunocytostaining of CD14, CD68, CD80, CD86, CD105, CD163, CD206, and HLA-DR. Geometric mean fluorescence intensity (gMFI) was plotted. Black line and error bar in the plot represent mean and standard deviation, respectively. * indicates a significance level of p ≤ 0.05 using Mann–Whitney test. Experiments were performed in four independent replicates.

Fig. 3. Quantitative analysis of cytokine secretion profile of M_IL-4/IL-13 and M_IL-10.

Cytokine secretion profile was analyzed from cell culture supernatants after 3 days of macrophage activation by a bead-based multiplex immunoassay using flow cytometry. Data are shown as a dot plot. Black line and error bar in the plot represent mean and standard deviation, respectively. * indicates a significance level of p ≤ 0.05 using Mann–Whitney test. Experiments were performed in six independent replicates.

Fig. 4. Alteration of fibroblast morphology and specific markers in the presence of TGF-β1 and co-culture with M2 macrophages subtypes.

a Representative images showing nuclei (blue), actin filaments (gray), and αSMA (green) (scale bar: 50 μm). Fibroblast differentiation was analyzed by b manual counting of αSMA-positive cells. At least 200 cells per condition were counted. Gene expression analysis of c αSMA, d Coll1a1, and e EDA-FN were performed using qPCR. Data are represented as mean ± SD; *a significance level of p ≤ 0.05 using a one-way ANOVA followed by Tukey’s post hoc analysis. The characters # and § represent the significance level of p ≤ 0.05 using a one-way ANOVA followed by Tukey’s post hoc analysis when compared to control, and treated samples with TGF-β1, respectively. Experiments were performed in four replicates.

Fig. 5. Matrix remodeling by fibroblasts in the presence of TGF-β1 and co-culture with M2 subtypes.

a Representative images of decellularized collagen matrices in mono and co-culture with M2 subtypes (scale bar: 50 μm). Decellularized collagen matrices were characterized regarding b matrix porosity characterized as mean pore size, c collagen fibril diameter, and d bulk matrix elastic modulus (data are represented as mean ± SD; *significance level of p ≤ 0.05 using a one-way ANOVA followed by Tukey’s post hoc analysis). The characters # and § represent the significance level of p ≤ 0.05 using a one-way ANOVA followed by Tukey’s post hoc analysis when compared to control, and treated samples with TGF-β1, respectively. For the quantification of matrix porosity and collagen fibril diameter, 10 different positions of each matrix condition were analyzed. Experiments were performed in four replicates.

Fig. 6. Alteration of myofibroblasts morphology and specific markers in the presence of IL-10 and co-culture with M2 macrophages subtypes.

a Representative images showing nuclei (blue), actin filaments (gray), and αSMA (green) (scale bar: 50 μm). Myofibroblast de-dedifferentiation were analyzed using b percentage of αSMA-positive cells by manual counting of cells with αSMA incorporation into actin stress fibers and quantitative analysis of c αSMA, d Coll1a1, and e EDA-FN gene expression. For quantitative analysis of αSMA-positive cells, at least 200 cells per condition were counted. Data are represented as mean ± SD; *significance level of p ≤ 0.05 using a one-way ANOVA followed by Tukey’s post hoc analysis). The characters # and § represent the significance level of p ≤ 0.05 using a one-way ANOVA followed by Tukey’s post hoc analysis when compared to control, and treated samples with IL-10, respectively. Experiments were performed in four replicates.

Fig. 7. Matrix remodeling by myofibroblasts in the presence of IL-10 and co-culture with M2 subtypes.

a Representative images of decellularized collagen matrices in mono and co-culture with M2 subtypes (scale bar: 50 μm). Decellularized collagen matrices were characterized regarding b matrix porosity characterized as mean pore size, c collagen fibril diameter, and d matrix elastic modulus. (Data are represented as mean ± SD; *significance level of p ≤ 0.05 using a one-way ANOVA followed by Tukey’s post hoc analysis.) The characters # and § represent the significance level of p ≤ 0.05 using a one-way ANOVA followed by Tukey’s post hoc analysis when compared to control, and treated samples with IL-10, respectively. For the quantification of matrix porosity and collagen fibril diameter, 10 different positions of each matrix condition were analyzed. Experiments were performed in four replicates.

Fig. 8. Migration of fibroblasts and myofibroblasts into 3D collagen matrices.

Cells were stained with Phalloidin conjugated with Alexa Fluor-488 and DAPI, for visualization and quantification of cell migration into 3D collagen matrices, respectively. Representative images of xz-view of a fibroblast and d myofibroblast into 3D collagen matrices in mono- and co-culture with macrophages. Quantitative analysis of b, e percentage of migrating cells and c, f maximum migration depth of fibroblast and myofibroblasts. Cells found >20 μm beneath the matrix surface were counted as migrating cells. The maximum migration distance was defined as the distance crossed by 10% of all cells. Data are represented as mean ± SD; *significance level of p ≤ 0.05 using a one-way ANOVA followed by Tukey’s post hoc analysis. The characters # and § represent the significance level of p ≤ 0.05 using a one-way ANOVA followed by Tukey’s post hoc analysis when compared to control, and treated samples with TGF-β1 or IL-10, respectively. Three different positions of each matrix condition were analyzed. Experiments were performed in four replicates.

Fig. 9. Schematic illustration of the proposed M2-associated functions in initiation and termination phases of tissue repair in 3D biomimetic wound-healing model.

M_IL-4/IL-13 macrophages control the initiation of tissue repair by modulating fibroblast differentiation via TGF-β1, while M_IL-10 macrophages terminate the tissue repair by de-differentiating myofibroblasts back into fibroblasts via IL-10.