Regulation of extracellular matrix assembly and structure by hybrid M1/M2 macrophages

Abstract

Aberrant extracellular matrix (ECM) assembly surrounding implanted biomaterials is the hallmark of the foreign body response, in which implants become encapsulated in thick fibrous tissue that prevents their proper function. While macrophages are known regulators of fibroblast behavior, how their phenotype influences ECM assembly and the progression of the foreign body response is poorly understood. In this study, we used in vitro models with physiologically relevant macrophage phenotypes, as well as controlled release of macrophage-modulating cytokines from gelatin hydrogels implanted subcutaneously in vivo to investigate the role of macrophages in ECM assembly. Primary human macrophages were polarized to four distinct phenotypes, which have each been associated with fibrosis, including pro-inflammatory M1, pro-healing M2, and a hybrid M1/M2, generated by exposing macrophages to M1-and M2-promoting stimuli simultaneously. Additionally, macrophages were first polarized to M1 and then to M2 (M1→M2) to generate a phenotype typically observed during normal wound healing. Human dermal fibroblasts that were cultured in macrophage-conditioned media upregulated numerous genes involved in regulation of ECM assembly, especially in M2-conditioned media. Hybrid M1/M2 macrophage-conditioned media caused fibroblasts to produce a matrix with thicker and less aligned fibers, while M2 macrophage-conditioned media caused the formation of a more aligned matrix with thinner fibers. Gelatin methacrylate hydrogels containing interleukin-4 (IL4) and IL13-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles were designed to promote the M2 phenotype in a murine subcutaneous in vivo model. NanoString multiplex gene expression analysis of hydrogel explants showed that hydrogels without cytokines caused mostly M1 phenotype markers to be highly expressed at an early time point (3 days), but the release of IL4+IL13 promoted upregulation of M2 markers and genes associated with regulation of ECM assembly, such as Col5a1 and Col6a1. Biochemical analysis and second harmonic generation microscopy showed that the release of IL4+IL13 increased total sulfated glycosaminoglycan content and decreased fibril alignment, which is typically associated with less fibrotic tissue. Together, these results show that hybrid M1/M2 macrophages regulate ECM assembly, and that shifting the balance towards M2 may promote architectural and compositional changes in ECM with enhanced potential for downstream remodeling.

Keywords: ECM assembly; Fibroblast; Fibrous capsule; Hydrogel; Macrophage phenotype.

Figure 1.

M2 macrophages upregulated genes associated with ECM assembly in vitro. A) Schematic of primary human monocyte to macrophage culture and subsequent polarization in vitro. B) Heatmap of all expressed genes represented as the row Z-score of log-transformed normalized data (normalized to negative and positive controls and housekeeping genes, GAPDH and TBP). Genes that were not expressed above background levels were: BGN, COL1A1, COL3A1, COL5A1, CCN2, CXCL12, DCN, FGF2, IGF1, VEGFC. Two representative differentially expressed genes from the categories related to C) M1, D) M2 markers, E) ECM, and F) additional ECM markers, and G) Angiogenesis markers. All genes are plotted in Suppl. Figs. 1-6. Data represented as mean ± standard deviation (SD), n=4 donors. **, ## p<0.01, ***, ### p<0.001, where # symbols indicate statistical significance compared to M0 control.

Figure 2.

Macrophage-conditioned media affects gene expression and ECM assembly by fibroblasts in vitro. A) Differentially expressed ECM-related and B) angiogenesis-related genes of fibroblasts cultured with primary macrophage-derived conditioned media for 14 days analyzed via NanoString, n=4 primary monocyte donors. C) Images of fibroblast-derived matrix after 14 days via maximum projections of confocal microscopy images. Image analysis (n=3 technical replicates per donor, n=3 donors) was used to determine D) matrix thickness, E) average matrix fiber diameter, and F) von Mises concentration analyzing matrix alignment. G) Indentation modulus of decellularized fibroblast-derived matrices, measured via atomic force microscopy nanoindentation. Each data point represents the average of n=10 experimental indents for each matrix per condition for each biological replicate (n=3 donors). Data represented as mean ± SD, with the exception of von Mises concentration, which is shown as κ with upper and lower limits of a 95% confidence interval. *, $ p<0.05, **, ## p<0.01, ***, ### p<0.001, where # indicates significance compared to the media-only fibroblast control, and $ indicates significance relative to fibroblasts cultured with M0 macrophage-conditioned media.

Figure 3.

IL4+IL13-loaded PLGA-GelMA hydrogels promote a dose dependent increase in ECM-related gene expression after 21 days in vivo. A) IL4+IL13-loaded PLGA microparticles were fabricated using a double emulsion technique and B) visualized with fluorescently labeled proteins via representative confocal microscopy. Scale bar = 25μm. C) IL4+IL13-loaded PLGA microparticles were embedded in UV-crosslinked GelMA hydrogels. D) Representative image of homogenous PLGA (round black dots) microparticle distribution throughout hydrogel. Scale bar = 500μm. E) Microparticle size distribution; n=3 batches of microparticles with approximately 25,000 particles analyzed in each sample. Data represent mean diameter (Blank: 8.8 ± 6.5μm, IL4+IL13: 9.7 ± 8.0μm). F) Cumulative release profiles of IL4 and IL13 from PLGA-GelMA hydrogels prepared with three different doses (Low, Medium (Med), and High), n=6 experimental replicates. G) Hydrogels were implanted subcutaneously in mice for 21 days. qRTPCR gene expression analysis of H) ECM genes, I) M1 genes, and J) M2 genes. Data represented as mean ± SD, *p<0.05, **p<0.01.

Figure 4.

IL4+IL13-releasing hydrogels promote upregulation in genes related to M2 markers, ECM assembly, and angiogenesis in vivo. A) Heatmap of all genes represented as the row Z-score of log-transformed normalized data, B) Principal component analysis, C-F) Differentially expressed genes organized by C) M2 markers, D) Immune signaling, E) ECM regulation, and F) Angiogenesis. *, # p<0.05, **, ## p<0.01, ***, ### p<0.001, where # symbols indicate significance compared to corresponding Day 3 data. All genes are shown in Suppl. Figs. 12-18. Genes that were not expressed were: Cxcl11, Ifna2, Il12a, Il2, Il1a, Mmp7. Data represented as mean ± SD, n=6 per group, per time point.

Figure 5.

IL4+IL13-releasing hydrogels promote a fibrous capsule with less aligned ECM and increased sulfated glycosaminoglycans (sGAGs). A-B) Representative images of tissue explants (skin-biomaterial interface) of Masson’s Trichrome staining. Scale bar = 100μm. C) Fibrous capsule characterization of thickness and area using image analysis. D-E) Second harmonic generation microscopy was used to analyze fibrillar collagen architecture of the fibrous capsule; regions of interest on each sample were isolated and quantitatively analyzed for F) % straight collagen fibers and fiber alignment. Biochemical analyses were performed on tissue explants for G) total collagen measured using the orthohydroxyproline assay and H) total sGAGs using the DMMB assay. Data represented as mean ± SD, n=6. *p<0.05.