Collagen Fibril Density Modulates Macrophage Activation and Cellular Functions during Tissue Repair

Abstract

Monocytes circulate in the bloodstream, extravasate into the tissue and differentiate into specific macrophage phenotypes to fulfill the immunological needs of tissues. During the tissue repair process, tissue density transits from loose to dense tissue. However, little is known on how changes in tissue density affects macrophage activation and their cellular functions. In this work, monocytic cell line THP-1 cells were embedded in three-dimensional (3D) collagen matrices with different fibril density and were then differentiated into uncommitted macrophages (MPMA) using phorbol-12-myristate-13-acetate (PMA). MPMA macrophages were subsequently activated into pro-inflammatory macrophages (MLPS/IFNγ) and anti-inflammatory macrophages (MIL-4/IL-13) using lipopolysaccharide and interferon-gamma (IFNγ), and interleukin 4 (IL-4) and IL-13, respectively. Although analysis of cell surface markers, on both gene and protein levels, was inconclusive, cytokine secretion profiles, however, demonstrated differences in macrophage phenotype. In the presence of differentiation activators, MLPS/IFNγ secreted high amounts of IL-1β and tumor necrosis factor alpha (TNFα), while M0PMA secreted similar cytokines to MIL-4/IL-13, but low IL-8. After removing the activators and further culture for 3 days in fresh cell culture media, the secretion of IL-6 was found in high concentrations by MIL-4/IL-13, followed by MLPS/IFNγ and MPMA. Interestingly, the secretion of cytokines is enhanced with an increase of fibril density. Through the investigation of macrophage-associated functions during tissue repair, we demonstrated that M1LPS/IFNγ has the potential to enhance monocyte infiltration into tissue, while MIL-4/IL-13 supported fibroblast differentiation into myofibroblasts via transforming growth factor beta 1 (TGF-β1) in dependence of fibril density, suggesting a M2a-like phenotype. Overall, our results suggest that collagen fibril density can modulate macrophage response to favor tissue functions. Understanding of immune response in such complex 3D microenvironments will contribute to the novel therapeutic strategies for improving tissue repair, as well as guidance of the design of immune-modulated materials.

Biomimetic 3D Models for Investigating the Role of Monocytes and Macrophages in Atherosclerosis

“…Contradicting our MFI data, we found slightly higher expression in the dense matrix in monocytes and macrophages, except M2 phenotypes. We postulate that this observation might be caused by an enhanced modulation of pro-inflammatory response by macrophages in a dense matrix, as previously reported [ 25 ]. Further evidence that collagen concentration can modulate the phenotype of macrophages is found in atherosclerotic plaque progression and regression studies [ 47 , 48 , 49 ], where progressive plaques (lower collagen content) contain more pro-inflammatory macrophages, while more anti-inflammatory macrophages are found in regressive plaques (higher collagen content).…”

“…3D collagen matrices at concentrations of 1 and 3 mg/mL were prepared as previously published [ 25 , 30 ]. In short, a collagen solution was prepared by mixing of type I rat tail collagen (Advanced BioMatrix, Inc., Carlsbad, CA, USA) with 250 mM of phosphate buffer and 0.1% acetic acid (both from Sigma-Aldrich, Inc., St. Louis, MO, USA).…”

“…Macrophage differentiation was performed using an established protocol [ 25 ]. In brief, THP-1 cells were differentiated into uncommitted macrophages (M0) by culturing in RPMI 1640 media without FBS supplemented with 300 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Inc.) for 6 h. To achieve pro-inflammatory (M1) and anti-inflammatory (M2) macrophages, PMA containing media was removed, and M0 were rested in RPMI-1640 cell culture media without FBS for 24 h. Afterward, cells were cultured in activating media for 48 h. For M1 activation, RPMI-1640 cell culture media without FBS was supplemented with 10 pg/mL lipopolysaccharide (LPS, Sigma-Aldrich, Inc.) and 20 ng/mL interferon-gamma IFN-γ (Biolegend, Inc., San Diego, CA, USA), while 20 ng/mL interleukin 4 (IL-4, Biolegend, Inc.) and 20 ng/mL interleukin 13 (IL-13, Biolegend, Inc.) was used for M2 activation.…”

“…Gene expression analysis was performed using an established protocol, as published [ 25 ]. Briefly, total RNA was extracted using TRIzol (Invitrogen, Thermo Fisher Scientific, Inc.) and converted into complementary DNA (cDNA) using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific, Inc.).…”

“…Moreover, aged or obese individuals are known to exhibit notably stiffened and thickened arteries [ 24 ]. Also, our previous work has specifically shown that macrophage functionality and immune phenotype is regulated by physical parameters of the surrounding ECM [ 25 ]. While the majority of biological readouts from 2D and 3D in vitro atherosclerosis models have focused on LDL uptake by the immune cells and secretion of specific pro-inflammatory cytokines, there is a lack of comprehensive secretome studies.…”