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.
Highlights
Macrophages are vital innate immune cells with remarkable plasticity that allow them to respond efficiently to pro- and anti-inflammatory signals within their microenvironment [1,2]
To bridge the phenotypical characteristics of macrophages with their cellular functions, we studied their capacity in modulating monocyte infiltration and fibroblast differentiation by co-culturing with THP-1 monocytes and human primary fibroblasts, respectively
There exists a lack of knowledge regarding how tissue density affects macrophage activation and functions, especially in the context of normal to cancerous tissue, wound to scar tissue, during tissue repair
Summary
Macrophages are vital innate immune cells with remarkable plasticity that allow them to respond efficiently to pro- and anti-inflammatory signals within their microenvironment [1,2] Their phenotype and physiology can be altered by the cytokine secretion profile of other cells in the microenvironment in order to modulate the innate and adaptive immune responses [3]. Pro-inflammatory macrophages were described as having inflammatory functions and were crucial for the resistance to pathogens and their elimination, whereas macrophages with anti-inflammatory functions were important for maintaining tissue integrity This simplified nomenclature can create the illusory perception of homogeneity of macrophage subpopulations, which is not shown to exist in vivo, since both cells can phenotypically span the pro-inflammatory–anti-inflammatory spectrum to serve different functions in response to microenvironmental cues [5,6]. While soluble cytokines and cell–cell interactions are well studied, there is still a gap of knowledge on the effects of tissue characteristics as a microenvironmental signal
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