Investigation of immunomodulation on myofibroblast activation: Implications for fibrotic development in wound healing

Endogenous mechanisms of wound healing and remodeling are a particularly attractive avenue for targeting traumatic injury and incomplete growth. Macrophages are highly involved in this process, and generally exhibit either an inflammatory (M1) phenotype, promoting cell debris clearance, or an anti-inflammatory (M2) phenotype, promoting tissue regeneration and remodeling, although fluidity exists in these phenotypes for injury repair in vivo. Type I collagen is also crucial to the repair process through development of extracellular matrix (ECM), which provides scaffolding for cellular and vascular growth as well as controlling cell differentiation later in the process. Myofibroblasts are the source of Type I collagen deposition, and differentiate from fibroblasts in the presence of transforming growth factor-beta (TGF-β), among other pathways. However, persistent myofibroblast activity can perpetuate fibrosis, leading to incomplete repair and scarring. While it is clear that macrophages and myofibroblasts are involved in the wound healing process, the interplay between these two populations has not been thoroughly investigated. Two in vitro experiments of M1, M2a, and M2c macrophage phenotypes with 10T1/2 fibroblasts were conducted. The first experiment involved fibroblasts interacting with cytokines produced by each macrophage phenotype at three different initial seeding densities: 500,000, 750,000, and 1 million cells per well. The second experiment involved fibroblasts and macrophages in a co-culture. Expression of alpha-smooth muscle actin (α-SMA), an indicator of myofibroblast activation, was probed via immunofluorescence after 72-hour incubation for both experiments. Through confocal microscopy and image analysis, it was determined that fibroblast interaction with M1 soluble factors lead to significantly higher levels of normalized α-SMA expression within fibroblasts compared to the M2a and M2c at a seeding density of 750,000. However, the co-culture model, with macrophages of different phenotypes interacting with fibroblasts in a contact dependent manner, saw no significant difference between these groups or the control. M2a and M2c may play an important role in promoting tissue regeneration over excessive fibrotic activity, whereas pro-inflammatory signaling from M1 may promote more collagen production. However, the lack of significant results from contact dependency may suggest different macrophage behavior. More investigation is necessary to comprehensively understand macrophage-fibroblast/myofibroblast interplay in wound healing.Undergraduat