Keloid refers to a fibroproliferative disorder characterized by an accumulation of extracellular matrix (ECM) components at the dermis level, overgrowth beyond initial wound, and formation of tumor-like nodule areas. Treating keloid is still an unmet clinical need and the lack of an efficient therapy is clearly related to limited knowledge about keloid etiology, despite the growing interest of the scientific community in this pathology. In past decades, keloids were often studied in vitro through the sole prism of fibroblasts considered as the major effector of ECM deposition. Nevertheless, development of keloids results from cross-interactions of keloid fibroblasts (KFs) and their surrounding microenvironment, including immune cells such as macrophages. Our study aimed to evaluate the effect of M1 and M2 monocyte-derived macrophages on KFs in vitro. We focused on the effects of the macrophage secretome on fibrosis-related criteria in KFs, including proliferation, migration, differentiation, and ECM synthesis. First, we demonstrated that M2-like macrophages enhanced the fibrogenic profile of KFs in culture. Then, we surprisingly founded that M1-like macrophages can have an anti-fibrogenic effect on KFs, even in a pro-fibrotic environment. These results demonstrate, for the first time, that M1 and M2 macrophage subsets differentially impact the fibrotic fate of KFs in vitro, and suggest that restoring the M1/M2 balance to favor M1 in keloids could be an efficient therapeutic lever to prevent or treat keloid fibrosis.

Keloid refers to a fibro-proliferative disorder characterized by an accumulation of extracellular matrix at the dermis level, overgrowing beyond the initial wound and forming tumor-like nodule areas. The absence of treatment for keloid is clearly related to limited knowledge about keloid etiology. In vitro, keloids were classically studied through fibroblasts monolayer culture, far from keloid in vivo complexity. Today, cell aggregates cultured as 3D spheroid have gained in popularity as new tools to mimic tissue in vitro. However, no previously published works on spheroids have specifically focused on keloids yet. Thus, we hypothesized that spheroids made of keloid fibroblasts (KFs) could be used to model fibrogenesis in vitro. Our objective was to qualify spheroids made from KFs and cultured in a basal or pro-fibrotic environment (+TGF-β1). As major parameters for fibrogenesis assessment, we evaluated apoptosis, myofibroblast differentiation and response to TGF-β1, extracellular matrix (ECM) synthesis, and ECM-related genes regulation in KFs spheroids. We surprisingly observed that fibrogenic features of KFs are strongly downregulated when cells are cultured in 3D. In conclusion, we believe that spheroid is not the most appropriate model to address fibrogenesis in keloid, but it constitutes an efficient model to study the deactivation of fibrotic cells.

In this study, we propose and validate a simple agent-based model to study cell-cell interactions and cell migration during in vitro scratch assays in the context of cutaneous fibrosis (keloid). For model parametrization, we collect data from in vitro experiments performed with healthy or keloid fibroblasts treated (or not) with type 1 or 2 macrophages secretome to mimic specific in vivo environments. All experiments were performed with mitomycin to inhibit cell proliferation, and subsequently isolate the sole contribution of migration to wound filling over time. The scratch assays are modeled within the cellular Potts model framework. The calibration process, via Levenberg-Maquart algorithm, gives a mean error of 4.53 ± 0.77% across the four modalities (healthy, control, M1 and M2 secretum) and the evaluation dataset gives a mean error of 10.55 ± 0.77%. With the help of this model, we test whether the hypothesis of contact inhibition of locomotion (CIL) can explain the movement of keloid fibroblasts. The simulation results and their comparison with the experimental data suggest that CIL might not characterize the movement of keloid fibroblasts, which is in contrast to the importance of CIL for the movement of healthy fibroblasts.