Miniaturized Drug Discovery Assays Targeting Macrophages in Fibrotic Diseases

Abstract

Macrophages have key regulatory functions in health and disease, such as fibrosis, and are therefore of high interest for drug discovery. Fibrosis is a result of chronic tissue damage leading to a deregulated wound healing process. Macrophages are involved in different phases of the fibrotic cascade and its onset is assumed to present a reaction to aberrant macrophage activation. Hence, there exists potential of modulating these cells for therapeutic benefits. In patients suffering from idiopathic pulmonary fibrosis (IPF), defective removal of apoptotic cells (efferocytosis) by macrophages was reported. Therefore, targeting macrophages and especially the modulation of their efferocytotic activity represents one strategy to diminish fibrotic processes in IPF. However, an impediment in drug discovery is the lack of physiologically relevant cellular in vitro models that can recapitulate the disease situation in patients. Most of the reported models lack relevant cells in sufficient quantity and thus, cannot be applied for screening campaigns. Hence, the aim of the presented work was to fill the gap of the unmet need for physiologically relevant in vitro drug discovery assays to target macrophage functions in fibrotic diseases. To access large numbers of model cells, an upscaled protocol was established for differentiation of human induced pluripotent stem cells (iPSCs) into progenitor cells and subsequent maturation into functional macrophages. These iPSC-derived macrophages (IDMs) resembled monocyte-derived macrophages (MDMs) both with respect to phenotypical and functional characteristics. To analyze macrophage functions in fibrotic diseases, a miniaturized high-content-imaging-based assay was established, enabling the analysis and quantification of both efferocytosis and phagocytosis for medium- to high-throughput applications. Utilizing IDMs and MDMs, the cells showed comparable pharmacology, as demonstrated by the analysis of Spleen tyrosine kinase (Syk) inhibitors, Dexamethasone, and a pro-fibrotic cocktail. Besides reduced efferocytotic function of macrophages, an accumulation of senescent cells is reported for IPF patients. To analyze the potential link between these two conditions, a miniaturized co-culture set-up was established. Using differently induced senescent epithelial cells, the inhibitory effect of senescence signals on efferocytosis and phagocytosis was shown in this context. On the contrary, senolytic treatment of senescent epithelial cells triggered their apoptosis induction and resulted in increased efferocytotic activity. The insights gained from this study imply that senescent cells may be a potential cause of reduced efferocytotic activity. Hence, addressing senescent cells and their communication with macrophages could present a promising therapeutical approach. In conclusion, the here established iPSC-derived macrophage model in combination with the miniaturized efferocytosis and phagocytosis assays can be implemented into large-scale screening campaigns and may open new routes to innovative therapeutic paths in the context of fibrosis and beyond.