Macrophage polarization critically contributes to a multitude of human pathologies. Hence, modulating macrophage polarization is a promising approach with enormous therapeutic potential. Macrophages are characterized by a remarkable functional and phenotypic plasticity, with pro-inflammatory (M1) and anti-inflammatory (M2) states at the extremes of a multidimensional polarization spectrum. Cell morphology is a major indicator for macrophage activation, describing M1(-like) (rounded) and M2(-like) (elongated) states by different cell shapes. Here, we introduced cell painting of macrophages to better reflect their multifaceted plasticity and associated phenotypes beyond the rigid dichotomous M1/M2 classification. Using high-content imaging, we established deep learning- and feature-based cell painting image analysis tools to elucidate cellular fingerprints that inform about subtle phenotypes of human blood monocyte-derived and iPSC-derived macrophages that are characterized as screening surrogate. Moreover, we show that cell painting feature profiling is suitable for identifying inter-donor variance to describe the relevance of the morphology feature 'cell roundness' and dissect distinct macrophage polarization signatures after stimulation with known biological or small-molecule modulators of macrophage (re-)polarization. Our novel established AI-fueled cell painting analysis tools provide a resource for high-content-based drug screening and candidate profiling, which set the stage for identifying novel modulators for macrophage (re-)polarization in health and disease.

Endocrine disrupting chemicals (EDCs) such as bisphenol S (BPS) are xenobiotic compounds that can disrupt endocrine signaling due to steric similarities to endogenous hormones. EDCs have been shown to induce disruptions in normal epigenetic programming (epimutations) and differentially expressed genes (DEGs) that predispose disease states. Most interestingly, the prevalence of epimutations following exposure to many EDCs persists over multiple generations. Many studies have described direct and prolonged effects of EDC exposure in animal models, but many questions remain about molecular mechanisms by which EDC-induced epimutations are introduced or subsequently propagated, whether there are cell type-specific susceptibilities to the same EDC, and whether this correlates with differential expression of relevant hormone receptors. We exposed cultured pluripotent (iPS), somatic (Sertoli and granulosa), and primordial germ cell-like (PGCLC) cells to BPS and found that differential incidences of BPS-induced epimutations and DEGs correlated with differential expression of relevant hormone receptors inducing epimutations near relevant hormone response elements in somatic and pluripotent, but not germ cell types. Most interestingly, we found that when iPS cells were exposed to BPS and then induced to differentiate into PGCLCs, the prevalence of epimutations and DEGs was largely retained, however, >90% of the specific epimutations and DEGs were replaced by novel epimutations and DEGs. These results suggest a unique mechanism by which an EDC-induced epimutated state may be propagated transgenerationally.
© 2024, Lehle et al.