Using macrophages derived from human induced pluripotent stem cells to identify activators of inflammation in fibrodysplasia ossificans progressiva
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BACKGROUND: Inflammation is a key regulator in skeletal homeostasis during normal growth and tissue repair. However, the role that inflammation plays in skeletal processes is not well understood. Previous studies showed that damage associated molecular pattern (DAMP) molecules released after injury may contribute to immune activation and subsequent fibrosis. OBJECTIVE: This project aims to elucidate the link between tissue damage caused by trauma and the subsequent inflammatory response in a genetic condition of bone morphogenetic protein (BMP) pathway over activation. METHODS: We investigated this potential link by examining immune cells from patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of endochondral heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1). Patients with FOP show sensitivity to trauma, elevated serum cytokines and abnormal cytokine/chemokine secretion from monocytes and macrophages when stimulated with lipopolysaccharide in vitro. This suggested that BMP pathway activation may alter immune responses in patients with FOP. We studied macrophages derived from peripheral blood monocytes or created from human induced pluripotent stem cells (iPSC) from FOP and control subjects. Macrophages were evaluated by gene expression and culture media by multiplex cytokine analysis after stimulation with key DAMPs that were previously identified to be released after tissue injury. These DAMPs act as endogenous activators of inflammation. RESULTS: Monocyte derived macrophages from control subjects showed increased expression of pro-inflammatory cytokines in response to stimulation with DAMPs, HMGB1 and S100A8/A9. FOP monocyte-derived macrophages treated with each DAMP showed elevated production of CCL22, IL-8, CCL3, and CCL8 when compared to control macrophages. However, both control and FOP macrophages showed increased production of pro-inflammatory cytokines in response to DAMPs compared to non-stimulated conditions. RNA expression profiles of FOP iPSC derived macrophages did not show significantly increased responsiveness to DAMPs compared to control. Surprisingly, control patient iPSC derived macrophages show elevated expression of TNF-a and IL-1B CONCLUSIONS: Macrophages derived from peripheral blood monocytes show that DAMPs may be responsible for macrophage activation and the development of inflammatory complications in patients with FOP. Control iPSC derived macrophages showed similarity to monocyte derived macrophages in their response to DAMPs, suggesting that our iPSC derived macrophages are an applicable model for investigating the human immune system. The dissimilarity in FOP macrophage responsiveness to endogenous activators of our two macrophage models, suggest that iPSC derived macrophages may be affected by the different differentiation and polarization methods, and needs to be characterized further. Similarly, RNA expression profiles may not reflect cytokine production patterns of stimulated iPSC macrophages and warrants further studies.