Pharmacologic modulation of innate immune dysfunction in heterotopic ossification

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PROJECT SUMMARY/ABSTRACT Heterotopic ossification is a rare but devastating condition of inappropriate bone formation. Although immune system activity is a critical contributor to heterotopic ossification, we lack a clear understanding of how the immune system drives bone formation. Fibrodysplasia ossificans progressiva (FOP) is a genetic disease of massive heterotopic ossification that is associated with inflammatory “flares” that can occur after injury. We recently found that sera from FOP patients have increased levels of pro-inflammatory cytokines at baseline. Furthermore, primary FOP monocytes showed increased responsiveness to LPS, a ligand that stimulates the TLR4 pathway, but not to other ligands that activate other TLR receptors. These preliminary results lead to our central hypothesis that FOP is an autoimmune disease caused by inappropriate activation of the innate immune system. Although there are currently no effective treatments for FOP, retinoic acid receptor-γ (RAR-γ) agonists can inhibit heterotopic ossification in mouse models. UCSF is part of an ongoing multicenter Phase II/III program sponsored by Clementia Pharmaceuticals to study the efficacy of the RAR-γ agonist palovarotene in blocking bone formation in FOP. Preliminary results from a completed randomized study (NCT02190747) and ongoing open label followup study (NCT02279095) indicate that palovarotene can decrease the amount of heterotopic bone formation in FOP subjects. The ongoing open label PVO-1A-202 (NCT02279095) and Phase III PVO-1A-301 (NCT03312634) interventional trials will be the parent studies for the proposed unique and time-sensitive opportunity to elucidate the immune mechanisms in FOP. In Aim 1, we will elucidate how FOP primary monocytes respond to endogenous TLR4 activation. We will assay FOP sera for known endogenous TLR4 activators that can be expressed after trauma, and test if FOP monocytes show increased responsiveness to the TLR4 activators such as HMGB1. In Aim 2, we will test if FOP primary monocytes sensitize endothelial cells to inflammation. We will use co-cultures to assay if FOP monocytes show increased chemotaxis towards FOP endothelial cells and if this is dampened by palovarotene. We will also test if FOP endothelial cells show enhanced pro-inflammatory cytokine expression in the presence of FOP monocytes. These assessments will also show if palovarotene affects immune cell function. In Aim 3, we will determine how ACVR1 and palovarotene change innate immune cell activation. We will use single cell RNAseq to elucidate how the ACVR1 R206H mutation changes the composition of peripheral immune cells, if palovarotene can normalize this profile, and if changes in cytokine production can be linked to inflammation and FOP clinical heterotopic bone formation. Our results will reveal critical inflammatory mechanisms that may provide the first mechanistic and diagnostic biomarkers for disease activity in FOP. The results may also improve our treatment strategies by identifying novel inflammatory targets that may be relevant to other conditions of heterotopic ossification.