Interaction of fibroblasts with cell corpses increases collagen synthesis during lung repair

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PROJECT SUMMARY Repair after injury is a fundamental biologic process that is critical for maintaining lung health. Fibroblasts must perform key pro-repair functions after injury including proliferation and synthesis of new extracellular matrix, particularly collagens, that scaffold wound closure. Dead cells (collectively termed cell corpses) are produced during lung injury and inflammation and have been shown to promote the transition from inflammation to repair. Prior research has focused on understanding macrophage interactions with apoptotic corpses. However, non- professional phagocytes including fibroblasts can also interact with cell corpses and the consequence of these interactions in the lung has not been studied. This proposal seeks to address unknowns regarding fibroblast- corpse interactions and their role in healthy lung repair. We have found that recognition of cell corpses by fibroblasts causes fibroblasts to increase collagen protein synthesis. Our preliminary data support a mechanism where the phosphatidylserine receptor Axl binds cells corpses, activates polyamine metabolite synthesis, increasing intracellular spermidine levels, which fuel hypusination. Hypusination occurs on a single protein: the ribosomal subunit eiF5a. When hypusinated, eiF5a is able to stabilize translation of poly-proline-repeat proteins including collagen. Hypusination is also important for cell proliferation. Blocking polyamine synthesis prevented hypusination of eiF5a in response to corpse recognition. Blocking Axl or hypusine-eiF5a prevented the increase in collagen in response to corpse recognition. We also show that, in vivo, hypusine-eiF5a is increased in fibroblasts after lung injury and that collagen deposition and alveolar-capillary repair are reduced when hypusination is blocked with the inhibitor GC7 or through targeted deletion of the hypusination enzyme deoxyhypusine synthase in fibroblasts. This led us to hypothesize that interaction of fibroblasts with cell corpses is a central cue that activates fibroblast proliferation and collagen synthesis following lung injury, facilitating repair. We will use three aims to test if 1) corpse recognition via Axl triggers a translationally-regulated increase in collagen protein, 2) corpse recognition drives polyamine synthesis that increases spermidine that increases hypusine-eiF5a and stabilizes collagen translation, and 3) to fully activate pro-repair programming fibroblasts require the ability to sense corpses via Axl, synthesize polyamines via ornithine decarboxylase, and hypusinate eiF5a via deoxyhypusine synthase in order to proliferate and synthesize new extracellular matrix after lung injury.