Microfibril-associated glycoproteins attenuating pulmonary fibrosis

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Project Summary/Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited treatment options and unclear pathogenesis. Extracellular matrix (ECM) deposition by proliferating fibroblast populations drives the progressive airflow limitation and hypoxia characteristic of IPF, mediated by the cytokine transforming growth factor beta (TGFB). Direct inhibition of TGFB leads to intolerable side effects, and there is increased interest in therapies that instead modulate its regulation. Microfibril-associated glycoproteins 1 and 2 (Magp1 and Magp2, or Magps) are fibroblast-produced proteins which are secreted to ECM, anchor to microfibrils, and bind TGFB to limit its signaling under basal conditions, but have no known role in fibrosis. Magp knockout (KO) mice exhibit increased TGFB signaling in various tissues and have immune cell alterations due to bone marrow TGFB effects. In a single cell RNASeq screen, the genes encoding these proteins were markedly upregulated in fibroblasts in fibrotic lungs, and combined KO of Magp1 and 2 led to more severe fibrosis after bleomycin injury in mice. This suggests that Magps act as inhibitors of fibrotic signaling and follow up studies in Magp1 and 2 individual KO mice show that deficiency of both proteins is required to produce a pro-fibrotic phenotype. Which fibroblasts populations produce Magps and how they limit fibrosis is unknown. We hypothesize that Magp expression is an adaptive response to lung injury and limits fibrosis by sequestering TGFB in ECM and will explore this central hypothesis through three specific aims: 1) Evaluate anti-fibrotic contributions of Magp1 and 2 in pulmonary fibrosis. 2) Evaluate Magp effects on TGFB signaling and immune responses in lung fibrosis. 3) Characterize Magp-producing fibroblast populations. Using combinations of KO and inducible/tissue-specific KO mice, we will determine the individual relationships of these proteins to fibrosis and whether they exhibit redundant antifibrotic functions in vivo. We will analyze alterations in TGFB signaling and immune cell infiltration in lung as possible underlying mechanisms of Magp protection from pulmonary fibrosis. Finally, we will employ our recently developed single nucleus RNASeq approach along with single nucleus assay for transposase-accessible chromatin (snATAC)-Seq to characterize the fibroblast populations that express Magp1 and 2 and evaluate how lung-wide transcriptomes and epigenomes change in the absence of Magps during fibrosis. This proposal will provide Dr. Koenitzer with the mentored training in single cell sequencing and bioinformatics, fibroblast and ECM biology, and lung histology/imaging needed to achieve his goal of independence as a physician-scientist. Under the oversight of an expert scientific advisory committee, He will realize career milestones, complete formal coursework, and develop lasting collaborations. Findings from this work will have implications beyond IPF in other interstitial lung diseases and enable Dr. Koenitzer to develop new approaches to the treatment of lung disease as an independent investigator.