Project description:Pulmonary fibrosis (PF) is a chronic, progressive condition that represents the end-stage of many interstitial lung diseases (ILDs). Single-cell transcriptomic studies have revealed disease-emergent epithelial, fibroblast, and macrophage cell types/states in PF lungs, but the spatial contexts wherein these cells contribute to disease pathogenesis has remained uncertain. Using image-based spatial transcriptomics to profile gene expression changes in-situ across 28 lung samples from control and PF lungs, we characterized the expression of 343 genes in over 1 million nuclei at subcellular resolution. Using both cell-based and cell-agnostic approaches, we observed a diversity of distinct molecularly-defined spatial niches in control and PF lungs. Overlaying these computationally-defined niches with disease-associated histopathologic features, we identified novel patterns of dysregulation in alveoli informed by spatial context. We computationally segmented individual air spaces and using cell composition, we ordered airspaces from homeostatic to most dysregulated. Using this ordering we identified a series of stepwise molecular changes associated with progressive distal lung remodeling. Together, these results advance our understanding of the molecular programs underlying progressive PF.
Project description:Pulmonary fibrosis (PF) is a chronic, progressive condition that represents the end-stage of many interstitial lung diseases (ILDs). Single-cell transcriptomic studies have revealed disease-emergent epithelial, fibroblast, and macrophage cell types/states in PF lungs, but the spatial contexts wherein these cells contribute to disease pathogenesis has remained uncertain. Using image-based spatial transcriptomics to profile gene expression changes in-situ across 28 lung samples from control and PF lungs, we characterized the expression of 343 genes in over 1 million nuclei at subcellular resolution. Using both cell-based and cell-agnostic approaches, we observed a diversity of distinct molecularly-defined spatial niches in control and PF lungs. Overlaying these computationally-defined niches with disease-associated histopathologic features, we identified novel patterns of dysregulation in alveoli informed by spatial context. We computationally segmented individual air spaces and using cell composition, we ordered airspaces from homeostatic to most dysregulated. Using this ordering we identified a series of stepwise molecular changes associated with progressive distal lung remodeling. Together, these results advance our understanding of the molecular programs underlying progressive PF.
Project description:To identify genes regulating the jamming transition in healthy distal airway epithelia compared to the dysregulated state in idiopathic pulmonary fibrosis epithelia (IPF) we analyzed RNA from three (3) healthy and four (4) IPF patient cells grown at air-liquid-interface (ALI). This bulk analysis of 3 timepointes (days 4, 8, and 14 of ALI) spanning the jamming transition revealed an enrichment for ERBB- and YAP-related genes.
Project description:Aberrant expansion of KRT5+ basal cells in the distal lung accompanies progressive alveolar epithelial cell loss and tissue remodelling during fibrogenesis in idiopathic pulmonary fibrosis (IPF). The mechanisms determining activity of KRT5+ cells in IPF have not been delineated. Here, we show an association between KRT5+ cells in human fibrotic lung and regional differences in collagen topography. In vitro, KRT5+ cell migratory characteristics and expression of remodelling genes are modulated by extracellular matrix (ECM) composition and organisation. Mass spectrometry-based proteomics revealed compositional differences in the matrisome secreted by primary human lung fibroblasts (HLF) from IPF patients compared to controls. Over-expression of ECM glycoprotein, Secreted Protein Acidic and Cysteine Rich (SPARC) in the IPF HLF matrisome restricts KRT5+ cell migration in vitro. Together, our findings demonstrate that changes to the ECM in IPF directly influence KRT5+ cell behaviour and function contributing to remodelling events in the fibrotic niche.
Project description:Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative disorder limited to the lung. New findings, starting from our proteomics studies on IPF, suggest that systemic involvement with altered molecular mechanisms and metabolic disorder is an underlying cause of fibrosis. The role of metabolic dysregulation in the pathogenesis of IPF has not been extensively studied, despite a recent surge of interest. In particular, our studies on bronchoalveolar lavage fluid have shown that the renin-angiotensin-aldosterone system (RAAS), the hypoxia/oxidative stress response, and changes in iron and lipid metabolism are involved in onset of IPF. These processes appear to interact in an intricate manner and to be related to different fibrosing pathologies not directly linked to the lung environment. The disordered metabolism of carbohydrates, lipids, proteins and hormones has been documented in lung, liver, and kidney fibrosis. Correcting these metabolic alterations may offer a new strategy for treating fibrosis. This paper focuses on the role of metabolic dysregulation in the pathogenesis of IPF and is a continuation of our previous studies, investigating metabolic dysregulation as a new target for fibrosis therapy.
Project description:The development of mirror-image biology systems and related applications is hindered by the lack of effective methods to sequence mirror-image (D-) proteins. Although natural-chirality (L-) proteins can be sequenced by bottom–up liquid chromatography–tandem mass spectrometry (LC–MS/MS), the sequencing of long D-peptides and D-proteins with the same strategy requires digestion by a site-specific D-protease before mass analysis. Here we apply solid-phase peptide synthesis and native chemical ligation to chemically synthesize a mirror-image version of trypsin, a widely used protease for site-specific protein digestion. Using mirror-image trypsin digestion and LC–MS/MS, we sequence a mirror-image large subunit ribosomal protein (L25) and a mirror-image Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and distinguish between different mutants of D-Dpo4. We also perform writing and reading of digital information in a long D-peptide of 50 amino acids. Thus, mirror-image trypsin digestion in conjunction with LC–MS/MS may facilitate practical applications of D-peptides and D-proteins as potential therapeutic and informational tools.