Characterization of transient and progressive pulmonary fibrosis by spatially correlated phase contrast microCT, classical histopathology and atomic force microscopy

: Pulmonary fibrosis (PF) is a severe and progressive condition in which the lung becomes scarred over time resulting in pulmonary function impairment. Classical histopathology remains an important tool for micro-structural tissue assessment in the diagnosis of PF. A novel workflow based on spatial correlated propagation-based phase-contrast micro computed tomography (PBI-microCT), atomic force microscopy (AFM) and histopathology was developed and applied to two different preclinical mouse models of PF - the commonly used and well characterized Bleomycin-induced PF and a novel mouse model for progressive PF caused by conditional Nedd4-2 KO. The aim was to integrate structural and mechanical features from hallmarks of fibrotic lung tissue remodeling. PBI-microCT was used to assess structural alteration in whole fixed and paraffin embedded lungs, allowing for identification of fibrotic foci within the 3D context of the entire organ and facilitating targeted microtome sectioning of planes of interest for subsequent histopathology. Subsequently, these sections of interest were subjected to AFM to assess changes in the local tissue stiffness of previously identified structures of interest. 3D whole organ analysis showed clear morphological differences in 3D tissue porosity between transient and progressive PF and control lungs. By integrating the results obtained from targeted AFM analysis, it was possible to discriminate between the Bleomycin model and the novel conditional Nedd4-2 KO model using agglomerative cluster analysis. As our workflow for 3D spatial correlation of PBI, targeted histopathology and subsequent AFM is tailored around the standard procedure of formalin-fixed paraffin-embedded (FFPE) tissue specimens, it may be a powerful tool for the comprehensive tissue assessment beyond the scope of PF and preclinical research

Congenital Deletion of Nedd4-2 in Lung Epithelial Cells Causes Progressive Alveolitis and Pulmonary Fibrosis in Neonatal Mice

Recent studies found that expression of NEDD4-2 is reduced in lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and that the conditional deletion of Nedd4-2 in lung epithelial cells causes IPF-like disease in adult mice via multiple defects, including dysregulation of the epithelial Na+ channel (ENaC), TGFβ signaling and the biosynthesis of surfactant protein-C proprotein (proSP-C). However, knowledge of the impact of congenital deletion of Nedd4-2 on the lung phenotype remains limited. In this study, we therefore determined the effects of congenital deletion of Nedd4-2 in the lung epithelial cells of neonatal doxycycline-induced triple transgenic Nedd4-2fl/fl/CCSP-rtTA2S-M2/LC1 mice, with a focus on clinical phenotype, survival, lung morphology, inflammation markers in BAL, mucin expression, ENaC function and proSP-C trafficking. We found that the congenital deletion of Nedd4-2 caused a rapidly progressive lung disease in neonatal mice that shares key features with interstitial lung diseases in children (chILD), including hypoxemia, growth failure, sterile pneumonitis, fibrotic lung remodeling and high mortality. The congenital deletion of Nedd4-2 in lung epithelial cells caused increased expression of Muc5b and mucus plugging of distal airways, increased ENaC activity and proSP-C mistrafficking. This model of congenital deletion of Nedd4-2 may support studies of the pathogenesis and preclinical development of therapies for chILD

The Mechanism of Prion Inhibition by HET-S

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Antibiotic use during pregnancy is linked to offspring gut microbial dysbiosis, barrier disruption, and altered immunity along the gut–lung axis

Antibiotic use during pregnancy is associated with increased asthma risk in children. Since approximately 25% of women use antibiotics during pregnancy, it is important to identify the pathways involved in this phenomenon. We investigate how mother-to-offspring transfer of antibiotic-induced gut microbial dysbiosis influences immune system development along the gut–lung axis. Using a mouse model of maternal antibiotic exposure during pregnancy, we immunophenotyped offspring in early life and after asthma induction. In early life, prenatal-antibiotic exposed offspring exhibited gut microbial dysbiosis, intestinal inflammation (increased fecal lipocalin-2 and IgA), and dysregulated intestinal ILC3 subtypes. Intestinal barrier dysfunction in the offspring was indicated by a FITC-dextran intestinal permeability assay and circulating lipopolysaccharide. This was accompanied by increased T-helper (Th)17 cell percentages in the offspring's blood and lungs in both early life and after allergy induction. Lung tissue additionally showed increased percentages of RORγt T-regulatory (Treg) cells at both time points. Our investigation of the gut–lung axis identifies early-life gut dysbiosis, intestinal inflammation, and barrier dysfunction as a possible developmental programming event promoting increased expression of RORγt in blood and lung CD4+ T cells that may contribute to increased asthma risk.Fil: Alhasan, Moumen M.. Universität zu Berlin; AlemaniaFil: Hölsken, Oliver. Freie Universität Berlin; AlemaniaFil: Duerr, Claudia. Freie Universität Berlin; AlemaniaFil: Helfrich, Sofia. Freie Universität Berlin; AlemaniaFil: Branzk, Nora. Freie Universität Berlin; AlemaniaFil: Philipp, Alina. Freie Universität Berlin; AlemaniaFil: Leitz, Dominik. Freie Universität Berlin; AlemaniaFil: Duerr, Julia. Freie Universität Berlin; AlemaniaFil: Almousa, Yahia. Freie Universität Berlin; AlemaniaFil: Barrientos, Gabriela Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Hospital Aleman; ArgentinaFil: Mohn, William W.. University of British Columbia; CanadáFil: Gamradt, Stefanie. Freie Universität Berlin; AlemaniaFil: Conrad, Melanie L.. Freie Universität Berlin; Alemani

The Mechanism of Prion Inhibition by HET-S

HET-S (97% identical to HET-s) has an N-terminal globular domain that exerts a prion-inhibitory effect in cis on its own prion-forming domain (PFD) and in trans on HET-s prion propagation. We show that HET-S fails to form fibrils in vitro and that it inhibits HET-s PFD fibrillization in trans. In vivo analyses indicate that β-structuring of the HET-S PFD is required for HET-S activity. The crystal structures of the globular domains of HET-s and HET-S are highly similar, comprising a helical fold, while NMR-based characterizations revealed no differences in the conformations of the PFDs. We conclude that prion inhibition is not encoded by structure but rather in stability and oligomerization properties: when HET-S forms a prion seed or is incorporated into a HET-s fibril via its PFD, the β-structuring in this domain induces a change in its globular domain, generating a molecular species that is incompetent for fibril growth