title: Localization and pneumococcal alteration of junction proteins in the human alveolar-capillary compartment.
abstract: Loss of alveolar barrier function with subsequent respiratory failure is a hallmark of severe pneumonia. Although junctions between endo- and epithelial cells regulate paracellular fluid flux, little is known about their composition and regulation in the human alveolar compartment. High autofluorescence of human lung tissue in particular complicates the determination of subcellular protein localization. By comparing conventional channel mode confocal imaging with spectral imaging and linear unmixing, we demonstrate that background fluorescent spectra and fluorophore signals could be rigorously separated resulting in complete recovery of the specific signal at a high signal-to-noise ratio. Using this technique and Western blotting, we show the expression patterns of tight junction proteins occludin, ZO-1 as well as claudin-3, -4, -5 and -18 and adherence junction protein VE-cadherin in naive or Streptococcus pneumoniae-infected human lung tissue. In uninfected tissues, occludin and ZO-1 formed band-like structures in alveolar epithelial cells type I (AEC I), alveolar epithelial cells type II (AEC II) and lung capillaries, whereas claudin-3, -4 and -18 were visualised in AEC II. Claudin-5 was detected in the endothelium only. Claudin-3, -5, -18 displayed continuous band-like structures, while claudin-4 showed a dot-like expression. Pneumococcal infection reduced alveolar occludin, ZO-1, claudin-5 and VE-cadherin but did not change the presence of claudin-3, -4 and -18. Spectral confocal microscopy allows for the subcellular structural analysis of proteins in highly autofluorescent human lung tissue. The thereby observed deterioration of lung alveolar junctional organisation gives a structural explanation for alveolar barrier disruption in severe pneumococcal pneumonia.
authors.affiliation: Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.|Department for Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355, Berlin, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Peter
authors.affiliation: Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Fatykhova
authors.affiliation: Department of Veterinary Pathology, Freie Universität Berlin, Robert-von-Ostertag-Strasse 15, 14163, Berlin, Germany.
authors.forename: Olivia
authors.initials: O
authors.lastname: Kershaw
authors.affiliation: Department of Veterinary Pathology, Freie Universität Berlin, Robert-von-Ostertag-Strasse 15, 14163, Berlin, Germany.
authors.forename: Achim D
authors.initials: AD
authors.lastname: Gruber
authors.affiliation: Department of General, Visceral, Vascular and Thoracic Surgery, Charité-Universitätsmedizin Berlin, Chariteplatz 1, 10117, Berlin, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Rueckert
authors.affiliation: Department of General, Visceral, Vascular and Thoracic Surgery, Charité-Universitätsmedizin Berlin, Chariteplatz 1, 10117, Berlin, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Neudecker
authors.affiliation: Lungenklinik Heckeshorn, HELIOS Klinikum Emil von Behring, Walterhöferstrasse 11, 14165, Berlin, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Toennies
authors.affiliation: Lungenklinik Heckeshorn, HELIOS Klinikum Emil von Behring, Walterhöferstrasse 11, 14165, Berlin, Germany.
authors.forename: Torsten T
authors.initials: TT
authors.lastname: Bauer
authors.affiliation: Department for General and Thoracic Surgery, DRK Clinics, Drontheimer Strasse 39-40, 13359, Berlin, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Schneider
authors.affiliation: Vivantes Netzwerk für Gesundheit, Klinikum Neukölln, Klinik für Thoraxchirurgie, Berlin, Rudower Straße 48, 12351, Berlin, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Schimek
authors.affiliation: Vivantes Netzwerk für Gesundheit, Klinikum Neukölln, Klinik für Thoraxchirurgie, Berlin, Rudower Straße 48, 12351, Berlin, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Eggeling
authors.affiliation: Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Suttorp
authors.affiliation: Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Andreas C
authors.initials: AC
authors.lastname: Hocke
authors.affiliation: Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany. stefan.hippenstiel@charite.de.|Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353, Berlin, Germany. stefan.hippenstiel@charite.de.
authors.forename: Stefan
authors.identifier: http://orcid.org/0000-0002-5146-1064
authors.initials: S
authors.lastname: Hippenstiel
mesh_terms: D015820:Cadherins / Q000378:metabolism*; D006801:Humans; D010547:Persistent Fetal Circulation Syndrome / Q000378:metabolism* / Q000382:microbiology; D011008:Pneumococcal Infections / Q000378:metabolism* / Q000382:microbiology; D011650:Pulmonary Alveoli / Q000002:abnormalities* / Q000378:metabolism / Q000382:microbiology; D013296:Streptococcus pneumoniae
chemical_list: D015820:Cadherins
keywords: Claudin; Occludin; Pneumonia; Spectral confocal microscopy; Streptococcus pneumoniae; Tight junctions