Protein Signatures of Remodeled Airways in Transplanted Lungs with Bronchiolitis Obliterans Syndrome Obtained Using Laser-Capture Microdissection

• DOI: 10.1016/j.ajpath.2021.05.014
• Publication Date: 2021-06-07T15:05:53Z
• Authors: Catharina Müller; Oskar Rosmark; Emma Åhrman; Hans Brunnström; Katharina Wassilew; Annika Nybom; Barbora Michaliková; Hillevi Larsson; Leif T. Eriksson; Hans H. Schultz; Michael Perch; Johan Malmström; Jenny Wigén; Martin Iversen; Gunilla Westergre...
• Keywords: Laser capture microdissection; Microdissection

Bronchiolitis obliterans syndrome, a common form of chronic lung allograft dysfunction, is the major limitation to long-term survival after transplantation. The histologic correlate progressive, fibrotic occlusion small airways, obliterative bronchiolitis lesions, which ultimately lead organ failure. molecular composition these lesions unknown. In this sutdy, protein in explanted lungs from four end-stage syndrome patients was analyzed using laser-capture microdissection and optimized sample preparation protocols for mass spectrometry. Immunohistochemistry immunofluorescence were used determine spatial distribution commonly identified proteins on tissue level, signatures 14 established. A set 39 proteins, >75% included distinct structural (collagen types IV VI) cellular components (actins, vimentin, tryptase). Each respective lesion exhibited unique (on average, n = 66 proteins), thereby mirroring morphologic variation lesions. Antibody-based staining confirmed spectrometry–based findings. showed variations their content, but also features. This study provides insights into development rejection patterns correlated pathways extracellular matrix organization, development, wound healing processes. Since first transplantations late 1980s, (BOS) progressive airway obstruction has been recognized as most important pathology associated with adverse outcome. definition guideline BOS issued 1993,1Cooper J.D. Billingham M. Egan T. Hertz M.I. Higenbottam Lynch J. Mauer Paradis I. Patterson G.A. Smith C. Trulock E.P. Vreim Yousem S. International Society Heart Lung TransplantationA working formulation standardization nomenclature clinical staging dysfunction allografts.J Transplant. 1993; 12: 713-716PubMed Google Scholar revisions 2002,2Estenne Maurer J.R. Boehler A. J.J. Frost Mallory G.B. Snell G.I. 2001: an update diagnostic criteria.J 2002; 21: 297-310Abstract Full Text PDF PubMed Scopus (1120) 2014,3Meyer K.C. Raghu G. Verleden G.M. Corris P.A. Aurora P. Wilson Brozek Glanville A.R. An international ISHLT/ATS/ERS practice guideline: diagnosis management syndrome.Eur Respir 2014; 44: 1479-1503Crossref (324) 2019.4Verleden Lease E.D. Fisher A.J. Calabrese F. Ensor C.R. Gottlieb Hachem R.R. Lama V. Martinu Neil D.A.H. Singer L.G. Vos R. Chronic dysfunction: definition, criteria, approaches treatment-a consensus report Pulmonary Council ISHLT.J Transpl. 2019; 38: 493-503Abstract (199) term defined causing >20% decline forced expiratory volume 1 second no obvious reason, like infection or other macroscopic pathologic (OB) These were, however, patchy could not be diagnosed reliably radiology transbronchial biopsies.3Meyer demonstrated both single-center studies5Burton C.M. Carlsen Mortensen Andersen C.B. Milman N. Iversen Long-term transplantation depends severity syndrome.J 2007; 26: 681-686Abstract (104) Transplantation registry studies6Kulkarni H.S. Cherikh W.S. Chambers D.C. Garcia V.C. Kreisel D. Puri Kozower B.D. Byers D.E. Witt C.A. Alexander-Brett Aguilar P.R. Tague L.K. Furuya Y. 3rd, Yusen R.D. syndrome-free transplantation: Thoracic Transplant Registry analysis.J 5-16Abstract (45) afflict nearly all transplant patients. median BOS-free 3 4 years, 10 years transplantation, BOS, some degree, present 90% initially synonymously type dysfunction. However, subtypes are now by parameters histopathologic features, prominently restrictive dysfunction/restrictive syndrome.7Verleden Meyer new classification system dysfunction.J 33: 127-133Abstract (360) Scholar, 8Sato Waddell T.K. Wagnetz U. Roberts H.C. Hwang D.M. Haroon Chaparro Hutcheon M.A. Keshavjee Restrictive (RAS): novel 2011; 30: 735-742Abstract (307) 9Estenne human transplantation.Am J Crit Care Med. 166: 440-444Crossref (333) 10Todd J.L. Palmer S.M. syndrome: final frontier transplantation.Chest. 140: 502-508Abstract (143) focused Studies have suggested that immunologic processes non-BOS different BOS.11Vandermeulen E. Lammertyn S.E. Ruttens Bellon H. Ricciardi Somers Bracke K.R. Van Den Eynde K. Tousseyn Brusselle G.G. Verbeken E.K. Verschakelen Emonds M.P. Raemdonck Vanaudenaerde B.M. Immunological diversity phenotypes comprehensive immunohistochemical analysis.Transpl Int. 2017; 134-143Crossref (31) Scholar,12von der Thusen J.H. Vandermeulen Weynand B. histomorphological spectrum implications prognosis.Mod Pathol. 2018; 31: 780-790Crossref (21) correlates OB develop remodeling although events leading occlusions completely understood.13Verleden Vasilescu McDonough J.E. Geenens Wuyts W.A. Sokolow Knoop Cooper Hogg J.C. Linking changes structure.Eur 2015; 46: 1430-1439Crossref (40) Epithelial cell death key event BOS14Levy L. Tigert Huszti Saito Mitsakakis Moshkelgosha Joe Boonstra K.M. Tikkanen J.M. Juvet S.C. markers bronchoalveolar lavage recipients-a retrospective cohort study.Transpl 32: 965-973Crossref (8) may trigger exaggerated healing, where mesenchymal activities, including buildup turnover matrix, subsequently obliterate airway.15Andersson-Sjoland Thiman Nihlberg Hallgren O. Rolandsson Skog Mared Hansson Eriksson Bjermer Westergren-Thorsson Fibroblast activity changed process transplantation.J 945-954Abstract (22) innovative study, micro–computed tomography demonstrate nature lesions.16Verleden Willems Hostens Rondelet Decramer site 189: 292-300Crossref (64) minute, affecting mainly peripheral airways diameter <2 mm. Furthermore, appeared heterogeneous, probably due developmental stages. seeks characterize exhibiting combination microscopy (LCM), spectrometry (MS), antibody-based combine state-of-the art identification information level. Thereby, it snapshot view ongoing Molecular profiling desirable elucidate initiating driving disease, aim find tools intervene.17Weigt S.S. Shino M.Y. Wang X. diagnostics rejection: pipeline pipe dream?.J 514-515Abstract (2) Thorough studies advanced knowledge field complex imaging methods, analysis, gene analysis.16Verleden Scholar,18Verleden Schoemans Dubbeldam Boone M.N. Hoorebeke Phenotypical morphology graft vs. host disease stem transplantation.Mod 817-829Crossref (4) 19Jonigk Izykowski Rische Braubach Kuhnel Warnecke Lippmann Kreipe Haverich Welte Laenger biopsies pulmonary allografts predict dysfunction.Am 185: 3178-3188Abstract (29) 20Kuehnel Maegel Vogel-Claussen Robertus Jonigk Airway remodelling transplanted lung.Cell Tissue Res. 367: 663-675Crossref (10) 21Morgan D.L. Merrick B.A. Gerrish K.E. Stockton P.S. Foley J.F. Gwinn W.M. Kelly F.L. Ton T.V. Flake G.P. Gene expression bronchiolitis-like 2,3-pentanedione-exposed rats.PLoS One. 10: e0118459Crossref (12) Characterization challenging because focal nature, scarcity lung, amount material available analysis. Herein, we use approach increase depth MS describe pathologically remodeled exploratory exquisitely dissecting out carefully excluding confounding surrounding lesion, involving warrant further investigation, finding therapeutic targets. obtained two centers (Skåne University Hospital, Lund, Sweden; Copenhagen Rigshospitalet, Copenhagen, Denmark). had complication retransplantation. Patient characteristics shown Supplemental Table S1. Informed consent ethical permit at center (Lund dnr 2015-89), data Scandiatransplant (dnr 2007-58-0008). Every patient consented research. Healthy Sahlgrenska Gothenburg, Sweden (ethical permit, 2008/413), informed obtained. based explant tissue. formalin-fixed underwent thorough examination, extensively sampled, typically least one central (containing bronchus) blocks per lobe. samples dehydrated paraffin embedded according routine procedures. Using microtome, sections (4 μm thick) produced placed glass slides (Menzel Superfrost Plus; VWR, Stockholm, Sweden) hematoxylin/eosin (HE) staining, immunohistochemistry (IHC), (IF), LCM [in Auto–laser pressure catapulting (LPC) mode]; polyethylene (PEN)–membrane slide (Zeiss, Membrane Slide 1.0 PEN, 415101-4401-000) (in Robo-LPC mode). Before usage, PEN activated 30 minutes UV irradiation (254 nm) Spectrolinker/Crosslinker XL-1000 (DOT Scientific Inc., Burton, MI). research study. large processed, submitted protocol examination lungs. During processing, alternating PEN-membrane (Supplemental Figure S1). Because high cost, feasible material. Depending consecutive, HE- elastica van Gieson–stained sections, lifted during slides. According our instrument manufacturer's (Zeiss), does affect quality retrieval LCM. containing pathologist (H.B. Lund K.W. Copenhagen). total 50 100 each block, systematic screening performed HE-stained regular intervals depths specimen (Histolab, Askim, Mayer's hematoxylin/eosin, instructions). Once identified, consecutive LCM, IF, IHC (Figure 1). Identification procedure done Biology Group, then reviewed independently pathologists Only pathologists' assessments concordance Lesions precise delineation technical difficulties excluded. No considered when satisfactory number reached (only study) lack well-delineated suitable Consecutive ones HE selected interest, previously published.22Rosmark Ahrman Muller Elowsson Rendin Malmstrom Larsson-Callerfelt A.K. Quantifying scaffold cultures.Sci Rep. 8: 5409Crossref After deparaffinization, heat-induced epitope PT Link (Histolab) citrate buffer. collagen α 2 (IV) chains tryptase EnVision Dual System (K4065; Dako, Glostrup, Denmark), instructions, horseradish peroxidase–coupled secondary antibodies counterstaining hematoxylin visualize nuclei. IF periostin, α-1 (VI) chain, Ki-67, α-smooth muscle actin (α-SMA; aortic smooth actin) incubation primary hour fluorochrome-coupled 45 minutes. Sections mounted ProLong Gold Antifade Mountant DAPI (Invitrogen, Carlsbad, CA) Nonspecific assessed omitting (Table 1).Table 1Antibody Specifications Proteins Detected HistologyAntibodyDescriptionManufacturer, catalog no.DilutionMethodSecondary antibodyCollagen α-2 chainsBasement membraneAbcam (Cambridge, UK), ab65861:4000IHCDAB+Collagen chainStructural proteinAbcam, ab65881:1000IFA-21246PeriostinGlycoproteinAbcam, ab799461:100IFA-21246α-SMACytoskeletonSigma (St. Louis, MO), C61981:2500IFDirectly conjugatedKi-67Proliferation markerAbcam, ab155801:1000IFA-21246TryptaseMast cellDako, Kod M70521:100IHCDAB+A, Alexa Fluor (Thermo Scientific, Waltham, MA), 1:200; DAB+, diaminobenzidine positive [Dako + System-HRP (K4065)]; immunofluorescence; IHC, immunohistochemistry; α-SMA, actin. Open table tab A, Images Zeiss fluorescence microscope VS120 virtual scanning (Olympus, Tokyo, Japan), either bright-field mode (hematoxylin/eosin IHC) fluorescent (IF). From scanned slides, representative images acquired OlyVIA soſtware 2.8 (Olympus). Exposure times, acquisition settings, image brightness adjustments consistently negative controls, apart VI periostin stainings healthy control subjects later time point. intended deparaffinized (2× xylene 5 minutes, 2× absolute ethanol 95% 70% distilled water). reported contain abundance hyaluronan; thus, hyaluronidase digest (0.2% bovine testicular hyaluronidase; Sigma, St. MO; I-S) acetate buffer (20 mmol/L sodium 150 chloride, pH 5.2) 37°C, enable effective extraction microdissected material.23Todd Sugimoto Kennedy V.E. Zhang H.L. Pavlisko E.N. Huang Gelman A.E. Hyaluronan contributes stimulates through activation innate immunity.Am 556-566Crossref (60) Predigested stained seconds three water, (2 × minutes), air dried, stored –20°C sealed containers. allowed equilibrate room temperature, avoiding condensation. PALM MicroBeam (Carl MicroImaging, Jena, Germany) Robo 4.6 Pro Software MicroImaging), 10× objective. whether glass, (defined larger structures cut piece laser) For object glasses, Auto-LPC (small fragments directly off discrete laser pulses) submucosal fibrosis narrowing bronchiolar lumen, within ring collected. Dissected catapulted lids adhesive cap tubes (Zeiss Adhesive Cap opaque 415190-9201-000; 500 μL). AxioCamICc1 CapCheck function successful collection dissected lid. Samples prepared Braakman et al,24Braakman R.B. Stingl Tilanus-Linthorst M.M. Deurzen C.H. Timmermans Smid Foekens J.A. Luider T.M. Martens J.W. Umar Proteomic characterization breast environment protein-level overview malignant transformation.Proteomics. 17: 1600213Crossref (6) modifications 8 mol/L urea SP3 beads peptide desalting. dissolved 20 μL/lid 0.1% RapiGest w/v (Waters, Milford, MA; 186001861) ammonium bicarbonate incubated temperature Subsequently, lysate centrifuged bottom tube (30 seconds/6082 g; Biofuge pico, Heraeus; DJB, Buckinghamshire, UK). slices sonicated Bioruptor Plus (Diagenode SA, Seraing, Belgium) 4°C cycles, 15 on/off. denatured 99°C, 300 rpm, reduced dithiothreitol 60°C alkylated iodoacetamide diluted 1.6 concentration digested μg Lys-C/Trypsin mix (Promega, Madison, WI) overnight orbital shaker mm orbit, rpm. digestion stopped addition 10% trifluoroacetic acid 0.5% (v/v), degraded 37°C 60 desalted beads, described Hughes al.25Hughes C.S. Foehr Garfield D.A. Furlong E.E. Steinmetz L.M. Krijgsveld Ultrasensitive proteome analysis paramagnetic bead technology.Mol Syst Biol. 757Crossref (411) As starting peptides, cleanup followed. Briefly, acidified supplemented μL acetonitrile added >95%. washed 100% acetonitrile. Peptides eluted aqueous 2% dimethyl sulfoxide, followed sonication. Peptide mixed 1:1 (v/v) (4% 0.4% formic acid), giving acetonitrile, 0.2% acid, 1% sulfoxide. proteomics deposited ProteomeXchange Consortium via Proteomics Database partner repository (PRIDE, https://www.ebi.ac.uk/pride/archive/projects/PXD014171, publication date, May 19, 2021). separated EASY-nLC 1200 high-performance liquid chromatography Waltham MA) 50-cm EASY-spray PepMap RSLC C18 column Scientific). separation, following segmented gradient solvent B (0.1% 80% acetonitrile) over acid) used: 3% 11 30% 87 45% 22 flow 350 nL/minute. Data acquisitions Q-Exactive HF-X spectrometer data-dependent acquisition, full scans resolution 120,000 200 m/z, automatic gain target million, maximum injection milliseconds range 1650 m/z. scan MS/MS fragmentation top abundant ions 30,000 3e6, milliseconds. Analysis searches raw files MaxQuant version 1.6.3.3 (https://www.maxquant.org, last accessed 1, 2019) Andromeda toward UniProt database standard contaminants (downloaded November 17, 2015). Default settings summed intensities peptides group. Search trypsin missed cleavages 4.5 parts million precursors fragment ions. Carbamidomethylation fixed modification, methionine oxidation variable. false discovery rate peptides. PRIDE (https://www.ebi.ac.uk/pride/archive, June 7, identifier (PXD014171).26Vizcaino Cote R.G. 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