Dataset Information

Spatial resolution of proteomic changes in lung tissue from a flexible AAV-DTR/DT mouse model of acute cell deletion mimicking epithelial lung injury

ABSTRACT: Lung epithelial injury leads to different pathologies, all associated with severe outcome and increased mortality. These pathologies are divided into chronic and acute diseases like idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) or the acute lung distress syndrome (ARDS). Hallmarks of both groups of lung diseases are a breakdown of the epithelial barrier and impairment of lung function. Repeated epithelial injury leads to chronic inflammation, fibroblast activation and ultimately to scarring and stiffening of the lung. The observed decline in lung function is the first symptom of IPF patients. As these changes are occurring slowly over time, patients only present to clinicians at a rather late stage. Therefore, disease driving mechanisms can only be estimated based on observed changes in biomarkers, the transcriptome or proteome. However, alterations in signalling pathways at a very early stage of disease development can only be investigated in vitro or in animal models. In this study a novel and flexible DTR/DT model of acute epithelial lung injury driven by AAV6.2 mediated human diphtheria toxin receptor (hDTR) expression was developed and characterized. The hDTR is sensitive for diphtheria toxin (DT), thereby inducing apoptosis by blocking protein synthesis. In contrast, a polymorphism of the murine DTR protects mice from DT mediated toxicity. The AAV6.2 variant transduces specifically into bronchial epithelial and alveolar epithelial type II cells leading to their depletion after DT administration. Using laser-capture microdissection different epithelial cell regions (bronchial epithelium and alveolar epithelium) were isolated from formalin-fixed and paraffin-embedded lung tissue of AAV-stuffer and AAV-hDTR mice, which were administered intratracheally with 100 ng DT for 24 h, and analyzed using mass spectrometry.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Lung

SUBMITTER: Eva Griesser

LAB HEAD: Kerstin Geillinger-Kaestle

PROVIDER: PXD025712 | Pride | 2022-06-30

REPOSITORIES: Pride

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Characterization of a flexible AAV-DTR/DT mouse model of acute epithelial lung injury.

Griesser Eva E Schönberger Tanja T Stierstorfer Birgit B Wyatt Hannah H Rist Wolfgang W Lamla Thorsten T Thomas Matthew James MJ Lamb David D Geillinger-Kästle Kerstin K

American journal of physiology. Lung cellular and molecular physiology 20220628 2

Animal models are important to mimic certain pathways or biological aspects of human pathologies including acute and chronic pulmonary diseases. We developed a novel and flexible mouse model of acute epithelial lung injury based on adeno-associated virus (AAV) variant 6.2-mediated expression of the human diphtheria toxin receptor (DTR). Following intratracheal administration of diphtheria toxin (DT), a cell-specific death of bronchial and alveolar epithelial cells can be observed. In contrast to ...[more]

PMID: 35762632

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Project description:Injury of the lung epithelium is one of the initial events driving acute lung diseases such as acute lung injury and acute respiratory distress syndrome. Repeated epithelial injury followed by aberrant repair leads to fibroblast activation and extracellular matrix deposition and is therefore considered as the major cause of chronic pulmonary diseases, such as idiopathic pulmonary fibrosis. In vitro models as well as in vivo animal models are necessary to investigate early disease mechanisms and altered signalling pathways of epithelial cells. One way to target specific cells is the diphtheria toxin receptor/diphtheria toxin (DTR/DT) system. The human DTR (hDTR) is sensitive to DT, thereby inducing cell death by blocking protein synthesis, while the murine DTR is at least 10^5 times more resistant to DT. Consequently, expression of the hDTR and application of DT lead to targeted cell depletion. Therefore, a novel and flexible DTR/DT model of acute epithelial lung injury was established and described recently, which is driven by adeno-associated virus (AAV) variant 6.2 mediated hDTR expression. The AAV6.2 vector transduces specifically into bronchial epithelial and alveolar epithelial type II cells leading to their depletion after DT administration. In this study we analysed the bulk lung proteome of the recently established AAV-DTR/DT mouse model of acute epithelial lung injury, providing a detailed insight into proteomic changes upon injury. Frozen pulverized lung tissue samples (~10 mg per sample) from AAV-stuffer control (1 E11 viral genome, n=7) and AAV-hDTR (0.3 E11 viral genome, n=7) treated mice 24 h after intratracheal application of 100 ng DT were analysed in a TMTpro-based workflow. TMTpro-labelled peptides were fractionated into 24 fractions using off-line high pH reversed phase chromatography. Fractions were analysed on an Orbitrap Eclipse Tribrid mass spectrometer in combination with the FAIMS Pro Interface (Thermo Scientific) using a SPS-MS3 based method including real-time search.

			Action	DRS
	DTR_BE_8F.msf	Msf
	DTR_BE_F1.raw	Raw
	DTR_BE_F2.raw	Raw
	DTR_BE_F3.raw	Raw
	DTR_BE_F4.raw	Raw