Validation and application of human in vitro models for investigating bronchial response to cigarette smoke

Mastalerz, Michal (2022): Validation and application of human in vitro models for investigating bronchial response to cigarette smoke. Dissertation, LMU München: Medizinische Fakultät

Abstract

Cigarette smoke (CS) is the single most deadly and preventable cause of death. It profoundly affects smokers’ lungs, by altering genes expression profiles, epigenetic modifications, causes DNA damage and changes cell function and morphology [1]. The respiratory tract is the main lung compartment exposed to CS. The bronchial epithelium lining the airway tracts subjected to chronic smoking often leads to incurable diseases such as lung cancers or chronic obstructive pulmonary disease (COPD). Due to its complicated nature and profound impact on human health, the response to CS has been extensively studied in both in vivo and in vitro settings. While CS effects can be investigated in numerous distinct experimental set-ups, little has been done in terms of standardization or validation of these methods. Here, a comprehensive direct comparison between different in vitro CS exposures has been established and analyzed. The cell-delivered dose and the expression profile of genes typically upregulated among smokers were assessed between models, also in relation to expression profile in human lungs. Three surprisingly dissimilar models, namely acute submerged basal cells exposure to cigarette smoke extract (CSE), chronic basolateral CSE ex-posure and acute whole cigarette smoke exposure, yielded responses that were substantially better than any other investigated experimental set-up. Despite the cell-delivered doses varying substantially between these three models, each of them significantly upregulated at least six of out 10 analyzed genes in the primary human bronchial epithelial cells (phBECs). Conclusions from validation study helped choosing the right model, which was later used in the next study, in the proteomic differential expression analysis. Chronic basolateral CSE exposure was the only model that successfully upregulated seven out of 10 genes typically upregulated in smokers. Results of the proteomic analysis further validated the physiological relevance of the model by identifying activation of the molecular pathways characteristic for the CS exposure, such as activation of xenobiotic metabolism pathways and inhibited sirtuin 1 pathway [2]. Interestingly, by using advanced pathway analysis software, a new potential ferroptotic regulator was found, namely nuclear factor 1 (NUPR1). Overall, this study reported a first evidence of critical ferroptosis repressor being aberrantly changed by the CS in phBECs derived from healthy donors. The final topic of this study addressed the question whether the differentiated phBECs from ex- and current smokers exhibit transient and persistent changes caused by smoking that can be seen in vivo. Here, the advantage was taken from proteomic study performed on bronchoalveolar lavage samples, which were derived from never-, ex- and current smokers. After identifying sev-eral genes which expression changes were either transient or persistent after smoking cessation, the basal expression levels of these genes was analyzed on transcript level in differentiated phBECs in vitro, derived also from never-, ex- and current smokers. Surprisingly, the in vitro anal-ysis revealed lower constitutive expression of analyzed genes in phBECs from patients from his-tory of smoking, which did not reflect changes seen in BALF study. Taken together, this thesis presents a successful validation of the several CS exposure models on phBECs. One of them, namely chronic basolateral CSE exposure, was further validated by the proteomic analysis, which, for the first time, revealed NUPR1, a crucial ferroptosis regulator [3], as a CS-regulated gene. This study establishes practical technique of validating CS exposure models, which can be used in in vitro studies, despite possibly different basal genes expressions of CS-regulated genes.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Validation, cigarette smoke, primary cells, CSE, bronchial epithelial cells, air liquid interface, cigarette smoke exposure, proteomics, molecular pathways, differentiation, smoking
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 2. November 2022
• 1. Bericht­erstatter:in: Staab-Weijnitz, Claudia