Estratégias de bioengenharia e Novas Abordagens Metodológicas (NAMs) aplicadas à avaliação do potencial de sensibilização pulmonar de toxicantes inalados
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2022-11-10
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Universidade Federal de Goiás
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Introduction: Respiratory sensitization encompasses a group of inflammatory diseases that manifest through airway hyperresponsiveness and airflow limitation. Although the chemical respiratory allergy (CRA) induced by Low Molecular Weight (LMW) sensitizers is a major concern, especially in terms of the regulatory framework, to date there are no methods available for preclinically addressing this toxicological outcome, as its mechanistic background is not fully understood at molecular or cellular levels. Objectives: The objective of this work is to apply several in vitro and in chemico approaches in order to address physiologically relevant aspects of human response to LMW respiratory allergens, aiming to contribute to the elucidation of this toxicant class mode of action and further evaluation regarding the preclinical stage. Methodology: First, we evaluated the interaction between LMW sensitizers and lung mucus by employing the mucin spectroscopic profile assessment in the presence and absence of seven chemical respiratory allergens. Then, we performed the evaluation of inflammatory and functional parameters in different respiratory tract cell types, including bronchial epithelial (BEAS-2B), lung fibroblasts (MRC-5), endothelial (EA.hy926) and monocytic cells (THP-1), after exposure to sub-cytotoxic concentrations of each sensitizer. Following the monolayer cell-based studies, we integrated the four mentioned cell tupes in a 3D bronchial co-culture model and exposed it to maleic anhydride aerosols in an air-liquid interface (ALI) for further evaluating inflammatory and functional tissue parameters. Finally, we also proposed the development of a bronchial epithelial model using the porcine descellularized bronchial wall as a bioscaffold, for which we evaluated different obtention methods, as well as the cell behavior when cultivated upon the obtained matrix. Results/Discussion: The results showed that some of the sensitizers evaluated interact with mucin, the main protein mucus component, but the toxicant-mucin complex formation does not seem to be a common feature of different chemical classes of allergens. At a cellular level, sensitizers promoted an increase in IL-8, IL-6, and IL-1β production in terms of the different evaluated cell types. It also impaired the MUC1 expression by bronchial cells BEAS-2B and activated endothelial cells (EA.hy926), thereby increasing the ICAM-1 surface detection. It has been also demonstrated an increased expression of dendritic cell activation/maturation surface biomarkers (CD86, HLA-DR and CD11b) in THP-1 monocytic cells after exposure to chemical allergens. In parallel, the 3D bronchial co-culture model was successfully reconstructed using by cultivating the four aforementioned cell types at an ALI, and maleic anhydride aerosols exposure increased the inflammatory biomarkers production, as well as the apoptosis in epithelial layer and THP-1 dendritic cell activation. Besides, the decellularized porcine bronchial matrix allowed the cultivation of Calu-3 cells, which regularly expressed the airway epithelium biomarkers after 7 and 14 days of cultivation in ALI. Conclusion: Taken together, our results showed that these aforementioned cell types participate in the CRA Adverse Outcome Pathway and must be considered when developing testing strategies. The integration of different cell types in an unique co-culture model allowed the obtention of a global toxicant response, enabling the aerosol exposure, which emulates more realistically the airway contact with external aggressors. Finally, the 3D bronchial bioscaffold yielded the obtention of an epithelial model that morphologically and phenotypically resembles human airway epithelium, being a useful alternative for addressing lung response to inhaled toxicants. Thus, the work subsidizes the employment of inflammatory and functional biomarkers, as well as of bioengineering-derived in vitro models for addressing the respiratory sensitization potential of LMW allergens at a preclinical stage.
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SILVA, A. C. G. Estratégias de bioengenharia e Novas Abordagens Metodológicas (NAMs) aplicadas à avaliação do potencial de sensibilização pulmonar de toxicantes inalados. 2022. 117 f. Tese (Doutorado em Ciências Farmacêuticas) - Universidade Federal de Goiás, Goiânia, 2022.