Unit:
Faculty of MedicineLibrary of the School of Health Sciences
Author:
Tzani-Tzanopoulou Panagiota
Dissertation committee:
Νικόλαος Παπαδόπουλος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Μαρία Τσολιά, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Γρηγόριος Καλτσάς, Καθηγητής, Σχολή Μηχανικών, ΠΑΔΑ
Παρασκευή Ξεπαπαδάκη, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Αλεξάνδρα Σολδάτου, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Αθανάσιος Μίχος, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Εμμανουήλ Φλεμετάκης, Αναπληρωτής Καθηγητής, Τμήμα Βιοτεχνολογίας, ΓΠΑ
Original Title:
Ανάλυση των αλληλεπιδράσεων βακτηριοφάγων, βακτηρίων και επιθηλιακών κυττάρων του αναπνευστικού συστήματος μέσω ενός in vitro συστήματος μικρορευστομηχανικής
Translated title:
Analysis of the interactions between bacteriophages, bacteria and airway epithelial cells through an in vitro microfluidic system
Summary:
The airway epithelium is the primary site where the inhaled and resident microbiota interact with the host and may play an important role in the development and pathophysiology of allergic asthma. With the advent of culture-independent molecular techniques and high-throughput technologies, the complex composition and diversity of respiratory bacterial communities has been well documented, and the notion of lung sterility has been definitively discarded. Recent studies indicate that the microbial composition of the airways of asthmatics differs markedly from that of healthy individuals across the spectrum of disease severity. For example, the airways of healthy individuals are reported to be dominated mainly by bacteria of the genera Prevotella (Prevotella melaninogenica, Prevotella nanceiensis, Prevotella salivae) and Veilonella (Veillonella alcalescens, Veillonella parvula, Veillonella dispar). In contrast, the bacteriome of asthmatic airways is dominated by Haemophilus (Haemophilus influenzae B, non-typeable Haemophilus influenzae), Neisseria and Moraxella catarrhalis together with Streptococcus pneumoniae, Acinetobacter baumannii, Staphylococcus aureus and Pseudomonas aeruginosa. In parallel, there is growing body of evidence suggesting that bacterial viruses (bacteriophages or simply phages), which regulate bacterial populations, can be found in almost every niche of the human body and can interact directly with eukaryotic cells. The triptych of respiratory epithelial cells, bacterial symbionts and resident phages should be considered as a functional and interdependent entity with direct effects on respiration and general homeostasis. While the role of epithelial cells in the pathophysiology of asthma is well established, the tripartite interactions between epithelial cells, bacteria and phages should be further investigated, to better understand asthma as a system disorder and to explore potential interventions. In this study, we focused on the isolation of lytic phages for the target bacteria M. catarrhalis, A. baumannni, S. aureus and P. aeruginosa from nasopharyngeal samples from healthy donors. We have also developed a human cell-based homeostasis model between a clinically isolated strain of S. aureus 141 and active phages for this strain (PYOSa141) isolated from the commercial Pyophage cocktail (PYO). The cocktail is produced by Eliava BioPreparations Ltd (Tbilisi, Georgia) and is used as an add-on therapy for bacterial infections, mainly in Georgia and Russia. For this study, the human bronchial epithelial cell line BEAS-2B and healthy primary nasal epithelial cells derived from healthy donors were selected to investigate the physiological co-existence of S. aureus 141 and PYOSa141. The focus was on mimicking a homeostasis model between S. aureus, lytic phages and airway epithelial cells and on developing novel strategies to study physical interactions of more complex systems on different in vitro culture platforms (2- or 3-dimensional systems and microfluidic systems). To construct the three-part in vitro model between bacteria, phages and airway epithelial cells, we studied each pairwise system separately by time- and dose-dependent analysis. Finally, we aimed to maintain the viability and number of each selected biological entity during triplicate culture and to evaluate the downstream physiological effect of the proposed model on cell inflammation, cell viability (apoptosis and necrosis levels), oxidative stress and cell membrane integrity by multiple time-course analyses after treatment. Inflammatory mediators (IL-8, CCL5/RANTES, IL-6 and IL-1β) were measured in the culture supernatant by enzyme-linked immunosorbent assay. The extent of cell viability and oxidative stress was estimated using flow cytometric analysis and the cytotoxic effect of the model on epithelial cells was assessed using a crystal violet staining assay. The epithelial cell integrity of differentiated nasal epithelial cells under air-liquid interface conditions was estimated by trans-epithelial electrical resistance measurements. PYOSa141 was found to have a prophylactic effect on airway epithelial cells exposed to S. aureus 141 by effectively down-regulating bacterial-induced inflammation, cytotoxicity, apoptosis levels and epithelial barrier disruption in a time-dependent manner. The effect of the triptych model on epithelial barrier protection was similar for cells cultured on 3-dimensional culture platforms and on the in-house fabricated microfluidic system. Overall, the proposed model represents an advance in the way multi-component biological systems can be simulated in vitro and further evaluated by adding more experimental parameters and using different culture platforms.
Main subject category:
Health Sciences
Keywords:
Bacteriophages, Staphylococcus aureus, Bronchial epithelial cells, Nasal epithelial cells, Triptych homeostasis model, In vitro culture systems, Microfluidics, Cytotoxicity, Inflammation, Trans-epithelial electrical resistance, Apoptosis, Necrosis, Oxidative stress
Number of references:
521
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