Darstellung und funktionelle Analyse von extrazellulären Vesikeln im Kontext epithelialer Aktivierungsprozesse bei Asthma bronchiale

The major aim of the present study referred to the question, whether extracellular vesicles (EVs), that are released by cells, play a role in intercellular communication mechanisms and thereby contribute to the emergence and propagation of inflammatory processes in the airways during asthma. The respective analyses were conducted in vitro using cells of the human bronchial epithelial cell line BEAS-­2B. EVs were obtained from cell culture supernatant of healthy and asthmatic primary bronchial epithelial cells cultured under air-­liquid-­interface conditions. EVs were concentrated by a combination of ultrafiltration and size exclusion chromatography. The results of preliminary experiments suggested that this method was well suited to concentrate EVs in a sample without compromising their morphological and functional properties. Within the scope of this study, initially the hypothesis should be examined, that EVs are taken up by recipient epithelial cells. Therefore, EVs were labelled by different fluorescence dyes. Of the tested dyes carboxyfluorescein-­succinimidyl-­ester (CFSE) proved to be the most promising labeling approach for extracellular vesicles originating from the apical and basal compartment of primary bronchial epithelial cells. Thereafter the uptake of CFSE-­labelled apical EVs was investigated by confocal microscopy. It was shown that EVs seemed to become attached to the cell surface rather than having been incorporated by recipient cells after six hours of incubation time. The second hypothesis to be tested in this study implied, that apical EVs from asthmatic cells in contrast to EVs from healthy cells induce an inflammatory response in healthy recipient cells. For this purpose, BEAS-­2B cells were incubated with EVs of healthy or asthmatic origin for 24 hours. The extend of a potential inflammatory response of the recipient cells was examined by real-­time-­quantitative-­polymerase-­chain-­reaction (RT-­qPCR) to detect the expression of a variety of related genes, enzyme-­linked immunosorbent assay (ELISA) for Interleukin 8 and by indirect quantification of nitric oxide according to Griess. In each case the results after stimulation by EVs were compared to unstimulated negative and lipopolysaccharide-­ stimulated positive control samples. For all selected genes of tight-­junction-­proteins a significant decrease of expression as compared to the negative control was detected after stimulation by EVs from asthmatic patients. This might hint to an impaired barrier function and an increased epithelial permeability during asthmatic airway inflammation. For the selected epithelial inflammatory markers as for the PYD and CARD domain containing gene a decreased or unaltered expression was detected after stimulation by EVs of asthmatic origin. In contrast to EVs from asthmatic patients EVs form healthy donors did not cause a significant change in the expression of any of the selected genes. Only the translation of Interleukin 8 was decreased after stimulation by EVs from healthy donors. In summary, the results suggest a potential role of EVs in intercellular signaling processes in asthma. They seem to contribute to emergence and propagation of asthmatic inflammatory processes. Hence further examination of extracellular vesicle function might substantially add to our understanding of underlying pathomechanisms of asthma. Prospectively it might be helpful to specifically analyze EVs from asthmatic patients affected by different endotypes of the disease to evaluate results on a more substantiated basis as heterogenic and multiple pathomechanisms possibly induce different forms of inflammatory responses. Further experiments with other experimental systems and perhaps other recipient cells are necessary to verify the reproducibility of obtained results suggesting a signal transmitting function of EVs in asthma. Furthermore, associated non-­EV samples should be analyzed in parallel to identify their functional contribution to signal transmission processes. Conceivably a synergistic interaction of EVs and soluble components like proteins might be crucial to exploit the full signal transmitting potential of EVs. A more profound knowledge of EV function may certainly contribute to further improve our understanding of pathomechanisms and to identify players involved in asthma development. Should EVs be proven to significantly contribute to asthmatic inflammatory processes, their inhibition might represent a novel approach for individualized concepts of therapy. In addition, EVs might also serve as tools for a better diagnosis of different asthma endotypes, since the information they carry depends largely on the status of their originating cell. Furthermore, there are attempts to instrumentalize EVs as a promising system for drug delivery into the human body. Certainly, further improvement of methods will have a major impact on advances on the still emerging but promising field of EV research.