In summary, our study aimed to characterize the immune modulatory activity of a natural plant extract against antibiotic-resistant bacteria as an alternative handling or prophylactic approach. With this data, we could strongly suggest that these plant products could enhance the immune system by boosting human neutrophils to form NETs to entrap and to kill bacteria, as shown for GUKUBA, VEOEBA, BYCRBA, and vitexin. Finally, this project might identify new therapeutic targets based on natural products, which can be further developed as new therapeutic treatment strategies against bacterial infections. However, the described detrimental effects of NETs need to be considered.
In the future, it is important to determine the detailed function of these natural products in the immune system by characterizing their biochemical mechanisms.
Based on the results presented in this thesis, further research should be conducted in vivo to gain a better understanding of how these natural products function in a more complex setting. One approach that can be done to clarify the changes in the aforementioned cells is to look at the gene expression in neutrophils; this gene expression can be altered upon treatment with the plant extracts. Indeed, most of the genes in the immune cells are very well characterized and their function in disease and health is well investigated. Upon treatment with the plant extracts, the upregulation or the downregulation of specific genes could lead to the identification of a pathway that is activated to boost the immune response of the cells. Furthermore, a proteomic and lipidomic analysis of the neutrophils and monocytes will be done in parallel to support that data obtained from the gene expression to determine the pathways that are affected under the BYCRBA and vitexin. In addition, it is important to verify that the boosting of immune cells does not lead to a reduction in the number of immune cells in the blood, since it is still unknown if the activated and NET releasing cells die after they have performed their function. Showing that NET release is revisable in immune cells and that the number of immune cells is not affected under the treatment with the herbals is necessary to strengthen the case for a potential use of the herbals as a treatment against bacterial infections. Here, I have shown that the mechanism of NET release is not only dependent but also ROS-independent. Therefore, if the herbals do not initiate ROS production, the cells stay
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viable after the treatment with the plant extracts. This in addition can be investigated by testing some enzymes that are involved in classical NETosis, such as chromatin decondensation, dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO).
The results presented here show that plant extracts can modulate cellular levels of cholesterol and induce NETs formation. However they do not induce ROS production which may influence the oxidation of lipids such as cholesterol. Recent research has shown that oxidized cholesterols, such as 7-ketocholesterol, can induce NET formation (Wang et al., 2017). Further research can be conducted into oxidized cholesterols to potentially differentiate between the ROS dependent and independent NETs forming pathways.
Finally, we propose that our plant extracts have direct effects on the cytokine production in the cells which might affect recruitment of immune cells. To test this hypothesis the following approaches will be performed: selected candidate extracts will be tested in a well-established subcutaneous wound infection using the mouse model for staphylococcal infections (Loof et al., 2015; Berends et al., 2010; Von Köckritz-Blickwede et al., 2008). The survival times of S. aureus in infected/treated mice will be measured. In addition the size of skin lesions caused by S. aureus and the bacterial load in organs and skin of the mice will be analyses. The inflammatory cytokines such as (IL-1, IL-6, and TNF- α) will be measured in the blood and in the organs by ELISA. Finally, especially the in vivo data will help to verify the protective effects of the compounds and help to verify new therapeutic targets.
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Acknowledgements
I would like to express my sincere gratitude to my supervisor, Prof. Dr. Maren von Köckritz-Blickwede, who gave me this great opportunity to launch this journey. Thank you for your continuous support, encouragement and for your patience, motivation guidance along the way and for helping me in all the time of research and writing of this thesis and everything in my life. Without your support it would not be possible to conduct this research.
I owe my deepest gratitude to Prof. Dr. Hassan Naim for his continuous support and allowing me perform my work in his department. Thank you for your suggestions, fruitful discussions.
I want to thank, Prof. Dr. Hani Moubasher, Prof. Dr. William N. Setzer, Dr. Katja Branitzki-Heinemann, Hadeel Shammas, Dalanda Wanes, Dr. Graham Brogden, and especially Dr. Mohamad Toutounji for all the suggestions, thoughtful discussion, feedback and sharp ideas, from which I learnt to be an independent thinker. I would also like to thank my committee members for their time and efforts specially Prof. Dr.
Corinna Kehrenberg.
I would like to thank Mrs. Maritta Ledwoch and Mrs. Johanna Kroll from TiHO international office, for offering me their support and their friendship.
To all the members in the lab for offering me the help, and for who are close to me every day, I want to thank you all especially to those who were involved in making my time in the lab enjoyable.
I would like to thank the University of Aleppo and the Professor and doctors for teaching me and supporting me.
Last but not least and as an honored to be one of the recipients of the Friedrich-Ebert-Stiftung scholarship to your generous support that has not only offered academic challenges, but has also helped me realize the value of a doctoral research.
I am forever thankful to my father who supports me from the first day that I opened my eyes until now without stopping, and for always pushing me forward and
94 encouraging me to be the best that I can be.
My deep and sincere gratitude are to my family for the continuous and unparalleled love, help and support. To my great husband Moustafa and my children Haifaa, Ibrahim and the little one Maren (who changed our life to the best), thank you from my heart for your continuous support you have offered in my life. I would like to thank my mother Alia, for loving me unconditionally and for giving birth to me at the first place and supporting me spiritually throughout my life. This journey would not have been possible if not for them, and I dedicate this milestone to them.
Especially thank to my grandmother Aziza for her love and advice, I miss you every moment.
I am grateful to my uncle Ahmad who encourages me to do this journey. I am very glad to my sister Haifa for always being there for me as a friend and my sister Raja also. Thank you to my brothers Yaseen, Dirar, Modar, Ahmad, Anwar (Ali) and the little one Qusai, I will forever hold you near and dear to my heart.
Prof. Monika Ganseforth, I express my gratitude for her friendship, support and for being there, when I need. Each day I say alhamdulillah for these people in my life.
Ragheda Yaseen Hannover, 21.09.2018