University of Connecticut OpenCommons@UConn Master's Theses University of Connecticut Graduate School 5-10-2020 Altered Gene Expression in Circulating Immune Cells Following a 24-Hour Passive Dehydration Aidan Fiol [email protected] Follow this and additional works at: https://opencommons.uconn.edu/gs_theses Recommended Citation Fiol, Aidan, "Altered Gene Expression in Circulating Immune Cells Following a 24-Hour Passive Dehydration" (2020). Master's Theses. 1480. https://opencommons.uconn.edu/gs_theses/1480 This work is brought to you for free and open access by the University of Connecticut Graduate School at OpenCommons@UConn. It has been accepted for inclusion in Master's Theses by an authorized administrator of OpenCommons@UConn. For more information, please contact [email protected]. Altered Gene Expression in Circulating Immune Cells Following a 24- Hour Passive Dehydration Aidan Fiol B.S., University of Connecticut, 2018 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science At the University of Connecticut 2020 i copyright by Aidan Fiol 2020 ii APPROVAL PAGE Master of Science Thesis Altered Gene Expression in Circulating Immune Cells Following a 24- Hour Passive Dehydration Presented by Aidan Fiol, B.S. Major Advisor__________________________________________________________________ Elaine Choung-Hee Lee, Ph.D. Associate Advisor_______________________________________________________________ Douglas J. Casa, Ph.D. Associate Advisor_______________________________________________________________ Robert A. Huggins, Ph.D. University of Connecticut 2020 iii ACKNOWLEDGEMENTS I’d like to thank my committee members. Dr. Lee, you have been an amazing advisor, mentor and friend to me these past few years. Your advice, whether it was how to be a better writer or scientist, or just general life advice given on our way to get coffee, has helped me grow as a researcher and as a person. Dr. Casa and Dr. Huggins, it has been great getting to know you two better over the last few years. Thank you both for your help as I tried to figure out what it was I wanted to do for my thesis. You both have an infectious love for this field, and I look forward to years of collaboration and Ragnar medals down the road. I’d like to thank all the current graduate students in the department, but especially Mike Syzmanski and Jeb Struder. You have both been excellent friends to me, and I can’t wait to continue to work together in the coming years. I’d also like to thank Coach Greg Roy for giving me the chance to compete for University of Connecticut six years ago. My time spent with the track and field program, both as an athlete and as a coach, has taught me invaluable lessons that will continue to shape me in the years to come. Tough times never last, but tough people do. A big thank you goes out to the boys of UConn track and field, especially Patrick, Mike, Kyle, Chris, Barber, PJ, O’D and Parker. You guys have been with me at some of my lowest lows and highest highs, and I’m the person I am today because I was constantly surrounded by amazing individuals always pushing me to be the best version of myself. Finally, I’d like to dedicate this thesis to Mom, Dad, Nina and Devin. Thank you for being unwavering in your support during my time at UConn. You are the best family I could ever ask for. iv TABLE OF CONTENTS Abstract 1 Literature Review and Introduction 2 1. Whole body dehydration results in a decreased plasma osmolality 2 2. Endocrine regulation of plasma osmolality 2 3. Subtle plasma osmolality increases induce cellular changes. 3 4. Cells respond to hypertonic stress-induced reductions in volume and DNA 5 and protein damage Research Aim and Hypothesis 5 Methods 5 Results 7 Discussion 12 Conclusion 14 Appendices 15 References 49 v FIGURES AND TABLES Figure 1: Regulation of water permeability in renal cells 3 Figure 2: Cellular adaptation to hypertonic stress 4 Figure 3: Study design 6 Figure 4: Plasma osmolality changes 8 Figure 5: Plasma osmolality fold changes 8 Figure 6: Body mass changes 9 Figure 7: Percent of body mass losses 9 Figure 8: 24h urine specific gravity 10 Figure 9: 24h urine osmolality 10 Figure 10: 24h urine color 11 Figure 11a: GOSlim analysis of upregulated genes 12 Figure 11b: GOSlim analysis of downregulated genes 12 Table 1: All significantly changed genes following 24h fluid restriction 15 Table 2: Significantly upregulated genes 28 Table 3: Significantly downregulated genes 38 Table 4: DAVID analysis of biological process GO Terms 42 Table 5: DAVID analysis of molecular function GO Terms 44 Table 6: DAVID analysis of cellular component GO Terms 44 Table 7: WEBGESTALT GO Term analysis: granulocyte activation 45 Table 8: WEBGESTALT GO Term analysis: regulation of body fluid 46 levels Table 9: WEBGESTALT GO Term analysis: platelet degranulation 47 Table 10: WEBGESTALT GO Term analysis: humoral immune 48 response Table 11: WEBGESTALT GO Term analysis: coagulation 48 Table 12: WEBGESTALT GO Term analysis: neutrophil mediated 49 immunity Table 13: WEBGESTALT GO Term analysis: response to interferon- 50 gamma Table 14: WEBGESTALT GO Term analysis: lymphocyte mediated 51 immunity Table 15: WEBGESTALT GO Term analysis: interferon-gamma- 51 mediated signaling pathway Table 16: WEBGESTALT GO Term analysis: innate immune response 52 vi ABSTRACT Dehydration has many deleterious effects ranging from impaired cognitive function to decreased aerobic performance. This study evaluated the effects of a mild, passive dehydration on the transcriptional responses of circulating immune cells. We pursued research questions about how whole-body dehydration and small changes in plasma osmolality may affect the greater concept of immune resiliency or susceptibility to infection through acute gene expression changes that may persist during chronic stress. Our approach was to precisely quantify hypertonicity experienced by circulating peripheral blood mononuclear cells and neutrophils and quantify transcriptomic changes in cell lysates using RNAseq. RNAseq revealed 373 total genes changing significantly in the level of expression following a 24-hour fluid restriction protocol in 18 male subjects (23±3.28 years, 80.09±9.6 kg, 175.78±5.68 cm). Upregulated biological process GO Terms included coagulation, humoral immune response, granulocyte activation, neutrophil-mediated immunity, and regulation of body fluid levels. Downregulated biological process GO Terms included response to interferon-gamma, interferon-gamma-signaling pathway, innate immune response, and lymphocyte mediated immunity. Further research should be done on specific genes within the set to determine their candidacy as novel biomarkers related to dehydration. vii LITERATURE REVIEW AND INTRODUCTION Much is known about conserved cell volume responses and cellular responses to hypertonic stress in cell culture and renal tissue models [1]. However, there is little known about how subtle shifts in osmolality affect circulating immune cells, the surveillance system and often first-line defense against infection. We pursue research questions about how whole-body dehydration and small changes in plasma osmolality may affect the greater concept of immune resiliency or susceptibility to infection through acute gene expression changes that may persist during chronic stress. Our approach is to precisely quantify hypertonicity experienced by circulating peripheral blood mononuclear cells and neutrophils and quantify transcriptomic changes in cell lysates using RNAseq. This brief literature review outlines current understanding in 4 areas: whole body dehydration effects on plasma osmolality, how endocrine regulation (in circulation) regulates plasma osmolality, current understanding of cellular response to osmotic stress, and cellular damage experienced during osmotic/hypertonic stress. 1. Whole body dehydration results in increased plasma osmolality Hypohydration is a state in which body water deficit is greater than normal daily fluctuation (typically >2% of body mass loss) [2]. Dehydration (i.e., losing water) is the process that results in hypohydration. The degree of dehydration is measured indirectly by acute changes in body mass, plasma osmolality, plasma volume, urine color, and urine output [3, 4]. Changes to hydration state can modify vascular fluid compartments [5]. Plasma osmolality increases with dehydration [6-8]. Plasma osmolality (Posm) is well correlated with changes in acute body mass loss as a result of dehydration. A -1% ∆BM is significantly related to increased serum osmolality in individuals exercising in the heat [9]. Subjects exercising at -2.5% of their euhydrated body 1 mass had significantly higher serum osmolality than a control group that drank to replace sweat loss and maintain euhydration [10]. Posm is also sensitive to acute rehydration in dehydrated individuals, and significantly changes within 30 minutes of fluid repletion [6]. During a passive fluid restriction protocol, Posm increases relative to time spent in a hypohydrated state [11]. Dehydration due to passive dehydration, exercise, and exercise heat stress decrease plasma volume as a result of sweating and further stimulate the hormonal response to plasma osmolality [12-16]. The sensation of thirst is a reproducible effect of hyperosmolality [17, 18]. Posm hyperosmolality that correlates to <2% loss of body water triggers the sensation of thirst and the release of AVP. As plasma osmolality increases, the sensation of thirst increases linearly [19]. Plasma