LLoouuiissiiaannaa SSttaattee UUnniivveerrssiittyy LLSSUU DDiiggiittaall CCoommmmoonnss LSU Doctoral Dissertations Graduate School 2014 TThhee AAnnttii--IInnflflaammmmaattoorryy EEffffeeccttss ooff VViittaammiinn DD aanndd EExxeerrcciissee Laura Ann Forney Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Kinesiology Commons RReeccoommmmeennddeedd CCiittaattiioonn Forney, Laura Ann, "The Anti-Inflammatory Effects of Vitamin D and Exercise" (2014). LSU Doctoral Dissertations. 3667. https://digitalcommons.lsu.edu/gradschool_dissertations/3667 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. THE ANTI-INFLAMMATORY EFFECTS OF VITAMIN D AND EXERCISE A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The School of Kinesiology by Laura Ann Forney B.S., Purdue University, 2010 M.S., Louisiana State University, 2012 December 2014 ACKNOWLEDGMENTS I would first like to thank my mentor, Dr. Laura Stewart, for her guidance over the past four and a half years. I would not be where I am today or feel as confident as I do taking the next step in my career without her help. Not many graduate students are blessed with a mentor whom they can also consider a friend. I would also like to thank my committee members – Dr. Arnold Nelson, Dr. Neil Johannsen, Dr. Georgianna Tuuri, and Dr. Tara Henagan – for their assistance through the program at LSU. Their keen discernment and ability to shed new light on my projects has most definitely made me a better researcher. Finally, I’d like to thank my family and friends for putting up with me as I finished my project. They offered their care and sympathy during times of stress and shared in my joy upon my completion. I’d especially like to thank my husband, Matt, for putting up with many less- than-delicious dinners and knowing when to offer to pick up carryout after a long day in the lab. ii TABLE OF CONTENTS ACKNOWLEDGMENTS .............................................................................................................. ii ABSTRACT .................................................................................................................................... v CHAPTER 1 – INTRODUCTION ................................................................................................. 1 1.1 Specific Aims ........................................................................................................................ 2 CHAPTER 2 – LITERATURE REVIEW ...................................................................................... 3 2.1 Inflammation and Disease..................................................................................................... 3 2.1.1 Exercise and Inflammation ............................................................................................ 4 2.2 Inflammatory Markers: Interleukin-6 ................................................................................... 6 2.2.1 Interleukin-6 Response to Exercise ............................................................................... 7 2.3 Inflammatory Markers: C-Reactive Protein .......................................................................... 9 2.3.1 C-Reactive Protein Response to Exercise ...................................................................... 9 2.4 The Role of Monocytes in the Inflammatory Process ........................................................ 11 2.5 The Toll-Like Receptor Pathway ........................................................................................ 11 2.6 Monocyte and Toll-Like Receptor 4 Response to Exercise ................................................ 12 2.7 Vitamin D............................................................................................................................ 13 2.7.1 Vitamin D Isoforms ..................................................................................................... 13 2.7.2 Vitamin D Metabolism ................................................................................................ 14 2.7.3 Influences on Vitamin D Status ................................................................................... 16 2.7.4 Environmental Influences on Vitamin D Concentrations ............................................ 16 2.7.5 Individual Factors Influencing Vitamin D Status ........................................................ 17 2.7.6 Dietary Influences on Vitamin D Concentrations ........................................................ 18 2.7.7 Vitamin D Requirements ............................................................................................. 20 2.7.8 Vitamin D and Health Outcomes in Older Populations ............................................... 24 2.7.9 Vitamin D and Exercise Performance.......................................................................... 27 2.7.10 Vitamin D and Inflammation ..................................................................................... 29 CHAPTER 3 – METHODS .......................................................................................................... 32 3.1 Study Design ....................................................................................................................... 32 3.2 Subjects ............................................................................................................................... 32 3.3 Study Visit Description ....................................................................................................... 33 3.4 Blood Analysis .................................................................................................................... 37 3.5 Statistical Analysis .............................................................................................................. 39 CHAPTER 4 – RESULTS ............................................................................................................ 40 4.1 Descriptive Measures .......................................................................................................... 40 4.2 Vitamin D Status: Measures of Intake and Serum Content ................................................ 40 4.3 Exercise Performance Measures ......................................................................................... 43 4.4 Serum CRP and Stimulated Cytokine Production .............................................................. 44 4.5 Monocyte Phenotype and TLR4 Surface Expression ......................................................... 46 CHAPTER 5 – DISCUSSION ...................................................................................................... 52 5.1 Chapter Overview ............................................................................................................... 52 iii 5.2 Descriptive Measures of the Overall Population ................................................................ 52 5.3 Vitamin D Status: Measures of Intake and Serum Content ................................................ 53 5.4 Exercise Performance Measures ......................................................................................... 55 5.5 Serum CRP and Stimulated Cytokine Production .............................................................. 57 5.5.1 Resting CRP Concentrations........................................................................................ 57 5.5.2 Stimulated IL-6 Production.......................................................................................... 58 5.6 Monocyte Phenotype and TLR4 Expression ...................................................................... 60 5.7 Limitations .......................................................................................................................... 65 5.8 Future Directions ................................................................................................................ 67 5.8.1 Tumor Necrosis Factor Alpha ...................................................................................... 68 5.8.2 Vitamin D Receptor ..................................................................................................... 69 5.9 Conclusion .......................................................................................................................... 71 REFERENCES ............................................................................................................................. 73 APPENDIX 1 – CONSENT FORMS ........................................................................................... 89 1.1 LSU IRB Approval ............................................................................................................. 89 1.2 Medical History Form ......................................................................................................... 94 1.3 Physical Activity Readiness Questionnaire ...................................................................... 102 1.4 International Physical Activity Questionnaire .................................................................. 103 1.5 Sun Exposure Survey ........................................................................................................ 105 1.6 Diet Log ............................................................................................................................ 106 APPENDIX 2 – RAW DATA .................................................................................................... 107 APPENDIX 3 – PROTOCOLS .................................................................................................. 135 3.1 FACS Analysis.................................................................................................................. 135 3.2 LPS Cell Stimulation ........................................................................................................ 136 APPENDIX 4 – ABBREVIATIONS.......................................................................................... 139 VITA ........................................................................................................................................... 141 iv ABSTRACT Elevated inflammation is associated with several chronic diseases, including obesity. Exercise is an established effective treatment of this condition by decreasing adiposity and independently regulating inflammatory pathways. The potential for vitamin D to confer anti-inflammatory benefits has been explored in cell culture studies, but few have explored its action at the whole body level. PURPOSE: To investigate the relationship between inflammatory markers in trained and untrained individuals with vitamin D levels either above or below a suggested optimal concentration. METHODS: College-aged females (N = 63), both trained and untrained, reported to the lab four times: to assess body size and composition, for blood collection, for a maximal aerobic test, and a test of anaerobic power. Blood was analyzed for serum 25OHD and CRP concentrations, stimulated with LPS to assess IL-6 production. Samples were prepared for FACS analysis for CD14, CD16, and TLR4 expression. RESULTS: Trained individuals presented with higher 25OHD levels, even prior to stratification into high and low groups (p = 0.015). VO peak was significantly higher (p < 2 0.0001) and fatigue during the test for anaerobic power was significantly lower (p = 0.021) in trained individuals. Untrained individuals had a higher average body weight (p = 0.039) and estimated percent body fat (p = 0.011) compared to trained individuals, although the average estimated percent body fat of both groups was higher than the recommended level for this age group. Additionally, measures of sun exposure were negatively correlated with measures of body size and composition, although these relationships did not exist between serum 25OHD. CONCLUSION: In this study, regular physical activity was associated with higher serum 25OHD, lower BMI, waist circumference, and estimated percent body fat as well as reduced LPS-stimulated IL-6 production. Optimal vitamin D status did not appear to provide any additional health related or anti-inflammatory benefit in those with regular physical activity v habits. However, in individuals not participating in a regular exercise program, the potential for vitamin D to mediate inflammation appeared more likely. More specifically, untrained people with optimal vitamin D status had lower numbers of total monocytes, CD14+CD16- cells, and decreased TLR4 expression on CD14+CD16+ cells; however, these differences did not translate into a change in overall cell function or markers of systemic inflammation as there was no difference between optimal and suboptimal groups with respect to LPS-stimulated IL-6 production or resting CRP concentrations. An expanded exploration of the relationship between vitamin D and inflammation may include assessing other inflammatory biomarkers, immune cell types, the vitamin D receptor, and the role of adipose tissue. vi CHAPTER 1 – INTRODUCTION Chronic, systemic inflammation is associated with diseases such as obesity, diabetes, cardiovascular disease, and others (1, 2). While there are a number of pharmaceutical anti- inflammatory treatments, many are associated with a multitude of negative side effects (3). Consequently, many experts speculate that altering the diet and increasing exercise may be the most beneficial treatment options for decreasing inflammation (4). Exercise training plays a role in mediating the inflammatory response at both an acute and chronic level, and while inflammation is peaked after an acute bout of exercise, it leads to decreased basal levels after long-term exercise training (5). Vitamin D is most commonly known for its importance in calcium homeostasis, but new research indicates the potential for this nutrient to mediate inflammation (6, 7). Research has identified the many benefits related to adequate vitamin D status, which is commonly evaluated using serum 25-hydroxyvitamin D (25OHD), including decreasing the risk for certain types of cancer, reducing the symptoms of depression, decreasing inflammation, and altering body composition (8). Despite the ability of vitamin D to potentially ameliorate inflammation, its mechanisms of action in this process are unclear. Because high levels of inflammatory markers such as interleukin-6 (IL-6) and C-reactive protein (CRP) are associated with chronic diseases, understanding how both vitamin D and physical activity are capable of decreasing the levels of circulating inflammatory markers could provide a potential treatment for these conditions (6, 9). Accordingly, the purpose of this study is to investigate the influence of training status and vitamin D status on circulating inflammatory markers, and monocyte number and function. Briefly, trained and untrained women with either high or low levels of serum 25OHD will be recruited in this study. Whole blood samples will be stimulated with the bacterial endotoxin lipopolysaccharide (LPS), which has been shown to elicit 1 an inflammatory response in monocytes through the toll-like receptor 4 (TLR4) (10). Baseline and circulating concentrations of IL-6 following stimulation, and resting CRP concentrations will be assessed using enzyme-linked immunosorbent assay (ELISA). Monocyte phenotype, classified as the classical CD14+CD16- or the non-classical CD14+CD16+, will be assessed in blood samples via flow cytometry. Data from trained and untrained subjects will be compared, as will the results from those with suboptimal 25OHD compared to those who have optimal 25OHD serum content in both trained and untrained groups. 1.1 Specific Aims Aim 1: Are vitamin D and training status related to measures of aerobic fitness, anaerobic power, and the presence of the inflammatory biomarker, CRP? Aim 2: Are vitamin D and training status related to the phenotype and function of monocytes? 2 CHAPTER 2 – LITERATURE REVIEW Chronic, systemic, low-grade inflammation is tightly linked to the development and progression of obesity, type 2 diabetes, and cardiovascular disease (1). Inflammation can be assessed using a number of different biomarkers, including IL-6 and CRP, and a number of interventions have been evaluated in an effort to reduce the concentrations of these agents (11, 12). Significant, transient increases in these inflammatory biomarkers have been observed during and immediately following acute bouts of exercise; however chronic bouts of exercise have been shown to reduce systemic inflammation (6, 9). Vitamin D has also emerged as a significant mediator in the inflammatory process (7, 12, 13). This literature review will provide a summary of the inflammatory process associated with chronic disease, the inflammatory response to acute and chronic exercise with a focus on the role of monocytes and TLR4, and how exercise and vitamin D can modulate the inflammatory response. 2.1 Inflammation and Disease Inflammation is an immune response elicited by the body during stress, characterized by the production of cytokines from immune and non-immune cells that mediate the inflammatory reaction (14). Inflammation can arise from a number of different stimuli, including traumatic events, sickness, or injury; alternatively, acute increases in inflammation also occur after an intense bout of exercise (14). Inflammation is also exacerbated over long periods of time in cases of obesity (1). This consistent stimulus for cell damage may lead to the progression of a number of obesity-related diseases, including insulin resistance, atherosclerosis and cardiovascular disease (CVD) (14, 15). Adipose tissue, skeletal muscle, hepatocytes, and immune cells such as monocytes and macrophages, are stimulated via integrated pathways that results in co-activation of inflammatory pathways and increased levels of cytokines in circulation (15). Of particular 3