VITAMIN D MEDIATED INDUCTION OF INNATE IMMUNITY IN GINGIVAL EPITHELIAL CELLS By WILLIAM N RUDDICK A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIR EMENTS FOR THE D EGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017
2017 William N Ruddick
To m y wife and f amily
4 ACKNOWLEDGMENTS I would like to thank my grandparents, William and Bonita Jones, without whom none of this would have been possible. I could not have been raised by two more loving and caring parents, and I owe my success my sanity, and my sense of humor to the both of you. I will forever look fondly back on our days spent in Walt Disney World, and I loo k forward to the vacations that are yet to come. I would also like to thank my committee chair and mentor Dr. Gill Diamond and his wife and colleague Dr. Lisa Ryan for supporting me scientifically, financially, and emotionally throughout my graduate adven ture. The two of you are the kindest and most understanding mentors I could have asked for, and I wish you and your family only the best in the future. I would like to thank my committee members, Dr. Mary Ellen Davey, Dr. Corwin Nelson, and Dr. Luciana Sh addox. Your passion for your fields of study and the professionalism with which you work have set an example for me that I will try follow in my future career I would like to thank all of the present and former members of the Diamond lab, our summer dent al research students and undergraduate researchers, and all of my friends made along this journey. Special thanks go to David Brice, for always doing your best to make everyone else happy and for knowing more about immunology than I ever could hope to, as well as to Theodore Harris and S. John Calise. You three are the best 3 p.m. coffee crew a guy could ever ask for. life for the better. I love you Amy, and I will work my hardest from here on out to show you just how much I care
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIS T OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 VITAMIN D: A REVIEW ................................ ................................ .......................... 11 Intro duction ................................ ................................ ................................ ............. 11 Brief History ................................ ................................ ................................ ...... 11 Vitamin D 2 and D 3 ................................ ................................ ............................. 12 Cytochrome P450 Enzymes and Vitamin D Conversion ................................ .. 1 2 The Vitamin D Receptor ................................ ................................ ................... 14 Genome Wide Studies ................................ ................................ ..................... 14 Vitamin D Status, Treatment, and Supplementation ................................ ............... 15 Sufficiency, Deficiency, Toxicity ................................ ................................ ....... 15 Food and Nutritio n ................................ ................................ ............................ 16 Vitamin D 2 and D 3 Drug Interactions, Bioavailability ................................ ........ 18 Immunity, Inflammation, and Infection ................................ ................................ .... 19 Immunity ................................ ................................ ................................ ........... 19 Inflammation ................................ ................................ ................................ ..... 19 Infection and AMPs ................................ ................................ .......................... 21 Racial, Territorial, Sexual, Age Dependent and Organismal Differences ............... 21 Racial and Ethnic Variability ................................ ................................ ............. 22 Worldwide Populations ................................ ................................ ..................... 24 Sexuality and Pregnancy ................................ ................................ .................. 26 From Infancy to Elderly ................................ ................................ ..................... 28 The Animal Kingdom and Beyond ................................ ................................ .... 29 Disease and Oral Health ................................ ................................ ......................... 31 2 VITAMIN D: CONVERSION WITHIN HUMAN GINGIVAL EPIT HELIAL CELLS .... 33 Background and Purpose ................................ ................................ ....................... 33 Methods ................................ ................................ ................................ .................. 34 Cell Cultur e ................................ ................................ ................................ ....... 34 Oligonucleotide Primers ................................ ................................ ................... 34 Cytochrome P450 Enzyme Presence ................................ ............................... 34 Vitamin D Anti Inflammatory and Anti Microbial Effects ................................ ... 36 Results ................................ ................................ ................................ .................... 38
6 Conversion of Inactive Vitamin D by Gingival Epithelial Cells .......................... 38 Anti Inflammatory Effects of Vitamin D in Gingival Epithelial Cells ................... 39 Antimicrobial Effects of Vitamin D in Gingival E pithelial Cells .......................... 39 3 DISCUSSION OF RESULTS ................................ ................................ .................. 50 LIST OF REFERENCES ................................ ................................ ............................... 56 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 70
7 LIST OF TABLES Table page 2 1 List of Primers Used ................................ ................................ ........................... 41 2 2 25 D 3 and 1,25 D 3 Quantification in OKF6/Tert 1 Radioimmunoassay ............ 44
8 LIST OF FIGURES Figure page 2 1 Cytochrome P450 Enzymes qPCR ................................ ................................ .. 42 2 2 Cytochrome P450 Enzymes PCR and Gel Electrophoresis ............................ 43 2 3 Inflammatory Markers and 1,25 D 3 qPCR ................................ ........................ 45 2 4 Inflammatory Regulation in Mice qPCR ................................ ............................ 46 2 5 Export of LL 37 ELISA ................................ ................................ ...................... 47 2 6 Vitamin D and Porphyromonas gingivalis Invasion Assay ................................ 48 2 7 Pg 16s rRNA Total Bacterial Load qPCR ................................ ...................... 49
9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science VITAMIN D MEDIATED INDUCTION OF INNATE IMMUNITY IN GINGIVAL EPITHELIAL CELLS By William N Ruddick December 2017 Chair: Gill Diamond Major: Medical Sciences Vitamin D, while classically thought of as important for the health and development of bones, is now known to play an important part immune regulation. My goal was to show if vitamin D contributed to immune regulation of gingival epithelial cells. Using OKF6/Tert 1 cell cultures as a model of gingival epithelial cells (GEC), I provide evidence that GEC have the cytochrome P450 enzyme machinery required to convert vitamin D 3 to active 1,25 dihydroxyvitamin D 3 (1,25 D 3 ) using RT qPCR, and PCR OKF6/Tert 1 cells convert 25 dihydroxyvitamin D 3 (25 D 3 ) to 1,25 D 3 as demonstrated with measurement of 1,25 D 3 in culture media and increased CYP24A1 expression The 1,25 D 3 regulated key antimicrobial and inflammatory responses of GECs. Pro inflammatory cytokines IL 6 and IL 8 w ere decreased in OKF6/Tert 1 cells following treatment with 10nm 1,25 D 3 Similarly, IL 1A expression was decreased in C57BL/6 mice with treatment of 10um 1,25 D 3 The antimicrobial peptide (AMP) LL 37 was exported from OKF6/Tert 1 cells at an increased ra te following 10nm 1,25 D 3 treatment. Porphyro monas gingivalis ( Pg ) CFUs and 16s rRNA expression were
10 decreased upon invasion of TIGK and OKF6/Tert 1 cells respectively following treatment with 1,25 D 3 These results show that treatment with topical vitamin D 3 may be effective in regulating innate immunity within human gingival epithelial cells and the or al environment.
11 CHAPTER 1 VITAMIN D: A REVIEW Introduction Vitamin D, classically thought of as important for the growth and development o f bones and assoc iated with calcium absorption has now gained acceptance as having a far wider reaching scope of importance to the human body Vitamin D is now recognized as playing a role in overall systemic health and functions within immunity, inflammation, and infecti on. Vitamin D deficiency affects health on a global level, and assessing how to best treat the problem has grown increasingly complex and involved with recent literature showing distinct differences in vitamin D functions between men and women, those of di f f ering ethnic and racial backgrounds the young and the elderly, as well as in different locations around the world. The treatment outlook and continued supplementation for vitamin D deficiency or associated disease is determinant on many factors includin g: type of vitamin D or analog compound used, delivery system including storage and half life issues, drug interactions and effects on a systemic level, and even Brief History The history of vitamin D begins with the discovery of vitamin A by McCollum and Davis in mid 1913 which led to further studies focusing on vitamin discovery 1 Vitamins was coined by McCollum in a paper published in June of 1922. 2 Wi thin the paper, McCollum builds off the previous work of Mellanby and describes vitamin D in terms of the effects of increased calcium deposition within sufferers of rickets. 2,3 The vitamin D compound was first isolated by Askew through distillation in 1930 and then further by crystallization in 1932 4,5 T he
12 compound discovered was what we k now now as vitamin D 2 It was not until 1936 when the compound we now describe as vitamin D 3 was identified by Windhaus. 6 While both vitamin D 2 and vitamin D 3 had a simil ar effect in treating rickets, the differences in form as well as function began to emerge with further research. Vitamin D 2 and D 3 Vitamin D 2 or ergocalciferol, was used to treat rickets by Hess in 1930 in the while vitamin D 3 or cholecalciferol, was used in the original Mellanby experiments in the form of cod liver oil 3,7 In general, D 2 is found mainly in plants or yeasts (occasionally in mushrooms) and vitamin D 3 is the natural form of vitamin D foun d in most primates an d other animals 8 10 The vitamin D 3 precursor, 7 dehydrocholesterol (DHCR7) absorbs UVB radiation through the skin and isomerizes into vitamin D 3 9 12 While vitamin D 2 can be ingested from other sources and utilized within the human body the natural presence of vitamin D 3 within humans has led to further research on the two showing that D 2 is less effective as supplement in form and function when compared to vitamin D 3 8,13 15 In order to develop a further understand in g of the complex cellular machinery required, and the process undergone by the human body to convert this natural vitamin D 3 into an active and useable form it is necessary to discuss what are known as cytochrome P450 enzymes. Cytochrome P450 Enzymes and Vitamin D Conversion The process to activate vitamin D 3 to a biologically active metabolite is a sequence of hydroxylation reactions involving many different cytochrome P450 (cyp) enzymes. 16 18 Following conversion of DHCR7 within the skin, Vitamin D 3 is transported to the liver where it is stored and further converted to 25 hydroxyvitamin D 3 (25 D 3 ); this conve rsion is a result of 25 hydroxylation by Cyp2R1 and Cyp27A1. 16,19 22 The 25 D 3 is
13 the primary cir culating form of vitamin D 3 and it is further converted hydroxylation to the active form 1 ,25 dihydroxyvitamin D 3 (1,25 D 3 ) by Cyp27B1 upon transport to the kidney. 23,24 The concentration of active 1,25 D 3 present within a system are regulated by the 24 hydroxylase Cyp24A1 which converts 1,25 D 3 to 1,24,25 D 3 to recirculate for future degradation. 16,22,25 27 Within all of these steps, the transport of vitamin D i s facilitated by the vitamin D binding protein (DBP). Due to being responsible for the entirety of vitamin D transport, the concentration of DBP present within a subject can affect concentration of certain vitamin D forms as well as having an effect on imm une response. 28 31 Finnish researchers c oncluded that variation in DBP concentration may be affecting levels of bone mass within adolescents. 31 Changes in concentration of DBP can also affect serum 25 D 3 concentration 29 The concentration of DBP should be considered as well as the levels of vitamin D itself when trying to determine if an individual is vitamin D deficient. 30 Although classi hydroxylation of 25 D 3 takes place within the kidney, it has hydroxylase enzyme Cyp27B1 and may convert circulating 25 D 3 to 1,25 D 3 at other such sites. 32 34 This extra renal conversion has been shown to also exist within respiratory epithelium gingival fibroblasts, and periodontal ligament cells 35 37 F ollowing conversion, the DBP and active 1,25 D 3 complex enters into cells via endocytosis following binding interactions with the megalin receptor. 38 1,25 D 3 undergoes active function and gene regulation within cells by binding to a specialized transcription factor known as the vitamin D receptor.
14 The Vita min D Receptor The vitamin D receptor (VDR) is a ligand specific transcription factor that is present in most tissues and immune cells in the human body. 39 42 The 1,25 D 3 is the most potent ligand for the VDR and upon ligation to the VDR it binds to the retinoid X receptor (RXR) which accompanies VDR translocation to the nucleus and localization at specific vitamin D response elements (VDREs) located in promoter and enhancer regi ons of many genes 40,42,43 The VDR and RXR have zinc finger protein regions that act as DNA binding domains on a potential VDRE. 42 VDREs generally consist of a PGTTCA region that will bind VDR followed by a three to six base spacer region and a PGGTCA region to bind with RXR. 42 These binding events allow for regulation both up and down for man y gene products including the VDR itself as well as many products with immune function. 40,4 2 44 Genome Wide Studies Through the usage of techniques such as chromatin immunoprecipitation and sequencing ( ChIP seq ) and microarray analysis, it is has been possible to find VDREs throughout the human genome and determine exactly which genes and gene products may be effected by vitamin D regulation. 45 48 One study has sh own greater than 1 000 chromatin domains that contain one or more VDR binding sites as well as the potential for up to 13,000 VDREs total within the human genome while another has shown differing numbers between 1,800 to 2,700 total binding domains in var ious immune cells. 45,46 It has also been shown that 1,25 D 3 does bind to these potential VDREs by showing location shifts of VDR to be within proximity o f potential VDREs. 49 Overal l, genomic studies and indentifying VDREs has shown that vitamin D binding events may have a large effect on both innate and adaptive immunity, antigen presentation,
15 inflammation, and expression of antimicrobials responsible for fig hting potential local and systemic infection. 47 Vitamin D Status, Treatment, and Supplementation Sufficiency, Deficiency, Toxicity Numerous efforts have been made in many different ways to try and better establish a standard for proper concentration of vitamin D in humans, but with multiple influences ranging from sun exposure and skin pigmentation to age and weight, it is difficult to determine if what is good for one individual will be good for another. 50 Knowing this, the recommended limits of vitamin D 3 supplementation recommended by some studies remain around 1,000 IU/day or 10,000IU/week, while others show that 500 700IU/day may be sufficient. 51,52 One international unit (IU) of vitamin D is equivalent to 0.025 g or 25ng of vitamin D 3 53 This would make the 1,000IU/day amount equivalent to 25 g/day of vitamin D. Another statistical approach has been attempted to determine the risk benefit assessment and discover the method of treatment that will allow for the maximum net health gains resultant from vitamin D supplementation. 55 Utilizing Quality of Life for Benefit Risk Assessment (QALIBRA) software, an advanced analysis takes place using an equation created to weigh loss versus gain as in an actuarial table to determine exactly how much vitamin D should be supplemented. 55 The completed equation given as an example in the study goes on to show that a serum concentration of 25 D 3 of 87.5nmol/L provides the lowest risk of mortality in a human population 55 This estimate can be directly compared to other studies which show estimates of >50nmol/L, 75nmol/L, or even as high as 90 100nmol/L. 55 Judging the study bas ed purely on the numerical proximity to other studies, this statistical method of analysis to develop a
16 individual method. Another potential pitfall of trying to reach sufficient levels of vitamin D in a population with so much variability is the toxicity that arises from overconsumption or supplementation. Vitamin D created in the skin is not an issue because the body will regulate excess levels of the hormone but ch ronic consumption of ~40,000IU/day in supplements will result in vitamin D toxicity and hypercalcemia. 56 Symptoms arising from vitamin D poisoning include: severe gastrointestinal pain (and possible nausea, vomiting, constipation, and diarrhea), bone pain, heart beat irregularity, joint and muscle pain, loss of appetite, frequent urination and thirst, nervousness and itching, and potenti al kidney stones. 56 However, in the case of an accidental overdose of 2,000,000 IU within two elderly nursing home residents, no short term results aside from slightly elevated plasma calcium levels were observed. 57 This does not, however, exclude the potential for a dosage of that size to result in long term vitamin D toxicity. 57 Food and Nutrition While there are foods like milk, which provide adequate amou nts of vitamin D for the prevention of rickets and foods historically proven to be rich in vitamin D, like the being experimentally fortified with vitamin D. 3,58,59 The fortification of various foods with vitamin D is an important potential option for the treatment of deficiency. Fortification in most cases requires a stable carrier for the compound to be introduced, and study has shown that the milk protein alpha lactalbumin is a good candidate for en capsulation with vitamin D 3 and subsequent use a carrier in fortified foods. 60
17 Hens that have been fortified with vitamin D lay vitamin D enriched eggs, and the effectiveness of these egg s on the vitamin D status of humans who consume them is an important topic of research. 61 63 Consumption of seven total vitamin D enhanced eggs a week over the course of an 8 week winter has shown complete s tabilization of serum 25 D 3 concentration when compared to the significantly lower concentration of 25 D 3 in individuals in the control group. 61 Another positive aspect is that the fortification of the eggs does not have any negative implications or detrimental results on the production process. 62 This process also improves the antioxidant quality of the eggs to be consumed. 63 Eggs may be successful as a vitamin D fortified food, but fortification is still required in many countries to assist in increasing serum 25 D 3 to healthy levels. In Ireland, a 10 year study was performed to assess the intake of citizens, and while it did not involve measured amounts of serum 25 D 3 taken from patients, the survey showed that residents of Ireland had a slight increase in vitamin D consumed over the 10 year period. 64 Their intake, however, was still nowhere near the recommended amount of intake for healthy adult vitamin D status. 64 A somewhat overlooked and non fortified option for dietary vitamin D intake is the mushroom. Most mushrooms develop large amounts of vitamin D 2 when exposed to sunlight or UV radiation, and while vitamin D 2 may not be as useful or potent a treatment as vitamin D 3 it is an often underestimated source to control potential serum vitamin D concentration 65 a nd even so there are even some mushrooms that can also produce vitamin D 3 66 Shitake mushrooms in particular can form small amounts of vitamin D 3 as well as vitamin D 2 when exposed to UV light, and the important part of
18 mushrooms as a potential source of vitamin D is that they bioavailability and amount gained from intake is equal to an oral vitamin D supplement of the same IU dosage. 66 Vitamin D 2 and D 3 Drug Interactions, Bioavailability Supplementation with vitamin D has been attemp ted with D 2 as well as vitamin D 3 and while vitamin D 3 is clearly the better option due to increasing serum 25 D 3 concentration in greater amount, vitamin D 2 as a supplement may have its own interesting implications. 67 Even at lower values of increase, vitamin D 2 supplementation has shown increases in DBP and the 24,25 D degradation product, and those increases may allow for similar increases in available 25 D and 1,25 D. 68 It is important to understand potential drug interactions that vitamin D may have within the body if taken as a supplement. Atorvastatin intake lowers serum 25 D 3 concentration while subs equent supplementation with 25 D 3 will lower Atorvastatin concentrations in kind. 69 Thiazide diuretics in combination with vitamin D or calcium supplementation will result in hypercalcemia within suffers of hyperparathyroidism, decreased kidney function, or in the elderly. 69 Aside from the two interactions mentioned, evidence of other drugs interfering with vitamin D supplementation status was not of sufficient quality of level to prove a connection. 69 The bioavailability of vitamin D is the final important issue to deal with when it comes to supplementation and amounts necessary. Bioavailability is the amount of a supplement that will be absorbed within the gastrointestinal tract following digestion, and it is almost always greatly lower than the amount of the compound that is originally taken due to factors like how much interaction and degradation there is when met in direct contact wit h other recently ingested compounds. 70 There is little known data about the absorption of vitamin D 3 compounds, but it is fat soluble and 25 D 3 is known to be
19 a bsorbed in greater quantities than non hydroxylated D 3 or D 2 70 The amount of fat ingested with supplementation does not seem to have an effect, but tetrahydro lipstatin (orlistat) and sucrose polyester compounds such as Olestra most likely lower the amount of vitamin D absorbed. 70 Immunity, Inflammation, and Infection Immunity Vitamin D signaling events occur within antigen presenting cells (APCs) such as dendritic cells (DCs), adap tive immune cells like T cells and B cells, and even innate cells such as monocytes macrophages and neutrophils. 71 74 In regards to antigen presentation and adaptive immune activity 1,25 D 3 functions in a generally immunosuppressive manner and primes dendritic cells which present antigen and encourage the development of T regulatory cells (Tregs) which function to regulate immunity through tolerance and suppression of active effector T cells 75 77 To further show potential as a potential anti inflammatory substance, vitamin D as an oral supplement in humans has also been shown to increase and decrease levels of C D 3 8+ cells and circulating I L 17+ T cells respectively 78 The innate immune protection provided by neutrophils, monocytes, and macrophages can be t hought of as result of the chemoattractant properties of antimicrobial peptides (AMPs) such as defensins and the human cathelicidin LL 37 71 74 The aforementioned regulatory effects on the immune system showcase vitamin D as a rather potent anti inflammatory substance. Inflammation As previously mentioned, the le vels of anti inflammatory IL 10 producing Tregs and pro inflammatory IL 17+ T cells can be regulated by vitamin D. 77,78 These ILs, or interleukins, are small protein products known as cytokines that can control and affect
20 the cells and tissue environments around them. 79 ILs, as well as chemokines, interferons, and tumor necrosis factor may be produced and released from the various cells a ffected by vitamin D such as T cells B cells, macrophages, and neutrophils. 79 Much like the binding of vitamin D to VDR, these cytokines function through di stinct specific bind events on the surface or internally within cells and release further cytokine and chemokine produ cts which result in a complicated interplay of control and self regulation. 79 Mast cells have been shown to be capable of metabolizing 25 D 3 which in turn reduces mast cell activation and allergic inflammation 80 Following the discovery of VDR binding, many synthetic VDR agonists have been produced, and several of these vitamin D analogs have shown to reduce release of CXCL10, a potent inflammatory chemokine. 81,82 1,25 D 3 and 25 D 3 both reduc e levels of pro inflammatory IL 6 released from human B and T cells. 83 In a study of an opposite approach, many pro inflammatory cytokines (IL 2, IL TNF and IFN circulation within adults with significant vitamin D deficiency. 84 Within mice, it was shown that vitamin D deficiency was responsible for disruption of normal fecal microbiota and resulted in increased intestinal inflammation. 85 1,25 D 3 and 25 D 3 were both responsible for inhibition of pro inflammatory cytokines IL 6 and TNF within human monocytes treated with LPS to stimulate inflammati on. 86 1,25 D 3 treatment lowers levels of IL TNF, IFN 8 in monocytes treated with heat killed bacteria 87 In many cases, such bacteria and other such microbes may infect and cause harmful inflammation whi ch can be readily prevented utilizing vitamin D.
21 Infection and AMPs Vitamin D treatment may increase antimicrobial activity in certain circumstances and vitamin D deficiency is known to have an association with increased infection incidence in the respira tory system, the GI tract, and many other places. 88 92 As previously discussed, AMPs like LL 37 defensin can be a powerful weapon to fight against infection both dir ectly and indirectly. 71 74 LL defensin 2 both contain a VDRE binding region and are positively regulated by 1,25 D 3 to fight against infection. 93 Suppleme ntation with 1,25 D 3 and 25 D 3 leading to increased levels of LL 37 can be used to treat many different types of i nfection which may result from vitamin D deficiency. 94 100 Vitamin D 3 treatment in patients with sepsis or septic shock shows increased levels of LL 37. 94 Low concentrations of 25 D 3 and 1,25 D 3 are commonplace within victims of community acquired pneumonia as well as those suffering from sepsis. 95,96 There is also a trend of severe vitamin D deficiency within those suffering from infection in areas in proximity to prost hetic attachments and implants. 97 Increased levels of LL 37 a nd other antimicrobials from 1,25 D 3 treatment has been shown to inhibit and control infection of Mycobacterium species marinum and tuberculosis respectively. 98,99 However, vitamin D cannot be involved in all infection p rocesses. It was shown that there is no association between genetic polymorphisms in the vitamin D pathway and incidence of Chlamydia trachomatis infection. Racial, Territorial, Sexual, Age Dependent and Organismal Differences The evolution of pigment with in the skin of human beings is a story that involves the importance of vitamin D to the human condition, survival, and development of strong immune function and bone and calcium homeostasis. 101 103 The development of lighte r
22 skin pigments as well as beneficial mutations of 7 dehydrocholesterol ( DHCR7 ) allowed for human beings to move away from the equator to more northern latitudes where UVB radiation was not plentiful enough to properly regulate vitamin D in non mutated ind ividuals of dark skin. 104 These variations in latit ude dependent UVB radiation as well as the tendency for individuals to move around the globe to locations where their skin color, diet, or life style may not be suitable for proper vitamin D development are a growing concern to the deficiency status of our global community. Racial and Ethnic Variability Vitamin D deficiency is more prevalent in those of African descent and darker pigment when compared to those of European descent with lighter pigment. 105,106 A darker pigme nt of skin is more concentrated in melanin and thus is more resistant to incoming UVB radiation and thus it is more difficult for darker skinned individuals to make vitamin D 3 within their skin. 107 In Ameri ca, where there are so many individuals of differing descent and skin color living in similar areas, it is thought that the darker skinned individuals will natural have l ower vitamin D intake from the s un and thus more foods fortified in vitamin D should b e made available to the general public. 106 Studies have shown that even with vitamin D fortified milk available, non Hispanic whites still have the highest dietary intake levels of total vitamin D. 106 That would mean that the population that latitude affects the least is also getting the highest levels of dietary assistance towards fighting vitamin D deficiency. One study shows that seru m concentrations of DBP are similar within black versus white individuals of the same area, so it can be said that the problem lies within total amount of vitamin D 3 available and not the levels of associated, required compounds. 108
23 This deficiency in vitamin D among individuals of darker skin can be compounded synergistically when other problems that are greater among darker pigmented individuals are taken into account. African American men are diagnose d with prostate cancer more often than European American men, and it has been shown that deficiency in vitamin D 3 also increases mortality in prostate cancer. 109 This comes coupled with the fact that far more African American men are deficient in vitamin D than European American men, and it presents a te rrible problem for the health of individuals based purely upon the color of their skin. 109 Efforts have been made to supplement and normalize the vitamin D levels of African Americans, and the research has led to evaluating dosage requirements for helping those suffering from deficiency. 110 It should also be noted, however, that even among individuals of the same pigment and living in the same area there are great di screpancies in vitamin D concentration 111 This was shown recently in a study of over 1,800 middle aged Caucasian French adults who still showed varying levels of vitamin D deficiency that seemed to be independent of dietary intake. 111 Adding to that fact, Caucasian individuals in more northern latitudes have their own problems when it comes to vitamin D production due to the longer winters and lesser levels of sunlight overall in addition to lower UVB radiation. 112 These issues hav e led to individuals in places like Norway needing to supplement vitamin D by scheduling therapy trips to tanning salons, solar simulators, sunbeds, and other such locations with UVB lamps available. 112 These problems arising from location are not distinct to Scandinavia, and vitamin D deficiency and status vary on a global level.
24 Worldwide Populations The health of populations and in dividuals on worldwide scale is affected by potential vitamin D deficiency and the varying degree by which they are able to supplement and/or prevent the problems associated with said deficiency. 113 123 The highest concentration s of serum 25 D 3 are present in North America, and this is most likely due to the fortification of foods present in North American with vitamin D when compared to many other countries. 113 In Iran, studies have shown that vitamin D deficiency is commonplace in young men and women duri ng the winter months, but status improved greatly during the summer months and also had a minor improvement with weekly supplementation. 114 Office workers both male and female were observed in Ankara, Turkey, as it could be assumed those who work indoors receive less essential sunlight when compared to others around them. 115 Results showed that the majority of workers were greatly deficient in vitamin D even within the summer months and it should be considered a significant problem to public health. 115 In a place like Saudi Arabia, the summer months can be the greatest time of deficiency and cause for alarm due to the tendency to avoid the sun and the intense heat. 116 Research has gone as far as to equate the most efficient times for Saudi citizens to expose themselves to the sun and the summer elements to more optimally create their essential vitamin D 3 116 Even in the tropical country of Malaysia, where again the sunny climate may lead one to thinking there could be cause for worry or alarm, vitamin D deficiency among women is an issue. 117 Women residing both in urban and rural areas were observed for deficiency, and a clear difference was observed as the urban women, who were most likely exposed to less sun, were a great deal more vitamin D deficient than tho se
25 residing in rural areas. 117 In New Zealand, citizens of non heritage are far more deficient than those of a background, and this is even true when both parties are taking weekly vitamin D supplements. 118 On the Mediterranean island of Crete, efforts have been made to observe the mean 25 D 3 concentration present within an isolated population and to redefine what deficiency meant to them based on health issues without making com parison of their deficiency to outside populations. 119 The study showed that a serum concentration of around 20ng/mL was the cut off where deficiency would be determined. 119 In Nor way, adolescent populations of natives as well as immigrants were sampled for vitamin D deficiency and to determine potential childhood health risks. 120,121 Deficiency was show to be much more common during the school year, especially in young boys, d ue to long hours spent indoors when compared to their summer months away from school. 120 Among the immigrant children observed, very few were found to be deficient by the standards of Norway, but almost half of them were under a level 121 A large part of the problem of deficiency comes from the inability to come to sunlight to combat this deficiency. 122 This is a great problem in Europe where many small countries lie in close proximity to one another and yet have very different standards of care and treatment when it comes to vitamin D defic iency and supplementation. 122 The problem that lies within Olmsted County, Minnesota is not one of deficiency, but rather of concentrations of vitamin D high enough to result in toxicity and illness due
26 to hypercalcemia 123 Over a 10 year period of study, 9.2% of individuals sampled had concentrations of serum 25 D 3 high enough to be consid ered toxic. 123 It is thought that this trend toward a toxic concentration of vitamin D present within the population may be a result of over treatment with high dose prescription supplements. 123 It is no surprise that this may happen in world tha t is finally growing informed of the potential health issues caused by vitamin D deficiency. Sexuality and Pregnancy While the majority of studies on vitamin D deficiency in relation to the sex of an individual may be focused on pregnant females, there are still several issues that pertain explicitly to male sufferers. In one study, a significant portion of patients suffering from erectile dysfunction (ED) were shown to be deficient in vitamin D. 124 It is believed this trend towards erectile dysfunction may have been due to vitamin D being necessary for the health of endothelial tissue within the penis and surrounding areas. 124 It is unlikely that it may be caused by low testosterone, because vitamin D supplementation has not had significant results on increasing testosterone concentrations in clinical trials. 125 Vitamin D is importan t in the reproductive system, and this has been shown in males where data has suggested a relationship between serum 25 D 3 concentration and the ability of semen to cause pregnancy during vaginal intercourse. 126 Vitamin D is still more important to the female, however, in regards to pregnancy and the health of a fetus. Levels of vitamin D, and supplementation if required, are important to a healthy ou tcome during pregnancy and post pregnancy for both the expectant mother and the unborn chil d. While already thought of as necessary for the development of healthy bones within a growing fetus, the status of vitamin D is also important balancing
27 antimicrobi al protection for both mother and child while being sure to regulate immune self tolerance to prevent fetal rejection and miscarriage. 127,128 Vitamin D deficiency leads to an increase chance of miscarriage during the first trimester of pregnancy. 129 This may be due to a lack of self tolerance as a result of impaired Treg function within vitamin D deficient pr egnant women. 130 Misregulation of vitamin D levels and subsequent deficiency may lead to problems such as infection, gestational diabetes, small fetal size, preterm birth, and preeclampasia. 131 Preeclampasia, or pregnancy induced hypertension, has shown to be increased in mothers of ethnic minorities that suffer from low concentration of serum 25 D 3 132 Requirements for supplementation to combat these iss ues have been tested and while doses in the 1,000IU/day range have shown little to no effect, dosage of 4,000 IU/day within pregnant females has been shown to normalize serum concentration s of vitamin D and improve chances of a normal, healthy birth. 133 135 In an almost opposition as to what one would normally expect, vitamin D supplementation slightly suppress es levels of the beneficial LL 37 AMP with in the macrophages of newborns 136 However, this suppression does not seem to affe ct the killing capacity of macrophages. 136 Post pregnancy, the psychological hea lth and mood of new mothers may also be directly affected by vitamin D deficiency but differ ent studies have shown opposing res ults In 2015, a study showed little to no links of post partum depression (PPD) to vitamin D deficiency, while a study in 2017 resulted in 76% of surveyed PPD sufferers being deficiency in vitamin D. 137,138 In addition to vitamin D supplementation having a positive effec t on weight at birth, bone mass and fetal calcium levels the kidney health of a individual may also show benefits from increased vitamin D even into early
28 childhood. 139 Foll owing birth, infants still require and benefit from vitamin D in a number of ways as do adults and the elderly From Infan cy to Elderly Previously, vitamin D supplementation in infant children has been only to combat rickets and thus no standard treatment levels have been agreed upon to eliminate the health problems that may apply to the youth of today. 140 In Finland, researchers are busy evaluating the levels of vitamin D necessary to allow for strong bones, a strong immune system and resistance to infection, less incidence of allergy and asthma, and greater cogni tive development of infants 140 As previously discussed, ch ildren around the world may be deficient in vitamin D. 121 This deficiency may lead to problems with bone health as well as many non skeletal issues which may affect a child well into the ons et of puberty or even into adulthood. 141 The most dangerous of these problems may very well be respiratory tract infections (RTIs) such as pneumonia which can lead to severe illness and death within a great number of children. 142 Efforts are being taken elimina te the onset of these RTIs with vitamin D supplementation, but proper treatment amounts are not well defined or documented for each potential infection. 142 Until proper supplementation amounts are agreed upon, this will continue to be a problem for children as they may suffer well into adulthood and even old age as a result of ill developed immune s yst ems caused by vitamin D deficiency. While discussing populations around the world, it was shown that the elderly in New Zealand suffer from high levels of vitamin D deficiency, and this is also true for those of increasing age in all walks of life. 118,143 This trend towards sub optimal concentration s of circulating serum vitamin D in an aging world population may lead to
2 9 increased bone fractures, as well as several o ther problems that may lead to early mortality unless the deficiency is treated. 143 Chronic pain is a problem for most, if not all, individuals of advanced age, and low concentrations of 1,25 D 3 are associated with severe bouts of chronic pain in men over the age of 70. 144 Increasing levels of chronic inflammation and swelling are also a problem for the elderly, and individuals over the age of 65 undergoing treatment for chronic inflammation show significant links between low levels of vitamin D and high levels of pro inflammatory cytokines. 145 The decline of cognitive function, onset of dementia, and d evelopment of brain abnormalities is a problem that many older individuals must confront. Low concentration s of serum 25 D 3 are present within African American and Hispanic persons of increased age and are associated with a greater rate of cognitive declin e. 146 It is yet to be determined i f v itamin D supplementation can assist in lowering the rate of this decline. 146 Individuals of all races, locales, ages, and sexes have shown to be challenged in various ways when it comes to the issue of vitamin D deficiency, but it is not just humans that deal with the associated health proble ms and further issues related to vitamin D. The Animal Kingdom and Beyond While the knowledge base of vitamin D in research and the effects of deficiency within other creatures may not be as great as it is in humans, many important things can be learned fr om looking into the lives and vitamin D status of other organisms. Canines and felines are very different from humans in that they lack the ability to create vitamin D 3 within their skin. 147,148 The diet of such creatures is important, as vitamin D 3 will be gained from a mainly animal based diet while D 2 will be acquire d if plant sources are consumed. 147,148 Felines in particular hav e a hard time utilizing D 2 as
30 well as vitamin D 3 though canines may utilize both forms efficiently. 147,148 Canines and felines may well be a potential species for research to be compared to humans, as many of the same conditions and diseases suffered by humans from vitamin D deficiency extend to cats and dogs as well. 147,148 Farm animals have also been the subjects of vitamin D rese arch for their own health as well as the health of the human population that may feed upon such animals. Vitamin D metabolism has been well researched within dai ry cattle, and much like humans their immune health, bone formation, and reproductive health ar e affected by daily sun exposure and varying concentration s of serum 25 D 3 149 In opposition to cattle, which have been studied for benefit to their own health, research has been performed on pigs to improve the health of the human population. 150 In order to explore another avenue for potential vitamin D fortification within the food we eat, experiments have been performed in order to determine the optimal conditions for the format ion of vitamin D 3 within the skin of pigs. 150 Pork or other foodstuffs that contain pig skin may be fortified with vitamin D through the use of UV producing LED lights within living area s and processing plants for pigs. 150 Some species of fish contain great levels of dietary vitamin D 3 and research has been performed to determine whether this vitamin D 3 is from a dieta ry source or converted in the skin through UV radiation as within humans. 151 Rainbow trout have been found to form vitamin D 3 within their skin, but the potential for formation of vitamin D 3 within species of fish that live within extremely deep waters is unknown 151 Although it is thought that plants mainly contain vitamin D 2 certain plants with in the Solanaceae or nightshade family also contain vitamin D 3 which may have the
31 potential for consumption and supplementation of vitamin D within individuals that do not consume animal products. 152 T he tomato, potato, eggplant, bell pepper, and other plants commonly eaten as food belong to the nightshade family. 152 While the fruits and other commonly eaten parts of such plants have not shown significant or any sign of vitamin D 3 it has been shown that small amounts of vitamin D 3 25 D 3 and 1,2 5 D 3 exist within the leaves of tomato and bell pepper plants exposed to UV radiation. 152 Th is is a problem for potential plant based fortification, as the leaves of such plants are extremely poisonous in large quantities, but perhaps future research will show possible vitamin D treatment or supplementation from plants. 152 Disease and Oral Health Vitamin D def iciency is associated with systemic health problems as well as common diseases Vitamin D is a excellent localized topical for the treatment of psoriasis and certain skin diseases. 153 Vitamin D and calcium are necessary in the proper amounts if the human body is to develop, r emodel, and grow a healthy bone structure. 154 Within muscle, vitamin D is important to strength, flexibility and proper function, and vitamin D deficiency may result in muscle weakness, and even muscle atrophy. 155,156 Vitamin D deficiency is associated with obesity in that serum concentration s of 25 D 3 are lower in obese individuals than the non obese. 157,158 Deficient concentration s of serum 25 D 3 are associated with both type 1 diabetes (T1D) and type 2 diabetes (T2D). 159,160 Within cystic fibrosis, treatment with vitamin D may be used to regulate anti inflammatory and anti microbial activity in t he airway. 161 Patients with liver disease are commonly vitamin D deficient and this applies to alcoholics, non drin kers, adults, and children suffering from liver disease. 162 As VDR is expressed mammary gland s and in many breast cancers, 1,25 D 3 treatment and the resultant anti
32 inflammatory and pro apoptotic effects from induced immune regulation are well documented as beneficial to breast cancer patients. 163,164 This trend of vitamin D deficiency as a symptom, or even causal agent of disease can be directly related to my current research interests of periodontal disease and oral health. Characterized by both chronic inflammation and infection as well as bone and tooth loss, periodontal disease has symptoms that mimic the normal effects of vitamin D deficiency. 165 Low serum concentration s of 25 D 3 may have an association with periodontal disease, and a strong association has been shown between increasing 1,25 D 3 concentration after alleviation of periodontal disease symptoms and reduction of inflammation. 166,167 1,25 D 3 treatment inhibits the expression of pro inflamm atory cytokine IL 8 within a cell culture model of periodontal disease, and 25 D 3 can downregulate TLR4 and the JAK1/STAT3 pathway to have potential implications in further alleviating periodontal inflammation. 168,169 25 hydroxylase activity that can convert vitamin D 3 to 25 D 3 has been confirmed in human gingiv al fibroblasts and periodontal ligament cells, and this is a positive discovery because 25 D 3 treatment in human oral keratinocytes can increase expression of the AMP LL 37. 37,170 25 D 3 has also shown upregulation of VDR and downregulation of NF kB which resulted potent anti inflammatory effects in mice. 171 Overall, vitamin D is a candidate for research on immune regulation within oral health.
33 CHAPTER 2 VITAMIN D: CONVERSION WITHIN HUMAN GINGIVAL EPITHELIAL CELLS Background and Purpose As described in chapter 1, vitamin D is important to the regulation of immunity, alleviating harmful inflammation, and fighting infection within the human body. 73,74,82,88 90,172 Within a great number of diseases, vitamin D is at levels below what could be considered sufficient to proper immune function. 173 Along with its recent role as an immune regulator, vitamin D is classically associated with successful bone formation and continued bone health. 154,174 176 As a disease that involves both harmful inflammation and bone/tooth loss, periodontitis is a prime candidate for potential phenotypic alleviatio n following treatment with vitamin D. 165 Serum 25 D 3 concentration s are lower in those suffering from periodontitis and treatment with 25 D 3 in diabetic mice with periodontitis has been shown to ameliorate symptoms of harmful inflammat ion and potential alveolar bone loss. 169 ,171,177 25 D 3 and 1,25 D 3 induce innate immunity and increase antimicrobial activity within human gingival epithelium. 72,170 While 1,25 D 3 is a n effective treatment, the enzyme Cyp24A1 exists to metabolize, negatively regulate, and degrade the hormone which gives it a short half life of ~6 8 hours. 27 Topical treatment of peridontitis with the more stable, and readily available over the counter, vitamin D 3 may be a better treatm ent option if it can be shown to convert to active 1,25 D 3 within an oral environment. While p resence of the cytochrome P450 machinery required to convert vitamin D 3 to active 1,25 D has been shown in gingival fibr oblasts and ligament cells, is has not bee n shown within gingival epithelium. 37 The overall goal of my research is to show the presence of c ytochrome P450 enzymes w ithin human gingival epithelium as evidence of
34 vitamin D conversion and increased innate immune regulation following topical vitamin D treatment. I hypothesize that vitamin D 3 is converted to active 1,25 D 3 within human gingival epithelium and that vitamin D 3 treatment will lower the expression of pro inflammatory cytokines in human gingival epithelium. Methods Cell Culture Cell lines used within the following experiments were the human oral k eratinocytes OKF6/Tert 1 and telomerase immortalized gingival keratinocytes (TIGK). 178,179 Cells were grown to confluency in 6 well or 12 well plates at 37C and 5% CO2 within keratinocyte serum free media (KSFM) supplemented with 10% FBS, L glutami ne, and penicillin/st reptomycin. All cell line experiments utilized both biological and assay triplicates. Oligonucleotide Primers All primers used within the following experiments were designed and ordered through Integrated DNA Technologies (IDT, Coralville, IA) and are list ed in table 2 1 following the results section. Primers were validated with the first derivative of melt curve analysis following RT qPCR containing a single peak, and products were run on agarose gels to reveal bands of predicted amplicon size by design. C ytochrome P450 Enzyme Presence Cytochrome P450 enzyme presence was examined by RT qPCR. OKF6/Tert 1 cells were treated in triplicate with 10nm vitamin D 3 10nm 1,25 D 3 or EtOH vehicle control for 6 hours at which point RNA was isolated from the cells acco rding to the RNeasy Plus Minikit (Qiagen, Valencia, CA). Genomic DNA contamination was minimized by running the samples through gDNA Eliminator spin columns (Qiagen)
35 prior to RNA purification. RNA was reverse transcribed using iScript Reverse Transcription Supermix for RT qPCR (Bio Rad, Hercules, CA). RT qPCR was carried out with SYBR Green Supermix (Bio Rad) and individual qPCR primers for CYP 2R1, CYP 27A1, CYP 27B1, and CYP 24A1. Data was analyzed using Bio Rad CFX Manager Software. actin was used as a con trol. For cytochrome p450 enzyme presence by PCR, OKF6/Tert 1 cells were treated in triplicate with EtOH, 10nm vitamin D 3 or 10nm 1,25 D 3 RNA was purified from cells and reverse transcribed to cDNA with the absence of reverse transcriptase (RT ) as a neg ative control for potential genomic DNA contamination. The cDNA and oligonucleotide primers for enzymes CYP 2R1, CYP 27A1, and CYP 27B1 were used in PCR amplification using an iProof High Fidelity Master Mix (Bio Rad) and then run on a 3% agarose gel with gel loading buffer in each sample and 100bp Quickload ladder for determining presence or absence by amplicon size. To assess concentration s of total 1,25 D 3 or 25 D 3 for the purposes of ascertaining if direct conversion was taking place, OKF6/Tert 1 cells wer e treated in triplicate with vitamin D 3 25 D 3 or EtOH vehicle control for 6 or 24 hours. Media fractions were removed and kept at 80C until samples were sent for analysis of total 1,25 D 3 or 25 D 3 content by radioimmunoassay (Heartland Assays, Ames, IA ). Untreated media was used as a control. Vitamin D 3 treated samples were analyzed for total 25 D 3 content, 25 D 3 treated samples were analyzed for total 1,25 D 3 content, and EtOH samples as well as untreated media control were analyzed for 25 D 3 and 1,25 D 3 content for purposes of control. The assay to measure 25 D 3 had a lower detection limit
36 of 2.5 ng/mL while the assay to measure 1,25 D 3 content had a lower detection limit of 5.5 pg/mL and an upper detection limit of 210 pg/mL. Vita min D Anti Inflammato ry and Anti M icrobial Effects Pro inflammatory cytokine expression was observed using RT qPCR. OKF6/Tert 1 cells were treated in triplicate for 6 hours with either 10nm 1,25 D 3 or EtOH vehicle control in the presence or absence of polyinosinic:polycytidyli c acid ( poly(I:C) ) as an inflammatory stimulus Poly (I:C) interacts with TLR3 unlike the TLR2 interactions with Pg in periodontitis, but it was used as a stimulus because both pathways can function to increase inflammation via NF kB activation. 180 182 RNA purification, reverse transcript ion, and RT qPCR were carried out as described previously but using primers for IL 6 and IL 8 as they are both pro inflammatory cytokines. actin was used as a control. Regulatory effects on pro inflammatory cytokines were also observed in mice. C57BL/6 mice (n = 15) were treated with an oral rinse of either 1 vitamin D 3 1 1,25 D 3 or EtOH vehicle control all of which were suspended i n mineral oil to allow for lengthened treatment through continual adhesion to the gingival surface. 6 hours following treatment, mice were euthanized by CO 2 exposure and all gingival epithelial tissue was harvested and placed in RNAlater Stabilization Solu tion (Thermo Fisher) for stability and then utilized for RT qPCR as previously described. CYP 24A1 expression was examined as a control of vitamin D activity and IL 1A expression was quantified to deter mine regulation of inflammation as it is commonly expr essed in inflammatory activated epithelial cells. actin was used as a control For quantifying levels of the antimicrobial peptide LL 37 OKF6/Tert 1 cells were treated in triplicate with 10nm 1,25 D 3 or EtOH vehicle control for 24 hours. Media
37 fra ctions were removed and kept for analysis while cells were washed 2x in PBS and lysed with cell scraping following the addition of cell lysis buffer. LL 37 levels were measured and the ratio between levels in both cell a nd media fractions to determine cell ular LL 37 export was determined by Sandwich ELISA with a kit from MyBioSource (San Diego, CA). To view the effects of vitamin D treatment on Pg invasion, untreated TIGK cells in triplicate and TIGK cells pre treated in triplicate for 24 hours with 10nm 1 ,25 D 3 or EtOH were washed 4x with PBS and infected with Pg (MOI = 100) and incubated for 90 minutes. Within the previously untreated TIGK cells, this infection also came with a concurrent 90 minute treatment of either 10nm 1,25 D 3 or EtOH, while the pre t reated cells received no treatment along with the infection. Cells were then washed 4x again in PBS and treated with metronidazole/gentamicin in media for another 60 minute incubation to kill externally adherent Pg that had not yet invaded. Cells were wash ed 4x more in PBS, scraped on ice with ice cold PBS, and lysates transferred to cryovials containing DMSO and Pg growth media and kept at 80C until CFU counts were to be determined by spot plating of lysate serial dilutions. For assessment of total bact erial load by visualization of Pg 16s rRNA, OKF6/Tert 1 cells were treated in triplicate with either 10nm vitamin D 3 or 10nm 1,25 D 3 for 24 hours, and washed 4x in PBS followed by infection with Pg (MOI = 100). Cells were then supplied with plain media or media supplemented with additional 10nm vitamin D 3 or 1,25 D 3 and allowed to incubate for a further 24hours, washed 4x in PBS, at which point RNA was purified, reverse transcribed, and RT qPCR was carried out as previously described but using primers for P g 16s rRNA to show total bacterial load.
38 Primers were developed to identify Pg exclusively and not other potential microbes. 183 Human 18s rRNA was used as a control. Results Conversion of Inactive Vitamin D by Gingival Epithelial Cells In RT qPCR for the presence of cytochrome P450 machinery (Fig 2 1 ) RNA for CYP 24A1, CYP 27A1, CYP 27B1, an d CYP 2R1 was present in OKF6/Tert 1 cells whether treated with EtOH, vitamin D 3 or 1,25 D 3 for 6 hours. Expression of CYP 24A1 was significantly increased in vitamin D 3 (Fig 2 1A) and 1,25 D 3 (Fig 2 1B) treated samples when compared to EtOH vehicle control samples. No significant difference was measured in the expression levels of CYP 27A1, CYP 2R1, or CYP 27B1 amongst all treatments. test with a p value of < 0.05 showing significance. Error bars represent standard e rror. PCR for cytochrome P450 presence (Fig 2 2) resulted in bands of proper amplicon size ( CYP 2R1: ~110bp, CYP 27A1: ~285bp, CYP 27B1: ~245bp) being present for each gene observed. CYP 2R1 lanes showed single banding and no genomic DNA contamination in RT c ontrol. CYP 27A1 lanes showed faint alternate banding at approximately 400bp but nothing in RT control. CYP 27B1 show s strong presence in all treatments, but also 2 more unexpected bands at ~335bp and <100bp and contamination from genomic DNA in RT contro l treatments at ~335bp as well. Radioimmunoassay for the presence of 25 D 3 and 1,25 D 3 varied by treatment type, but not by treatment length Plain media as a control, as well as samples treated with vitamin D 3 and EtOH vehicle control for 6 or 24 hours, sh owed no measureable amount of 25 D 3 present within the limits of the assay (<2.5ng/mL) ( Table 2 2 ). Samples treated with 25 D 3 for 6 and 24 hours showed 1,25 D 3 present in levels above the upper
39 limits of the assay (>210pg/mL), while media and EtOH showed no 1,25 D 3 present within lower limits of the assay (< 5.5pg/mL) (Table 2 2 ). Anti Inflammatory Effects of Vitamin D in Gingival Epithelial Cells RT qPCR for immune markers of inflammation result ed in no significant difference in the levels of IL 6 or IL 8 between samples treated with EtOH or 1,25 D 3 but IL 6 and IL 8 levels were significantly decreased in 1,25 D 3 treated samples that had undergone prior inflammatory stimulus with poly(I :C) versus EtOH treated samples (Fig 2 3 ). Statistics were performed us test with a p value < 0.05 showing significance. Error bars represent standard error. RT qPCR within mice showed significant differences in expression of CYP 24A1 in 1,25 D 3 treated v ersu s EtOH and vitamin D 3 treated mice, and significant di fferences in expression of IL 1A in EtOH treated versus vitamin D 3 and 1,25 D 3 treated mice. CYP 24A1 was ~300 fold upregulated in 1,25 D 3 treated mice when compared to other treatments (Fig 2 4A) IL 1A was appreciably downregulated within both 1,25 D 3 (Fi g 2 4A) and vitamin D 3 (Fig 2 4B) treated mice compared to those treated with EtOH. test with a p value < 0.05 showing significance. Error bars represent standard error. Antimicrobial Effects of Vitamin D in Ging ival Epithelial Cells ELISA showed a significantly higher level of free LL 37 in growth media than in the cell lysate fraction of samples treated with 1,25 D 3 when compared to EtOH treated samples (Fig 2 test with a p value < 0.05 showing significance. Error bars represent standard error. To determine the effects on antimicrobial activity within gingival epithelium, cells were treated with vitamin D and infected with Pg CFUs of Pg present follo wing invasion
40 a nd re plating were decreased 2 3 times in samples treated with 1,25 D 3 than samples treated with EtOH L evels of overall Pg present were lower in pre treated samples than samples treated with 1,25 D 3 and EtOH concurrent to the introduction and invasion of Pg (Fig 2 6A). CFU counts were lower in re plated samples of the same dilution treated with 1,25 D 3 (Fig 2 6B) versus EtOH (Fig 2 6C). Statistics were performed using test with a p value < 0.05 showing significance. Error bars represent standar d error. RT qPCR to estimate total bacterial load showed significantly lower levels of 16s rRNA expression following dual treatment in vitamin D 3 (2 7A) and 1,25 D 3 treated cells when compared to cells that were only pre treated with either A greater redu ction in 16s rRNA levels was shown in the 1,25 D 3 treated samples when compared to those treated with vitamin D 3 test with a p value < 0.05 showing significance. Error bars represent standard error.
41 Table 2 1. List of Primers Used
42 Figure 2 1. Cytochrome P450 Enzymes qPCR OKF6/Tert 1 cells were treated in triplicate with EtOH, 10nm vitamin D 3 or 10nm 1,25 D 3 for 6 hours RNA was purified and reverse transcribed to cDNA for use in RT qPCR to observe the p resence and expression of CYP 24A1, CYP 27A1, CYP 27B1, and CYP actin was used as a control. All cytochrome P450 enzymes observed were present while CYP 24A1 was significantly upregulated within both vitamin D 3 ( A) and 1,25D 3 ( B) treated cells when compared to EtOH control The presence of Cyp family enzymes all ows for the potential conversion of vitamin D 3 to active 1,25 D 3 within gingival epithelium and the significant increase in CYP 24A1 shows active degradation of 1,25 D 3 which provides evidence for the possibility of conversion with in the vitamin D 3 treated cells. test with a p value of < 0.05 showing significance. Error bars represent standard error.
43 Figure 2 2. Cytochrome P450 Enzymes PCR and Gel Electrophoresis Gel electrophoresis was performed on PCR prod ucts to show cytochrome p450 enzyme presence within OKF6/Tert 1 cells. OKF6/Tert 1 cells were treated in triplicate with EtOH, 10nm vitamin D 3 or 10nm 1,25 D 3 RNA was purified from cells and reverse transcribed to cDNA with the absence of reverse transcr iptase (RT ) as a negative control for potential genomic DNA contamination cDNA and oligonucleotide primers for enzymes CYP 2R1, CYP 27A1, and CYP 27B1 were used in PCR amplification and run on a 3% agarose gel for determining presence or absence by amplicon size. Bands of proper amplicon size ( CYP 2R1: ~110bp, CYP 27A1: ~285bp, CYP 27B1: ~245bp) were found for each gene observed. CYP 2R1 lanes showed single banding and no genomic DNA contamination in RT control. CYP 27A1 lanes showed faint alternate banding at a pproximately 400bp but nothing in RT control. CYP 27B1 show s strong presence in all treatments, but also 2 more unexpected bands at ~335bp and <100bp and contamination from genomic DNA in RT control treatments at ~335bp as well. Amplification of PCR produ cts shows no quantifiable difference between EtOH, vitamin D 3 and 1,25 D 3 treatments, but overall presence of gene products is observed in all RT+ treatment groups which shows the presence of RNA that would code for the enzymes in OKF6/Tert 1 cells. Futur e study should excise and pu rify gel bands to send out for S anger sequencing to ensure the observed bands were the proper amplicon sequences and thus the observed gene products.
44 Table 2 2 25 D 3 and 1,25 D 3 Quantification in OKF6/Tert 1 Radioimmunoassa y To quantitatively observe vitamin D conversion, OKF6/Tert 1 cells were treated in triplicate for 6 or 24 hours with EtOH, 10nm vitamin D 3 or 10nm 25 D 3 respectively. Media fractions were collected and kept at 80C until analyzed by radioimmunoassay fo r total concentration of either 25 D 3 or 1,25 D 3 Untreated media was used as an additional control. D 3 or 1,25 D 3 if D 3 or 1,25 D 3 if u nder the detection limit of the assay. Cells treated with media, EtOH, and vitamin D 3 showed no detectable 25 D 3 within the limits of the assay (<2.5 ng/mL). Cells treated with media and EtOH showed no detectable 1,25 D 3 (<5.5 pg/mL), but 25 D 3 treated cel ls showed 1,25 D 3 presence above the limits of the assay (>210 pg/mL). This potentially shows conversion of 25 D 3 to 1,25 D 3 within gingival epithelium, but not conversion of vitamin D 3 to 25 D 3 This is possibly because all of the vitamin D 3 used within t reatment fully underwent conversion to 1,25 D 3 within both the 6 and 24 hour timepoints. In that case, a shorter timepoint may have shown detectable 25 D 3 within the vitamin D 3 treated cells. Another potential explanation may be that the 10nm vitamin D 3 tr eatment used is close to being under the detection limit (2.5ng/mL) of the 25 D 3 assay. Future study should use 100nm concentration as a treatment level to ensure that initial treatment is over the lower limit of the radioimmunoassay. Vitamin D 3 treated ce lls have not been tested for 1,25 D 3 presence yet d ue to time constraints and cost.
45 Figure 2 3. Inflammatory Markers and 1,25 D 3 qPCR OKF6/Tert 1 cells in were treated in triplicate for 6 hours with EtOH or 10nm 1,25 D 3 in the presence or absence of 1ug/mL poly(I:C) as an inflammatory stimulus. While poly(I:C) may be a TLR3 agonist, it can activate the NF kB pathway much like TLR2 activation by Pg within periodontitis, which makes it adequate for our stimulation of inflammation. RT qPCR was used t o view expression of pro inflammatory cytokines IL 6 and IL 8 relative to actin as a control Both IL 6 and IL 8 were significantly increased in poly(I:C) treated cells when compared to those not exposed to inflammatory stimulus. Levels of IL 6 and IL 8 were significantly reduced in the samples treated with poly(I:C) and 1,25 D 3 when compared to the poly(I:C) stimulated cells treated with EtOH The reduction of IL 6 and IL 8 pro inflammatory cytokines in OKF6/Tert 1 cells treated with 1,25 D 3 following th e induction of inflammation by poly(I:C) shows the potential for amelioration of symptoms within an inflammatory phenotypic environment. Statistics test with a p value < 0.05 showing significance. Error bars represent stand ard error. 0 1 2 3 4 5 6 7 8 9 IL 6 IL 8 Expression Relative to B actin EtOH 1,25 D3 Poly(I:C) + EtOH Poly(I:C) + 1,25 D3 P<0.05 ____*____ ____*____ ____ ___ ____ ___
46 Figure 2 4. Inflammatory Regulation in Mice qPCR C57BL/6 Mice (n = 15) were treated with an oral rinse of EtOH, 1um vitamin D 3 or 1um 1,25 D 3 for 6 hours to further observe innate immune regulation by vitamin D treatment. Mouse gingival ti ssue was harvested and RT qPCR was performed to determine the levels of CYP 24A1 as a measure of vitamin D activity and IL 1A as a pro inflammatory cytokine that is present in inflammatory epithelial tissue. Mouse actin was used a control. Results showed significant increase of CYP 24A1 in 1,25 D 3 treated mice ( A) and significant reduction of pro inflammatory cytokine IL 1A in vitamin D 3 ( B) and 1,25 D 3 treated ( A) mice. The increase i n CYP 24A1 provides evidence of 1,25 D 3 activity, and the decreased levels of IL 1A can be attributed to the anti inflammatory effects of vitamin D treatment. S test with a p value of < 0.05 showing significance. E rror bars represent standard er ror.
47 Figure 2 5. Export of LL 37 ELISA OKF6/Tert 1 cells were treated in triplicate for 24 hours with either 10nm 1,25 D 3 or EtOH vehicle control to observe the effects of vitamin D treatment on the levels of the antimicrobial peptide LL 37. Media an d cell lysate fractions were taken from cell samples and used in sandwich ELISA to show levels of total LL 37. Results showed that a greater ratio of total free LL 37 resided in the media fraction within the 1,25 D 3 treated cells when compared to the EtOH treated cells. This shows greater levels of LL 37 export from vitamin D treated cells when compared to EtOH control treated cells. This increased level of extra cellular LL 37 could mean potentially increased antimicrobial activity in the oral cavity upon vitamin D treatment of human gingival epithelium. test with a p value of < 0.05 showing significance. Error bars represent standard error.
48 Figure 2 6. Vitamin D and Porphyromonas gingivalis Invasion Assay TI GK cells initially left untreated and TIGK cells pre treated for 24 hours with 10nm 1,25 D 3 or EtOH were infected in triplicate with Pg (MOI=100) to show the antimicrobial effects of vitamin D tre atment. The initially untreated cells then received a 90 min ute treatment with either 10nm 1,25 D 3 or EtOH while undergoing Pg invasion. Following invasion, cells were washed, lysed to release invaded Pg and plated to count CFU s to determine viable Pg CFUs were 2 3 times lower in all vitamin D treated cells ( A) w hich shows potential antimicrobial activity with overall levels of Pg being higher in cells that were not pre treated with either 1,25 D 3 or EtOH Plate images show cells pre treated with 1,25 D 3 ( B) or EtOH ( C) at equivalent dilutions. It is yet unclear whether vitamin D is acting directly or indirectly to fight Pg test with a p value of < 0.05 showing significance. Error bars represent standard error.
49 Figure 2 7. P g 16s rRNA Total Bacterial Load qPCR To observe the synergy of dual (pre and post invasion vitamin D treatment), OKF6/Tert 1 cells were treated in triplicate with either 10nm vitamin D 3 or 10nm 1,25 D 3 for 24 hours, infected with Pg (MOI=100), and incubated for 24 hours or supplemented with a dditional 10nm vitamin D 3 or 1,25 D 3 and then incubated 24 hour s. RNA was purified and RT qPCR was then carried to observe expression levels of Pg 16s rRNA to show total bacterial load Human 18s rRNA was used as a control. 16s rRNA expression was signific antly lower with dual treatment in both vitamin D 3 ( A) and 1,25 D 3 ( B) treated cells when compared to cells o nly pre treated with either This may mean that vitamin D treatment has both active and passive/bolstering effects on anti microbial activity and im munity that contribute synergistically Statistics were performed using test with a p value of < 0.05 showing significance. Error bars represent standard error.
50 CHAPTER 3 DISCUSSION OF RESULTS The presence of cytochrome P450 enzymes within hum an gingival epithelium was successfully shown by RT qPCR and PCR. While this does not explicitly mean that conversion is taking place, the presence of local conversion machinery means that there is a good chance for vitamin D 3 applied for treatment to be h ydroxylated to 1,25 D 3 If vitamin D 3 is transformed to 1,25 D 3 it would mean that vitamin D 3 treatment alone could result in VDR and RXR binding that would facilitate the inflammatory regulation that occurs upon interactions with vitamin D response eleme nts in specific genes. In the case of an inflammatory disease like periodontitis, this would mean vitamin D 3 may be effective local/topical treatment for symptom alleviation. Within the RT qPCR in figure 2 1 the insignificant difference in the levels of C YP 2R1, CYP 27A1, and CYP 27B1 is to be expected, as the hydroxylation events that take place with the conversion of vitamin D 3 and 25 D 3 would not require additional machinery except to potentially speed up the reaction. While CYP 2R1 and CYP 27A1 levels are n ot affected by treatment, CYP 27B1 as way to maintain calcium homeostasis, is known to be downregulated by Ca 2+ ions that result from increased vitamin D activity 16 The upregulation of CYP 24A1 levels in accordance with vitamin D treatment can be explained by the increased need to degrade active 1,25 D 3 to prevent further binding to VDR and unnecessary events of immune regulation in the case of excess 1,25 D 3 16 In the case of vitamin D 3 treated cells, the CYP 24A1 upregulation would result from conversion of vitamin D 3 to 1,25 D 3 which would require the enzyme to degrade excess vitamin D Samples in the future should also be treated with 25 D 3 to ensure similar data amongst all vitamin D treatment types.
51 Amplification of PCR products shows no quantifiable difference between EtOH, vitamin D 3 and 1,25 D 3 treatments, but overall presence of gene products is observed in all RT+ treatment groups which shows the presence of RNA that would code for the enzymes in OKF6/Tert 1 cells. CYP 27B1 bands are dark and thus levels are higher hydroxylation and the importance of converting 25 D 3 to 1,25 D 3 to take place in VDR binding events for potential genetic regulation 43 The comparatively low le vels of the CYP 27A1 and CYP 2R1 enzymes, may be due to cells so far away from the normal site of 25 hydroxylation in the liver not requiring much if any at all of the 25 hydroxylase enzymes. This would also explain the higher levels of CYP 27B1, as the gingival cells would have developed seeing vitamin D mainly in the serum 25 D 3 form upon cellular localization for conversion to active 1,25 D 3 following hydroxylation in th e liver Future study should excise and purify gel bands to send out for Sanger sequencing to ensure the observed bands were the proper amplicon sequences and thus the observed gene products. It is also of note that more specific primers must be developed to eliminate the 1 or 2 bands that are not specific to our target amplicon size and also to further lower amplification of genomic DNA with our primers. The attempt to directly show conversion o f vitamin D by radioimmunoassay in table 2 2 showed that treat ment with 25 D 3 resulted in conversion to 1,25 D 3 but treatment with vitamin D 3 did not show presence of 25 D 3 The reasons for this are most likely because vitamin D 3 was fully converted to 1,25 D 3 in both the 6 and 24 hour treatments and there was no re sidual 25 D 3 left. A shorter timepoint (2 hours, 4 hours) may subvert these issues and allow 25 D 3 to be detected. Another thought and potential
52 explanation is that our treatment concentration of 10nm vitamin D 3 may be too close to the lower limit of detec tion (2.5 ng/mL) to be observed by the assay. In that case, we should raise our treatment level to 100nm concentration of vitamin D 3 and retest to see if vitamin D 3 can be detected. As our treatment samples are on both the upper and lower limits of the ass ay used, it is important to think of the results as more of a happening but not truly quantify the extent of conversion. If vitamin D 3 does fully convert to 1,25 D 3 as it was shown with the 25 D 3 treated cells then it is good evidence of our hypothesis of vitamin D conversion taking place within human gingival epithelium. This would result in being able to potentially use over the counter vitamin D 3 as a topical treatment for periodontitis or other inflammatory diseases wit hin the oral cavity. Future analysis should analyze vitamin D 3 treated cells for 1,25 D 3 concentration Assuming conversion is successful, it is also important to sh ow the potential anti inflammatory and/or antimicrobial effects of active 1,25 D 3 treatment within gingival epithelium. This has been shown by v isualizing levels of pro inflammatory cytokines following vitamin D treatment as well as cellular export levels of the antimicrobial peptide LL 37 72,173 Antimicrobial activity can also be observed by testing the effects of vitamin D on the periodontal pathogen Pg in regards to cellular invasion and survival The decre ased levels of pro inflammatory cytokines in figure 2 3 as a result of vitamin D treatment in the cells that have under gone inflammatory stimulation are a direct example of the potential for vitamin D 3 to topically treat harmful inflammation. While the pol y(I:C) used is not directly relevant to periodontal disease as it is a TLR3 agonist and not a TLR2 agonist, both pathways can lead to induction of inflammation
53 through NF kB. 180 182 IL 6 and IL 8 were shown to be downregulated as in previous studies but other pro inflammatory cytokines s uch as IL 1B IL 1A and TNF must be observed to see if on site topical vitamin D treatment is truly responsible for alleviating harmful inflammation. 83,86,87 Figure 2 4 continues to show the decrease of harmful inflammation but within mice. The increas ed levels of CYP 24A 1 indicate that 1,25 D 3 is being utilized, but the cytokine is not upregulated in the vitamin D 3 treated mice which would have supported our working hypothesis and shown conversion to active vitamin D within mice as well. The significant downregulation of the pro inflammatory cytokine IL 1A in mice treated with EtOH compared to 1,25 D 3 treated mice shows active vitamin D to be anti inflammatory The mice treated with vitamin D 3 also showed si gnificant IL 1A downregulation which may further support vitamin D 3 as an anti inflammatory treatment. Further mouse experiments should be expanded to include inflammatory stimulus prior to vitamin D treatment as well as viewing other cytokines such as IL 1B, TNF, IL 6, and IL 8. The increase in the levels of the antimi crobial peptide LL 37 in the external media fraction of cells treate d with vitamin D within figure 2 5 shows a potential increase in the release of antimicrobial peptides from cells to combat potentially pathogenic bacteria. This export of LL 37 from cells may function to fight against Pg in periodontitis, but it must be recognized that Pg is known to utilize peptides similar in form to LL 37 as a protein source. 184 The effects of direct application of LL 37 to Pg growth and survival must be observed in a future experiment to ensure that this upregulation of cellular antimicrobial activity is relevant to periodontitis.
54 Vitamin D treatment affects the survival and viability of Pg invasion a s shown in figure 2 6 The CFU counts of viable Pg are reduced 2 3 times from treatment with 1,25 D 3 as both a pre treatment prior to invasion as well as a treatment during potential invasion. This shows proper treatment with and/or maintaining sufficient levels of vitamin D may have a bolstering effect to immunity that can prevent Pg invasion as a pre emptive deterrent within healthy individuals as well as when used as a topical treatment in those suffering from periodontitis. The differences in CFU counts between the pre and concurrent treatment levels are most likely due to unintended differences in levels of viable Pg introduced for the invasion assay, but future studies will make an attempt to standardize the invasion assay further and only use living/ replicating organisms. The difference between 1,25 D 3 and EtOH treatment and the effects on Pg colony viability are clear enough, but it remains to be seen whether vitamin D is acting as a direct or indirect antimicrobial on Pg so that will need to researc hed in the future. Another way of exploring the potential for vitamin D treatment of periodontitis with action on Pg is to measure Pg 16s rRNA levels for total bacterial load. Figure 2 7 attempts to perform this measurement as well as observe the synergy b etween pre emptive immune bolstering with vitamin D treatment in a healthy cellular environment and treatment post invasion of Pg when harmful inflammation would be present. Pg 16s rRNA levels are reduced following treatment with both vitamin D 3 and 1,25 D 3 and this shows again that vitamin D 3 treatment may have an active effect that is nearly comparable to that of treatment with 1,25 D 3 A good direction for 16s rRNA studies and a meaningful future experiment would be to directly correlate the levels of 1 6s rRNA to viable Pg colonies in a sample in order to be able to more easily quantify the effects of
55 vitamin D treatment on Pg without needing to re plate and determine CFU values by colony counting. While conversion from inactive to active vitamin D has been previously shown in gingival fibroblasts and ligament cells, it has never before been shown in gingival epithelium. 37 The data presented shows the potential for vitamin D conversion w ithin human gingival epithelium and thus a direct topical application of vitamin D 3 may be viable as an anti inflammatory an d anti microbial treatment in periodontitis. The idea of a topical treatment brings many new c hallenges however, such as determining the necessary concentration with which to treat humans, how to ensure the treatment stays localized to the affected areas f ollowing application, or the potential for harmful side effects related to the direct application of a tightly regulated hormone to a single/specific bodily surface. The implications are great, but further study must be performed on the mechanics of potent ial conversion within the human oral environment as well as on the direct effects that vitamin D may have on invading periodontal pathogens and alleviation of harmful inflammation within human patient s suffering from periodontitis. What was once thought o ff as a simple regulator of bone deposition is now showing proof as an integral regulatory substance in immune function throughout the human body. W ith vitamin D proving itself as much more than a simple hormone produced in the skin, the field of vitamin D research is wide open and continued studies will lead to the improved immune health of all suffering the harmful effects of vitamin D deficiency.
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70 BIOGRAPHICAL SKETCH William Ruddick was born in Hinsdale, Illinois and raised by his grandparents in Oak Forest, Illinois. He currently resides in Cle rmont, Florida with his wife Amy He completed his primary and secondary educ atio n while continuing to reside in Oak Forest degree in science at M oraine Valley Community College, in Palos Hills, Illinois, and his University, in University Park, Illinois. William worked for a short time as a laboratory chemist before deciding to further his education He was admitted to the Interdisciplinary Program in Biomedical Sciences at the Universit y of Florida in the fall of 2014 His scientific fields of interest include immunology, microbiology, ecology, and ornithology. Outside of science his interests include film, gaming, hunting, fishing, spelunking, skydiving, reading, theme parks, music, singing, crochet, cooking, board games, baseball, and golf.