Source: FAIRFIELD UNIVERSITY submitted to NRP
EGG EFFECTS ON THE IMMUNOMODULATORY PROPERTIES OF HDL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1014925
Grant No.
2018-67018-27508
Cumulative Award Amt.
$149,419.00
Proposal No.
2017-06937
Multistate No.
(N/A)
Project Start Date
Mar 1, 2018
Project End Date
Feb 28, 2022
Grant Year
2018
Program Code
[A1341]- Food Safety, Nutrition, and Health: Function and Efficacy of Nutrients
Recipient Organization
FAIRFIELD UNIVERSITY
1073 N BENSON RD
FAIRFIELD,CT 06824
Performing Department
Biology
Non Technical Summary
Chicken eggs contain a wide variety of bioactive compounds in yolk and white fractions that possess lipid- and immune-modulating properties. Our previous research has demonstrated that consumption of whole eggs vs. egg whites differentially affects immune cell inflammation in metabolic syndrome subjects, and that differences may be dependent upon changes in HDL composition and efflux capacity. Therefore, the long-term goal of this project is to further elucidate the effects of bioactive egg components on the immunomodulatory properties of HDL in a healthy population using a translational, mechanistic approach. In this randomized crossover study, we will compare the effects of 1) an egg-free diet, 2) an egg white-rich diet, and 3) a whole egg-rich diet on HDL size, lipid composition, and proteomic profiles (Objective 1); the anti-inflammatory and cholesterol efflux properties of HDL (Objective 2); and the inflammatory potential and cholesterol dynamics of subject-derived primary leukocytes (Objective 3). We hypothesize that bioactive lipids in whole eggs will drive enrichment of HDL with yolk-derived lipids, leading to increased HDL size, anti-inflammatory shifts in the HDL proteome, and increased efflux capacity that will suppress immune cell inflammation, as compared to the egg-free and egg white-rich diet. We further anticipate that intake of an egg-free and egg white-rich diet will have minimal impact on HDL size, lipid composition, proteome and efflux capacity, leading to relatively greater pro-inflammatory immune responses. This project supports U.S. Agriculture by contributing to our fundamental understanding of the role that eggs play in human health, and the mechanisms by which they exert these effects.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70232701010100%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
3270 - Eggs;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
Chicken eggs contain a wide variety of bioactive compounds in yolk and white fractions that possess lipid- and immune-modulating properties. Consumption of whole eggs (yolk + white) has consistently yielded beneficial shifts in HDL-cholesterol and HDL particle size, closely mimicking the effects of egg-derived phospholipid supplementation. Conversely, the effects of whole egg/yolk components and egg white proteins on inflammation remains somewhat controversial. Previous work funded by a USDA Predoctoral Fellowship Award to C.J. Andersen (Project Director) suggests that consumption of whole eggs vs. egg whites differentially affects immune cell inflammation in metabolic syndrome subjects following a moderate-carbohydrate-restricted diet, and that observed differences may be dependent upon changes in HDL function. HDL is known to exert anti-inflammatory activity, which has predominantly been attributed to 1) the capacity of HDL to mediate cellular cholesterol efflux, leading to plasma membrane raft depletion and diminished inflammatory receptor signaling; and 2) the dynamic distribution of lipid species, particles sizes, and the HDL proteome, which dictate cholesterol efflux capacity, sequestration of inflammatory ligands (e.g. lipopolysaccharide, LPS), and immune activation. Thus, further investigation is essential to 1) determine the relationship between egg-derived components, HDL function, and immune cell inflammation during weight maintenance in metabolically healthy adults - individuals who differ from metabolic syndrome subjects in regard to HDL metabolism and basal inflammation, 2) elucidate whether egg-induced changes in immune inflammation are truly dependent upon HDL properties using a translational, mechanistic approach, and 3) determine whether the HDL-immune relationship can be manipulated by various, commonly-consumed egg diets within a single individual.Therefore, the long-term goal of this project is to investigate the effects of bioactive egg components on the immunomodulatory properties of HDL. We will address our long-term goal by comparing intake of 1) an egg-free diet, 2) an egg white-rich diet, and 3) a whole egg-rich diet using a crossover study design in healthy adults. Egg effects on the immunomodulatory properties of HDL will be assessed by:OBJECTIVE 1: Characterization of HDL size, lipid composition, and proteomes. We will test the hypothesis that yolk-derived components provided by whole egg promotes anti- inflammatory and efflux-promoting shifts in HDL compositional profiles as compared to an egg-free and egg white-rich diet. HDL particle size, lipid species composition, and associated proteins determine the capacity of HDL to facilitate cholesterol efflux and regulate immune cell activity. Therefore, prior to testing the functional capacities of HDL on immune cells, we will investigate the effects of an egg-free vs. egg white vs. whole egg diet on 1) HDL particle size, 2) HDL lipid species composition, and 3) the global HDL proteome - a novel and rapidly- developing field of research.OBJECTIVE 2: Evaluating anti-inflammatory and cholesterol efflux properties of HDL. We will test the hypothesis that yolk-derived components provided by whole egg will increase the anti-inflammatory and efflux functionality of HDL as compared to egg-free and egg white diets, which will induce relatively greater immune inflammation by not altering HDL efflux capacity. We will test the biological significance of HDL compositional profiles by functional ex vivo assays. We anticipate that differences in cellular inflammation will be associated with shifts in HDL proteomic profiles and dependent upon HDL-mediated efflux. Therefore, we will investigate the effects of an egg-free vs. egg white vs. whole egg diet on 1) ex vivo cholesterol efflux to subject HDL and whole serum from THP-1 macrophages and Jurkat T lymphocytes, 2) LPS-induced inflammation and proteomic profiling of THP-1 and Jurkat cells in the presence of subject HDL and serum, 3) the contribution of ABCA1-, ABCG1-, and SR-BI- mediated efflux on LPS-induced inflammation using selective inhibitors.OBJECTIVE 3: Characterizing the inflammatory potential and cholesterol dynamics of subject-derived primary leukocytes. We will test the hypothesis that yolk-derived components provided by whole egg will reduce peripheral blood mononuclear cell (PBMC) inflammation as compared to egg-free and egg white-rich diets, and that these changes will be associated with reduced total cellular cholesterol and lipid rafts content. We anticipate that alterations in PBMC inflammation will be associated with corresponding changes in the cholesterol efflux capacity, anti-inflammatory properties of HDL, and leukocyte cholesterol content. Thus, we will test the effects of an egg-free vs. egg white vs. whole egg diet on 1) clinical complete blood cell counts, 2) subject-derived PBMC proteomic profiling and expression of genes related to inflammation and cholesterol metabolism, 3) quantification of total PBMC cholesterol and lipid rafts, and 4) ex vivo LPS stimulation of subject PBMCs.We hypothesize that bioactive lipids in whole eggs will drive enrichment of HDL with yolk-derived lipids and increase HDL size, leading to anti-inflammatory shifts in the HDL proteome and increased efflux capacity that will suppress immune cell inflammation, as compared to the egg-free and egg white-rich diet. Further, we hypothesize that intake of an egg- free and egg white-rich diet will have no effect on HDL size, lipid composition, proteomes and efflux capacity, leading to greater pro-inflammatory immune responses as compared to whole egg intake. This study will elucidate a novel role for bioactive egg compounds in the regulation of the HDL-immune relationship.
Project Methods
We will address our long-term goal of elucidating the effects of bioactive egg components on the immunomodulatory properties of HDL through the following methods:Study Design: 28 healthy adults (age 18-30y, n = 14 women, n = 14 men) will be recruited to participate in a 16-week randomized crossover study. Upon enrollment, all subjects will enter a 4-week run-in egg-free period where they will refrain from consuming any egg-based foods (Period 1). Subjects will then be randomly assigned to consume 3 whole eggs/day or 3 egg whites/day for 4 weeks (Period 2). Afterwards, subjects will enter a 4-week washout period where egg-based foods are restricted (Period 3), followed by assignment to the alternative whole egg- or egg white-based treatment for 4 weeks (Period 4). Fasting blood samples will be collected at the end of Period 1, 2, and 4 for isolation of plasma, serum, and PBMCs under sterile conditions for analyses pertaining to each objective.OBJECTIVE 1: Characterizing egg effects on HDL size, lipid composition, and proteomes. Serum lipids will be measured in fasted serum samples, thus allowing for associations to be observed between clinical lipid biomarkers and experimental measures of HDL function. HDL subfractions (d = 1.063-1.21) will be isolated from fasted plasma by sequential ultracentrifugation for analysis of 1) HDL particle size by nuclear magnetic resonance spectroscopy, 2) HDL proteomic profiles by mass spectrometry, 3) HDL lipid composition and sphingosine 1-phosphate content by mass spectrometry and biochemical assays.OBJECTIVE 2: Evaluating egg effects on the anti-inflammatory and cholesterol efflux properties of HDL.?To determine the effects of egg intake on HDL efflux capacity, ex vivo cholesterol efflux from THP-1 macrophages and Jurkat T lymphocytes to subject HDL and whole serum will be measured. Inflammatory challenge assay will be performed using THP-1 and Jurkat cells in the presence of subject HDL or whole serum, with or without the presence of inhibitors for HDL transporters. Cells will additionally be collected for whole cell profiling and inflammatory phenotyping by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Given the controversial findings related to egg feeding and trimethylamine-N-oxide (TMAO), plasma levels of choline and TMAO will be measured.OBJECTIVE 3: Characterizing egg effects on the inflammatory potential and cholesterol dynamics of subject-derived primary leukocytes. Complete blood cell counts will be analyzed, allowing for comparisons of our experimental results to routine clinical immune cell biomarkers. mRNA and proteins expression of genes related to HDL metabolism and inflammation will be measured in subject-derived PBMCs. PBMC cholesterol and lipid raft will additionally be quantified, as will the inflammatory potential of PBMCs following an LPS challenge.Together, these measures will elucidate the relationship between egg intake, HDL metabolism, and immune inflammation.

Progress 03/01/18 to 02/28/22

Outputs
Target Audience:Our research efforts have reached various scientific and student groups over the course of this project, and will continue to do so beyond the defined reporting periods. Target audiences have and will include: 1)The broader scientific community, as work stemming from our research efforts have been, and will continue to be, disseminated via journal articles, book chapters, and invited talks by the PD and Co-PD. 2)Communities associated with related scientific and professional societies, including the American Society for Nutrition, the American Nutrition Association, the Federation of American Societies for Experimental Biology, the National Lipid Association, the American Heart Association, and the American Chemical Society. 3) Fairfield University undergraduate students, including the undergraduate research students of the PD and Co-PD who have been directly involved in experimental analysis related to this project, as well as undergraduate students enrolled in the PD's nutrition courses, where this project is highlighted as an example when discussing clinical trials and nutrition research. 4) The greater Fairfield University community, which include faculty across campus, administrators, alumni, students, and students' families who attended the annual Sigma Xi Research Society poster symposium in 2019, 2020, and 2021, where the PD's undergraduate research students presented their research findings. The Co-PD's students presented their work in the Department of Chemistry & Biochemistry's weekly research student seminar. The project was additionally showcased on the weekly News@Fairfield Newsletter and the College of Arts and Sciences Dean's Newsletter. Changes/Problems:The project duration and original timeline was extended due to two unforeseen circumstances: 1) significant renovations to Fairfield University's Bannow Science Center (where PD and Co-PD's research laboratories were housed), and the subsequent move of the Co-PD's lab space, and 2) the COVID-19 pandemic, which resulted in the shutdown and slowdown of research activities. What opportunities for training and professional development has the project provided?This project served as an excellent platform for student training activities and professional development of the PD and Co-PD. Undergraduate research students working with the PD and Co-PD received hands-on training in dietary and physiological assessment, advanced laboratory techniques, and data analysis. Further, students developed skills in scientific writing and data presentation, with opportunities to serve as first, presenting author of numerous posters at international conferences, as well as posters and oral presentations at Fairfield University symposia. Numerous students received competitive scholarships from the Fairfield University Provost's Office to support their conference travel. Overall, the PD has supervised a total of 8 students in research related to this project, and the Co-PD has supervised 6 students - many of whom have since graduated and begun graduate school, medical school, or obtained competitive positions in their fields. This project has additionally facilitated, and will continue to facilitate, professional development of the PD and Co-PD by providing opportunities to establish new research collaborations, develop new research techniques and approaches (particularly in regard to whole-cell immune profiling, HDL compositional and functional analyses, and related data analysis approaches), pursue complementary funding mechanisms to acquire equipment and software for this project, generate preliminary data for subsequent grant proposals, disseminate research findings via publications, presentation at national conferences, and deliver invited talks. Products from this project further supported the PD's successful application for tenure and promotion to Associate Professor at Fairfield University. How have the results been disseminated to communities of interest?The results from this research project have been, and will continue to be, disseminated to target audiences and communities of interest by a variety of mechanisms, including: 1) publication of results in journal articles and a book chapter, 2) presentation at conferences, such as those hosted by the American Society for Nutrition, the American Nutrition Association, the Federation of American Societies for Experimental Biology, the National Lipid Association, the American Heart Association, and the American Chemical Society (a total of 11 abstracts have been presented, with 1 more currently under review), 3) invited talks by the PD and Co-PD at other academic intuitions and scientific organizations, 4) local and University-based events, research symposiums, and communications, and 5) the PD and Co-PD's teaching and research mentorship activities. This project was particularly impactful at Fairfield University, as it was the first dietary and ClinicalTrials.gov-registered human intervention trial to be conducted on Fairfield University campus. Further, given the translational nature of the project, it provided students with opportunities to understand and gain technical skills in the analysis of human lifestyle factors, clinical biomarkers, and advanced biochemical and molecular biology outcome measures. Given that Fairfield University didnot have dedicated nutritional sciences or agriculture-based academic or research programs at the time of this project, this researchprovided students with a unique opportunity to engage with these areas of science and technology. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? To address our long-term goal, a 16-week randomized crossover intervention trial was conducted in which young healthy men and women (18-35y, BMI < 30kg/m2or < 30% body fat for men and < 40% body fat for women, n = 26) consumed an egg-free diet for 4 weeks, followed by a 4-week diet containing either 3 whole eggs or 3 egg whites per day. Participants then followed a 4-week egg-free diet washout period, before switching to the alternative whole egg or egg white diet treatment. Fasted whole blood, serum, and peripheral blood mononuclear cells (PBMCs) were collected to measure outcomes for each of the following objectives: OBJECTIVE 1: Characterization of HDL size, lipid composition, and proteomes. 1) Major activities completed / experiments conducted: Clinical serum lipid profiles were measured by Quest Diagnostics. HDL particles size profiles were measured by nuclear magnetic resonance (NMR). HDL fractions were isolated ultracentrifugation for compositional analysis. Composition of HDL complex lipids were determined by untargeted CSH-ESI QTOF MS/MS. Global HDL proteomes were determined by untargeted UPLC-MS/MS. 2) Data collected: NMR profiling provided data on average HDL particle size, the total concentrations of HDL particles, the concentration of large HDL (9.6-13nm), medium HDL (8.1-9.5nm), small HDL (7.4-8.0nm), and 7 HDL subspecies. Agilent's MassHunter software and the LipidBlast library were utilized to identify HDL lipid species. Total spectral counts were obtained for HDL proteins, then analyzed using a normalized quantitative method based on average precursor intensity using Scaffold software. 3) Summary statistics and discussion of results: No changes in serum lipids were observed between egg diet periods, while the concentration of a large HDL subspecies, H6P (10.8nm range) was increased by whole egg intake. We also observed global and sex-specific shifts in the HDL lipidome, with the greatest changes observed following the whole egg diet in the distribution of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine species. We also observed numerous diet-induced and sex-specific changes in the global HDL proteome, including an overall increase in anti-microbial/immunomodulatory proteins following the whole egg diet period, yet a reduction in the systemic inflammation markers in women. 4) Key outcomes or other accomplishments realized: Despite a lack of changes in clinical serum lipids, and a minimal change in HDL particle size profiles, we observed global changes in the HDL lipidome and proteome following whole egg intake - some of which were sex-specific. Notably, proteomic changes following whole egg intake are indicative of increased pathogendefense. OBJECTIVE 2: Evaluating anti-inflammatory and cholesterol efflux properties of HDL. 1) Major activities completed / experiments conducted: The cholesterol efflux capacity of HDL was determined by measuringex vivocholesterol efflux to apoB-depleted participant serum from macrophages. The anti-inflammatory capacity of HDL was assessed by treatment of immune cells with participant-derived HDL in the presence or absence of LPS. We additionally measured serum paraoxonase 1 (PON1) lactonase activity - a measure of HDL antioxidant activity. 2) Data collected: The cholesterol efflux capacity of HDL was measured utilizing a fluorescence-based assay kit, and reported as % cholesterol efflux from cells to cell supernatant. In experiments determining the anti-inflammatory capacity of HDL, media and cell pellets were collected following incubation with HDL -/+ LPS, and media was assayed for cytokines by ELISA. 3) Summary statistics and discussion of results: We observed thatwhole egg intake increased cholesterol efflux capacity and PON-1 activity; however, these changes were observed in women only.After culturing macrophages with participant-derived HDL in the absence or presence of LPS, we found that HDL - regardless of diet treatment - had a suppressive effect on LPS-induced pro-inflammatory cytokine production. However, cytokine levels were increased following the whole egg diet as compared to the egg white diet. 4) Key outcomes or other accomplishments realized: Interestingly, we observed sex-specific increases in HDL efflux capacity and antioxidant activity following whole egg intake, whereas HDL-mediated suppression of LPS-induced pro-inflammatory cytokine production was reduced following the whole egg diet as compared to the egg white diet - further indicating that whole egg intake may increase inflammatory immune responsiveness to pathogens. OBJECTIVE 3: Characterizing the inflammatory potential and cholesterol dynamics of subject-derived primary leukocytes. 1) Major activities completed / experiments conducted: Clinical complete blood cell counts were measured by Quest Diagnostics. PBMCs were isolated from fasted blood by density-gradient centrifugation. RNA was isolated from fresh PBMCs for gene expression analysis, were as PBMC pellets and live PBMCs were cryopreserved for subsequent proteomic profiling and functional analysis. Expression of genes involved in immune regulation, inflammation, and cholesterol metabolism were determined by quantitative real-time PCR. Proteomic profiling of PBMCs was determined by MALDI-TOF MS. Inflammatory capacity of PBMCs was determined viaex vivoassays andtreatment with LPS. 2) Data collected: From clinical complete blood cell count reports, we obtained data on total white blood cells, in addition to absolute counts and % distribution of leukocyte subsets. Expression of genes involved in cholesterol metabolism (e.g., ABCA1, ABCG1, SCARBI, LDLR, HMGCR) and ~100 gene markers of distinct immune cell subsets and pathways were analyzed. Proteomic profiling of PBMCs by MALDI-TOF MS yielded global spectra of cell pellets relative to internal standards, which were subsequently analyzed by Shimadzu's eMSTAT software. In experiments determining the inflammatory capacity of PBMCs, media and cell pellets were collected following incubation with or without LPS, and media was assayed for cytokines by ELISA. Statistical analysis to evaluate correlations between HDL composition and measures of immune function/profiles was additionally performed. 3) Summary statistics and discussion of results: We did not observe significant changes in total white blood cell counts between diet periods, nor did we observe changes in absolute cell counts in leukocyte subsets. Similarly, no changes in pro-inflammatory serum GlycA were observed between diet periods. However, egg diets differentially altered several red blood cell parameters, which correlated with changes in serum lipids following the whole egg period. While we did not observe changes in cholesterol gene expression, whole egg consumption modulated the expression of gene markers of adaptive and innate immune responses.Proteomic profiling revealed unique clustering of spectral patterns in PBMCs following the whole egg diet, as compared to the egg-free and egg white diet periods.In the absence of LPS, pro-inflammatory cytokine production by PBMCs was reduced following the egg white diet, whereas a trend toward increased LPS-induced, pro-inflammatory cytokine production by PBMCs was observed following the whole egg diet.We found extensive and differential correlations between changes in HDL parameters vs. clinical and experimental measures of immune profiles between the different egg diet periods. 4) Key outcomes or other accomplishments realized: Whole egg intake induced transcriptomic and proteomic shifts in PBMCs, and trended toward increasingLPS-induced pro-inflammatory cytokine production. In line with the findings from our previous objectives, and the extensive correlations observed between HDL and immune parameters, we conclude that egg intake differentially modulates the HDL-immune relationship in healthy young adults, and that whole egg intake may promote immune-mediated pathogen defense.

Publications

  • Type: Conference Papers and Presentations Status: Under Review Year Published: 2022 Citation: Huang L, Garcia C, Corvino O, Woo H, Jang H, Zhang R, Woodruff R, Blesso C, Lee J-Y, Esposito CP, and Andersen CJ. Egg consumption induces sex-specific changes in HDL composition and function. American Heart Association Scientific Sessions 2022, November 2022 (In review).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Woodruff R, Jiang J, Esposito CP, and Andersen CJ. Egg consumption modulates expression of genes involved in T cell activation and differentiation that correlate with HDL profiles. Nutrition 2022, June 2022, Online.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Zhang R, Esposito CP, Greco JM, and Andersen CJ. Consumption of different egg-based diets minimally, yet differentially, impacts markers insulin resistance in young healthy adults. Nutrition 2022, June 2022, Online.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Greco J, Mis D, Esposito CP, and Andersen CJ. Consumption of different egg-based diets induce changes in HDL profiles that are associated with shifts in inflammatory immune markers. Federation of American Societies For Experimental Biology, The Nutrition, Immunity, and Inflammation Conference: From Model Systems to Human Trials. July 2021, Online.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Greco J, Mis D, Esposito CP, and Andersen CJ. Diet-induced shifts in large HDL concentrations are inversely correlated with immune markers. American Heart Association, Vascular Discovery: From Genes to Medicine 2021 Scientific Sessions. September 2021, Online.
  • Type: Journal Articles Status: Other Year Published: 2022 Citation: Andersen CJ, Greco JM, Esposito CP, Campbell C, Sloan A, Mis D, and VanDyke A. Changes in serum lipids from egg consumption are associated with clinical erythrocyte and platelet indices. (In Preparation for submission to Nutrients, 2022).
  • Type: Journal Articles Status: Other Year Published: 2022 Citation: Andersen CJ, Van Dyke AR, Woodruff R, Jiang J, Huang L, Jang H, Woo H, Corvino O, Lee, J-Y, and Esposito CP. Daily intake of egg whites vs. whole egg intake differentially alters immune profiles. (In Preparation for submission to the Journal of Nutritional Biochemistry, 2022).
  • Type: Journal Articles Status: Other Year Published: 2022 Citation: Andersen CJ, Huang L, Garcia C, Woodruff R, Corvino O, Woo H, Jang H, Zhang R, Blesso C, Lee, J-Y, and Esposito CP. Egg consumption modulates HDL composition and function in healthy young adults. (In preparation for submission to the Journal of Lipid Research, 2022).


Progress 03/01/20 to 02/28/21

Outputs
Target Audience:Target audiences of this project during this reporting period include: The American Society for Nutrition (ASN) scientific community, as data from this project were presented as a virtual poster at the Nutrition 2020 online conference. The American Nutrition Association (ANA) scientific community, as data from this project were presented as a poster at the online Personalized Nutrition 2020 conference. The National Lipid Association (NLA) scientific community, as data from this project were presented by PD Catherine Andersen via a live poster at the online National Lipid Association Scientific Sessions 2020 conference. The Fairfield University community, including the undergraduate research students of the PD and Co-PD who have been directly involved in experimental analysis related to this project, as well as undergraduate students enrolled in the PD's nutrition courses, where this project is highlighted as an example when discussing clinical trials and nutrition research. Target audiences additionally include faculty across campus, administrators, alumni, students, and students' families who attended the annual Sigma Xi Research Society poster symposium (held virtually in 2020), where the PD's undergraduate research students presented their ongoing findings. Changes/Problems:A one-year, no-cost extension was requested due to the COVID-19 pandemic, which caused delays in the project timeline. What opportunities for training and professional development has the project provided?This project continues to provide training and professional development opportunities for the PD, Co-PD, and undergraduate students involved in study analyses. Professional development opportunities include the ongoing development of new laboratory techniques/protocols, interdisciplinary collaborations, and dissemination and networking activities at national conferences. Products from this project further supported the PD's successful application for tenure and promotion to Associate Professor at Fairfield University. Undergraduate research students working with the PD and Co-PD have additionally received hands-on training in laboratory techniques, as well as data analysis and presentation, which has supported their successful applications to graduate programs. How have the results been disseminated to communities of interest?Results of this project were disseminated via virtual posters presentations at the American Society for Nutrition (ASN) Nutrition 2020 conference, American Nutrition Association (ANA) Personalized Nutrition 2020 conference, and National Lipid Association Scientific Sessions 2020 conference. Undergraduate students involved in this project additionally presented their work at the annual Fairfield University Sigma Xi Research Society Poster Symposium. What do you plan to do during the next reporting period to accomplish the goals?The COVID-19 pandemic significantly impacted the progress of this project - particularly experiments involving cell culture and proteomics. It is now expected that remaining analyses related to each project objective will continue as planned during the next reporting period in order to reach project goals.

Impacts
What was accomplished under these goals? Analysis related to project goals and objectives continued, despite significant obstacles and delays posed by the COVID-19 pandemic. Completed analyses and primary findings from this reporting period are as follows: Dietary intake and body composition analysis: Analysis of5-day diet records was completed to evaluate the impact of the egg-free, egg white, and whole egg diets on overall nutrient intake during treatment periods. Total, monounsaturated, and polyunsaturated fatintake was increased during the whole egg period, whereas animal protein intake was increased in both whole egg and egg white diet periods as compared to the egg-free diet period. Numerous dietary nutrients/components found in egg yolks were additionally increased during the whole egg period, including cholesterol, choline, vitamin D, selenium, and the carotenoids lutein + zeaxanthin. Body weight and composition data collected at the end of each dietary period were additionally analyzed. Body weight increased during the egg white period as compared to the whole egg period, whereas body fat mass increased during the whole egg period. No significant differences in body composition were observed between the whole egg vs. egg white period. Serum markers of choline status/metabolism and inflammation: In line with increased dietary choline intake during the whole egg period, serum choline and betaine were increased by whole egg intake compared to the other diet periods. Importantly, there were no differences in pro-atherogenic/inflammatory trimethylamine-N-oxide (TMAO) between diet periods. Similarly, no changes in pro-inflammatory serum GlycA were observed between diet periods. Lipoprotein particle profiles: HDL particle subclass/size profiles were analyzed, as were profiles for triglyceride-rich lipoproteins (TRL) and LDL. No changes were observed in the concentration of total HDL particles, or average HDL particle diameter. While no changes in the concentration of small (7.4-8.0 nm) or medium (8.1-9.5 nm) HDL particles was observed, the concentration of large HDL particles (10.8nm range) was increased by whole egg intake as compared to intake of egg whites. Weadditionally observed a trend toward increased apolipoprotein A1 (apoA1) during the whole egg diet period as compared to the egg-free diet period. In line with our hypothesis, changes in large HDL concentrations between whole egg vs. egg white periods were inversely correlated with total white blood cell counts and absolute lymphocyte counts. No significant changes in TRL, LDL, and apolipoprotein B (apoB) profiles or lipoprotein-insulin resistance indices were observed. Analysis of additional HDL parameters related to this project are ongoing. Inflammatory/immune gene expression: Gene expression arrays were performed on a subset of participant-derived peripheral blood mononuclear cell (PBMC) samples to determine whether the different egg-based diets impacted expression of genes involved in immune responses/inflammation. Numerous pro-inflammatory genes were found to be decreased in expression following the whole egg diet period. Changes in mRNA expression of CD8A between whole egg and egg white diet periods positively correlated with changes in total white blood cell and absolute lymphocyte counts, whereas CD8A expression was inversely correlated with concentrations of total and large HDL particles. Analysis of additional PBMC/immune cell line parameters related to this project are ongoing.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Andersen CJ, Greco J, Campbell C, Sloan A, Mis D, and Esposito CP. Egg consumption induces changes in clinical leukocyte markers that are dependent on sex and baseline white blood cell counts. National Lipid Association Scientific Sessions, December 2020, Online.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Andersen CJ, Greco J, Campbell C, Sloan A, Mis D, and Esposito CP. Egg consumption induces changes in clinical leukocyte markers that are dependent on sex and baseline white blood cell counts. American Nutrition Association meeting on Personalized Nutrition, November 2020, Online.


Progress 03/01/19 to 02/29/20

Outputs
Target Audience:Target audiences of this project during this reporting period include: 1)The American Society for Nutrition (ASN) scientific community, as Project Director (PD) Catherine Andersen delivered an oral presentation of preliminary results from this study at Nutrition 2019 in Baltimore, MD, and her undergraduate research student (Julia Greco) presented a poster. Ongoing research findings from this project was additionally submitted for presentation at Nutrition 2020. 2)The broader scientific community, as the PD was invited to be a Guest Editor for a Special Issue of Nutrients (Impact Factor 4.171) on this project's topic, entitled "Lipid Metabolism in Inflammation and Immune Function". The PD and Co-PD Aaron Van Dyke additionally published a book chapter on the project topic of eggs and inflammation. 3)The Fairfield University community, including faculty, administrators, alumni, and the general student body. This study has received notable attention on campus, as it is the first ClinicalTrials.gov-registered intervention trialto be conducted on Fairfield University campus. The project has been showcased on the weekly News@Fairfield Newsletter and the College of Arts and Sciences Dean's Newsletter. The PD has additionally used this project as an example during formal classroom instruction of undergraduate courses when covering topics related to human nutrition, lipid metabolism, nutritional immunology, diet quality, research design, and grant writing. Target audiences also included undergraduate research students of the PD and Co-PD who have been directly involved in experimental analysis related to this project and have received hands-on, laboratory-based training. Ongoing findings from this project were additionally disseminated via poster presentation by the PD's and Co-PD's undergraduate research students at the annual Sigma Xi Research Society poster symposium on Fairfield University campus. During the previous reporting period (2018-2019), target audiences additionally included the American Chemical Society scientific community, as Co-PD Aaron Van Dyke and his undergraduate research students presented their work related to optimization of whole-cell fingerprinting and lipid quantification of immune cells. Changes/Problems:A one-year, no-cost extension was requested due to significant renovations to Fairfield University's Bannow Science Center (where PD and Co-PD's research laboratorties are housed) that took place thispast year, which caused delays in the original project timeline. What opportunities for training and professional development has the project provided?This project has facilitated professional development of the PD and Co-PD by providing opportunities to establish new collaborations, pursue complementary funding mechanisms to acquire equipment and software for this project, and disseminate research findings at national conferences. Undergraduate research students working with the PD and Co-PD have additionally received hands-on training in laboratory techniques, as well as data analysis and presentation. To date, 8 undergraduate students have been trained in methods related to this project. How have the results been disseminated to communities of interest?Results of this project were presented in an oral and poster presentation at Nutrition 2019, and have been accepted for poster presentation Nutrition 2020. Undergraduate students involved in this project additionally presented their work at the annual Sigma Xi Research Society Poster Symposium on Fairfield University campus. What do you plan to do during the next reporting period to accomplish the goals?Analyses related to each project objective will continue as planned during the next reporting period in order to reach project goals.With the acquisition of Shimadzu eMSTAT statistical software, analysis capabilities of leukocyte whole cell profiles and inflammatory phenotyping by MALDI-TOF MS will be strengthened. Protocols will additionally be finalized for imaging PBMC lipid rafts using the newly-acquired Keyence BZ-X800 fluorescent microscope microscope. Given our findings that sex and white blood cell counts may influence responses to egg whites and while eggs, we will take these factors into consideration for future analyses.

Impacts
What was accomplished under these goals? This project aims to elucidate the effects of egg intake on HDL characteristics and immune function in healthy men and women. Findings from this study will contribute to scientific knowledge regarding the bioactive properties of eggs, and may help to inform dietary recommendations. In evaluating clinical biomarkers, we observed that serum total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides were not changed between the egg-free, egg white, or whole egg diet periods. The total cholesterol:HDL-cholesterol ratio was also similar between diet periods, and no differences in serum lipids in response to the egg diets were observed between male and female subjects. In regards to clinical leukocyte markers, we did not observe significant changes in total white blood cell counts between the egg-free, egg white, or whole egg diet periods, nor did we observe changes in absolute cell counts in leukocyte subsets (neutrophils, lymphocytes, monocytes, eosinophils, and basophils). In contrast to our preliminary observations, changes in serum lipid levels did not significantly correlate with changes in absolute white blood cell or leukocyte subset counts. However, when we divided male and female subjects, we observed sex-dependent effects of egg diets on leukocyte counts. Compared to female subjects, the percent change in absolute white blood cell and monocyte counts were significantly reduced by the egg white diet. Conversely, there was a trend toward greater increases in monocyte counts in women as compared to men following the whole egg diet. Interestingly, we further observed that individuals who had higher white blood cell counts after the initial egg-free diet displayed greater increases in %monocytes following both the egg-white and whole egg diet, whereas increases in %basophils were only observed following the egg white diet period. Given these observations, and the role of HDL in regulating erythropoiesis and platelet activity that has been reported by others, we evaluated whether there were associations between clinical lipid and erythrocyte and platelet markers. Interestingly, hematocrit level significantly increased following the whole egg diet as compared to the egg-free diet, whereas a trend toward increased red blood cell counts was additionally observed. Conversely, mean corpuscular volume was significantly increased by the egg white diet as compared to the egg-free diet, whereas platelet counts were significantly lowered by both egg white and whole egg diets. Interestingly, changes in total cholesterol positively correlated with changes in red blood cell counts, hematocrit, and hemoglobin following the egg white and whole egg period (with stronger associations observed with egg whites), whereas changes in HDL-cholesterol only correlated with changes in clinical erythrocyte markers during the egg white period. Together, these findings suggest that the effects of whole egg vs. egg white intake on clinical leukocyte markers may vary by sex and baseline white blood cell counts. Further, it appears that egg intake differentially impacts clinical erythrocyte and platelet markers, with stronger correlations with serum lipids observed following egg white consumption. Continued analysis of our functional assays will provide further insight into the mechanisms underlying the changes in these clinical profiles.

Publications

  • Type: Book Chapters Status: Published Year Published: 2019 Citation: Andersen C.J. and Van Dyke A. R. (2019). Modulation of inflammation by egg components, In J. Wu (Ed), Eggs as Functional Foods and Nutraceuticals for Human Health, Food Chemistry, Function and Analysis. Cambridge, UK: The Royal Society of Chemistry.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Lyons T, Hong E, Gaito A, Cintron-Rivera L, Dupree L, Andersen CJ, and Van Dyke AR. Molecular fingerprinting of K562 human leukemia cells via MALDI-TOF mass spectrometry. American Chemical Society National Meeting 2018.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Gilbertson J, Hong E, Gaito A, Cintron-Rivera L, Dupree L, Andersen CJ, and Van Dyke A. Lipid profiling in K562 human leukemia cells via gas chromatography-mass spectrometry. American Chemical Society National Meeting 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Greco J, Sloan A, Esposito CP, Van Dyke A, and Andersen CJ. Changes in serum lipids from egg consumption are associated with clinical erythrocyte indices. Nutrition 2019, Baltimore, MD. Current Developments in Nutrition, 2019, 3(Supplement 1): P08-093-19.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Andersen CJ, Esposito CP, Greco J, Sloan A, and Van Dyke A. Egg-induced changes in serum lipids are associated with clinical immune cell counts. Nutrition 2019, Baltimore, MD. Current Developments in Nutrition, 2019, 3(Supplement 1): OR12-04-19.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Greco J, Campbell C, Sloan A, Mis D, Esposito CP, and Andersen CJ. Consumption of different egg-based diets impacts body composition in young healthy adults. Nutrition 2020, Online.


Progress 03/01/18 to 02/28/19

Outputs
Target Audience:Target audiences ofthis project during this reporting period include: 1) USDA NIFA awardees and staff, as Catherine Andersen participated in the 2018 Project Directors meeting and presented an overview of the ongoing project. 2) The scientific community, as Catherine Andersen was invited to write a published review article inNutrients on the project topic, and preliminary data has been submittedfor presentation at the American Society for Nutrition 2019 meeting. 3) Project collaboratorsand undergraduate research students who have contributed to project analyses. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has providedthe Project Director and Co-Project Director with opportunities for professional scientific development in regard to establishing new experimental protocols, advancing skills and knowledge in the area of lipid metabolism and immune function, and the generation of preliminary data to present at the upcoming American Society for Nutrition 2019 meeting. This project has additionally provided undergraduate research students with training opportunities through participation in study analyses. How have the results been disseminated to communities of interest?Catherine Andersen participated in the 2018 NIFA Project Director's meeting to share an overview of the project, and preliminary data stemming from this project has been submittedfor presentationat theAmerican Society for Nutrition 2019 meeting. What do you plan to do during the next reporting period to accomplish the goals?Analyses related to each project objective will continue as planned during the next reporting period in order to reach project goals.

Impacts
What was accomplished under these goals? This project aims to elucidate the effects of egg intake on HDL characteristics and immune function in a healthy population. Findings from this study will contribute to scientific knowledge regarding the bioactive properties of eggs, and may help to inform dietary recommendations. The dietary intervention phase of the project was conducted during this reporting period, allowing for collection of all subject-derived samples needed for analyses related to Objective 1-3. OBJECTIVE 1: Characterization of HDL size, lipid composition, and proteomes. We will test the hypothesis that yolk-derived components provided by whole egg promotes anti- inflammatory and efflux-promoting shifts in HDL compositional profilesas compared to an egg-free and egg white-rich diet.Subject-derived serum samples have been collected and stored for isolation of HDL fractions that will be analyzed for particle size, lipid species composition, and global proteomes. Preliminary data demonstrates modest differential shifts in serum lipids following egg-free, whole egg, and egg white diets.Analyses related to this objective are ongoing. OBJECTIVE 2: Evaluating anti-inflammatory and cholesterol efflux properties of HDL. We will test the hypothesis that yolk-derived components provided by whole egg will increase the anti-inflammatory and efflux functionalityof HDL as compared to egg-free and egg white diets, which will induce relatively greater immune inflammation by not altering HDL efflux capacity.Subject-derived serum samples have been collected and stored for HDL isolation and cell culture analyses. Methods for proteomic profiling of established cell lines have been developed, as have protocols for cell culture assays utilizing selective inhibitors of cholesterol efflux. Analyses related to this objective are ongoing. OBJECTIVE 3: Characterizing the inflammatory potential and cholesterol dynamics of subject-derived primary leukocytes. We will test the hypothesis that yolk-derived components provided by whole egg will reduce peripheral blood mononuclear cell (PBMC) inflammation as compared to egg-free and egg white-rich diets, and that these changes will be associated with reduced total cellular cholesterol and lipid rafts content.Subject-derived PBMCs have been isolated and cryopreserved forex vivostimulation assays and lipidanalyses. PBMCs aliquots have additionally been stored for whole-cell proteomic profiling, for which the protocolhas been optimized. RNA has been extracted from fresh PBMC for real-time qPCR analysis of genes related to inflammation and cholesterol metabolism. Clinical complete blood cell counts have been measured, and preliminary data indicates that whole egg and egg white intake differentially impacts clinical leukocyte and red blood cell profiles, and that these changes correlate with changes in serum lipids.Analyses related to this objective are ongoing.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Andersen CJ. Impact of Dietary Cholesterol on the Pathophysiology of Infectious and Autoimmune Disease. Nutrients, 2018; 10, 764.