Research Project: Immunity, Inflammation, and Nutrition in Aging

Location: Jean Mayer Human Nutrition Research Center On Aging

2016 Annual Report

Objectives
LAB NAME: Nutritional Immunology 1: Determine the impact and underlying mechanisms of obesity during pregnancy on newborn's life-long immune and inflammatory responses and resistance to infectious diseases in appropriate animal models. 1A. To investigate the impact of maternal obesity on immune function and influenza infection in young offspring. 1B. To determine the role of inflammatory, oxidative stress, and selected epigenetic markers that are critical for immune response to influenza and the higher susceptibility of obese mothers’ offspring to influenza infection. 1C. To determine the impact of maternal obesity on immune function and resistance to infection through the life cycle. 2: Determine the effect and mechanisms of food components such as fruits and vegetables or whole grains influence on, and their interaction with, age on immune and inflammatory responses and related diseases in both animal models and human studies. 2A: To determine the impact of increased consumption of F&V on prevention of key age-related biologic dysfunctions and pathologies in a suitable animal model. 2B: To determine the effect of consumption of an isocaloric diet rich in whole grains (WG)[compared to refined grains (RG)] on gut microbiota, and immune and inflammatory responses. LAB NAME: Vascular Biology 1: Determine the effectiveness and underlying mechanisms of fruits/vegetables and Mediterranean style diets or their specific bioactive components in the prevention of atherosclerosis and survival using animal models of humans. 1A: Determine the effectiveness and underlying mechanisms of increased consumption F&V diets on prevention of atherosclerosis and survival using appropriate animal model of human atherosclerosis. 1B: Determine the effectiveness of natural bioactive polyphenols of foods and spices and their underlying mechanisms on prevention of atherosclerosis using appropriate animal models of human atherosclerosis. 2: Determine the efficacy of fruits/vegetables, avenanthramides from oats, and selected other bioactive compounds on alteration of microbiome and anti-microbial peptides, suppression of chronic inflammation of the GI tract, prevention of colon cancer and atherosclerosis in appropriate animal models. Under this objective, we will pursue the following sub-objectives: 2A: Determine the efficacy of bioactive phosphorylated tocopherols ('Tp) versus tocopherols on age-dependent decline of angiogenesis and suppression of atherosclerosis in vivo and ex-vivo. 2B: To determine the molecular signaling mechanisms by which 'TP induces angiogenesis. 3: Determine the effect of eight weeks consumption of an isocaloric diet enriched with whole grains (compared to refined grains) on gut microbiota, inflammatory and other risk predictors of atherosclerosis.

Approach
NUTRITIONAL IMMUNOLOGY: Aging is associated with dysregulation of immune and inflammatory responses, which contribute to several age-related diseases. Nutritional status including that during early life has been linked to health status of older adults, & nutritional interventions during different life stages including those during pregnancy are reported to have long-lasting effects on the health status of newborns. Long-term goal is to determine the underlying mechanisms of age-related immune & inflammatory dysregulation in order to develop nutritional interventions, which will prevent/reduce these alterations. [1] will determine the impact & underlying mechanisms of obesity during pregnancy on newborn's life-long immune & inflammatory responses & resistance to infectious diseases. To achieve this, we will use a diet-induced obesity mouse model and determine: 1) effect of maternal obesity on immune function and influenza infection in young offspring, 2) role of inflammatory, oxidative stress, and selected epigenetic markers in altered immune function and susceptibility to infection in offspring born to obese mothers, and 3) impact of maternal obesity on immune function and resistance to infection through the life cycle. [2] will determine the effect and mechanisms of food components such as fruits and vegetables or whole grains influence on, and their interaction with, age on immune and inflammatory responses and related diseases in both animal models and human studies. [2A], we will use a mouse model to determine whether the dietary supplementation with a variety of fruits and vegetables would impact health- and life-span through modulating oxidative stress, inflammation and the regulatory lipid, ceramide. [2B], we will conduct a dietary intervention trial in healthy middle-aged human participants to determine the effect of consumption of an isocaloric diet rich in whole grains compared to refined grains on gut microbiota, and immune and inflammatory responses. These studies will help develop effective strategies to improve health- and life-span of the aged through optimization of immune and inflammatory responses. VASCULAR BIOLOGY: Determine survival rates, prevention of atherosclerosis, and underlying mechanism(s) in LDLr-/- young to old mice using freeze-dried fruits, vegetables and cereal grains in a high fat diet. Further, we will use C57/BL and LDLr-/- mice to determine the causal relationship & underlying mechanism(s) of specific bioactive food components (such as curcumin, resveratrol, avenanthramides, and their combinations) on inhibition of dyslipidemia, fatty liver, inflammation, metabolic disorders, & atherosclerosis resulting from consuming a diet high in fat and/or sweeteners like sucrose & high fructose corn syrup. We plan to establish the causal relationship of increased consumption of whole grains versus refined grains and their capacity to improve oxidative & antioxidant status as well as surrogate markers of cardiovascular disease. We will investigate potential molecular mechanisms by which alpha-Tocopheryl phosphate (alpha-TP), a natural derivative of alpha-T, induces angiogenesis in vitro & in an in vivo animal model.

Progress Report
NUTRITIONAL IMMUNOLOGY LAB: As the obesity rate continues to increase, the negative impact of maternal obesity (MO) on offspring is becoming a concern. While studies have linked MO to certain immune disorders in offspring, it is not known how maternal obesity impacts the offspring resistance to infection. We conducted a study to address this gap. We initially used high fat-fed female C57BL/6 mice as dam, but significant cannibalism by the high fat fed dams prevented us from continuing the study. Thus, we switched to CD-1 mice. We successfully induced obesity by feeding high fat diet, generated offspring, and used them to determine the impact of maternal obesity on vaccine efficacy against influenza infection. Studies have suggested that obesity may be associated with impaired immune function as well as reduction in resistance to infection. The most commonly utilized animal model for diet-induced obesity involves feeding mice a high fat (HF) diet. Since HF diets are also known to affect immune cell function, it is difficult to determine whether, and to what extent, the reported findings are a result of the influence of obesity on immune function and resistance to infection or a direct result of the high fat content of the diet used. To address this, female CD-1 mice (1-mo) were weight-matched and fed either a HF diet or low fat (LF) control diet. After 5-mo feeding mice showed considerable variation in HF-induced weight gain and we thus further divided the HF fed mice into two sub-groups: HF lean (HFL, <40 g) and HF obese (HFO, >40 g). Mice were immunized with an influenza vaccine and boosted 3-wk later. They were then exposed to influenza virus. Vaccinated mice had less weight loss than unvaccinated mice after infection. Among vaccinated mice, LF and HFL mice largely maintained their weight while HFO mice demonstrated significant weight loss following exposure to the virus. HFO mice had lower lymphocyte proliferation and a strong trend toward higher interferon-gamma production compared to HFL mice. Our results suggest that obesity, rather than the high fat diet, is responsible for the diminished vaccine efficacy and altered immune responses. Observational studies suggest an association between consumption of whole grains (WG) and a decrease in inflammatory markers. However, results from limited interventional studies are controversial. We have completed a study to determine the effect of consuming high WG vs. refined grain (RG) diet in context of a weight maintenance diet for 8-weeks on immune and inflammatory responses, and gut microbiota in healthy adults (40-65 years). Results showed that compared to RG, WG group had increased plasma levels of alkylresorcinols (WG biomarkers), stool weight and frequency, short chain fatty acid (SCFA) producer Lachnospira, stool acetate and total SCFA, and increased, higher % total effector memory T cells and LPS-stimulated ex vivo production of TNF-a. On the other hand WG had decreased level of the pro-inflammatory Enterobacteriaceae. Our findings suggest that short-term consumption of WG in context of a weight maintenance diet has modest positive effects on gut microbiota, SCFA, and certain measure of the immune response. Aging is associated with dysregulation of immune and inflammatory responses, which contributes to increased morbidity and mortality in elderly. Also, obesity is one of the predisposing factors for inflammation and development of diseases. We conducted a study to determine the effect of replacing oils used in a typical American diet with extra virgin olive oil for 3 months on immune responses and cardio-metabolic risk factors in overweight and obese older adults. We found that consuming extra virgin olive oil reduced systolic blood pressure, increased HDL-cholesterol concentration (good lipoprotein) and enhanced certain functions of immune system. Our results indicate that substitution of oils used in a typical American diet with extra virgin olive oil in overweight and obese older adults may have cardio-metabolic and immunological health benefits. Although commensal bacteria are crucial in maintaining immune homeostasis of the intestine, little is known about commensal bacteria’s role in shaping the immune inductive function in a non-intestinal mucosal surface such as the lung. To address the gap in understanding the pulmonary microbiome as it relates to aging, we conducted a study to systematically look at the pulmonary microbiome of healthy older individuals through analysis of high-throughput 16SrDNA sequence data from broncho-alveolar lavage. In this prospective cohort study, we plan to enroll 15 young (21-30 years) and 15 older (65 years and older) participants with good physical performance. We have currently enrolled 6 young and 8 older participants; blood samples for certain immunological outcome measures have been analyzed. VASCULAR BIOLOGY LAB: Fruits and Vegetables Project: Cardiovascular disease (CVD) and associated chronic inflammation is the major cause of morbidity and mortality in the United States, and globally. Several epidemiological studies and meta-analyses have suggested that high intake of fruits and vegetables (F&V) or adopting a Mediterranean style diet rich in F&V is associated with reduced risk of obesity, diabetes, coronary heart disease (CHD), stroke, cancer and may contribute to longevity. The benefit of F&V and the Mediterranean style diet for prevention of these conditions has been documented most often as an “association in observational studies” and there is a dearth of data to demonstrate the “causal relationships.” Limited number of studies have investigated the impact of high intake of F&V on clinical end point of CVD and reported mixed results. According to current comprehensive reviews, there is a belief that high F&V consumption would prevent development of hypertension, CHD, stroke, and diabetes. However, the causality of F&V on clinical or intermediate endpoints of CVD such as atherosclerosis in humans is yet to be demonstrated. Thus, there is a need for prospective long-term studies to determine the impact of F&V on CVD. Because investigating the effect of long-term consumption of F&V on prevention of atherosclerosis in humans is not practical, to test the efficacy of F&V on prevention of heart disease we selected Ldlr-/- mice which are prone to atherosclerosis, displays aortic inflammation and lesions similar to humans. First we designed a pilot study to determine the most effective level of F&V to be used in the long-term study. Mice were fed either a control diet or a diet supplemented with the different levels of a freeze-dried mixture of 12 fruits and 12 vegetables that are most commonly used in the U.S. according to USDA database. We are monitoring the impact of the diets on food intake of mice, body weight gain, and body fat using magnetic resonance imaging (MRI) as well as on risk factors for atherosclerosis. The study is on going. However, thus far, we have noted that Ldlr-/- mice consume the synthetically prepared diet supplemented with up to 10% of F&V mixture. From this ongoing pilot study we have also learned that preparation of the F&V mixture for the larger study will be logistically very diffictult, given the quantities needed. Therefore, we have identified a vendor (Vandrunen Farm) to prepare it for us for the future studies. Upon completion of the pilot and once we obtain the mixture of F&V (there has been a delay in obtaining the mixture), we will determine the impact of increasing F&V consumption started from early life, compared to those started at mid- and late-life.

Accomplishments
1. NUTRITIONAL IMMUNOLOGY LAB: Long-term moderate calorie restriction inhibits inflammation without impairing immunity in non-obese humans. Calorie restriction (CR) inhibits inflammation and slows aging in many animal species, but at the same time, CR impairs immunity against certain pathogens in the experimental rodents. How CR impacts the immune response in humans in this regard is currently unknown and was the goal of our research. In a collaborative multi-center, randomized clinical trial, the ARS-funded researchers at the Jean Mayer USDA-Human Nutrition Research Center on Aging (HNRCA) at Tufts University in Boston, Massachusetts determined long-term (2 year) CR’s effect on inflammation and cell-mediated immunity in 218 healthy non-obese men and women (20-50 years). They found that CR induced a 10.4% weight loss over the 2-year period. Relative to the diet group that ate freely, CR reduced circulating inflammatory markers, including total numbers of white blood cells and lymphocytes (white cells fundamental to the immune system such a B cells and T cells), and several soluble substances known to be involved in chronic inflammation. CR had no effect on the two key indices of immune function closely relevant to the body’s defense against harmful micro-organisms: delayed-type hypersensitivity skin response (a test to determine T cell functions) and antibody titer levels (a test to determine both T and B cell functions) in response to vaccines, nor did it increase the risk of infection. These results represent the first evidence showing that long-term moderate CR without malnutrition in humans can suppress inflammation without compromising the vigor of immunity. Given the established role of inflammation in the development of multiple chronic diseases, CR may represent a promising strategy for improving health and alleviating age-related diseases.

2. NUTRITIONAL IMMUNOLOGY LAB: Zinc supplementation of older adults with low serum zinc level improves zinc status and T cell function. Zinc is essential for the regulation of immune response, and zinc supplementation has the potential to improve serum zinc concentrations and improve impaired immune function in the elderly. Tufts University researchers at USDA Boston, Massachusetts conducted a study to determine the effect of supplementation with 30 mg zinc/day for 3 months on serum zinc concentrations and immune function in zinc-deficient nursing home elderly. We found that zinc supplementation increased serum zinc levels, increased the number of peripheral T cells, and enhanced T-cell proliferation. These results indicate that zinc supplementation at 30 mg/day for 3 months is effective in increasing serum zinc concentrations in nursing home elderly, and this increase serum zinc level was associated with enhancement of T cell function mainly due to increase in number of T cells. Thus, older subjects with low serum zinc levels should consider increasing their zinc intake to promote their health.

3. VASCULAR BIOLOGY LAB: Short-term consumption of whole grains does not affect markers of cardiovascular disease. Several studies report an association between a diet rich in whole grains (WG) and reduced risk of cardiovascular disease (CVD). WG have been shown to slow down the absorption of carbohydrates and lower LDL (bad) cholesterol, total cholesterol, as well as blood pressure. The majority of these studies did not control the weight of the subjects and none controlled the other components of the diet. The current study investigated the effects of a diet rich in WG compared to refined grain (RG), independent of weight loss and other dietary components, on cardiovascular risk factors (inflammatory and oxidative stress markers, plasma lipids, and blood pressure) in healthy older adults. For a total of 6 weeks, 81 men and post-menopausal women were randomly assigned to either a WG or RG diet and CVD risk factors were assessed before and at the end of the dietary intervention period. In summary, while WG tended to reduce oxidative stress, the consumption of WG compared to RG independent of weight loss had no effect on blood pressure, or plasma lipids and inflammation markers. These results suggests that the reported beneficial effect of whole grains might be mainly due to its associated weight loss.

NUTRITIONAL IMMUNOLOGY LAB: None. VASCULAR BIOLOGY LAB: None.

Review Publications
Barnett, J., Dao, M.C., Hamer, D.H., Kandel, R., Brandeis, G., Wu, D., Dallal, G.E., Jacques, P.F., Schreiber, R., Kong, E., Meydani, S. 2016. Effect of zinc supplementation on serum zinc concentration and T cell proliferation in nursing home elderly:A randomized double-blind placebo-controlled trial. American Journal of Clinical Nutrition. 103(3):942-951. doi: 10.3945/ajcn.115.115188.
Azzi, A., Meydani, S., Meydani, M., Zingg, J.A. 2016. The rise, the fall and the renaissance of vitamin E. Archives Of Biochemistry and Biophysics. 595:100-108. doi: 10.1016/j.abb.2015.11.010.
Meydani, S.N., Das, S., Pieper, C.F., Lewis, M.R., Klein, S., Dixit, V.D., Gupta, A.K., Villareal, D.T., Bhapkar, M., Huang, M., Fuss, P.J., Roberts, S.B., Holloszy, J.O., Fontana, L. 2016. Long-term moderate calorie restriction inhibits inflammation without impairing cell-mediated immunity: a randomized controlled trial in non obese humans. Aging. 8(7). doi:10.18632/aging.100994.
Fontana, L., Villareal, D.T., Das, S., Smith, S.R., Meydani, S.N., Pittas, A.G., Klein, S., Bhapkar, M., Rochon, J., Ravussin, E., Holloszy, J.O. 2016. Effects of 2-year calorie restriction on circulating levels of IGF-1, IGF-binding proteins and cortisol in non-obese men and women: a randomized clinical trial. Aging Cell. 15:22-27.
Rozati, M., Barnett, J., Wu, D., Handelman, G., Saltzman, E., Wilson, T., Li, L., Wang, J., Marcos, A., Ordovas, J.M., Lee, Y., Meydani, M., Meydani, S.N. 2015. Cardio metabolic and immunological impacts of extra virgin olive oil consumption in overweight and obese older adults: a randomized controlled trial. Journal of Nutrition. 12:28. doi: 10.1186/s12986-015-0022-5.