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Title:Effect of bovine lactoferrin on T cell and NK cell populations and NK cell cytotoxicity in neonatal piglets
Author(s):Liu, Kilia
Advisor(s):Donovan, Sharon M.
Department / Program:Nutritional Sciences
Discipline:Nutritional Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Bovine lactoferrin
NK cells
Nenonatal innate immunity
mother's milk
neonatal pigs
formula feeding
Abstract:Neonates face a dramatic transition at birth as they move from the protected womb to an environment full of foreign antigens. Due to limited prenatal exposure to antigens, newborns initially rely on their innate immune system for protection. Natural killer (NK) cells are large granular lymphocytes that protect the body against infections due to their ability to kill target cells as well as secrete inflammatory cytokines to further induce themselves or the neighboring immune cells to mount a protective response. NK cell populations in the blood of breast-fed infants are higher than in formula-fed infants, leading to the hypothesis that human milk components influence the development of NK cells. Lactoferrin (Lf) is a multifunctional glycoprotein found in high concentrations in human milk (2.1 g/L), but at 20-fold lower levels (100 mg/L) in bovine milk and infant formula. Lf has been shown to increase human NK cell cytotoxic activity when the cells were isolated and stimulated with Lf in vitro; however, the effect of dietary Lf on NK cell cytotoxicity was not known. The goal of this thesis was to investigate the effect of bovine Lf and vaccination on T-cell subpopulations and NK cell development in the neonate using the piglet as a model. Newborn piglets received 4h of colostrum and then were randomized to sow milk replacer formula (FF), formula supplemented with 1 g/L bovine Lf (LF), or sow-reared (SR). Half of the piglets in each diet group were vaccinated using a pediatric dose (0.25 mL) of Fluzone™ at postnatal d7 followed by a booster on d14. On d21 postpartum, piglets were euthanized and blood and tissue samples were collected. Cells were isolated from blood, spleen and mesenteric lymph nodes (MLN) to identify T-cell subpopulations and NK cell populations by flow cytometry. NK cells were further isolated from peripheral blood mononuclear cells (PBMCs) and assessed in a NK cell cytotoxicity assay. Body weight, T-cell subpopulations and NK cell cytotoxicity were not affected by vaccination; thus vaccination data for these analyses were pooled by diet group. The average body weight of SR piglets was significantly higher (p<0.05) than that of FF or LF piglets on d3-21. On d21, average body weight of LF piglets was higher (p<0.05) than FF piglets, suggesting that bLf supplementation to formula may enhance growth of young animals. There was an interaction between the effect of diet and vaccination on NK cell population. Vaccination did not affect the number of NK cell population in PBMC of SR and LF animals. However, NK cell population size in PBMC changed after vaccination in FF animals. The NK cell population size of FF vaccinated (FFV) animals was significantly larger (p<0.05) than those of FF non-vaccinated (FFN), suggesting that PBMC NK cells from FF animals responded to Fluzone vaccination. However, diet did not affect NK cell population size in PBMC of the vaccinated animals. The percentage of NK cells in PBMC was not different between SR vaccinated (SRV), LF vaccinated (LFV) and FFV animals. Among non-vaccinated animals, diet had a significant effect on NK cell population size. SR non-vaccinated (SRN) animals had over twice as many NK cells than non-vaccinated FF (FFN) animals, whereas the NK cell population of non-vaccinated LF (LFN) was not significantly different from wither SRN or FFN. These data are consistent with data from human studies in which breast-fed infants had a higher percentage of PBMC NK cells than FF infants. These data suggest that NK cell population is strongly influenced by feeding mode. However, NK cell populations in MLN and spleen were not affected by diet or vaccination. In PBMC, the double positive (DP) memory T cell population size of LF piglets was smaller (p=0.0388) than those of FF piglets, with SR being intermediate, which indicates that Lf may play a regulatory role in immune cell activation. Diet did not affect the DP memory T cell population in MLN or spleen or CD4+ T helper cell population, CD8+ cytotoxic T cell population, and the CD4:CD8 T cell ratio in PBMC, MLN or spleen. SR, FF and LF samples were pooled by tissue to evaluate cell population sizes at different organs. The percent of NK cell was highest (p<0.0001) in PBMC followed by the spleen and lowest in MLN, whereas the percent of DP memory T cell was higher (p<0.0001) in MLN and spleen than PBMC. Furthermore, the percent of T helper cell was higher (p<0.0001) in MLN compared to PBMC and spleen. However, percent of cytotoxic T cell was not different among different tissues. Overall, the innate defenders, NK cells, predominate in the circulation, while responsive DP T-cells and CD4+ T-cells predominate at the body’s barrier, MLN. NK cells isolated from PBMC were stimulated with IL-2 (20 ng/mL) or bLf (25 μg/mL) and incubated 48 h with K562 target cells at 10:1 effector to target cell ratio. In general, IL-2 stimulated NK cells had higher cytotoxicity compared to bLf-stimulated or unstimulated NK cells. When stimulated with IL-2, NK cell cytotoxicity of LF animals was lower (p=0.0003) than that of SR animals with cytotoxicity of NK cells from FF piglets being intermediate. Even without IL-2 or bLf stimulation, NK cell cytotoxicity of LF animals was similar to FF piglets and significantly less (p<0.05) than SR piglets. A similar pattern was observed within the LF-stimulated NK cells; however, the differences were not significant (p=0.0595). We also measured interlectin-2 (ITLN-2, LF receptor) mRNA expression in NK cells and found that LF piglets had 2.5-fold higher (p<0.01) ITLN-2 mRNA expression in NK cells than SR or FF piglets. This suggests that oral supplementation of bLf may increase LF receptor expression on NK cells. However, this increase in expression of LF receptors in NK cells did not increase NK cell’s cytotoxicity to bLf stimulation, which suggests that Lf may be involved in immune regulation of NK cell activation. To investigate potential mechanisms for improved NK cell cytotoxicity, the mRNA expression of perforin, natural killer group 2 member D (NKG2D) receptors and IFN-α receptor in NK cells isolated from PBMCs were accessed. Perforin gene expression in NK cells from SR animals was 6-fold higher (p=0.0038) than FF animals and 3.5-fold higher (p=0.0038) than LF animals. The gene expression of perforin in NK cells from LF and FF animals was not significantly different. In addition, the expression of NKG2D in NK cell of SR animals was higher (p=0.0172) than that of FF animals, while the NKG2D gene expression in NK cell of LF animals was intermediate. However, the gene expression of IFN-α receptor was not affected by diet. The level of gene expression of perforin, NKG2D receptor, and IFN-α receptors in NK cells represents the activation status of NK cells. Overall, diet had a significant impact on NK cell population and activity. Piglets that were fed sow’s milk had larger NK cell population size as well as greater cytotoxic activity compared to those of FF. However, NK cell development was minimally affected by bLf supplementation. As NK cells are important first line defenders against infection, knowledge of factors to increase their numbers and improve their cytotoxic activity may enable us to further protect formula-fed infants from infection.
Issue Date:2012-07-24
Rights Information:Copyright 2012 Kilia Y Liu
Date Available in IDEALS:2012-07-24
Date Deposited:2012-08

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