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Title:Lactoferrin in neonatal development: effects on intestinal structure and intestinal function and immune response in a piglet model of systemic Staphylococcus aureus infection
Author(s):Reznikov, Elizabeth
Director of Research:Donovan, Sharon M.
Doctoral Committee Chair(s):Miller, Michael J.
Doctoral Committee Member(s):Donovan, Sharon M.; Gaskins, H. Rex; Dilger, Ryan N.
Department / Program:Nutritional Sciences
Discipline:Nutritional Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Infant Formula
Human Milk
Bifidobacteria Infantis
Neonatal Intestinal Development
Neonatal Immunity
Neonatal Piglet Model
Staphylococcus Aureus Infection
Abstract:Human milk is the best source of nutrition for the human infant. Breast-fed infants are more resistant to disease and infection early in life than formula-fed children. Breast-fed infants have decreased incidence of diarrhea and gastroenteritis, necrotizing enterocolitis, respiratory infection, urinary tract infections and neonatal sepsis (Stuebe 2009; Golding et al 1997). The difference in health outcomes observed between breast-fed and formula-fed infants is in part explained by immune factors present in human milk that provide specific and innate immune factors that protect against and directly interact with pathogens (Stuebe 2009). The consumption of human milk also impacts the development of the intestinal microbiota in infants, which has implications for immunological programming (Kainonen et al 2013; Jacobi and Odle, 2012). Lactoferrin (Lf), a bioactive protein found in high concentrations in human milk, when fed orally, has bacteriostatic activity against S. aureus in vivo (Bhimani et al 1999) and has been proven efficacious against neonatal septicemia when supplemented in infant formula (Manzoni et al 2009). In addition to its antioxidant, immune modulating and antibacterial properties, Lf promotes the growth of bifidobacteria, while inhibiting pathogenic bacteria. Bifidobacteria is the predominant fecal bacterium in breast-fed infants (Trebichavsky et al 2009), and contributes to development of the mucosal immune system, regulation of the systemic immune response, and decreased bacterial sepsis in preterm infants (Dong et al 2010; Cohen-Wolkowiez et al 2009). Lastly, Lf has a role on intestinal maturation and has been shown to have a proliferative effect at the intestine (Nguyen et al 2014; Buccigrossi et al 2007; Nichols et al 1987). It has been suggested intestinal maturation leads to improved local immunity that in turn affects the systemic immune system and overall health. Treatment of Staphylococcus aureus (S. aureus) infections is complicated by an increasing number of antibiotic resistant strains. Antibiotic resistant strains are a significant cause of morbidity and mortality in neonatal intensive care units, disproportionally affecting infants less than three months old (Zervou et al 2014; Maraqa et al 2011). One example of an antibiotic resistant strain is methicillin-resistant S. aureus, or MRSA, which results in more deaths annually than any other single infectious agent in the United States (Miller and Cho, 2011). Colonization of S. aureus is incompletely understood, but involves virulence factors which are structural to and secreted by S. aureus. The pathogen is able to adhere to host cells, evade the immune response, and persist intracellularly within epithelial cells and cells of the host immune system (Liu 2009). Newborn infants are unable to mount an effective immune response, making them a highly susceptible population in the hospital setting. The proposed research in this dissertation was to understand how Lf can modulate intestinal maturation and immune response. In addition, we looked at how oral intake of Lf and bifidobacteria attenuate a systemic S. aureus infection. While these bioactive ingredients are found naturally in human milk, and have been shown to be clinically efficacious in blood-born S. aureus infection, there is a lack of understanding of their biological activities in order to justify their addition to infant formulas. Thus, the findings in this research are a significant step in identifying the potential role of Lf to improve gastrointestinal maturation and immune response in newborn infants. Herein, we have three aims to investigate the effects of supplementing Lf on neonatal intestinal and immune development. Proliferative properties of bovine Lf (bLf) were explored through 5-ethynyl-2'-deoxyuridine (EdU) incorporation in vivo for aim one. Colostrum-deprived pigs were fed formula containing 0.4 (CON), 1 (LF1) or 3.6 (LF3) g bLf/L. On d7 or 14, pigs were intraperitoneally injected with 10mM EdU (1ml/kg body weight) 2 h prior to euthanasia. Proliferation was assessed ex vivo in formalin-fixed jejunal sections using Click-iT EdU Alexa Fluor 555 Kit. Sections were imaged with a confocal microscope. Staining was quantified with AxioVision, where area positive for EdU (proliferation) was normalized by the area positive for DAPI (total nuclei). Formalin-fixed jejunum sections were stained with haematoxylin and eosin for villus and crypt measurements. LF1 and LF3 pigs had a 1.5-fold increase in crypt cell proliferation compared to CON pigs. In LF3 pigs, crypt depth and area were greater (1.5-fold on d 7, 1.3-fold on d 14) than in CON pigs. This study supports in vitro studies that showed LF could stimulate proliferation and was the first to show that physiologically-relevant concentrations of milk-borne bLf stimulate crypt cell proliferation in pigs suggesting Lf plays a role in healthy GI development. The effect of bLf alone or in combination with B. infantis on the course of S. aureus infection was assessed. Colostrum-deprived pigs had umbilical catheters placed at birth and were fed formula with 4g/L bLf (LF) or whey protein (CON); half of the piglets in each group were further randomized to receive B. infantis (109 CFU/day, ATCC 15697). On d 7, all piglets were infected intravenously with S. aureus (105 CFU/kg BW, S54F9) and euthanized on d 12. The same study design was used to investigate aims two and three. In aim two, LF piglets had decreased (p<0.05) S. aureus load at the kidney, and LF tended to decrease S. aureus load at the lung and heart compared to CON, with no effect of B. infantis. LF tended to decrease the incidence of infection at the kidney, and when combined with B. infantis (COMB; LF + B. infantis) had the lowest incidence of infection at the lung. Furthermore, LF-fed animals had significantly increased IFNγ expression at the lung, significantly decreased IL-10 expression at the kidney, and when combined with B. infantis (COMB) ameliorated TLR2 expression at the kidney. Lastly, B. infantis significantly increased IFNγ expression at the spleen. Overall, bLf decreases translocation of S. aureus to tissues, which could potentially reduce organ dysfunction. In aim three, piglets had elevated (p<0.05) rectal temperature beginning at 36 h post-infection. LF piglets had elevated rectal temperatures and improved weight gain on d10, 72 h post-infection. LF piglets also had decreased serum IL-10 and increased lymphocyte percent in whole blood prior to infection. B. infantis increased serum IL-10 following infection, with a peak IL-10 concentration at 96 and 108 h post-infection. Despite these results, Foxp3 expression was actually depressed in Lf and B. infantis animals, and Foxp3 expression was restored in COMB animals relative to CON. Lastly, Lf decreased relative proportions of monocytes and B cells in the PBMC population, whereas B. infantis decreased memory T cells at 120 hours post-infection. Overall, B. infantis decreased inflammatory immune responses. The combination of Lf and B. infantis may improve the neonatal immune response, while decreasing potentially harmful over-inflammatory responses.
Issue Date:2014-09-16
Rights Information:Copyright 2014 Elizabeth A Reznikov
Date Available in IDEALS:2014-09-16
Date Deposited:2014-08

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