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The Lactoferrin Receptor May Mediate the Reduction of Eosinophils in the Duodenum of Pigs Consuming Milk Containing Recombinant Human Lactoferrin

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The Lactoferrin Receptor May Mediate the Reduction of Eosinophils in the Duodenum of Pigs Consuming Milk Containing Recombinant Human Lactoferrin Biometals DOI 10.1007/s10534-014-9778-8 The lactoferrin receptor may mediate the reduction of eosinophils in the duodenum of pigs consuming milk containing recombinant human lactoferrin Caitlin Cooper • Eric Nonnecke • Bo Lo¨nnerdal • James Murray Received: 11 February 2014 / Accepted: 16 July 2014 Ó Springer Science+Business Media New York 2014 Abstract Lactoferrin is part of the immune system effects of consumption of milk containing recombi- and multiple tissues including the gastrointestinal (GI) nant human lactoferrin (rhLF-milk) on small intestinal tract, liver, and lung contain receptors for lactoferrin. eosinophils and expression of eosinophilic cytokines. Lactoferrin has many functions, including antimicro- In addition, LFR localization was analyzed in duode- bial, immunomodulatory, and iron binding. Addition- num and circulating eosinophils to determine if the ally, lactoferrin inhibits the migration of eosinophils, LFR could play a role in lactoferrin’s ability to inhibit which are constitutively present in the GI tract, and eosinophil migration. In the duodenum there were increase during inflammation. Lactoferrin suppresses significantly fewer eosinophils/unit area in pigs fed eosinophil infiltration into the lungs and eosinophil rhLF-milk compared to pigs fed control milk migration in -vitro. Healthy pigs have a large popu- (p = 0.025); this was not seen in the ileum lation of eosinophils in their small intestine and like (p = 0.669). In the duodenum, no differences were humans, pigs have small intestinal lactoferrin recep- observed in expression of the LFR, or any eosinophil tors (LFR); thus, pigs were chosen to investigate the migratory cytokines, and the amount of LFR protein was not different (p = 0.386). Immunohistochemistry (IHC) showed that within the duodenum the LFR C. Cooper (&) Á J. Murray localized on the brush border of villi, crypts, and Department of Animal Science, University of California, within the lamina propria. Circulating eosinophils also Davis, CA, USA contained LFRs, which may be a mechanism allowing e-mail: [email protected] lactoferrin to directly inhibit eosinophil migration. J. Murray e-mail: [email protected] Keywords Eosinophils Á Gastrointestinal tract Á E. Nonnecke Á B. Lo¨nnerdal Human lactoferrin Á Inflammation Á Lactoferrin Department of Nutrition, University of California, Davis, receptor Á Transgenic CA, USA B. Lo¨nnerdal Departments of Internal Medicine, University of California, Davis, CA, USA Introduction J. Murray Lactoferrin is an 80 kDa iron binding protein that is Department of Population Health and Reproduction, University of California, One Shields Ave, Davis, secreted in milk and tears, and can also be found in CA 95616, USA neutrophil granules. Lactoferrin acts as part of the 123 Biometals immune system and has a variety of functions, acting Lactoferrin is not toxic to eosinophils and exerts a direct as an antimicrobial, immunomodulator, and antioxi- effect on eosinophils by inhibiting their migratory dant (Wakabayashi et al. 2006). Multiple tissue types ability in a concentration dependent manner without including the gastrointestinal (GI) tract, liver, and lung affecting transcription of chemoattractant molecules contain receptors for lactoferrin (Elfinger et al. 2007; (Bournazou et al. 2010; Curran and Bertics 2012). In Suzuki et al. 2005). Lactoferrin has targeted control of addition, lactoferrin reduces granulocyte macrophage some cellular processes and can act as a transcription colony-stimulating factor (GM-CSF) stimulated adhe- factor, regulating granulopoiesis and DNA synthesis in sion of eosinophils, which is possibly one of the certain cells types (Kanyshkova et al. 2001). Admin- mechanisms causing decreased eosinophil migration istration of lactoferrin is an effective treatment for (Curran and Bertics 2012). enterogenic endotoxinemia in rats (Nebermann et al. Pigs, like humans, have lactoferrin receptors 2001) and suppressed tumor formation in a transgenic throughout their small intestine (Liao et al. 2007). mouse lung tumor model (Tung et al. 2013). Lacto- Due to the high number of lactoferrin receptors located ferrin can also alter circulating levels of white blood in the small intestine, it is a logical target for cells (Cooper et al. 2012) and direct differentiation of lactoferrin mediated inhibition of eosinophil migra- monocytes (van der Does et al. 2012). tion. To determine the effects of consumption of Eosinophils are a class of immune cells often seen human lactoferrin on the population of eosinophils in during allergic reaction and other bouts of inflamma- the small intestine, young pigs were fed transgenic tion; however eosinophils are also constitutively present cows’ milk containing recombinant human lactoferrin in certain tissues, such as the GI tract. There is growing (rhLF-milk) or control cows’ milk. Pigs are an ideal evidence that eosinophils have a more complex role in model because, in addition to having small intestinal immunology than previously thought (Svensson-Frej lactoferrin receptors, healthy pigs are known to have 2011), including playing a role in tissue remodeling, high numbers of eosinophils in their GI tracts homeostasis, and the adaptive immune response. In (Tsukahara et al. 2007). The effects of feeding milk different tissues eosinophils have different phenotypes containing recombinant human lactoferrin (rhLF- and life spans (Masterson et al. 2011). Within the GI milk) on expression of key cytokines in the small tract eosinophils comprise a significant subset of the intestine related to eosinophil migration were also population of resident immune cells seen during steady investigated. To help elucidate if the lactoferrin state conditions, with notably more eosinophils in the receptor could play a role in inhibiting eosinophil duodenum. GI tract eosinophils typically live for at least migration the presence and localization of the lacto- 7 days, and increased longevity is correlated with ferrin receptor was also analyzed in circulating changes in cytokine expression (Svensson-Frej 2011). eosinophils and the small intestine which is a key Changes in eosinophil numbers can occur via sites that eosinophils migrate to. changes in eosinophil differentiation in the bone marrow leading to changes in the circulating pool, changes in migration from circulation into a target Materials and methods tissue, and changes in survival longevity within the tissue (Svensson-Frej 2011). The number of intestinal Milk collection and pasteurization eosinophils is significantly elevated in ulcerative colitis (UC) and Crohns disease, with prior research Transgenic cow’s milk containing recombinant human indicating that they are key to the pathologies of these lactoferrin (rhLF-milk) was provided by Pharming diseases. This may be caused by increased eosinophil Group NV from a second parity Holstein from their products like major basic protein (MBP) and eosino- herd in Wisconsin (USA). Milk was collected, pooled, phil cationic protein (ECP), which are found in UC frozen and then sent to the University of California tissue and correlated with increased disease severity Davis. A non-transgenic Holstein matched for parity (Maltby et al. 2010). and stage of lactation (mid-lactation) from the UC Lactoferrin can suppress airway inflammation and Davis dairy herd was selected and control milk was eosinophil infiltration to the lungs during experimen- collected and frozen. Both control and rhLF contain- tally induced lung inflammation (Zimecki et al. 2012). ing milk were pasteurized at 73.8 °C for ten seconds 123 Biometals then immediately cooled and stored at 4 °C until in a recumbent position on a V shaped table to restrict consumption by the pigs. Pre and post-pasteurisation their movement and blood was collected from the samples were collected and tested for concentrations cranial vena cava. Samples were collected into 10 mL of rhLF and bLF and for lactoferrin activity through a tubes containing EDTA (Becton–Dickinson Com- bacterial lysis assay. The rhLFmilk contained 1.2 g/L pany, Franklin Lakes, NJ). Blood samples were pooled of rhLF and 0.13 g/L bLF and retained 85 % of its and whole blood smears were made on lysine coated activity after pasteurisation. adhesive slides and fixed in either paraformaldehyde or 4 °C methanol for 10 min. Eosinophils were Animals, Necropsy, and Sample Collection isolated from the remaining whole blood according to Samoszuk (2006). Briefly, 2 mL of whole blood Male Hampshire Yorkshire crossbred pigs were was added to 40 mL of sterile distilled water in a obtained from the University of California specific 50 mL conical tube and slowly inverted for 1 min, pathogen free (SPF) swine facility. Pigs were weaned then 4 mL of sterile 10X PBS was added. The tubes at 3 weeks of age and then moved to a containment were centrifuged at 1,2009g for 5 min and the facility at 6 weeks of age. Pigs were weighed upon supernatant was aspirated, leaving a light tan pellet arrival and kept in a temperature-controlled room of eosinophils. The pellet was resuspended in 1 mL of between 25 and 27 °C with ad libitum access to food distilled water and transferred to a 2 mL centrifuge and water for the duration of the trials. The pigs were tube and centrifuged again at 1,2009g for 5 min. monitored twice daily for physical and general well- Supernatant was again aspirated and the pellet resus- being. The specific diet and rearing of the pigs used for pended in 0.2 mL of distilled water and 50 lL of the this study was previously described (Brundige et al. resuspended eosinophil pellet was applied to lysine 2008). Pigs were singly housed and randomly assigned coated adhesive slides and fixed in either paraformal- to groups and twice daily for one week fed 250 mL of dehyde or 4 °C methanol for 10 min. Slides were either pasteurized rhLF-milk from one transgenic cow examined under a confocal microscope to assess the (n = 8) or pasteurized milk from a non-transgenic purity of the isolated eosinophil samples. control cow (n = 8). Groups were balanced to have equal numbers of pigs from each litter. The amount of Histology milk was increased to 350 mL twice daily for the second week.
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