FNS  Vol.5 No.14 , August 2014
Human Milk Oligosaccharides Enhance Innate Immunity to Respiratory Syncytial Virus and Influenza in Vitro
Abstract: Human milk oligosaccharides (HMO) contribute to innate immunity by enhancing growth of beneficial bacteria, epithelial cell maturation and mucosal barrier integrity. They have immunomodulatory effects and can block pathogen binding to host cell surface glycans or receptors. We investigated the effects of 2’-fucosyllactose (2’FL), 6’-sialyllactose (6’SL), 3’-sialyllactose (3’SL) and lacto-N-neoTetraose (LNnT) on human respiratory epithelial cell lines or peripheral blood mononuclear cells (PBMCs) following respiratory viral infectionin vitro. Expression of cytokines and viral load were monitored in infected cells. These biomarkers of innate immunity were selected since viral load and cytokine levels (IP-10, MIP-1α, IL-6, IL-8, TNF-α) have been correlated with disease severity in respiratory syncytial virus (RSV) and influenza (IAV) virus infectionin vivo. 2’FL significantly decreased RSV viral load and cytokines associated with disease severity (IL-6, IL-8, MIP-1α) and inflammation (TNF-α, MCP-1) in airway epithelial cells. LNnT and 6’SL significantly decreased IAV viral load in airway epithelial cells. 6’SL dose-dependently down-regulated IP-10 and TNF-α in RSV infected PBMCs. HMO at or below levels found in breast milk enhance innate immunity to respiratory viruses in vitro and may interact directly with cells to modulate biomarkers of innate immunity.
Cite this paper: Duska-McEwen, G. , Senft, A. , Ruetschilling, T. , Barrett, E. and Buck, R. (2014) Human Milk Oligosaccharides Enhance Innate Immunity to Respiratory Syncytial Virus and Influenza in Vitro. Food and Nutrition Sciences, 5, 1387-1398. doi: 10.4236/fns.2014.514151.

[1]   Walker, W.A. (2013) Initial Intestinal Colonization in the Human Infant and Immune Homeostasis. Annals of Nutrition and Metabolism, 63, 8-15.

[2]   Yu, Z.T., Chen, C. and Newburg, D.S. (2013) Utilization of Major Fucosylated and Sialylated Human Milk Oligosaccharides by Isolated Human Gut Microbes. Glycobiology, 23, 1281-1292.

[3]   Chichlowshi, M., De Lartigue, G., German, J.B., Raybould, H.E. and Mills, D.A. (2012) Bifidobacteria Isolated from Infants and Cultured on Human Milk Oligosaccharides Affect Intestinal Epithelial Function. Journal of Pediatric Gastroenterology and Nutrition, 55, 321-327.

[4]   Holscher, H.D., Davis, S.R. and Tappenden, K.A. (2014) Human Milk Oligosaccharides Influence Maturation of Human Intestinal Caco-2Bbe and HT-29 Cell Lines. Journal of Nutrition, 144, 586-591.

[5]   Morrow, A.L., Ruiz-Palacios, G.M., Jiang, X. and Newburg, D.S. (2005) Human-Milk Glycans That Inhibit Pathogen Binding Protect Breast-Feeding Infants Against Infectious Diarrhea. Journal of Nutrition, 135, 1304-1307.

[6]   Crane, J.K., Azar, S.S., Stam, A. and Newburg, D.S. (1994) Oligosaccharides from Human Milk Block Binding and Activity of the Escherichia coli Heat-Stable Enterotoxin (STa) in T84 Intestinal Cells. Journal of Nutrition, 124, 2358-2364.

[7]   Yang, B., Chuang, H. and Chen, R.-F. (2012) Protection from Viral Infections by Human Milk Oligosaccharides: Direct Blockade and Indirect Modulation of Intestinal Ecology and Immune Reactions. Open Glycoscience, 5, 19-25.

[8]   Shang, J., Piskarev, V.E., Xia, M., Huang, P., Jiang, X., Likhosherstov, L.M., Novikova, O.S., Newburg, D.S. and Ratner, D.M. (2013) Identifying Human Milk Glycans That Inhibit Norovirus Binding Using Surface Plasmon Resonance. Glycobiology, 23, 1491-1498.

[9]   Hester, S.N., Chen, X., Li, M., Monaco, M.H., Comstock, S.S., Kuhlenschmidt, T.B., Kuhlenschmidt, M.S. and Donovan, S.M. (2013) Human Milk Oligosaccharides Inhibit Rotavirus Infectivity in Vitro and in Acutely Infected Piglets. British Journal of Nutrition, 110, 1233-1242.

[10]   Hong, P., Ninonuevo, M.R., Lee, B., Lebrilla, C. and Bode, L. (2009) Human Milk Oligosaccharides Reduce HIV-1-gp120 Binding to Dendritic Cell-Specific ICAM3-Grabbing Non-Integrin (DC-SIGN). British Journal of Nutrition, 101, 482-486.

[11]   Garofalo, R.P. and Haeberle, H. (2000) Epithelial Regulation of Innate Immunity to Respiratory Syncytial Virus. American Journal of Respiratory Cell and Molecular Biology, 23, 581-585.

[12]   Breary, S.P. and Smyth, R.L. (2007) Pathogenesis of RSV in Children. In: Cane, P., Ed., Respiratory Syncytial Virus, Vol. 14. Perspectives in Medical Virology, Elsevier, The Netherlands, 141-162.

[13]   Rossman, J.S. and Lamb, R.A. (2011) Influenza Virus Assembly and Budding. Virology, 411, 229-236.

[14]   Everard, M.L., Swarbrick, A., Wrightham, M., McIntyre, J., Dunkley, C., James, P.D., Sewell, H.F. and Milner, A.D. (1994) Analysis of Cells Obtained by Bronchial Lavage of Infants with Respiratory Syncytial Virus Infection. Archives of Disease in Childhood, 71, 428-432.

[15]   Joshi, P., Kakakios, A., Jayasekera, J. and Isaacs, D. (1998) A Comparison of IL-2 Levels in Nasopharyngeal and Endotracheal Aspirates of Babies with Respiratory Syncytial Viral Bronchiolitis. The Journal of Allergy and Clinical Immunology, 102, 618-620.

[16]   Bode, L. (2006) Recent Advances on Structure, Metabolism, and Function of Human Milk Oligosaccharides. Journal of Nutrition, 136, 2127-2130.

[17]   Bode, L., Rudloff, S., Kunz, C., Strobel, S. and Klein, N. (2004) Human Milk Oligosaccharides Reduce Platelet-Neutrophil Complex Formation Leading to a Decrease in Neutrophil β2 Integrin Expression. The Journal of Leukocyte Biology, 76, 820-826.

[18]   Bode, L., Kunz, C., Muhly-Reinholz, M., Mayer, K., Seeger, W. and Rudloff, S. (2004) Inhibition of Monocyte, Lymphocyte, and Neutrophil Adhesion to Endothelial Cells by Human Milk Oligosaccharides. Thrombsis and Haemostasis, 92, 1402-1410.

[19]   Obermeier, S., Rudloff, S., Pohlentz, G., Lentze, M.J. and Kunz, C. (1999) Secretion of 13C-Labelled Oligosaccharides into Human Milk and Infant’s Urine after an Oral 13C-Galactose Load. Isotopes in Environmental and Health Studies, 352, 119-125.

[20]   Rudloff, S., Pohlentz, G., Borsch, C., Lentze, M.J. and Kunz, C. (2012) Urinary Excretion of in Vivo 13C-Labelled Milk Oligosaccharides in Breastfed Infants. British Journal of Nutrition, 107, 957-963.

[21]   Gnoth, M.J., Rudloff, S., Kunz, C. and Kinne, R.K. (2001) Investigations of the in Vitro Transport of Human Milk Oligosaccharides by a Caco-2 Monolayer Using a Novel High Performance Liquid Chromatography-Mass Spectrometry Technique. Journal of Biological Chemistry, 276, 34363-34370.

[22]   Gnoth, M.J., Rudloff, S., Kunz, C., Kinne, R.K. (2002) Studies on the Intestinal Transport of Human Milk Oligosaccharides Using Caco-2 Cells. Food Research International, 35, 145-149.

[23]   DeVincenzo, J.P., El Saleeby, C.M. and Bush, A.J. (2005) Respiratory Syncytial Virus Load Predicts Disease Severity in Previously Healthy Infants. The Journal of Infectious Diseases, 191, 1861-1868.

[24]   Houben, M.L., Coenjaerts, F.E., Rossen, J.W., Belderbos, M.E., Hofland, R.W., Kimpen, J.L. and Bont, L. (2010) Disease Severity and Viral Load Are Correlated in Infants with Primary Respiratory Syncytial Virus Infection in the Community. Journal of Medical Virology, 82, 1266-1271.

[25]   DeVincenzo, J.P., Wilkinson, T., Vaishnaw, A., Cehelsky, J., Meyers, R., Nochur, S., Harrison, L., Meeking, P., Mann, A., Moane, E., Oxford, J., Pareek, R., Moore, R., Walsh, E., Studholme, R., Dorsett, P., Alvarez, R. and Lambkin-Williams, R. (2010) Viral Load Drives Disease in Humans Experimentally Infected with Respiratory Syncytial Virus. American Journal of Respiratory and Critical Care Medicine, 182, 1305-1314.

[26]   Harrison, A.M., Bonville, C.A., Rosenberg, H.F. and Domachowske, J.B. (1999) Respiratory Syncytial Virus-Induced Chemokine Expression in the Lower Airways: Eosinophil Recruitment and Degranulation. American Journal of Respiratory and Critical Care Medicine, 159, 1918-1924.

[27]   Sheeran, P., Jafri, H., Carubelli, C., Saavedra, J., Johnson, C., Krisher, K., Sánchez, P.J. and Ramilo, O. (1999) Elevated Cytokine Concentrations in the Nasopharyngeal and Tracheal Secretions of Children with Respiratory Syncytial Virus Disease. The Pediatric Infectious Disease Journal, 18, 115-122.

[28]   Tripp, R.A., Oshansky, C., Alvarez, R. (2005) Cytokines and Respiratory Syncytial Virus Infection. Proceedings of the American Thoracic Society, 2, 147-149.

[29]   Launes, C., Garcia-Garcia, J.J., Jordan, I., Selva, L., Rello, J., Muñoz-Almagro, C. (2012) Viral Load at Diagnosis and Influenza A H1N1 (2009) Disease Severity in Children. Influenza and Other Respiratory Viruses, 6, e89-e92.

[30]   Li, C.C., Wang, L., Eng, H.L., You, H.L., Chang, L.S., Tang, K.S., Lin, Y.J., Kuo, H.C., Lee, I.K., Liu, J.W., Huang, E.Y. and Yang, K.D. (2010) Correlation of Pandemic (H1N1) 2009 Viral Load to Disease Severity and Prolonged Viral Shedding in Children. Emerging Infectious Diseases, 16, 1265-1272.

[31]   Lee, N., Chan, P.K., Hui, D.S., Rainer, T.H., Wong, E., Choi, K.W., Lui, G.C., Wong, B.C., Wong, R.Y., Lam, W.Y., Chu, I.M., Lai, R.W., Cockram, C.S. and Sung, J.J. (2009) Viral Loads and Duration of Viral Shedding in Adult Patients Hospitalized with Influenza. The Journal of Infectious Diseases, 200, 492-500.

[32]   Kawashima, H., Go, S., Kashiwagi, Y., Morishima, Y., Miura, T., Ushio, M., Nishimata, S. and Takekuma, K. (2010) Cytokine Profiles of Suction Pulmonary Secretions from Children Infected with Pandemic Influenza A (H1N1) 2009. Critical Care, 14, 411.

[33]   Mitchell, J. and Smith, D.M. (1980) Aquametry, Part III (The Karl Fischer Reagent). 2nd Edition, John Wiley & Sons, New York.

[34]   Prieto, P.A. (2005) In Vitro and Clinical Experiences with a Human Milk Oligosaccharide, Lacto-N-Neotetraose and Fructooligosaccharides. Foods and Food Ingredients Journal of Japan Journal, 219, 1018-1030.

[35]   Cozens, A.L., Yezzi, M.J., Kunzelmann, K., Ohrui, T., Chin, L., Eng, K., Finkbeiner, W.E., Widdicombe, J.H. and Gruenert, D.C. (1994) CFTR Expression and Chloride Secretion in Polarized Immortal Human Bronchial Epithelial Cells. American Journal of Respiratory Cell and Molecular Biology, 10, 38-47.

[36]   Saedisomeolia, A., Wood, L.G., Garg, M.L, Gibson, P.G. and Wark, P.A.B. (2009) Anti-Inflammatory Effects of LongChain n-3 PUFA in Rhinovirus-Infected Cultured Airway Epithelial Cells. British Journal of Nutrition, 101, 533-540.

[37]   Harcourt, J.L., Caidi, H., Anderson, L.J. and Haynes, L.M. (2011) Evaluation of the Calu-3 Cell Line as a Model of in Vitro Respiratory Syncytial Virus Infection. The Journal of Virological Methods, 174, 144-149.

[38]   Douville, R.N., Bastien, N., Li, Y., Pochard, P., Simons, F.E.R. and Hayglass, K.T. (2006) Human Metapneumovirus Elicits Weak IFN-γ Memory Responses Compared with Respiratory Syncytial Virus. Journal of Immunology, 176, 5848-5855.

[39]   Boukhvalova, M.S., Yim, K.C., Prince, G.A. and Blanco, J.C.G. (2010) Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by Real-Time Reverse Transcription-PCR in Vivo: Detection of Abortive Viral Replication. Current Protocols in Cell Biology, Unit 26.6.

[40]   Boukhvalova, M.S., Prince, G.A. and Blanco, J.C. (2007) Respiratory Syncytial Virus Infects and Abortively Replicates in the Lungs in Spite of Pre-Existing Immunity. Journal of Virology, 81, 9443-9450.

[41]   Dewhurst-Maridor, G., Simonet, V., Bornand, J.E., Nicod, L.P. and Pache, J.C. (2004) Development of a Quantitative TaqMan RT-PCR for Respiratory Syncytial Virus. Journal of Virological Methods, 120, 41-49.

[42]   Weichert, S., Jennewein, S., Hüfner, E., Weiss, C., Borkowski, J., Putze, J. and Schroten, H. (2013) Bioengineered 2’-Fucosyllactose and 3-Fucosyllactose Inhibit the Adhesion of Pseudomonas aeruginosa and Enteric Pathogens to Human Intestinal and Respiratory Cell Lines. Nutrition Research, 33, 831-838.

[43]   Bourgeois, C., Bour, J.B., Lidholt, K., Gauthray, C. and Pothier, P. (1998) Heparin-Like Structures on Respiratory Syncytial Virus Are Involved in Its Infectivity in Vitro. Journal of Virology, 72, 7221-7227.

[44]   Domurat, F., Roberts Jr., N.J., Walsh, E.E. and Dagan, R. (1985) Respiratory Syncytial Virus Infection of Human Mononuclear Leukocytes in Vitro and in Vivo. Journal of Infectious Diseases, 152, 895-902.

[45]   Erney, R.M., Malone, W.T., Skelding, M.B., Marcon, A.A., Kleman-Leyer, K.M., O’Ryan, M.L., Ruiz-Palacios, G., Hilty, M.D., Pickering, L.K. and Prieto, P.A. (2000) Variability of Human Milk Neutral Oligosaccharides in a Diverse Population. Journal of Pediatric Gastroenterology & Nutrition, 30, 181-192.

[46]   Bao, Y., Zhu, L. and Newburg, D.S. (2007) Simultaneous Quantification of Sialyloligosaccharides from Human Milk by Capillary Electrophoresis. Analytical Biochemistry, 370, 206-214.

[47]   Lin, A.E., Autran, C.A,. Espanola, S.D, Bode, L. and Nizet, V. (2014) Human Milk Oligosaccharides Protect Bladder Epithelial Cells against Uropathogenic Escherichia coli Invasion and Cytotoxicity. Journal of Infectious Diseases, 209, 389-398.

[48]   Newburg, D.S. (2009) Neonatal Protection by an Innate Immune System of Human Milk Consisting of Oligosaccharides and Glycans. Journal of Animal Science, 87, 26-34. 10.2527/jas.2008-1347

[49]   Morrison, P.T., Sharland, M., Thomas, L.H., Manna, S., Handforth, J., Tibby, S. and Friedland, J.S. (2008) Chemokine-Receptor Upregulation and Disease Severity in Respiratory Syncytial Virus Infection. Clinical Immunology, 128, 85-93.

[50]   Midulla, F, Huang,Y.T., Gilbert, I.A., Cirino, N.M., McFadden Jr., E.R. and Panuska J.R. (1989) Respiratory Syncytial Virus Infection of Human Cord and Adult Blood Monocytes and Alveolar Macrophages. The American Review of Respiratory Disease, 140, 771-777.

[51]   Panuska, J.R., Cirino, N.M., Midulla, F., Despot, J.E., McFadden Jr., E.R. and Huang, Y.T. (1990) Productive Infection of Isolated Human Alveolar Macrophages by Respiratory Syncytial Virus. The Journal of Clinical Investigation, 86, 113-119.

[52]   Guerrero-Plata, A., Casola, A., Suarez, G., Yu, X., Spetch, L., Peeples, M.E. and Garofalo, R.P. (2006) Differential Response of Dendritic Cells to Human Metapneumovirus and Respiratory Syncytial Virus. American Journal of Respiratory Cell and Molecular Biology, 34, 320-329.

[53]   Qi, X.F., Kim, D.H., Yoon, Y.S., Jin, D., Huang, X.Z., Li, J.H., Deung, Y.K. and Lee, K.J. (2009) Essential Involvement of Cross-Talk between IFN-Gamma and TNF-Alpha in CXCL10 Production in Human THP-1 Monocytes. Journal of Cellular Physiology, 220, 690-697.

[54]   Bode, L. (2012) Human Milk Oligosaccharides: Every Baby Needs a Sugar Mama. Glycobiology, 22, 1147-1162.

[55]   Tayyari, F., Marchant, D., Moraes, T.J., Duan, W., Mastrangelo, P. and Hegele, R.G. (2011) Identification of Nucleolin as a Cellular Receptor for Human Respiratory Syncytial Virus. Nature Medicine, 17, 1132-1135.

[56]   Yu, Y., Mishra, S., Song, X., Lasanajak, Y., Bradley, K.C., Tappert, M.M., Air, G.M., Steinhauer, D.A., Halder, S., Cotmore, S., Tattersall, P., Agbandje-McKenna, M., Cummings, R.D. and Smith, D.F. (2012) Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers. The Journal of Biological Chemistry, 287, 44784-44799.

[57]   Galton Bachrach, V.R., Schwarz, E. and Bachrach, L.R. (2003) Breastfeeding and the Risk of Hospitalization for Respiratory Disease in Infancy. Archives of Pediatrics and Adolescent Medicine, 157, 237-243.

[58]   Duijts, L., Ramadhani, M.K. and Moll, H.A. (2009) Breastfeeding Protects against Infectious Diseases during Infancy in Industrialized Countries. A Systematic Review. Maternal & Child Nutrition, 5, 199-210.

[59]   Flanders Stepans, M.B., Wilhelm, S.L., Hertzog, M., Callahan Rodehorst, T.K, Blaney, S., Clemens, B., Polak III, J.J. and Newburg, D.S. (2006) Early Consumption of Human Milk Oligosaccharides Is Inversely Related to Subsequent Risk of Respiratory and Enteric Disease in Infants. Breastfeeding Medicine, 1, 207-215.

[60]   Tarrant, M., Kwok, M.K., Lam, T.H., Leung, G.M. and Schooling, C.M. (2010) Breast-Feeding and Childhood Hospitalizations for Infections. Epidemiology, 21, 847-854.