Protective Effects of Lactoferrin against SARS-CoV-2 Infection In Vitro
Claudio Salaris, Melania Scarpa, Marina Elli, Alice Bertolini, Simone Guglielmetti, Fabrizio Pregliasco, Corrado Blandizzi, Paola Brun, Ignazio Castagliuolo
Nutrients, doi:10.3390/nu13020328
SARS-CoV-2 is a newly emerging virus that currently lacks curative treatments. Lactoferrin (LF) is a naturally occurring non-toxic glycoprotein with broad-spectrum antiviral, immunomodulatory and anti-inflammatory effects. In this study, we assessed the potential of LF in the prevention of SARS-CoV-2 infection in vitro. Antiviral immune response gene expression was analyzed by qRT-PCR in uninfected Caco-2 intestinal epithelial cells treated with LF. An infection assay for SARS-CoV-2 was performed in Caco-2 cells treated or not with LF. SARS-CoV-2 titer was determined by qRT-PCR, plaque assay and immunostaining. Inflammatory and anti-inflammatory cytokine production was determined by qRT-PCR. LF significantly induced the expression of IFNA1, IFNB1, TLR3, TLR7, IRF3, IRF7 and MAVS genes. Furthermore, LF partially inhibited SARS-CoV-2 infection and replication in Caco-2 intestinal epithelial cells. Our in vitro data support LF as an immune modulator of the antiviral immune response with moderate effects against SARS-CoV-2 infection.
Conflicts of Interest: The authors declare no conflict of interest.
References
Baveye, Elass, Mazurier, Spik, Legrand, Lactoferrin: A Multifunctional Glycoprotein Involved in the Modulation of the Inflammatory Process, Clin. Chem. Lab. Med,
doi:10.1515/CCLM.1999.049
Buscarini, Manfredi, Brambilla, Menozzi, Londoni et al., GI Symptoms as Early Signs of COVID-19 in Hospitalised Italian Patients, Gut,
doi:10.1136/gutjnl-2020-321434
Campione, Cosio, Rosa, Lanna, Di Girolamo et al., Lactoferrin as Protective Natural Barrier of Respiratory and Intestinal Mucosa against Coronavirus Infection and Inflammation, Int. J. Mol. Sci
Caprio, Infante, Calanchini, Mammi, Fabbri et al., Not Just the Bone. Evidence for Beneficial Pleiotropic Extraskeletal Effects, Eat Weight Disord,
doi:10.1007/s40519-016-0312-6
Castillo, Entrenas Costa, Vaquero Barrios, Alcalá Díaz, López Miranda et al., Effect of Calcifediol Treatment and Best Available Therapy versus Best Available Therapy on Intensive Care Unit Admission and Mortality among Patients Hospitalized for COVID-19: A Pilot Randomized Clinical Study, J. Steroid Biochem. Mol. Biol,
doi:10.1016/j.jsbmb.2020.105751
Channappanavar, Fehr, Zheng, Wohlford-Lenane, Abrahante et al., IFN-I Response Timing Relative to Virus Replication Determines MERS Coronavirus Infection Outcomes, J. Clin. Investig,
doi:10.1172/JCI126363
Clausen, Sandoval, Spliid, Pihl, Perrett et al., SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2, Cell,
doi:10.1016/j.cell.2020.09.033
Convertino, Tuccori, Ferraro, Valdiserra, Cappello et al., Exploring Pharmacological Approaches for Managing Cytokine Storm Associated with Pneumonia and Acute Respiratory Distress Syndrome in COVID-19 Patients, Crit. Care,
doi:10.1186/s13054-020-03020-3
Elnagdy, Alkhazindar, The Potential of Antimicrobial Peptides as an Antiviral Therapy against COVID-19, ACS Pharm. Transl. Sci,
doi:10.1021/acsptsci.0c00059
Fabbri, Infante, Ricordi, Editorial-Vitamin D Status: A Key Modulator of Innate Immunity and Natural Defense from Acute Viral Respiratory Infections, Eur. Rev. Med. Pharm. Sci,
doi:10.26355/eurrev_202004_20876
Fornasa, Tsilingiri, Caprioli, Botti, Mapelli et al., Dichotomy of Short and Long Thymic Stromal Lymphopoietin Isoforms in Inflammatory Disorders of the Bowel and Skin, J. Allergy Clin. Immunol,
doi:10.1016/j.jaci.2015.04.011
Ghio, Carter, Dailey, Devlin, Samet, Respiratory Epithelial Cells Demonstrate Lactoferrin Receptors That Increase after Metal Exposure, Am. J. Physiol. Lung Cell. Mol. Physiol,
doi:10.1152/ajplung.1999.276.6.L933
Glanville, Treatments and Vaccines for COVID-19: Authorised Medicines
Hoffmann, Kleine-Weber, Schroeder, Krüger, Herrler et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor, Cell,
doi:10.1016/j.cell.2020.02.052
Honda, Taniguchi, IRFs: Master Regulators of Signalling by Toll-like Receptors and Cytosolic Pattern-Recognition Receptors, Nat. Rev. Immunol,
doi:10.1038/nri1900
Ikeda, Nozaki, Sugiyama, Tanaka, Naganuma et al., Characterization of Antiviral Activity of Lactoferrin against Hepatitis C Virus Infection in Human Cultured Cells, Virus Res,
doi:10.1016/S0168-1702(99)00121-5
Jain, Chaurasia, Sengar, Singh, Mahor et al., Analysis of Vitamin D Level among Asymptomatic and Critically Ill COVID-19 Patients and Its Correlation with Inflammatory Markers, Sci. Rep,
doi:10.1038/s41598-020-77093-z
Jiang, Lopez, Kelleher, Lönnerdal, Apo-and Holo-Lactoferrin Are Both Internalized by Lactoferrin Receptor via Clathrin-Mediated Endocytosis but Differentially Affect ERK-Signaling and Cell Proliferation in Caco-2 Cells, J. Cell Physiol,
doi:10.1002/jcp.22650
Kell, Heyden, Pretorius, The Biology of Lactoferrin, an Iron-Binding Protein That Can Help Defend Against Viruses and Bacteria, Front. Immunol,
doi:10.3389/fimmu.2020.01221
Lamers, Beumer, Van Der Vaart, Knoops, Puschhof et al., SARS-CoV-2 Productively Infects Human Gut Enterocytes, Science,
doi:10.1126/science.abc1669
Lang, Yang, Deng, Liu, Yang et al., Inhibition of SARS Pseudovirus Cell Entry by Lactoferrin Binding to Heparan Sulfate Proteoglycans, PLoS ONE,
doi:10.1371/journal.pone.0023710
Leung, Clinical Features of Deaths in the Novel Coronavirus Epidemic in China, Rev. Med Virol,
doi:10.1002/rmv.2103
Machnicki, Zimecki, Zagulski, Lactoferrin Regulates the Release of Tumour Necrosis Factor Alpha and Interleukin 6 in Vivo, Int. J. Exp. Pathol
Martinez, Compounds with Therapeutic Potential against Novel Respiratory 2019 Coronavirus, Antimicrob. Agents Chemother,
doi:10.1128/AAC.00399-20
Mehta, Mcauley, Brown, Sanchez, Tattersall et al., HLH Across Speciality Collaboration, UK. COVID-19: Consider Cytokine Storm Syndromes and Immunosuppression, Lancet,
doi:10.1016/S0140-6736(20)30628-0
Milewska, Zarebski, Nowak, Stozek, Potempa et al., Human Coronavirus NL63 Utilizes Heparan Sulfate Proteoglycans for Attachment to Target Cells, J. Virol,
doi:10.1128/JVI.02078-14
Mrityunjaya, Pavithra, Neelam, Janhavi, Halami et al., Immune-Boosting, Antioxidant and Anti-Inflammatory Food Supplements Targeting Pathogenesis of COVID-19, Front. Immunol,
doi:10.3389/fimmu.2020.570122
Ou, Liu, Lei, Li, Mi et al., Characterization of Spike Glycoprotein of SARS-CoV-2 on Virus Entry and Its Immune Cross-Reactivity with SARS-CoV, Nat. Commun,
doi:10.1038/s41467-020-15562-9
Pereira, Dantas Damascena, Galvão Azevedo, De Almeida Oliveira, Da Mota Santana et al., Deficiency Aggravates COVID-19: Systematic Review and Meta-Analysis, Crit. Rev. Food Sci. Nutr,
doi:10.1080/10408398.2020.1841090
Pišlar, Mitrović, Sabotič, Fonović, Nanut et al., The Role of Cysteine Peptidases in Coronavirus Cell Entry and Replication: The Therapeutic Potential of Cathepsin Inhibitors, PLoS Pathog,
doi:10.1371/journal.ppat.1009013
Sano, Miyauchi, Takakura, Yamauchi, Murata et al., Cysteine Protease Inhibitors in Various Milk Preparations and Its Importance as a Food, Food Res. Int,
doi:10.1016/j.foodres.2004.10.011
Scaldaferri, Ianiro, Privitera, Lopetuso, Vetrone et al., The Thrilling Journey of SARS-CoV-2 into the Intestine: From Pathogenesis to Future Clinical Implications, Inflamm. Bowel Dis,
doi:10.1093/ibd/izaa181
Serrano, Kochergina, Albors, Diaz, Oroval et al., Liposomal Lactoferrin as Potential Preventative and Cure for COVID-19, IJRHS,
doi:10.5530/ijrhs.8.1.3
Shin, Wakabayashi, Yamauchi, Yaeshima, Iwatsuki, Recombinant Human Intelectin Binds Bovine Lactoferrin and Its Peptides, Biol. Pharm Bull,
doi:10.1248/bpb.31.1605
Siqueiros-Cendón, Arévalo-Gallegos, Iglesias-Figueroa, García-Montoya, Salazar-Martínez et al., Immunomodulatory Effects of Lactoferrin, Acta Pharm. Sin,
doi:10.1038/aps.2013.200
Sun, Louie, Vannella, Wilke, Levine et al., New Concepts of IL-10-Induced Lung Fibrosis: Fibrocyte Recruitment and M2 Activation in a CCL2/CCR2 Axis, Am. J. Physiol. Lung Cell Mol. Physiol,
doi:10.1152/ajplung.00122.2010
Superti, Ammendolia, Valenti, Seganti, Antirotaviral Activity of Milk Proteins: Lactoferrin Prevents Rotavirus Infection in the Enterocyte-like Cell Line HT-29, Med. Microbiol. Immunol,
doi:10.1007/s004300050049
Van Doorn, Meijer, Frampton, Barclay, De Boer, Systematic Review with Meta-analysis: SARS-CoV-2 Stool Testing and the Potential for Faecal-oral Transmission, Aliment Pharm,
doi:10.1111/apt.16036
Van Splunter, Perdijk, Fick-Brinkhof, Feitsma, Floris-Vollenbroek et al., Bovine Lactoferrin Enhances TLR7-Mediated Responses in Plasmacytoid Dendritic Cells in Elderly Women: Results From a Nutritional Intervention Study With Bovine Lactoferrin, GOS and Vitamin D, Front. Immunol,
doi:10.3389/fimmu.2018.02677
Wang, Li, Zhao, Ren, Guo, Lactoferrin Stimulates the Expression of Vitamin D Receptor in Vitamin D Deficient Mice, J. Funct. Foods,
doi:10.1016/j.jff.2019.02.012
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