BACKGROUND: Lactoferrin (LF) is an 80 kDa glycoprotein which is known for its effects against bacteria, viruses and other pathogens. It also has a high potential in nutrition therapy and welfare of people and a variety of animals, including piglets. The ability to bind lipopolysaccharide (LPS) is one of the described anti-inflammatory mechanisms of LF. Previous studies suggested that cells can be stimulated even by LPS-free LF. Therefore, the aim of our study was to bring additional information about this possibility. Porcine monocyte derived macrophages (MDMF) and human embryonic kidney (HEK) cells were stimulated with unpurified LF in complex with LPS and with purified LF without bound LPS. RESULTS: Both cell types were stimulated with unpurified as well as purified LF. On the other hand, neither HEK0 cells not expressing any TLR nor HEK4a cells transfected with TLR4 produced any pro-inflammatory cytokine transcripts after stimulation with purified LF. This suggests that purified LF without LPS stimulates cells via another receptor than TLR4. An alternative, TLR4-independent, pathway was further confirmed by analyses of the NF-kappa-B-inducing kinase (NIK) activation. Western blot analyses showed NIK which activates different NFκB subunits compared to LF-LPS signaling via TLR4. Though, this confirmed an alternative pathway which is used by the purified LF free of LPS. This stimulation of MDMF led to low, but significant amounts of pro-inflammatory cytokines, which can be considered as a positive stimulation of the immune system. CONCLUSION: Our results suggest that LF's ability is not only to bind LPS, but LF itself may be a stimulant of pro-inflammatory pathways.

Faculty of Science Masaryk University Brno Czech Republic

Veterinary Research Institute Hudcova 296 70 Brno 621 00 Czech Republic

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Buckett NM, Luckas MJ, Gazvani MR, Aird IA, Lewis-Jones DI. Seminal plasma lactoferrin concentrations in normal and abnormal semen samples. J Androl. 1997;18:302–304. PubMed

Fullard RJ, Snyder C. Protein levels in nonstimulated and stimulated tears of normal human subjects. Invest Opthalmol Vis Sci. 1990;31:1119–1126. PubMed

Hayakawa T, Harada H, Noda A, Kondo T. Lactoferrin in pure pancreatic juice in chronic pancreatitis. Am J Gastroenterol. 1983;78:222–224. PubMed

Hetherington SV, Spitznagel JK, Quie PG. An enzyme-linked immunoassay (ELISA) for measurement of lactoferrin. J Immunol Methods. 1983;65:183–190. doi: 10.1016/0022-1759(83)90314-9. PubMed DOI

Lonnerdal B, Iyer S. Lactoferrin: Molecular structure and biological function. Annu Rev Nutr. 1995;15:93–110. doi: 10.1146/annurev.nu.15.070195.000521. PubMed DOI

Ronayne De Ferre PA, Baroni A, Sambucetti ME, López NE, Ceriani Cernadas JM. Lactoferrin levels in term and preterm milk. J Am Coll Nutr. 2000;19:370–373. doi: 10.1080/07315724.2000.10718933. PubMed DOI

Shugars DS, Watkins CA, Cowen HJ. Salivary concentration of secretory leukocyte protease inhibitor, an antimicrobial protein, is decreased with advanced age. Gerontology. 2001;47:246–253. doi: 10.1159/000052808. PubMed DOI

Kondori N, Baltzer L, Dolphin GT, Mattsby-Baltzer I. Fungicidal activity of human lactoferrin-derived peptides based on the antimicrobial αβ region. Int J Antimicrob Agents. 2011;37:51–57. doi: 10.1016/j.ijantimicag.2010.08.020. PubMed DOI

Wakabayashi H, Oda H, Yamauchi K, Abe F. Lactoferrin for prevention of common viral infections. J Infect Chemother. 2014;20:666–671. doi: 10.1016/j.jiac.2014.08.003. PubMed DOI

Yin C, Wong JH, Ng TB. Recent studies on the antimicrobial peptides lactoferricin and lactoferrampin. Curr Mol Med. 2014;14:1139–1154. doi: 10.2174/1566524014666141015151749. PubMed DOI

Adlerova L, Bartoskova A, Faldyna M. Lactoferrin: a review. Vet Med. 2008;53:457–468.

Hung YP, Yang YP, Wang HC, Liao JW, Hsu WL, Chang CC, Chang SC. Bovine lactoferrin and piroxicam as an adjunct treatment for lymphocytic-plasmatic gingivitis stomatitis in cats. Vet J. 2014;202:76–82. doi: 10.1016/j.tvjl.2014.06.006. PubMed DOI

Lim LY, Koh PY, Somani S, Al Robaian M, Karim R, Yean YL, Mitchell J, Tate RJ, Edrada-Ebel RA, Blatchford DR, Mullin M, Dufes C. Tumor regression following intravenous administration of lactoferrin- and lactoferricin-bearing dendriplexes. Nanomed Nanotech Biol Med. 2015;11:1445–1454. doi: 10.1016/j.nano.2015.04.006. PubMed DOI PMC

Zemann N, Klein P, Wetzel E, Huettinger F, Huettinger M. Lactoferrin induces growth arrest and nuclear accumulation of Smad-2 in HeLa cells. Biochimie. 2010;92:880–884. doi: 10.1016/j.biochi.2010.03.013. PubMed DOI

Van Berkel PH, Geerts ME, Van Veen HA, Mericskay M, De Boer HA, Nuijens JH. N-terminal stretch Arg2, Arg3, Arg4and Ar g5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA. Biochem J. 1997;328:145–151. doi: 10.1042/bj3280145. PubMed DOI PMC

Na YJ, Han SB, Kang JS, Yoon YD, Park SK, Kim HM, Yang KH, Joe CO. Lactoferrin works as a new LPS binding protein in inflammatory activation of macrophages. Int Immunopharmacol. 2004;4:1187–1199. doi: 10.1016/j.intimp.2004.05.009. PubMed DOI

Puddu P, Carollo MG, Belardelli F, Valenti P, Gessani S. Role of endogenous interferon and LPS in the immunomodulatory effects of bovine lactoferrin in murine peritoneal macrophages. J Leukoc Biol. 2007;82:347–353. doi: 10.1189/jlb.1106688. PubMed DOI

Curran CS, Demick KP, Mansfield JM. Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways. Cell Immunol. 2006;242:23–30. doi: 10.1016/j.cellimm.2006.08.006. PubMed DOI

Oh SM, Hahm DH, Kim IH, Choi SY. Human neutrophil lactoferrin trans-activates the matrixmetalloproteinase 1 gene through stress-activated MAPK signaling modules. J Biol Chem. 2001;276:42575–42579. doi: 10.1074/jbc.M107724200. PubMed DOI

Zhou Y, Zeng Z, Zhang W, Xiong W, Wu M, Tan Y, Yi W, Xiao L, Li X, Huang C, Cao L, Tang K, Li X, Shen S, Li G. Lactotransferrin: a candidate tumor suppressor-deficient expression in human nasopharyngeal carcinoma and inhibition of NPC cell proliferation by modulating the mitogen-activated protein kinase pathway. Int J Cancer. 2008;123:2065–2072. doi: 10.1002/ijc.23727. PubMed DOI

Malinin NL, Boldin MP, Kovalenko AV, Wallach D. MAP3K-related kinase involved in NF-kB induction by TNF, CD95 and IL-1. Nature. 1997;385:540–544. doi: 10.1038/385540a0. PubMed DOI

Xiao G, Harhaj EV, Sun SC. NF-kappa-B-inducing kinase regulates the processing of NF-kappaB2 p100. Mol Cell. 2001;7:401–409. doi: 10.1016/S1097-2765(01)00187-3. PubMed DOI

Zarnegar B, Yamazaki S, He JQ, Cheng G. Control of canonical NF-kappaB activation through the NIK-IKK complex pathway. Proc Natl Acad Sci U S A. 2008;105:3503–3508. doi: 10.1073/pnas.0707959105. PubMed DOI PMC

Pammi M, Abrams SA. Oral lactoferrin for the prevention of sepsis and necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev. 2015;20 PubMed

Nguyen DN, Li Y, Sangild PT, Bering SB, Chatterton DE. Effects of bovine lactoferrin on the immature porcine intestine. Br J Nutr. 2014;111:321–331. doi: 10.1017/S0007114513002456. PubMed DOI

Nguyen DN, Jiang P, Stensballe A, Bendixen E, Sangild PT, Chatterton DE. Bovine lactoferrin regulates cell survival, apoptosis and inflammation in intestinal epithelial cells and preterm pig intestine. J Proteomics. 2016;139:95–102. doi: 10.1016/j.jprot.2016.03.020. PubMed DOI

Choi BK, Actor JK, Rios S, d’Anjou M, Stadheim TA, Warburton S, Giaccone E, Cukan M, Li H, Kull A, Sharkey N, Gollnick P, Kocięba M, Artym J, Zimecki M, Kruzel ML, Wildt S. Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response. Glycoconj J. 2008;25:581–593. doi: 10.1007/s10719-008-9123-y. PubMed DOI PMC

Hwang SA, Kruzel ML, Actor JK. CHO expressed recombinant human lactoferrin as an adjuvant for BCG. Int J Immunopathol Pharmacol. 2015;28:452–468. doi: 10.1177/0394632015599832. PubMed DOI PMC

Bagwe S, Tharappel LJ, Kaur G, Buttar HS. Bovine colostrum: an emerging nutraceutical. J Complement Integr Med. 2015;12:175–185. doi: 10.1515/jcim-2014-0039. PubMed DOI

Vicenova M, Nechvatalova K, Chlebova K, Kucerova Z, Leva L, Stepanova H, Faldyna M. Anti-inflammatory activity of biologically active phospholipids endowed with anti-neoplastic potential in in vivo and in vitro porcine immune systems. BMC Complement Altern Med. 2014;1:339. doi: 10.1186/1472-6882-14-339. PubMed DOI PMC

Majka G, Śpiewak K, Kurpiewska K, Heczko P, Stochel G, Strus M, Brindell M. A high-thoughput method for the quantification of iron saturation in lactoferrin preparations. Anal Bioanal Chem. 2013;405:5191–5200. doi: 10.1007/s00216-013-6943-9. PubMed DOI PMC

Thu YM, Richmond A. NF-κB inducing kinase: A key regulator in the immune system and in cancer. Cytokine Growth Factor Rev. 2010;21:213–226. doi: 10.1016/j.cytogfr.2010.06.002. PubMed DOI PMC

Park GY, Wang X, Hu N, Pedchenko TV, Blackwell TS, Christman JV. NIK is involved in nucleosomal regulation by enhancing histone H3 phosphorylation by IKKalpha. J Biol Chem. 2006;281:18684–18690. doi: 10.1074/jbc.M600733200. PubMed DOI PMC

Oeckinghouse A, Hayden MS, Gosh S. Crosstalk in NF-kB signaling pathways. Nature Immunol. 2011;12:695–708. doi: 10.1038/ni.2065. PubMed DOI

Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis T, Arnold B, Stern DM, Nawroth PP. Understanding RAGE, the receptor for advanced glycation end products. J Mol Med. 2005;83:876–886. doi: 10.1007/s00109-005-0688-7. PubMed DOI

Veloso CA, Fernandes JS, Volpe CM, Fagundes-Netto FS, Reis JS, Chaves MM, Nogueira-Machado JA. TLR4 and RAGE: similar routes leading to inflammation in type 2 diabetic patients. Diabetes Metab. 2011;37:336–342. doi: 10.1016/j.diabet.2010.12.005. PubMed DOI

Sharif O, Knapp S. From expression to signaling: roles of TREM-1 and TREM-2 in innate immunity and bacterial infection. Immunobiology. 2008;213:701–713. doi: 10.1016/j.imbio.2008.07.008. PubMed DOI

Van Beijnum JR, Buurman WA, Griffioen AW. Convergence and amplification of toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1) Angiogenesis. 2008;11:91–99. doi: 10.1007/s10456-008-9093-5. PubMed DOI

Hosoda H, Kida S, Nagaoka I. Transcriptional regulation of mouse TREM-1 gene in RAW264.7 macrophage-like cells. Life Sci. 2011;89:115–122. doi: 10.1016/j.lfs.2011.05.007. PubMed DOI

Arts RJW, Joosten LAB, van den Meer JWM, Netea MG. TREM-1: intracellular signaling pathways and interaction with pattern recognition receptors. J Leukoc Biol. 2013;93:209–215. doi: 10.1189/jlb.0312145. PubMed DOI

Fortin CF, Lesur O, Fulop T., Jr Effects of TREM-1 activation in human neutrophils: activation of signalling pathways, recruitment into lipid rafts and association with TLR4. Int Immunol. 2007;19:41–50. doi: 10.1093/intimm/dxl119. PubMed DOI

Hwang SA, Kruzel ML, Actor JK. Immunomodulatory effects of recombinant lactoferrin during MRSA infection. Int Immunopharmacol. 2014;20:157–163. doi: 10.1016/j.intimp.2014.02.029. PubMed DOI PMC

Hwang SA, Actor JK. Lactoferrin modulation of BCG-infected dendritic cell functions. Int Immunol. 2009;21:1185–1197. doi: 10.1093/intimm/dxp084. PubMed DOI PMC

Kruzel ML, Harari Y, Chen CY, Castro GA. Lactoferrin protects gut mucosal integrity during endotoxemia induced by lipopolysaccharide in mice. Inflammation. 2000;24:33–44. doi: 10.1023/A:1006935908960. PubMed DOI

Hwang SA, Kruzel ML, Actor JK. Influence of bovine lactoferrin on expression of presentation molecules on BCG-infected bone marrow derived macrophages. Biochimie. 2009;91:76–85. doi: 10.1016/j.biochi.2008.04.008. PubMed DOI PMC

Manzoni P, De Luca D, Stronati M, Jacqz-Aigrain E, Ruffinazzi G, Luparia M, Tavella E, Boano E, Castagnola E, Mostert M, Farina D. Prevention of nosocomial infections in neonatal intensive care units. Am J Perinatol. 2013;30:81–88. doi: 10.1055/s-0032-1333131. PubMed DOI

Ochoa TJ, Pezo A, Cruz K, Chea-Woo E, Cleary TG. Clinical studies of lactoferrin in children. Biochem Cell Biol. 2012;90:457–467. doi: 10.1139/o11-087. PubMed DOI

Stronati M, Bollani L, Maragliano R, Ruffinazzi G, Manzoni P, Borghesi A. Neonatal sepsis: new preventive strategies. Minerva Pediatr Febbraio. 2013;65:103–110. PubMed

Shanbacher FL, Goodman RE, Talhouk RS. Bovine mammary lactoferrin: implications from messenger ribonucleic acid (mRNA) sequence and regulation contrary to other milk proteins. J Dairy Sci. 1992;76:3812–3831. doi: 10.3168/jds.S0022-0302(93)77725-5. PubMed DOI

Kruzel ML, Harari Y, Mailman D, Actor JK, Zimecki M. Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice. Clin Exp Immunol. 2002;130:25–31. doi: 10.1046/j.1365-2249.2002.01956.x. PubMed DOI PMC

Drago-Serrano ME, de la Garza-Amaya M, Serrano Luna J, Campos-Rodríguez R. Lactoferrin-lipopolysaccharide (LPS) binding as key to antibacterial and antiendotoxic effects. Int Immunopharmacol. 2012;12:1–9. doi: 10.1016/j.intimp.2011.11.002. PubMed DOI

Watanabe J, Miyazaki Y, Zimmerman GA, Albertine KH, McIntyre TM. Endotoxin contamination of ovalbumin suppresses murine immunologic responses and development of airway hyper-reactivity. J Biol Chem. 2003;278:42361–42368. doi: 10.1074/jbc.M307752200. PubMed DOI

Lin IC, Kuo CD. Pro-inflammatory effects of commercial alpha-lactalbumin on RAW 264.7 macrophages is due to endotoxin contamination. Food Chem Toxicol. 2010;48:2642–2649. doi: 10.1016/j.fct.2010.06.034. PubMed DOI

Cohen MS, Rasmussen GT, Serody JS, Britigan BE. Interaction of lactoferrin and lipopolysaccharide (LPS): effects of antioxidant property of lactoferrin and the ability of LPS to prime human neutrophils for enhanced superoxide formation. J Infect Dis. 1992;166:1375–1378. doi: 10.1093/infdis/166.6.1375. PubMed DOI

Stepanova H, Pavlova B, Stromerova N, Ondrackova P, Stejskal K, Slana I, Zdrahal Z, Pavlik I, Faldyna M. Different immune response of pigs to Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis infection. Vet Microbiol. 2012;159:343–350. doi: 10.1016/j.vetmic.2012.04.002. PubMed DOI

Zelnickova P, Matiasovic J, Pavlova B, Kudlackova H, Kovaru F, Faldyna M. Quantitative nitric oxide production by rat, bovine and porcine macrophages. Nitric Oxide. 2008;19:36–41. doi: 10.1016/j.niox.2008.04.001. PubMed DOI

Kyrova K, Stepanova H, Rychlik I, Faldyna M, Volf J. SPI-1 encoded genes of Salmonella Typhimurium influence differential polarization of porcine alveolar macrophages in vitro. BMC Vet Res. 2012;8:15. doi: 10.1186/1746-6148-8-115. PubMed DOI PMC

Pavlova B, Volf J, Ondrackova P, Matiasovic J, Stepanova H, Crhanova M, Karasova D, Faldyna M, Rychlik I. SPI-1 type III secretion system of Salmonella enterica is required for the suppression of porcine alveolar macrophage cytokine expression. Vet Res. 2011;42:1. doi: 10.1186/1297-9716-42-16. PubMed DOI PMC

Characterization of Porcine Monocyte-Derived Macrophages Cultured in Serum-Reduced Medium