title: Effects of recombinant flagellin B and its ND1 domain from Vibrio anguillarum on macrophages from gilthead seabream (Sparus aurata L.) and rainbow trout (Oncorhynchus mykiss, W.).
abstract: Flagellin is the principal component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in mammals and fish. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines and the co-stimulatory molecules present in antigen-presenting cells needed for the activation of T cells. In the present study, we report the production of two recombinant proteins of Vibrio anguillarum: i) a full length flagellin B (FlaB) (rFla) and ii) the amino-terminus of the D1 domain (rND1) of the same protein, the region mainly responsible for binding to TLR5 and for the immunostimulatory activity of flagellin. The effects of these recombinant proteins were assessed in vitro using head kidney macrophages of gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species, 3 h of stimulation with rFla and rND1 induced expression of the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and of the chemokine IL-8. In gilthead seabream macrophages stimulated with rFla and rND1, a 900- and 6-fold increase were observed for IL-1β transcription, while a 900- and 3-fold increase were recorded for IL-8 transcription, respectively, as compared to non-stimulated macrophages. In rainbow trout, rFla increased expression of IL-8 40-fold in macrophages, whereas rND1 increased expression of the chemokine 3-fold, as compared to non-stimulated cells. The results obtained for rFla and rND1 demonstrate their modulatory capabilities in vitro, suggesting that rFla and rND1 could be evaluated as immunostimulatory candidates for use in farmed fish. However, further in vivo studies are needed to confirm and expand on the present results.
authors.affiliation: Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; Laboratorio de Biotecnología y Patología Acuática, Instituto de Patología Animal, Universidad Austral de Chile, Valdivia, Chile; Centro FONDAP, Interdisciplinary Center for Aquaculture Research (INCAR), Chile. Electronic address: rgstegmaier@gmail.com.
authors.forename: Roxana
authors.initials: R
authors.lastname: González-Stegmaier
authors.affiliation: Laboratorio de Biotecnología y Patología Acuática, Instituto de Patología Animal, Universidad Austral de Chile, Valdivia, Chile; Centro FONDAP, Interdisciplinary Center for Aquaculture Research (INCAR), Chile.
authors.forename: Alex
authors.initials: A
authors.lastname: Romero
authors.affiliation: Institute of Molecular and Cell Biology, Universidad Miguel Hernandez de Elche, Spain.
authors.forename: Amparo
authors.initials: A
authors.lastname: Estepa
authors.affiliation: Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain.
authors.forename: Jana
authors.initials: J
authors.lastname: Montero
authors.affiliation: Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain.
authors.forename: Victoriano
authors.initials: V
authors.lastname: Mulero
authors.affiliation: Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile. Electronic address: lmercado@ucv.cl.
authors.forename: Luis
authors.initials: L
authors.lastname: Mercado
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D017756:Aquaculture / Q000379:methods; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D005408:Flagellin / Q000737:chemistry / Q000494:pharmacology*; D020869:Gene Expression Profiling; D060226:Head Kidney / Q000166:cytology / Q000187:drug effects; D053583:Interleukin-1beta / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects*; D008958:Models, Molecular; D017686:Oncorhynchus mykiss / Q000276:immunology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology*; D021541:Sea Bream / Q000276:immunology* / Q000378:metabolism; D051198:Toll-Like Receptor 5 / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D017931:DNA Primers; D053583:Interleukin-1beta; D011994:Recombinant Proteins; D051198:Toll-Like Receptor 5; D005408:Flagellin; C416532:flaB flagellin
keywords: Fish cytokines; Flagellin; TLR5; Teleost; Vibrio anguillarum