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1 Flagellin from Marinobacter algicola and Vibrio vulnificus activates the innate 2 immune response of gilthead seabream 3 4 Jana Monteroa, Eduardo Gómez-Casadob, Alicia García Alcázarc, José Meseguera, 5 Victoriano Muleroa* 6 7 a 8 and Institute of Biomedical Research of Murcia, Murcia, Spain 9 b Department of Cell Biology and Histology, Faculty of Biology, University of Murcia Department of Biotechnology, Instituto Nacional de Investigación y Tecnología Agraria 10 y Alimentaria (INIA), Madrid, Spain 11 c 12 Mazarrón, Murcia, Spain Oceanographic Centre of Murcia, Spanish Oceanographic Institute (IEO), Puerto de 13 14 *Corresponding author: Prof. Victoriano Mulero. Department of Cell Biology and 15 Histology, Faculty of Biology, University of Murcia. Campus Universitario de 16 Espinardo. 30100. Murcia. Spain. 17 Tel.: +34 868887581 18 Fax: +34 868883963 19 E-mail: [email protected] 20 1 21 Abstract 22 Adjuvants emerge as the better tool to enhance the efficacy of vaccination. 23 Traditional adjuvants used in aquaculture cause adverse alterations in fish. Thus, it is 24 necessary the development of new adjuvants able to stimulate the immune system and 25 generate high protection against infectious pathogens with minimal undesirable effects. 26 To this end, flagellin emerges as an attractive candidate due to its potency to stimulate 27 the immune response of fish. In the current study, we have evaluated the ability of 28 recombinant flagellin from Marinobacter algicola (MA) and Vibrio vulnificus (Vvul), a 29 non-pathogenic and a pathogenic bacteria, respectively, to stimulate the innate immune 30 system of gilthead seabream (Sparus aurata L.) in comparison with the classical 31 flagellin from Salmonella enterica serovar Thyphimurium (Salmonella Thyphimurium, 32 STF). Intraperitoneal injection of MA and Vvul resulted in a strong inflammatory 33 response characterized by increased reactive oxygen species production and the 34 infiltration of acidophilic granulocytes at the injection site. Interestingly, however, only 35 flagellin from MA consistently induced the expression of the gene encoding pro- 36 inflammatory interleukin-1. These effects were further confirmed in vitro, where a 37 dose-dependent activation of macrophages and acidophilic granulocytes by MA and 38 Vvul flagellins was observed. In contrast, STF flagellin was found to be less potent in 39 either in vivo or in vitro experiments. Our results suggest the potential use of MA and 40 Vvul flagellins as immunostimulants and adjuvants for fish vaccination. 41 42 Keywords: Flagellin, Adjuvant, Immunostimulants, Seabream, Teleosts 43 44 2 45 1. Introduction 46 Nowadays, vaccination appears as the most effective approach to prevent 47 infectious diseases. However, the majority of vaccines in aquaculture are usually not 48 able to confer an effective protection by themselves. A good way to enhance the 49 immune-stimulant effects of vaccines is the co-administration of adjuvants favoring the 50 antigen presentation to immune system. Commercial vaccines include mineral oil-based 51 adjuvants that cause undesirable alterations in fish (Afonso et al., 2005; 2008) and, 52 despite the success against bacterial pathogen, their ability in viral disease has been low 53 (Tafalla et al., 2013). So, it is necessary to understand the fish immune mechanism in a 54 vaccination context to elucidate optimal targets that trigger high immunogenicity with 55 minimal negative effects. 56 The innate immune system recognizes conserved pathogen-associated molecular 57 patterns (PAMPS) by the Toll-like receptor (TLRs) (Medzhitov, 2007). TLRs compound 58 a great family of receptors which stimulation leads to the activation of the transcription 59 factor NF-B and the subsequent expression of pro-inflammatory cytokines and co- 60 stimulatory molecules that end into the induction of the adaptive immune system 61 (Gewirtz et al., 2001; Kawai and Akira, 2007; Means et al., 2003; Rebl et al., 2010; 62 Tsujita et al., 2004). Although they present similar key characteristics in mammals and 63 fish, functional characterization of fish receptors shows differences in signaling 64 pathway and ligands (Phelan et al., 2005; Sepulcre et al., 2009), as the presence of a 65 specific-fish soluble TLR5 (TLR5S). This receptor has been already identified in a 66 diversity of fish species as puffer fish, rainbow trout, catfish or sea bream, but not in 67 mammals (Baoprasertkul et al., 2007; Bilodeau and Waldbieser, 2005; Munoz et al., 68 2013; Oshiumi et al., 2003; Tsujita et al., 2004). The soluble form has lost the typical 69 structure of TLR and only preserves the extracellular leucin-rich repeats domain. 3 70 Flagellin is the ligand of TLR5, and both forms of the receptor are able to recognize it 71 (Hayashi et al., 2001; Oshiumi et al., 2003; Tsujita et al., 2004). Their tissue expression 72 patterns differs and, although the majority of works hypothesize that a TLR5M/ TLR5S 73 interaction modulates the flagellin-mediated immune response, the exact role of TLR5S 74 in fish is very controversial and remains unclear (Hwang et al., 2010; Munoz et al., 75 2013; Sepulcre et al., 2007a). 76 Flagellin is the main structural component of flagella in gram positive and 77 negative bacteria, a filamentous appendage at the bacterial surface involved in their 78 motility, but also performing attachment or chemotaxis functions. It is one of the most 79 powerful PAMP described so far (Hayashi et al., 2001; Tafalla et al., 2013) due to its D1 80 domain involved in binding to the TLR5 and in immunostimulatory activity (Beatson et 81 al., 2006; Eaves-Pyles et al., 2001b; Smith et al., 2003; Takeda et al., 2003; Yonekura et 82 al., 2003; Yoon et al., 2012). Several studies have demonstrated the potent adjuvant 83 ability of flagellin against diverse pathogens in mammals (Bargieri et al., 2011; Bates et 84 al., 2009; Camacho et al., 2011; Cuadros et al., 2004; Honko et al., 2006; Huleatt et al., 85 2007; Lee et al., 2006; Leng et al., 2011; Liu et al., 2011; McDonald et al., 2007; 86 McNeilly et al., 2008; Munoz et al., 2010; Newton et al., 1989; Saha et al., 2006; 87 Skountzou et al., 2010; Strindelius et al., 2004; Turley et al., 2011; Weimer et al., 2009a; 88 Weimer et al., 2009b). This characteristic is the result of its capacity to activate a broad 89 amount of process that are critical for the development of either cellular and also 90 humoral immune responses (Mizel and Bates, 2010; Verma et al., 1995; Zheng et al., 91 2012). However, the use of flagellin for vaccines or immunostimulants has not been 92 widely studied in fish. 93 In this context, we evaluate the immunostimulant properties in vivo and in vitro of 94 three flagellins from different origin: Marinobacter algicola (MA), Salmonella 4 95 thyphimurium (STF) and Vibrio vulnificus (Vvul), in gilthead seabream (Sparus aurata). 96 M. algicola is a Gram-negative and non-pathogenic bacteria, present in marine flora 97 associated with dinoflagellates (Green et al., 2006). On the contrary, S. thyphimurium 98 and V. vulnificus are also Gram-negative but pathogenic species. 99 100 2. Materials and methods 101 102 2.1 Fish 103 Healthy specimens of the hermaphroditic protandrous marine fish gilthead 104 seabream (S. aurata L., Perciformes, Sparidae) were bred and maintained at the Centro 105 Oceanográfico de Murcia, Instituto Español de Oceanografía (IEO) (Mazarrón, Murcia). 106 Fish (approximately 40 gr mean weight) were kept in running seawater tanks (dissolved 107 oxygen 6 ppm, flow rate 20% aquarium volume/h) with natural temperature and 108 photoperiod, and fed twice a day with commercial pellet diet (Skretting, Burgos, Spain) 109 at a feeding rate of 1.5% of fish biomass. All experiments comply with the Guidelines 110 of the European Union Council (86/609/EU), the Spanish RD 53/2013, and the 111 Bioethical Committees of the University of Murcia and the IEO for the use of 112 laboratory animals. 113 114 2.2 Recombinant flagellins 115 The different recombinant flagellins were generated tested as previously reported 116 (Terron-Exposito et al., 2012; Lee Se et al., 2006). Briefly, recombinant pFastbacTM 117 plasmids were used to generate the recombinant baculovirus. Then, in order to express 118 the recombinant flagellins, Sf21 insect cells were infected with each specific 119 baculovirus and the recombinant proteins were purified by affinity chromatography on a 5 120 Co2+ resin (HisPur cobalt resin, Pierce) following the manufacturer’s recommendations. 121 122 2.3 Cell isolation 123 For peritoneal exudate, fish were injected intraperitoneally with 4 ml PBS 124 adjusted to gilthead seabream serum osmolarity (353.33 mOs) with 0.35% NaCl. Then 125 their abdomens were massaged for 10 minutes to dislodge tissue-attached cells into the 126 PBS solution. Incisions were made below of the lateral fin to access the peritoneum, and 127 the peritoneal exudate aspirated and collected into 15 ml Falcon tubes. Head kidney 128 leukocytes were isolated following the method previously described (Sepulcre et al., 129 2002). 130 Some experiments were conducted using purified acidophilic granulocytes 131 obtained by MACS and macrophages monolayers (Roca et al., 2006; Sepulcre et al., 132 2007a). Briefly, macrophages monolayers were obtained after overnight culture of total 133 head kidney leukocytes and the next day the non-adherent cells were removed. 134 Acidophilic granulocytes were purified by MACS following the manufacturer’s 135 instructions by using a monoclonal antibody specific against gilthead seabream 136 acidophilic granulocytes (G7 mAb) (Sepulcre et al., 2002) plus a commercial 137 micromagnetic bead-conjugated anti-mouse IgG antibody (Miltenyi Biotec). The purity 138 was confirmed by flow cytometry. 139 140 2.4 In vivo treatments 141 Fish were injected intraperitoneally with 100µl of phosphate-buffered saline 142 (PBS) alone or containing 1 µg of flagellin per fish. At 3 hours, 1 day, 3 days and 6 days 143 post-treatment, four specimens per treatment were anesthetized with clove oil, bled, and 144 the peritoneal exudate and head kidney were removed. Cell suspensions were then 6 145 obtained to perform respiratory burst assays, immunofluorescence staining and RNA 146 extraction (see below). 147 148 2.5 In vitro treatments 149 Macrophages and acidophilic granulocytes were maintained in RPMI-1640 150 culture medium (Life Technologies) adjusted to gilthead seabream serum osmolarity 151 (353.33 mOs) with 0.35% NaCl, supplemented with 0.1% fetal calf serum (FCS, Life 152 Technologies), 100 I.U./ml penicillin,100 µg/ml streptomycin and 1% L-glutamine, and 153 were disposed in 25 cm2 flask or 24-well plates respectively. Macrophages monolayers 154 were exposed during 3h and acidophilic granulocytes during 18 hour to different 155 concentrations of flagellin. After treatment, RNA was extracted from the cells as 156 described below. 157 158 159 2.6 Respiratory burst assay Respiratory burst activity was measured as the luminol-dependent 160 chemiluminescence produced by 0.6 x 106 cells (Mulero et al., 2001). This was 161 achieved by adding 100µM luminol (Sigma) and 1µg/ml phorbol myristate acetate 162 (PMA, Sigma-Aldrich), while the chemoluminiscence was recorded every 127 seconds 163 for 1 hour in a FLUOstart luminometer (BGM, LabTechnologies). The values reported 164 are the average of triple readings, expressed as the maximum of the reaction curve from 165 127 to 1016s, from which the apparatus background was subtracted. 166 167 2.7 Quantification of G7+ cells (acidophilic granulocytes) 168 The percentage of G7+ cells from head kidney or peritoneal exudate populations 169 was evaluated by using flow cytometry. In the case of head kidney, the G7+ cells 7 170 correspond to the R1 region (FSChigh, SSChigh) as have been already described (Sepulcre 171 et al., 2002). To analyze cells from the peritoneal exudate, 0.2 x 106 cells/well were 172 disposed in a 96-well plate and incubated with 1:1000 dilution of the G7 mAb for 1h at 173 4°C. After three washes, cells were incubated with commercial FITC-labeled anti- 174 mouse IgG, analyzed by a FACSCalibur flow cytometer. The data were analyzed using 175 FlowJo software. All data were obtained from triplicate biological samples to confirm 176 the results. 177 178 2.8 Analysis of gene expression 179 Total RNA was extracted using Trizol (Life Technologies) following the 180 manufacturer’s instructions and quantified with a spectrophotometer (Nanodrop, ND- 181 1000). RNA was then treated with DNAse I (amplification grade 1 unit/µg RNA, Life 182 Technologies), and SuperScript III RNAse H-Reverse Transcriptase (Life Technologies) 183 was used to synthesize first strand cDNA with oligo (dT)18 primer from 1 µg of total 184 RNA at 50 °C for 50 min. The levels of transcription of different genes were determined 185 through real-time PCR (RT-qPCR) with an ABI PRISM 7500 instrument (Applied 186 Biosystems) using SYBR Green PCR Core Reagents (Applied Biosystems). Reaction 187 mixtures were incubated for 10 min at 95 °C, followed by 40 cycles of 15s at 95 °C, 1 188 min at 60 °C and 15s at 95°C. For each mRNA, gene expression was corrected by the 189 ribosomal protein S18 (rps18) content in each sample using the comparative Ct method 190 (2-ΔΔCt). The primers used are shown in Table 1. All amplifications were performed in 191 triplicate to confirm the results. 192 193 194 2.9 Statistical analysis Data were analyzed by one-way ANOVA and a Tukey´s multiple range test to 8 195 determine differences between groups (p ≤ 0.05). For samples that do not follow a 196 Normal distribution, a Kruskal-Wallis non-parametric test and Dunn´s test were used. 197 198 3. Results 199 200 3.1 In vivo effect of the intraperitoneal injection of recombinant flagellins 201 After the intraperitoneal injection of MA, STF and Vvul flagellins, the production 202 of reactive oxygen species (ROS) was measured at different points in isolated cells from 203 head kidney and peritoneal exudates (Fig. 1). In the peritoneal exudate, a strong 204 enhancement on the ROS production was observed at day 1 after the injection with each 205 of the three flagellins, being MA the most powerful in comparison with PBS-injected 206 controls (Fig. 1A). In addition, MA and Vvul significantly increased ROS levels at 3h. 207 However, the response of head kidney leukocytes was not affected by flagellin injection 208 (Fig. 1B). These data suggest the induction of an inflammatory process at the injection 209 site rather than a systemic effect. 210 We next measured by immunofluorescence coupled to flow cytometry the 211 percentage of acidophilic granulocytes following flagellin injection. It was found that 212 flagellins increased the percentage of acidophilic granulocytes in the peritoneal exudate 213 (Fig. 2A). This increase was observed 3 days post-injection for all the three recombinant 214 flagellins. However, the rise was also significant for MA and Vvul at day 1 and for Vvul 215 only at 3h post-injection. Concerning head kidney acidophilic granulocytes, a non- 216 statistically significant reduced percentage was detected at an early time point after the 217 intraperitoneal injection in comparison with control (Fig. 2B). However, the situation 218 changed resulting in an enhanced percentage of acidophilic granulocytes that was more 219 evident for MA at day 3 and for Vvul at day 1 and 3 post-injection. This pattern could 9 220 be associated with the new production of acidophilic granulocytes in the head kidney, in 221 response to an initial migration of cells towards the inflammatory site. 222 The activation of TLR5 by flagellin triggers an inflammatory state, promoting the 223 expression of cytokines as well as ROS production (Sepulcre et al., 2002). Therefore, 224 we analyzed by RT-qPCR the mRNA levels of different genes encoding major pro- and 225 anti-inflammatory cytokines. Since the results obtained for ROS induction pointed to a 226 maximum effect of flagellins at day 1, we examined the mRNA levels of gene encoding 227 IL-1β at this time. Unexpectedly, although the differences among all studied flagellins 228 in ROS production and acidophilic granulocyte mobilization were relatively weak, MA 229 flagellin was the only one able to significantly increase the transcript levels of il1b gene 230 (Fig. 3A). In addition, no significant effects were observed at the other time points 231 analyzed respect to PBS-injected fish (Fig. 3B). For the rest of the pro-inflammatory 232 genes analyzed (il8 and tnfa), as well as for the anti-inflammatory one (il10) (Fig 3B), 233 there was not significant changes in comparison to the control. In general, there were 234 inductions on the mRNA transcription in response to all flagellins, but this occurred at 235 different points depending on the fish. Therefore, the kinetics of induction of every 236 single gene was particular for each individual. 237 238 3.2 The three recombinant flagellins possess different abilities to stimulate gilthead 239 seabream professional phagocytes in vitro 240 The in vivo results prompted us to determine the ability of the different flagellins 241 in the two types of professional phagocytes of this species (Fig. 4). In both cell types, 242 the expression pattern was similar, the effect being dose-dependent. A marked mRNA 243 induction was obtained with MA and Vvul at the major doses (0.1 µg/ml), reaching 244 levels even eight hundred times higher than the control. These results were obtained 10 245 both in granulocytes (Fig. 4A) and also in macrophages (Fig. 4B), although the relative 246 levels of expression were smaller in macrophages for all experimental groups (data not 247 shown). Notably, STF flagellin weakly, but significantly, induced the il1b transcript 248 levels. 249 250 4. Discussion 251 Vaccination results essential to prevent or ameliorate the effects of infectious 252 pathogens. Thank to it, the treatment of antibiotics and the losses related to diseases 253 have decreased in aquaculture (Sommerset et al., 2005). Nevertheless the use of live- 254 attenuated vaccines is limited due to safety problems, and inactivating vaccines do not 255 confer an appropriate protection. At this point, adjuvants emerge as the better tool to 256 produce effective results. Mineral oil-based adjuvants generates adverse affects as 257 granulomas or abdominal lesions (Mutoloki et al., 2004; Mutoloki et al., 2008; 258 Mutoloki et al., 2010; Mutoloki et al., 2006), thus is necessary the optimization of new 259 adjuvants. Through TLR5, flagellin is able to stimulate all models of action described 260 for adjuvants (Tafalla et al., 2013) and numerous studies shows the potent adjuvant 261 capacity of flagellin in stimulating the immune system and increasing the survival in 262 mammals (Bargieri et al., 2011; Bates et al., 2009; Camacho et al., 2011; Cuadros et al., 263 2004; Honko et al., 2006; Huleatt et al., 2007; Lee et al., 2006; Leng et al., 2011; Liu et 264 al., 2011; McDonald et al., 2007; McNeilly et al., 2008; Munoz et al., 2010; Newton et 265 al., 1989; Saha et al., 2006; Skountzou et al., 2010; Strindelius et al., 2004; Turley et al., 266 2011; Weimer et al., 2009a; Weimer et al., 2009b). However, just a few papers are 267 focused on its adjuvant effects in fish, and the results suggest that not all flagellins 268 results in the same protective immune response (Hynes et al., 2011; Jiao et al., 2010; 269 Jiao et al., 2009; Wilhelm et al., 2006). Furthermore, fish species have not equal 11 270 response to immunostimulants (Fierro-Castro et al., 2012; Fierro-Castro et al., 2013). 271 Therefore, studies aimed at identify alternative flagellin proteins and different strategies 272 of vaccination appear pertinent. In this context, we have determined the immune- 273 stimulant properties of MA flagellin (from M. algicola), Vvul flagellin (from V. 274 vulnificus) and the FljB flagellin protein (STF) (from S. thyphimurium) in vivo and in 275 vitro, as a first step to find a good candidate for using as adjuvant in vaccination in 276 gilthead seabream, a teleost specie with great commercial value. 277 In the in vivo experiment, we analyzed the production of reactive oxygen species 278 since it can be used as a reliable marker of the host immune response (Sepulcre et al., 279 2007b). Our results show that MA and Vvul flagellins were able to enhance ROS 280 production by peritoneal exudate cells as earlier as 3 hour post-injection, peaking the 281 effect 1 day post-injection. However, the effect of STF flagellin was only observed 1 282 day post-injection. Unexpectedly, flagellins did not produce any effect on the respiratory 283 burst of head kidney cells. The nitric oxide production or iNOS transcription induced by 284 flagellin treatment has been validated previously in numerous studies performed on 285 different cell types as monocytes, macrophages or ephitelial cells from mammals, with 286 bacteria as well as with recombinant flagellin (Eaves-Pyles et al., 2001b; Mizel et al., 287 2003; Moors et al., 2001; Sierro et al., 2001; Steiner et al., 2000). Eaves-Pyles et al 288 (Eaves-Pyles et al., 2001a) also detected NO liberation after a systemic inflammatory 289 response produced by administration of purified flagellin from Salmonella dublin. In 290 fish, purified flagellin from Bacillus subtilis highly raises the oxidative burst of 291 acidophilic granulocytes in vitro (Sepulcre et al., 2007a). However this is the first study 292 examining the respiratory burst of professional phagocytes in response to flagellin in 293 vivo administration. 294 Acidophilic granulocytes are the major cell type implies in ROS production in 12 295 gilthead seabream (Sepulcre et al., 2007a) and have been considered functionally 296 equivalent to mammalian neutrophils (Chaves-Pozo et al., 2004; Sepulcre et al., 2007a; 297 Sepulcre et al., 2011; Sepulcre et al., 2002). The percentage of these cells (G7+ cells) 298 was measured, showing a marked increased in the injection site from 3 hour to day 6 299 post-injection in the case of MA and Vvul. Once again, STF resulted in a lower 300 response, which was statistically significant only at day 1 respect to the control. 301 Similarly, in mammals, flagellin induces the recruitment of high number of neutrophils 302 to the injection site (Eaves-Pyles et al., 2001a; Honko and Mizel, 2004; Liaudet et al., 303 2003; Neely et al., 2014), and this infiltration is concomitant with the greatest levels of 304 NO (Zgair, 2012). All these results demonstrate the existence of a pro-inflammatory 305 response caused by flagellins at the site of injection characterized by the infiltration and 306 stimulation of professional phagocytic granulocytes together with the production pro- 307 inflammatory cytokines, such as IL-1 (see below), that would be responsible for the 308 recruitment of these cells from the head kidney and its subsequent activation. 309 It is known that flagellin stimulates cytokine production in mammals and fish 310 (Mizel and Bates, 2010) (Tafalla et al., 2013). IL-1β is a pro-inflammatory cytokine 311 whose expression is enhanced in gilthead seabream macrophages and acidophilic 312 granulocytes, following stimulation with different PAMPs, including flagellin (Sepulcre 313 et al., 2002). Surprisingly, in cells from the peritoneal exudates, a slight induction of IL- 314 1β was observed one day post-injection with MA flagellin, while the other flagellins 315 tested failed to do so. For this reason, we focused our attention in MA flagellin but, 316 unexpectedly, there were not differences for tnfa, il8 or il10 genes. In fish, flagellin 317 increased the ability of macrophages to phagocyte, produce nitric oxide or generate 318 respiratory burst (Hardie et al., 1994; Tafalla et al., 2001; Whyte, 2007), including 319 gilthead seabream (Garcia-Castillo et al., 2004). Hynes et al (Hynes et al., 2011) 13 320 reported low levels of IL-1β and IL-8 after injection of two different version of FlaD 321 from Vibrio anguillarum in Atlantic salmon, but quite high for TNF-α. In the present 322 study, we did not observe a consistent induction of TNF-, Il-8 and IL-10 and it is 323 noticeable the great variations among individuals. These differences could reflect a 324 suboptimal dose of flagellin and, probably, higher concentrations would result in more 325 consistent data. 326 To further clarify the mechanisms of action and potency of the different flagellins, 327 we studied their ability to induce in vitro the expression of IL-1β in the two types of 328 professional phagocytes of the gilthead seabream. Both macrophages and acidophilic 329 granulocytes showed a dose-dependent induction of IL-1 gene in response to flagellin 330 stimulation. Once again, MA and Vvul flagellins induced a higher stimulation than STF, 331 confirming the in vivo results. Taking into account that the doses of recombinant 332 flagellin used to stimulate mammalian immune cells are in the range of 1 to 10 µg/ml 333 (Hynes et al., 2011; Purcell et al., 2006; Sepulcre et al., 2007a; Tsujita et al., 2006; 334 Tsujita et al., 2004; Tsukada et al., 2005), it is surprising the strong effect observed in 335 gilthead seabream acidophilic granulocytes and macrophages using as low as 1 ng/ml 336 MA and Vvul flagellins. 337 In conclusion, we have determined the ability of two new recombinant proteins 338 (MA and Vvul) to stimulate the innate immune system of gilthead seabream and 339 demonstrated their greater potency than the classical STF flagellin. The mobilization 340 and activation of professional phagocytes following intraperitoneal injection of these 341 flagellins, together with the powerful activation of macrophages and acidophilic 342 granulocytes in vitro by them, suggests the use of these flagellins as adjuvants for fish 343 vaccination. Further work aimed at elucidating their roles in adaptive immunity is 344 guaranteed. 14 345 Acknowledgements 346 The authors would like to thank to Inma Fuentes for expert technical assistance. 347 Jana Montero wants to thank the Spanish Ministry of Economy and Competence for her 348 Juan de la Cierva research contract. This work was supported by the Spanish Ministry 349 of Economy and Competence (grant BIO2011-23400, co-funded with Fondos Europeos 350 de Desarrollo Regional/European Regional Development Funds), Fundación Séneca- 351 Murcia (grant 04538/GERM/06) and from the European Commission under the 7th 352 Framework Programme for Research and Technological Development (FP7) of the 353 European Union (grant agreement TARGETFISH 311993). 354 355 References 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 Abos, B., Castro, R., Pignatelli, J., Luque, A., Gonzalez, L., Tafalla, C., 2013. 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Primers used for RT-qPCR analysis. 593 Gene Acc. number rps18 AM490061 Primer sequence (5´→ 3´) F: AGGGTGTTGGCAGACGTTAC R: CTTCTGCCTGTTGAGGAACC F: GGGCTGAACAACAGCACTCTC il1b AJ277166 R: TTAACACTCTCCACCCTCCA F: TCGTTCAGAGTCTCCTGCAG tnfa AJ413189 R: CATGGACTCTGAGTAGCGCGA F: GCCACTCTGAAGAGGACAGG il8 AM765841 R: TTTGGTTGTCTTTGGTCGAA F: TGGAGGGCTTTCCTGTCAGA il10 FG261948 R: TGCTTCGTAGAAGTCTCGGATGT 594 595 596 597 598 599 20 600 601 Figure Legends 602 Figure 1. In vivo effect of the intraperitoneal injection of MA, STF or Vvul 603 flagellins on the respiratory burst. Levels of ROS production were measured in 604 isolated cells from peritoneal exudate (A) or head kidney (B) at different times after 605 intraperitoneal injection of 1µg/fish MA, STF or Vvul. Data represents means of four 606 individuals ± S.E.M. Different letters denote statistically significant differences among 607 the groups in the peritoneal exudate or head kidney according to the Tukey test and the 608 Dunns test respectively (p ≤ 0.05). 609 610 Figure 2. In vivo effect of MA, STF or Vvul flagellins on acidophilic granulocytes 611 mobilization. The presence of granulocytes were measured by the percentage of G7+ 612 cells in the peritoneal exudate (A) or R1 (FSChigh, SSChigh) head kidney (B) at different 613 times after intraperitoneal injection of 1µg/fish MA, STF or Vvul. Data represents 614 means of four individuals ± S.E.M. Different letters denote statistically significant 615 differences among the groups according to the Tukey test (p ≤ 0.05). 616 617 Figure 3. In vivo effect of MA, STF or Vvul flagellins on the cytokines expression. 618 Cytokines gene expression in peritoneal exudate cells from fish intraperitoneally 619 injected with 1µg/fish MA, STF or Vvul flagellins. The mRNA levels of il1b were 620 analyzed by RT-qPCR at day 1 post-injection for MA, STF and Vvul (A) or at the 621 indicated times for MA (B). The mRNA levels of il8, tnfa and il10 genes were also 622 measured at the indicated times for MA flagellin (C). The values were normalized to the 623 expression of rps18 and the results are expressed as the fold change compared with the 624 control group (injected with PBS). Data represents means of four individuals ± S.E.M. * 625 Different letters denote statistically significant differences among the groups according 21 626 to the Tukey test (p ≤ 0.05). 627 628 Figure 4. In vitro effect of MA, STF or Vvul flagellins on expression of IL-1 in 629 professional phagocytes. 630 The mRNA levels were determined by RT-qPCR in acidophilic granulocytes (A) and 631 macrophages (B) incubated for 3h or 24h, respectively, with flagellins at 0.1, 0.01 and 632 0.001 µg/ml. The values were normalized to the expression of rps18 and the results are 633 expressed as the fold change compared with the control group (without stimulation). 634 Data represents means of a pool of three fish ± S.E.M. of triplicate samples. *Values 635 significantly higher than those obtained in the control (p ≤ 0.05). 636 22