Download Flagellin from Marinobacter algicola and Vibrio vulnificus activates

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. Transcriptional
heterogeneity of IgM+ cells in rainbow trout (Oncorhynchus mykiss) tissues. PloS one 8, e82737.
Afonso, A., Gomes, S., da Silva, J., Marques, F., Henrique, M., 2005. Side effects in sea bass
(Dicentrarchus labrax L.) due to intraperitoneal vaccination against vibriosis and pasteurellosis.
Fish & shellfish immunology 19, 1-16.
Baoprasertkul, P., Xu, P., Peatman, E., Kucuktas, H., Liu, Z., 2007. Divergent Toll-like receptors in
catfish (Ictalurus punctatus): TLR5S, TLR20, TLR21. Fish & shellfish immunology 23, 12181230.
Bargieri, D.Y., Soares, I.S., Costa, F.T., Braga, C.J., Ferreira, L.C., Rodrigues, M.M., 2011. Malaria
Vaccine Development: Are Bacterial Flagellin Fusion Proteins the Bridge between Mouse and
Humans? Journal of parasitology research 2011, 965369.
Bates, J.T., Uematsu, S., Akira, S., Mizel, S.B., 2009. Direct stimulation of tlr5+/+ CD11c+ cells is
necessary for the adjuvant activity of flagellin. J Immunol 182, 7539-7547.
Beatson, S.A., Minamino, T., Pallen, M.J., 2006. Variation in bacterial flagellins: from sequence to
structure. Trends in microbiology 14, 151-155.
Bilodeau, A.L., Waldbieser, G.C., 2005. Activation of TLR3 and TLR5 in channel catfish exposed to
virulent Edwardsiella ictaluri. Developmental and comparative immunology 29, 713-721.
Camacho, A.G., Teixeira, L.H., Bargieri, D.Y., Boscardin, S.B., Soares, I.S., Nussenzweig, R.S.,
Nussenzweig, V., Rodrigues, M.M., 2011. TLR5-dependent immunogenicity of a recombinant
fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and
the FliC flagellin of Salmonella Typhimurium. Memorias do Instituto Oswaldo Cruz 106 Suppl 1,
167-171.
Cuadros, C., Lopez-Hernandez, F.J., Dominguez, A.L., McClelland, M., Lustgarten, J., 2004. Flagellin
fusion proteins as adjuvants or vaccines induce specific immune responses. Infection and
immunity 72, 2810-2816.
Chaves-Pozo, E., Pelegrin, P., Garcia-Castillo, J., Garcia-Ayala, A., Mulero, V., Meseguer, J., 2004.
Acidophilic granulocytes of the marine fish gilthead seabream (Sparus aurata L.) produce
interleukin-1beta following infection with Vibrio anguillarum. Cell and tissue research 316, 189195.
Eaves-Pyles, T., Murthy, K., Liaudet, L., Virag, L., Ross, G., Soriano, F.G., Szabo, C., Salzman, A.L.,
2001a. Flagellin, a novel mediator of Salmonella-induced epithelial activation and systemic
inflammation: I kappa B alpha degradation, induction of nitric oxide synthase, induction of
proinflammatory mediators, and cardiovascular dysfunction. J Immunol 166, 1248-1260.
Eaves-Pyles, T.D., Wong, H.R., Odoms, K., Pyles, R.B., 2001b. Salmonella flagellin-dependent
15
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
proinflammatory responses are localized to the conserved amino and carboxyl regions of the
protein. J Immunol 167, 7009-7016.
Fierro-Castro, C., Barrioluengo, L., Lopez-Fierro, P., Razquin, B.E., Carracedo, B., Villena, A.J., 2012.
Fish cell cultures as in vitro models of pro-inflammatory responses elicited by immunostimulants.
Fish & shellfish immunology 33, 389-400.
Fierro-Castro, C., Barrioluengo, L., Lopez-Fierro, P., Razquin, B.E., Villena, A.J., 2013. Fish cell cultures
as in vitro models of inflammatory responses elicited by immunostimulants. Expression of
regulatory genes of the innate immune response. Fish & shellfish immunology 35, 979-987.
Garcia-Castillo, J., Chaves-Pozo, E., Olivares, P., Pelegrin, P., Meseguer, J., Mulero, V., 2004. The tumor
necrosis factor alpha of the bony fish seabream exhibits the in vivo proinflammatory and
proliferative activities of its mammalian counterparts, yet it functions in a species-specific manner.
Cellular and molecular life sciences : CMLS 61, 1331-1340.
Gewirtz, A.T., Simon, P.O., Jr., Schmitt, C.K., Taylor, L.J., Hagedorn, C.H., O'Brien, A.D., Neish, A.S.,
Madara, J.L., 2001. Salmonella typhimurium translocates flagellin across intestinal epithelia,
inducing a proinflammatory response. The Journal of clinical investigation 107, 99-109.
Green, D.H., Bowman, J.P., Smith, E.A., Gutierrez, T., Bolch, C.J., 2006. Marinobacter algicola sp. nov.,
isolated from laboratory cultures of paralytic shellfish toxin-producing dinoflagellates.
International journal of systematic and evolutionary microbiology 56, 523-527.
Hardie, L.J., Chappell, L.H., Secombes, C.J., 1994. Human tumor necrosis factor alpha influences
rainbow trout Oncorhynchus mykiss leucocyte responses. Veterinary immunology and
immunopathology 40, 73-84.
Hayashi, F., Smith, K.D., Ozinsky, A., Hawn, T.R., Yi, E.C., Goodlett, D.R., Eng, J.K., Akira, S.,
Underhill, D.M., Aderem, A., 2001. The innate immune response to bacterial flagellin is mediated
by Toll-like receptor 5. Nature 410, 1099-1103.
Honko, A.N., Mizel, S.B., 2004. Mucosal administration of flagellin induces innate immunity in the
mouse lung. Infection and immunity 72, 6676-6679.
Honko, A.N., Sriranganathan, N., Lees, C.J., Mizel, S.B., 2006. Flagellin is an effective adjuvant for
immunization against lethal respiratory challenge with Yersinia pestis. Infection and immunity 74,
1113-1120.
Huleatt, J.W., Jacobs, A.R., Tang, J., Desai, P., Kopp, E.B., Huang, Y., Song, L., Nakaar, V., Powell, T.J.,
2007. Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands
induces rapid cellular and humoral immunity. Vaccine 25, 763-775.
Hwang, S.D., Asahi, T., Kondo, H., Hirono, I., Aoki, T., 2010. Molecular cloning and expression study on
Toll-like receptor 5 paralogs in Japanese flounder, Paralichthys olivaceus. Fish & shellfish
immunology 29, 630-638.
Hynes, N.A., Furnes, C., Fredriksen, B.N., Winther, T., Bogwald, J., Larsen, A.N., Dalmo, R.A., 2011.
Immune response of Atlantic salmon to recombinant flagellin. Vaccine 29, 7678-7687.
Jiao, X.D., Hu, Y.H., Sun, L., 2010. Dissection and localization of the immunostimulating domain of
Edwardsiella tarda FliC. Vaccine 28, 5635-5640.
Jiao, X.D., Zhang, M., Hu, Y.H., Sun, L., 2009. Construction and evaluation of DNA vaccines encoding
Edwardsiella tarda antigens. Vaccine 27, 5195-5202.
Kawai, T., Akira, S., 2007. Signaling to NF-kappaB by Toll-like receptors. Trends in molecular medicine
13, 460-469.
Lee, S.E., Kim, S.Y., Jeong, B.C., Kim, Y.R., Bae, S.J., Ahn, O.S., Lee, J.J., Song, H.C., Kim, J.M., Choy,
H.E., Chung, S.S., Kweon, M.N., Rhee, J.H., 2006. A bacterial flagellin, Vibrio vulnificus FlaB,
has a strong mucosal adjuvant activity to induce protective immunity. Infection and immunity 74,
694-702.
Leng, J., Stout-Delgado, H.W., Kavita, U., Jacobs, A., Tang, J., Du, W., Tussey, L., Goldstein, D.R., 2011.
Efficacy of a vaccine that links viral epitopes to flagellin in protecting aged mice from influenza
viral infection. Vaccine 29, 8147-8155.
Liaudet, L., Szabo, C., Evgenov, O.V., Murthy, K.G., Pacher, P., Virag, L., Mabley, J.G., Marton, A.,
Soriano, F.G., Kirov, M.Y., Bjertnaes, L.J., Salzman, A.L., 2003. Flagellin from gram-negative
bacteria is a potent mediator of acute pulmonary inflammation in sepsis. Shock 19, 131-137.
Liu, G., Tarbet, B., Song, L., Reiserova, L., Weaver, B., Chen, Y., Li, H., Hou, F., Liu, X., Parent, J.,
Umlauf, S., Shaw, A., Tussey, L., 2011. Immunogenicity and efficacy of flagellin-fused vaccine
candidates targeting 2009 pandemic H1N1 influenza in mice. PloS one 6, e20928.
McDonald, W.F., Huleatt, J.W., Foellmer, H.G., Hewitt, D., Tang, J., Desai, P., Price, A., Jacobs, A.,
Takahashi, V.N., Huang, Y., Nakaar, V., Alexopoulou, L., Fikrig, E., Powell, T.J., 2007. A West
Nile virus recombinant protein vaccine that coactivates innate and adaptive immunity. The Journal
of infectious diseases 195, 1607-1617.
16
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
McNeilly, T.N., Naylor, S.W., Mahajan, A., Mitchell, M.C., McAteer, S., Deane, D., Smith, D.G., Low,
J.C., Gally, D.L., Huntley, J.F., 2008. Escherichia coli O157:H7 colonization in cattle following
systemic and mucosal immunization with purified H7 flagellin. Infection and immunity 76, 25942602.
Means, T.K., Hayashi, F., Smith, K.D., Aderem, A., Luster, A.D., 2003. The Toll-like receptor 5 stimulus
bacterial flagellin induces maturation and chemokine production in human dendritic cells. J
Immunol 170, 5165-5175.
Medzhitov, R., 2007. TLR-mediated innate immune recognition. Seminars in immunology 19, 1-2.
Mizel, S.B., Bates, J.T., 2010. Flagellin as an adjuvant: cellular mechanisms and potential. J Immunol
185, 5677-5682.
Mizel, S.B., Honko, A.N., Moors, M.A., Smith, P.S., West, A.P., 2003. Induction of macrophage nitric
oxide production by Gram-negative flagellin involves signaling via heteromeric Toll-like receptor
5/Toll-like receptor 4 complexes. J Immunol 170, 6217-6223.
Moors, M.A., Li, L., Mizel, S.B., 2001. Activation of interleukin-1 receptor-associated kinase by gramnegative flagellin. Infection and immunity 69, 4424-4429.
Mulero, V., Pelegrin, P., Sepulcre, M.P., Munoz, J., Meseguer, J., 2001. A fish cell surface receptor
defined by a mAb mediates leukocyte aggregation and deactivation. Developmental and
comparative immunology 25, 619-627.
Munoz, I., Sepulcre, M.P., Meseguer, J., Mulero, V., 2013. Molecular cloning, phylogenetic analysis and
functional characterization of soluble Toll-like receptor 5 in gilthead seabream, Sparus aurata. Fish
& shellfish immunology 35, 36-45.
Munoz, N., Van Maele, L., Marques, J.M., Rial, A., Sirard, J.C., Chabalgoity, J.A., 2010. Mucosal
administration of flagellin protects mice from Streptococcus pneumoniae lung infection. Infection
and immunity 78, 4226-4233.
Mutoloki, S., Alexandersen, S., Evensen, O., 2004. Sequential study of antigen persistence and
concomitant inflammatory reactions relative to side-effects and growth of Atlantic salmon (Salmo
salar L.) following intraperitoneal injection with oil-adjuvanted vaccines. Fish & shellfish
immunology 16, 633-644.
Mutoloki, S., Alexandersen, S., Gravningen, K., Evensen, O., 2008. Time-course study of injection site
inflammatory reactions following intraperitoneal injection of Atlantic cod (Gadus morhua L.) with
oil-adjuvanted vaccines. Fish & shellfish immunology 24, 386-393.
Mutoloki, S., Cooper, G.A., Marjara, I.S., Koop, B.F., Evensen, O., 2010. High gene expression of
inflammatory markers and IL-17A correlates with severity of injection site reactions of Atlantic
salmon vaccinated with oil-adjuvanted vaccines. BMC genomics 11, 336.
Mutoloki, S., Reite, O.B., Brudeseth, B., Tverdal, A., Evensen, O., 2006. A comparative
immunopathological study of injection site reactions in salmonids following intraperitoneal
injection with oil-adjuvanted vaccines. Vaccine 24, 578-588.
Neely, C.J., Kartchner, L.B., Mendoza, A.E., Linz, B.M., Frelinger, J.A., Wolfgang, M.C., Maile, R.,
Cairns, B.A., 2014. Flagellin treatment prevents increased susceptibility to systemic bacterial
infection after injury by inhibiting anti-inflammatory IL-10+ IL-12- neutrophil polarization. PloS
one 9, e85623.
Newton, S.M., Jacob, C.O., Stocker, B.A., 1989. Immune response to cholera toxin epitope inserted in
Salmonella flagellin. Science 244, 70-72.
Oshiumi, H., Tsujita, T., Shida, K., Matsumoto, M., Ikeo, K., Seya, T., 2003. Prediction of the prototype
of the human Toll-like receptor gene family from the pufferfish, Fugu rubripes, genome.
Immunogenetics 54, 791-800.
Phelan, P.E., Mellon, M.T., Kim, C.H., 2005. Functional characterization of full-length TLR3, IRAK-4,
and TRAF6 in zebrafish (Danio rerio). Molecular immunology 42, 1057-1071.
Purcell, M.K., Smith, K.D., Hood, L., Winton, J.R., Roach, J.C., 2006. Conservation of Toll-Like
Receptor Signaling Pathways in Teleost Fish. Comparative biochemistry and physiology. Part D,
Genomics & proteomics 1, 77-88.
Rebl, A., Goldammer, T., Seyfert, H.M., 2010. Toll-like receptor signaling in bony fish. Veterinary
immunology and immunopathology 134, 139-150.
Roca, F.J., Sepulcre, M.A., Lopez-Castejon, G., Meseguer, J., Mulero, V., 2006. The colony-stimulating
factor-1 receptor is a specific marker of macrophages from the bony fish gilthead seabream.
Molecular immunology 43, 1418-1423.
Saha, S., Takeshita, F., Sasaki, S., Matsuda, T., Tanaka, T., Tozuka, M., Takase, K., Matsumoto, T.,
Okuda, K., Ishii, N., Yamaguchi, K., Klinman, D.M., Xin, K.Q., 2006. Multivalent DNA vaccine
protects mice against pulmonary infection caused by Pseudomonas aeruginosa. Vaccine 24, 62406249.
17
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
Sepulcre, M.P., Alcaraz-Perez, F., Lopez-Munoz, A., Roca, F.J., Meseguer, J., Cayuela, M.L., Mulero, V.,
2009. Evolution of lipopolysaccharide (LPS) recognition and signaling: fish TLR4 does not
recognize LPS and negatively regulates NF-kappaB activation. J Immunol 182, 1836-1845.
Sepulcre, M.P., Lopez-Castejon, G., Meseguer, J., Mulero, V., 2007a. The activation of gilthead seabream
professional phagocytes by different PAMPs underlines the behavioural diversity of the main
innate immune cells of bony fish. Molecular immunology 44, 2009-2016.
Sepulcre, M.P., Lopez-Munoz, A., Angosto, D., Garcia-Alcazar, A., Meseguer, J., Mulero, V., 2011. TLR
agonists extend the functional lifespan of professional phagocytic granulocytes in the bony fish
gilthead seabream and direct precursor differentiation towards the production of granulocytes.
Molecular immunology 48, 846-859.
Sepulcre, M.P., Pelegrin, P., Mulero, V., Meseguer, J., 2002. Characterisation of gilthead seabream
acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in
fish phagocytic response. Cell and tissue research 308, 97-102.
Sepulcre, M.P., Sarropoulou, E., Kotoulas, G., Meseguer, J., Mulero, V., 2007b. Vibrio anguillarum evades
the immune response of the bony fish sea bass (Dicentrarchus labrax L.) through the inhibition of
leukocyte respiratory burst and down-regulation of apoptotic caspases. Molecular immunology 44,
3751-3757.
Sierro, F., Dubois, B., Coste, A., Kaiserlian, D., Kraehenbuhl, J.P., Sirard, J.C., 2001. Flagellin
stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells.
Proceedings of the National Academy of Sciences of the United States of America 98, 1372213727.
Skountzou, I., Martin Mdel, P., Wang, B., Ye, L., Koutsonanos, D., Weldon, W., Jacob, J., Compans,
R.W., 2010. Salmonella flagellins are potent adjuvants for intranasally administered whole
inactivated influenza vaccine. Vaccine 28, 4103-4112.
Smith, K.D., Andersen-Nissen, E., Hayashi, F., Strobe, K., Bergman, M.A., Barrett, S.L., Cookson, B.T.,
Aderem, A., 2003. Toll-like receptor 5 recognizes a conserved site on flagellin required for
protofilament formation and bacterial motility. Nature immunology 4, 1247-1253.
Sommerset, I., Krossoy, B., Biering, E., Frost, P., 2005. Vaccines for fish in aquaculture. Expert review of
vaccines 4, 89-101.
Steiner, T.S., Nataro, J.P., Poteet-Smith, C.E., Smith, J.A., Guerrant, R.L., 2000. Enteroaggregative
Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells.
The Journal of clinical investigation 105, 1769-1777.
Strindelius, L., Filler, M., Sjoholm, I., 2004. Mucosal immunization with purified flagellin from
Salmonella induces systemic and mucosal immune responses in C3H/HeJ mice. Vaccine 22, 37973808.
Tafalla, C., Bogwald, J., Dalmo, R.A., 2013. Adjuvants and immunostimulants in fish vaccines: current
knowledge and future perspectives. Fish & shellfish immunology 35, 1740-1750.
Tafalla, C., Figueras, A., Novoa, B., 2001. Viral hemorrhagic septicemia virus alters turbot Scophthalmus
maximus macrophage nitric oxide production. Diseases of aquatic organisms 47, 101-107.
Takeda, K., Kaisho, T., Akira, S., 2003. Toll-like receptors. Annual review of immunology 21, 335-376.
Terron-Exposito, R., Dudognon, B., Galindo, I., Quetglas, J.I., Coll, J.M., Escribano, J.M., GomezCasado, E., 2012. Antibodies against Marinobacter algicola and Salmonella typhimurium flagellins
do not cross-neutralize TLR5 activation. PloS one 7, e48466.
Tsujita, T., Ishii, A., Tsukada, H., Matsumoto, M., Che, F.S., Seya, T., 2006. Fish soluble Toll-like
receptor (TLR)5 amplifies human TLR5 response via physical binding to flagellin. Vaccine 24,
2193-2199.
Tsujita, T., Tsukada, H., Nakao, M., Oshiumi, H., Matsumoto, M., Seya, T., 2004. Sensing bacterial
flagellin by membrane and soluble orthologs of Toll-like receptor 5 in rainbow trout
(Onchorhynchus mikiss). The Journal of biological chemistry 279, 48588-48597.
Tsukada, H., Fukui, A., Tsujita, T., Matsumoto, M., Iida, T., Seya, T., 2005. Fish soluble Toll-like receptor
5 (TLR5S) is an acute-phase protein with integral flagellin-recognition activity. International
journal of molecular medicine 15, 519-525.
Turley, C.B., Rupp, R.E., Johnson, C., Taylor, D.N., Wolfson, J., Tussey, L., Kavita, U., Stanberry, L.,
Shaw, A., 2011. Safety and immunogenicity of a recombinant M2e-flagellin influenza vaccine
(STF2.4xM2e) in healthy adults. Vaccine 29, 5145-5152.
Verma, N.K., Ziegler, H.K., Stocker, B.A., Schoolnik, G.K., 1995. Induction of a cellular immune
response to a defined T-cell epitope as an insert in the flagellin of a live vaccine strain of
Salmonella. Vaccine 13, 235-244.
Weimer, E.T., Ervin, S.E., Wozniak, D.J., Mizel, S.B., 2009a. Immunization of young African green
monkeys with OprF epitope 8-OprI-type A- and B-flagellin fusion proteins promotes the
18
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
production of protective antibodies against nonmucoid Pseudomonas aeruginosa. Vaccine 27,
6762-6769.
Weimer, E.T., Lu, H., Kock, N.D., Wozniak, D.J., Mizel, S.B., 2009b. A fusion protein vaccine containing
OprF epitope 8, OprI, and type A and B flagellins promotes enhanced clearance of nonmucoid
Pseudomonas aeruginosa. Infection and immunity 77, 2356-2366.
Whyte, S.K., 2007. The innate immune response of finfish--a review of current knowledge. Fish &
shellfish immunology 23, 1127-1151.
Wilhelm, V., Miquel, A., Burzio, L.O., Rosemblatt, M., Engel, E., Valenzuela, S., Parada, G., Valenzuela,
P.D., 2006. A vaccine against the salmonid pathogen Piscirickettsia salmonis based on recombinant
proteins. Vaccine 24, 5083-5091.
Yonekura, K., Maki-Yonekura, S., Namba, K., 2003. Complete atomic model of the bacterial flagellar
filament by electron cryomicroscopy. Nature 424, 643-650.
Yoon, S.I., Kurnasov, O., Natarajan, V., Hong, M., Gudkov, A.V., Osterman, A.L., Wilson, I.A., 2012.
Structural basis of TLR5-flagellin recognition and signaling. Science 335, 859-864.
Zgair, A.K., 2012. Escherichia coli flagellin stimulates pro-inflammatory immune response. World journal
of microbiology & biotechnology 28, 2139-2146.
Zheng, Z., Yingeng, W., Qingyin, W., Nannan, D., Meijie, L., Jiangbo, Q., Bin, L., Lan, W., 2012. Study
on the immune enhancement of different immunoadjuvants used in the pentavalent vaccine for
turbots. Fish & shellfish immunology 32, 391-395.
19
592
Table 1. 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