Healing and mucosal immunity in the skin of experimentally wounded gilthead seabream (Sparus aurata L)

Accepted Manuscript Healing and mucosal immunity in the skin of experimentally wounded gilthead seabream (Sparus aurata L) Diana Ceballos-Francisco, Héctor Cordero, Francisco A. Guardiola, Alberto Cuesta, María Ángeles Esteban PII:

S1050-4648(17)30617-4

DOI:

10.1016/j.fsi.2017.10.017

Reference:

YFSIM 4888

To appear in:

Fish and Shellfish Immunology

Received Date: 31 July 2017 Revised Date:

10 September 2017

Accepted Date: 7 October 2017

Please cite this article as: Ceballos-Francisco D, Cordero Hé, Guardiola FA, Cuesta A, Esteban MaríÁ, Healing and mucosal immunity in the skin of experimentally wounded gilthead seabream (Sparus aurata L), Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.10.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Wound tracking

RI PT

Three experimental groups

SC

Wound above LL (A)

EP

TE D

Mucus

Immune parameters

AC C

No wound

M AN U

Wound below LL (B)

&

Skin

Gene expression

ACCEPTED MANUSCRIPT 1 2 3

Healing and mucosal immunity in the skin of experimentally wounded

5

gilthead seabream (Sparus aurata L)

RI PT

4

6

Diana Ceballos-Francisco1, Héctor Cordero1, Francisco A. Guardiola2, Alberto Cuesta1,

8

María Ángeles Esteban1*

SC

7

M AN U

9 10

1.

11

Faculty of Biology, University of Murcia, 30100, Murcia, Spain.

12

2.

13

Marinha e Ambiental (CIIMAR), University of Porto, Terminal de Cruzeiros do Porto

14

de Leixões, Av. General Norton de Matos s/n, 4450-208, Porto, Portugal.

Fish Innate Immune System Group, Department of Cell Biology and Histology,

15 16 17

EP

TE D

Fish Nutrition & Immunobiology Group. Centro Interdisciplinar de Investigação

18

*

19

Faculty of Biology, Campus Regional de Excelencia Internacional “Campus Mare

20

Nostrum”, University of Murcia. 30100 Murcia, Spain

21

E-mail address: [email protected]

AC C

Corresponding author: M.A. Esteban, Department of Cell Biology and Histology,

22 23

1

ACCEPTED MANUSCRIPT Abstract

25

Skin lesions are very common in fisheries, increasing the risk of pathogens entering

26

through the wounded skin of the fish. In the present assay, the progression of wound

27

healing was studied over a 7 day period in gilthead seabream (Sparus aurata L.) after

28

making experimental wounds in two different locations: above (group A) or below

29

(group B) the lateral line. Macroscopic observation confirmed faster wound healing of

30

the wounds of fish from group B. Furthermore, several immune-related components

31

were studied in the skin mucus of wounded fish to ascertain whether wounding altered

32

the mucus composition compared with the values obtained from non-wounded fish

33

(group C, control). Significant variations were detected depending on both the site of

34

the wound and the studied parameter. At the same time, the gene expression profile of

35

several immune-relevant genes, including pro-inflammatory (il1b,il6, tnfa), anti-

36

inflamamtory (tgfb, il10), immunoglobulins (ighm, ight), involved in oxidative stress

37

(sod, cat) and in skin regeneration (krt1and grhl1) were studied in the three groups of

38

fish (A, B and C). The results throw further light on the complex process of skin wound

39

healing in fish, since substantial changes in the skin mucus and in the skin gene

40

expression originated by the presence of wounds were observed. This work underline

41

some important differences depending on the place of the fish body where the wound is

42

located. Of particular note was the fact that such changes depended on the site of the

43

wound.

AC C

EP

TE D

M AN U

SC

RI PT

24

44 45

Keywords: Wound healing; skin; mucus; mucosal immunity; gilthead seabream

46

(Sparus aurata L.); teleosts; aquaculture.

47

2

ACCEPTED MANUSCRIPT 1. Introduction

49

Teleost skin is a living non-keratinized organ that covers the body and protects it not

50

only from the entry of pathogens or allergens, but also from the leakage of water,

51

solutes or nutrients [1,2]. As in other vertebrates, fish skin has a well conserved

52

organization, being composed of the epidermis, dermis and hypodermis [1,3], the

53

outermost layer of the skin, the epidermis, separating the individual from its

54

environment [4]. Thus, fish skin is the first line of defence against biological, chemical

55

and physical injuries such as wounds or ulcers [5]. Covering the fish skin, a mucus layer

56

confers immune protection against pathogen entry [3]. Physiologically, fish skin is

57

involved in several important functions, including immunity [6]. In fact, restriction of

58

entry to the epidermal cells and/or colonization by microbial pathogens occurs on the

59

skin

60

immunoglobulins,

61

antiproteases, esterases, alkaline phosphatase and lysozyme), and numerous

62

antimicrobial peptides are present, not only within the epidermal intercellular fluid but

63

also on the mucus layer [5,7-9].

64

Epidermal integrity is vital for fish defence because any breach in the normal barrier

65

function of the skin may allow colonization by commensal (typically with low

66

pathogenicity) and opportunist pathogenic microorganisms [10], which are always

67

present in the aquatic environment. However, in farmed fish, skin lesions, abrasions or

68

ulcers are produced routinely as a result of poor husbandry practices or some traumatic

69

processes during the handling [11]. After injury, the skin begins to heal in a highly

70

complex biological process, comprising a series of sequential events aiming at repairing

71

the injured tissue [12]. These events can roughly be divided into three phases: 1) a

72

coagulation/fibrinolysis phase, during which a fibrin clot is formed and subsequently

where

many immune-related

C-reactive

proteins,

substances

enzymes

(e.g.:

such

as

peroxidase,

agglutinins, protease,

AC C

EP

TE D

surface,

M AN U

SC

RI PT

48

3

ACCEPTED MANUSCRIPT dissolved; 2) an inflammatory phase, involving the recruitment of cells that fight

74

potential bacterial contamination of the wound and activate the secretion of some

75

cytokines to trigger epidermal and dermal repair processes; and 3) a final phase of repair

76

aimed at re-establishing tissue integrity and functions [13-15]. Previous findings

77

concerning wound healing in the mucosal epithelia of terrestrial vertebrates (such as

78

corneal, gingival, and tracheal tissue) have demonstrated that the highest healing rate

79

occurs in a moist environment, compared to the process occurring in keratinized

80

epithelia [16]. In this same context, as mentioned above, all the layers of the fish skin

81

are formed by living cells, due which they are highly susceptible to both acute and

82

chronic injuries, and, since fish live in an aquatic environment (skin is constantly

83

exposed not to a moist substrate but to water), they have developed fast healing

84

capabilities as a vital defence and survival mechanism [2,16,17]. Indeed, it has been

85

hypothesized that the wound healing process in aquatic species is faster and more

86

efficient compared to that of terrestrial vertebrates [16-18-21].

87

Furthermore, fish integument anatomy can vary with many factors, including species,

88

sex, life stage, season, reproductive condition, nutrition, water quality, location on the

89

body and general health status [16,20-22]. Regarding the body location, two very recent

90

papers reported differences in the gene expression profile between dorsal and ventral

91

skin explants/cells of fish after in vitro exposure to both probiotics and pathogens

92

[23,24]. However, the immune mechanism trigger during skin wound healing still

93

remains unknown in fish. Taken into account all these considerations, the aim of the

94

present work was to study the skin healing progress during 7 post-wound days after

95

making experimental wounds above and below the lateral line on gilthead seabream (S.

96

aurata L.) specimens. Concomitantly, the mucosal immunity was studied by analyzing

97

some important immune parameters in skin mucus as well as the gene expression profile

AC C

EP

TE D

M AN U

SC

RI PT

73

4

ACCEPTED MANUSCRIPT 98

of some immune-relevant genes and others involved in cell regeneration in skin

99

wounded samples. The healing process and the changes originated by the wound in

100

mucus composition and skin gene expression are discussed, taking in consideration the

101

place of the wound in the fish body.

RI PT

102 103

2. Materials and Methods

105

2.1. Animals

106

Fifty four gilthead seabream (S. aurata) specimens (138 ± 10 g and 19 ± 1 cm) obtained

107

from a local farm (San Pedro del Pinatar, Murcia, Spain), were kept in re-circulating

108

seawater aquaria (400 L), with a flow rate of 900 L h -1 at 22 ± 2 °C and 28% salinity in

109

the Marine Fish Facilities at the University of Murcia. A commercial diet (Skretting)

110

was administered at a rate of 2% body weight day-1. The photoperiod was 12 h light:12

111

h dark. All the experimental protocols were approved by the Ethical Committee of the

112

University of Murcia, following the guidelines of European Union for animal handling

113

(2010/63/EU).

114

2.2. Experimental trial and wounds

115

Before making the experimental wounds, fish were sedated with 20 mg L-1 of clove oil

116

(Guinama®) and were afterwards divided into three experimental groups of 18

117

specimens each: the first group (control group or non-wounded group, C) did not

118

receive any wound although fish were manipulated in the same way as the other two

119

groups; all the fish from the second and the third groups were wounded by using a

120

metallic circular biopsy punch with a diameter of 8 mm and 2 mm depth (Stiekel),

AC C

EP

TE D

M AN U

SC

104

5

ACCEPTED MANUSCRIPT either above or below the lateral line: groups A (above) and B (below), respectively. All

122

wounds were made by the same researcher and in the same part of the fish (always in

123

the middle of the left side). The trial was performed in accordance with the wound

124

healing process, the day the wound was made being taken as day 0. The fish were

125

sampled every 24 h during 7 days [25,26], as described below.

126

2.3. Image analysis

127

For macroscopic observation of the healing process, daily images of the wounds were

128

taken with a Canon 7D camera with a wide-angle lens of 22 mm 4.5 (Canon EF)

129

coupled to a ring flash with a tripod. The images were analysed using the Leica QWin

130

image analysis software (Leica Microsystems Ltd.) to determine the wound area (mm2)

131

[27].

132

2.4. Skin mucus immunity

133

2.4.1. Mucus collection

134

Skin mucus was gently collected with a cell scraper (Sigma-Aldrich) from the whole

135

left lateral skin surface of fish, avoiding blood, urine and faeces during collection [28].

136

Mucus samples were vigorously shaken and centrifuged (1,400 g, 10 min, 4 °C) and the

137

supernatants were kept frozen at -20 °C until use. The protein concentration in each

138

sample was determined by the Bradford method [29].

139

2.4.2. Protease activity

140

Protease activity was quantified using the azocasein hydrolysis assay [9]. Briefly, 100

141

µl of skin mucus was incubated with 100 mM ammonium bicarbonate buffer containing

142

100 µl of 0.7%, azocasein (Sigma-Aldrich) for 24 h at 30 °C. The reaction was stopped

AC C

EP

TE D

M AN U

SC

RI PT

121

6

ACCEPTED MANUSCRIPT by adding 250 µl of 4.6% trichloroacetic acid (TCA) and the mixture was centrifuged

144

(6,000 g, 5 min). The supernatants were transferred to a 96-well plate in triplicate

145

containing 100 µl well-1 of 0.5 N sodium hydroxide (NaOH), and the optical density

146

(OD) was read at 450 nm using a plate reader (BMG Labtech). Skin mucus was

147

replaced by trypsin solution (5 mg ml-1, Sigma-Aldrich), as positive control (100% of

148

protease activity), or by buffer, as negative control (0% activity).

149

2.4.3. Antiprotease activity

150

Total antiprotease activity was determined by the capacity of the skin mucus to inhibit

151

trypsin activity [30]. Briefly, 10 µl of skin mucus samples were incubated (10 min,

152

22°C) with the same volume of standard trypsin solution (5 mg ml-1). After adding 100

153

µl of 100 mM ammonium bicarbonate buffer and 125 µl of 0.7% azocasein, the samples

154

were incubated (2 h, 30 °C) and, following the addition of 250 µl, 4.6% TCA, a new

155

incubation (30 min, 30 °C) was carried out. The mixture was then centrifuged (13,000

156

g, 5 min) and the supernatants were transferred to 96-well plates in triplicate containing

157

100 µl well 0.5N NaOH, and the OD was read at 450 nm using a plate reader. For a

158

positive control, buffer replaced skin mucus (100% protease and 0% antiprotease

159

activity), and for a negative control, buffer replaced the trypsin (0% protease and 100%

160

antiprotease activity). The percentage of inhibition of trypsin activity by each sample

161

was calculated.

162

2.4.4. Peroxidase activity

163

The peroxidase activity in skin mucus samples was measured according to [31]. Briefly,

164

10 µl of skin mucus was diluted with 40 µl of Hank's buffer (HBSS) without Ca+2 or

165

Mg+2 in flat-bottomed 96-well plates. As substrates, 100 µl of 10 mM 3,3’,5,5’-

166

tetramethylbenzidine hydrochloride (TMB, Sigma-Aldrich) and 0.015% H2O2 were

AC C

EP

TE D

M AN U

SC

RI PT

143

7

ACCEPTED MANUSCRIPT 167

added. The colour-change reaction was stopped after 2 min by adding 50 µl of 2 M of

168

sulphuric acid (H2SO4) and the OD was read at 450 nm in a plate reader. Standard

169

samples without skin mucus were used as blanks. One unit was defined as the amount

170

producing an absorbance change of 1 and the activity was expressed as U mg

171

proteins.

172

2.4.5. Esterase activity

173

Esterase activity was determined by a colorimetric method [9]. An equal volume of skin

174

mucus samples was incubated with 0.4 mM p-nitrophenylmyristate substrate (Fluka),

175

previously heated at 65 ºC for 10 min and then cooled in 100 mM ammonium

176

bicarbonate buffer containing 0.5% Triton X-100 (pH 7.8, 30°C). The OD was

177

continuously measured at 1 min intervals over 1 h at 405 nm in a plate reader. Standard

178

samples without mucus were used as blanks. The initial rate of the reaction was used to

179

calculate the activity. The activity was expressed as U ml1, which was defined as the

180

amount of enzyme required to release 1 µmol of p-nitrophenylmyristate substrate

181

product in 1 min.

182

2.4.6. Total immunoglobulin M levels

183

Total IgM levels were analysed for gilthead seabream using the enzyme-linked

184

immunosorbent assay (ELISA) [32]. Thus, 10 µg well-1 of skin mucus were placed in

185

flat-bottomed 96-well plates in triplicate coating the proteins by overnight incubation at

186

4 °C with 100 µl of carbonate-bicarbonate buffer (35 mM NaHCO3 and 15 mM

187

Na2CO3, pH 9.6). After three rinses with PBS-T [phosphate buffer saline (PBS) and

188

0.05% Tween 20], plates were blocked for 2 h at room temperature with 200 µl per well

189

of blocking buffer with 3% bovine serum albumin (BSA, Sigma-Aldrich) in PBS-T, and

190

rinsed three times with PBS-T. The plates were then incubated for 1 h with 100 µl per

mucus

AC C

EP

TE D

M AN U

SC

RI PT

-1

8

ACCEPTED MANUSCRIPT well of mouse anti-gilthead seabream IgM monoclonal antibody (1:100 in blocking

192

buffer; Aquatic Diagnostics Ltd.), washed and incubated for 1 h with the secondary

193

antibody anti-mouse IgG-HRP (1:1,000 in blocking buffer; Sigma-Aldrich). After

194

exhaustive rinsing with PBS-T the samples were developed using 100 µl of a 0.42 mM

195

solution of TMB (Sigma-Aldrich), prepared daily in distilled water containing 0.01%

196

hydrogen peroxide (H2O2). The reaction was allowed to proceed for 10 min and stopped

197

by the addition of 50 µl of 2M (H2SO4). The plates were read at 450 nm in a plate

198

reader. Negative controls consisted of samples without skin mucus samples or without

199

primary antibody, whose optical density (OD) values were subtracted for each sample

200

value.

201

2.5. Skin gene expression analysis

202

Skin samples taken from around the wounds were placed in TRIzol® reagent (Life

203

Technologies) and stored at -80 °C for later RNA extraction. RNA from the samples

204

was extracted as indicated by the manufacturer’s instructions, and quantified with

205

Nanodrop®. The RNA was then treated with DNase I (Promega) to remove genomic

206

DNA contamination. Complementary DNA (cDNA) was synthesized from 1 µg of total

207

RNA using the SuperScriptIV reverse transcriptase (Life Technologies) with an oligo-

208

dT18 primer (Life Technologies). The expression of the selected genes was analysed by

209

real-time PCR (qPCR), which was performed with an ABI PRISM 7500 Instrument

210

(Applied Biosystems) as described elsewhere [33] and using the 2 ∆∆Ct method [34]. For

211

each mRNA, gene expression was corrected by both the elongation factor 1 alpha

212

(ef1a) and the ribosomal protein S18 (rps18) RNA content in each sample. Details of

213

primers are listed in Table 1.

214

2.6. Statistical analysis

AC C

EP

TE D

M AN U

SC

RI PT

191

9

ACCEPTED MANUSCRIPT The results were expressed as mean ± SEM. All data were analysed by one- or two-way

216

ANOVA and a Tukey's post-hoc test to determine differences among groups. Normality

217

of the data was previously assessed using a Shapiro-Wilk test and homogeneity of

218

variance was also verified using the Levene test. In the case of non-parametric data, a

219

Kruskal-Wallis H test was performed. For gene expression, data were expressed as fold

220

change obtained by dividing each sample value by the mean control value at the same

221

sampling time. Values higher than 1 express an increase, while values lower than 1

222

express a decrease in the indicated gene [35]. All the statistical analyses were conducted

223

using Statistical Package for Social Science (SPSS for Windows version 19.0) and

224

differences were considered statistically significant when p < 0.05.

M AN U

SC

RI PT

215

225 226

3. Results

228

3.1 Image analysis of wound healing

229

Experimental wounds were made above (A) or below (B) the lateral line of the gilthead

230

seabream specimens and the wounds were photographed daily over a period of 7 days to

231

study the healing progress in both group of fish (Fig. 1). The initial and final stages of

232

wound healing in groups A and B are shown in more detail in Fig. 2.

233

Wound areas were measured from the macroscopic photographs by image analysis. The

234

results indicate that the area of the wounds made in group A increased until 2 days post-

235

wounding and then start to decrease until day 7 post-wounding. However, the area of

236

the wound in group B continued to increase until day 4 post-wounding before beginning

237

to decrease (Fig. 2). However, no significant differences were found between the areas

AC C

EP

TE D

227

10

ACCEPTED MANUSCRIPT of the wounds in A and B fish on any experimental day, although the wound areas in

239

group B were always smaller than those of group A.

240

3.2. Skin mucus immune parameters

241

Different enzymes related to innate immunity were determined in the mucus of

242

wounded fish and the values were compared to those obtained in mucus of the control,

243

non-wounded fish (C). No significant differences were observed between the protease

244

activity in the skin mucus of gilthead seabream from groups A and B compared to the

245

values obtained for the control fish (C) (Fig. 3a). Seven days post wounding the

246

antiprotease activity of group B fish was significantly lower than that observed in

247

group C (Fig. 3b). As regards peroxidase activity, statistically significant decreases and

248

increases , respectively, were detected in mucus from groups B and A 1 or 2 days post-

249

wounding compared with the values recorded in skin mucus from the control group

250

(Fig. 3c). Esterase activity increased in the mucus from group B and was significantly

251

higher 2 days post-wounding, again compared with the values of control group ( (Fig.

252

3d). Finally, total IgM levels in the skin mucus of gilthead seabream were significantly

253

lower in groups B and A 1 and 7 days post-wounding, respectively, with respect to the

254

values recorded in mucus of control fish (Fig. 4).

255

3.3. Gene expression profile in skin

256

The expression profile of eleven genes [five pro-inflammatory or anti-inflammatory

257

cytokines (interleukin 1beta, il1b, interleukin 6, il6, tumor necrosis factor alpha, tnfa,

258

transforming growth factor beta, tgfb, interleukin 10, l10), two involved in wound

259

healing (grainyhead-like transcription factor 1, grhl1 and keratin type 1, krt1), two

260

immunoglobulins (immunoglobulin M heavy chain, ighm, and immunoglobulin T heavy

AC C

EP

TE D

M AN U

SC

RI PT

238

11

ACCEPTED MANUSCRIPT chain, ight) and two involved in oxidative stress (Cu-Zn superoxide dismutase, sod, and

262

catalase, cat)] was studied by real-time PCR in the skin of A, B and C fish. As regards

263

expression of the genes involved in immunity, the transcription of the pro-inflammatory

264

genes (il1b, il6 and tnfa) was very similar. The expression of il1b was significantly up-

265

regulated at day 0, 1 and 2 post-wound in fish from group A. However in B group, the

266

expression increased on day 0 and decreased on the other days studied although none of

267

the variations was statistically significant with respect to the values recorded in fish

268

from group C (Fig. 5a). Similarly, the expression of il6 gene increased in fish from

269

group A sampled on days 0, 1, 2, and 3 post-wounding. By contrast, in group B

270

significant increases were only recorded in fish sampled on day 0 post-wounding (Fig.

271

5b). In the case of tnfa, non-significant increases or decreases were detected in skin

272

from fish of groups A and B, respectively, at all tested times. However, significant

273

differences were detected in the expression of tnfa between fish from groups A and B 7

274

days post-wounding (Fig. 5c).

275

The expression of two anti-inflammatory genes (tgfb and il10) was also studied. The

276

expression of tgfb was significantly increased in fish from A groups sampled at 1 and 7

277

days post-wound. However, the expression in skin samples from fish of B group was

278

always decreased, although the detected decrements never reach significance (Fig. 6a).

279

Finally, regarding il10 the expression significantly increased in A fish sampled 2 days

280

post-wound, respect to the values recorded for fish from A group sampled at other

281

experimental times (Fig. 6b). On the contrary, the expression of il10 in B group always

282

decreased, except in those fish sampled at 3 days post-wound. However, the detected

283

differences were not statistically significant (Fig. 6b).

AC C

EP

TE D

M AN U

SC

RI PT

261

12

ACCEPTED MANUSCRIPT The expression of two anti-inflammatory genes (tgfb and il10) was also studied. The

285

expression of tgfb was significantly higher in fish from group A sampled on days 1 and

286

7 post-wounding while the expression in skin of samples from group B fish was always

287

lower, although the detected decreases never reach significance, always compared with

288

the control fish (Fig. 7a). Finally, the expression of il10 was significantly higher in

289

group A fish sampled 2 days post-wounding than at other experimental times, (Fig. 7b).

290

In contrast, the expression of il10 in group B always decreased, except in those fish

291

sampled at 3 days post-wounding. However, the detected differences were not

292

statistically significant (Fig. 7b).

293

Regarding immunoglobulins, ighm gene expression was higher in the skin of groups A

294

and B than in group C, the decrease always being greater in fish from group A, although

295

the variations observed were not statistically significant (Fig. 7a). However, the

296

expression of gene ight was higher in all wounded fish than in the control fish although

297

the variation was only statistically significant in group A 1 day post-wounding.

298

Furthermore, the expression of this last gene was decreased in a non-significant way in

299

skin from fish of B group at 1 and 7 days post-wound (Fig. 7b).

300

The expression of grhl1 gene was up-regulated (in samples taken from A group) and

301

down-regulated or up-regulated in fish from B group compared with group C.

302

Significant variations were detected in the expression at days 0 and 7 post-wounding in

303

fish from group B. Furthermore, statistically significant differences were detected in the

304

expression of this gene between the samples taken from fish in groups A and B 3 days

305

post-wounding (Fig. 8a). The expression of gene krt1 during the wound healing process

306

was significantly lower in the fish of group A group than in control group fish (from

307

day 0 until 3 days post-wounding). In skin from group B fish significant variations were

AC C

EP

TE D

M AN U

SC

RI PT

284

13

ACCEPTED MANUSCRIPT observed in the down-regulation of this gene expression in samples taken 3 and 7 days

309

post-wound (Fig. 8b).

310

Finally, as regards the expression of genes involved in oxidative stress, the expression

311

of sod was higer in fish from A group (at all the experimental times) while lower in

312

samples from B group although in any case the detected differences were statistically

313

significant (Fig. 9a). Similarly, the expression of cat was higher in samples from A

314

group at all the tested times and in B group in samples taken at 0 and 1 days post-

315

wound. Afterwards (from day 2 till 7 post-wound), the expression of cat decreased

316

although never reached significant extends (Fig. 9b).

M AN U

SC

RI PT

308

317

318

4. Discussion

320

Different types of lesions, abrasions or ulcers may appear not only as a physical process

321

among fish but also as a result of poor husbandry practices or traumatic processes due to

322

the confined environment since farmed fish are often maintained at high densities [10].

323

Skin lesions act as entry sites for pathogens and so fish welfare greatly depends on skin

324

integrity [16,36]. Before carrying out a protocol of prevention or treatment of acute or

325

chronic injuries, it is important to know how skin behaves and responds during the

326

healing process [16]. To the best of our knowledge, only two previous works have

327

studied differences in the skin of two fish species depending on the body location. One

328

was developed using Atlantic cod epidermal cells taken from the dorsal and ventral

329

areas, which were incubated with the probiotics Pseudomonas sp. (GP21) and

330

Psychrobacter sp. (GP12) and with the pathogen (Vibrio anguillarum). The results

AC C

EP

TE D

319

14

ACCEPTED MANUSCRIPT demonstrated differences in the gene expression of the skin according to body location

332

after incubation with the mentioned bacteria, as well as a reduction in cellular apoptosis

333

induced by V. anguillarum in the epidermal cells [23]. More recently, our group

334

demonstrated higher modulation and susceptibility of ventral skin (compared with

335

dorsal skin) to inhibit the cytokine expression profile after in vitro exposure of gilthead

336

seabream skin explants to a pathogen (Photobacterium damselae) and a probiotic

337

(Shewanella putrefaciens, known as SpPdp11) [24]. It is important to underline that

338

both studies were carried out in vitro. However, the present work represents an in vivo

339

study using a widely farmed fish species, gilthead seabream, which was used as a model

340

because of the propensity of farmed fish to suffer skin lesions [10,16] mainly due to the

341

high number of fish that are usually confined in the reduced volume of the tanks or

342

cages. Furthermore, due to the differences detected in vitro between skin cells from

343

different body areas, an in vivo study was made also using gilthead seabream as a fish

344

model [37]. In the above study, the gilthead seabream skin from two different areas

345

(above and below the lateral line, named dorsal and ventral in the manuscript) were

346

compared by studying the isolated epithelial cells and their cell cycle by flow

347

cytometry, as well as the skin histology by scanning electron microscopy and the

348

transcription level of some immune-relevant genes using RT-PCR. As regard

349

morphological differences between both parts of the skin, the results obtained

350

demonstrated that no differences existed in the cell cycle of isolated cells from both

351

zones of the skin or in the gene expression of the genes studied in both epidermal zones.

352

Nevertheless, the cell size and area of microridges in the apical part of the dorsal

353

epidermal cells were larger than in ventral skin epidermal cells, as detected by scanning

354

electron microscopy. Furthermore, the epidermis thickness of the ventral skin was

355

higher than that of the dorsal skin, as demonstrated by image analysis using light

AC C

EP

TE D

M AN U

SC

RI PT

331

15

ACCEPTED MANUSCRIPT microscopy [37]. Additionally, for functional characterization, experimental wounds

357

were made to compare the wound healing rate between the dorsal and ventral regions of

358

skin over the time. The results showed a higher ratio of wound healing in the ventral

359

region, whose wounds were closed after 15 days, compared to dorsal region of skin

360

[37]. These previous results led us to develop the present study in which our attention

361

focused on the first 7 days post-wounding. For this, experimental open wounds were

362

made in two different body locations (above and below the lateral line), in order to

363

establish possible differences between these two skin zones regarding the healing

364

process (now studied by image analysis) as well as the possible influence of the wound

365

on the mucus constituents of the surrounding skin. Taking into account that the

366

morphology of the skin can vary depending on several factors such as location in the

367

body, species, sex, life stage, season, reproductive condition, nutrition and water quality

368

[11,16,20-22,36], the study was developed using fish of the same origin and size and

369

subjected to the same handling procedures.

370

Some authors have described that chronic in wound-healing models wounding with

371

biopsy punch or excisions tends to form non-homogeneous wounds [25]. However,

372

based on the fact that most experimental wounds in animals are made using excisional

373

methods [26,38,39], we have also applied wounds with a biopsy punch since it is the

374

most suitable and reproducible method that we have found. According to the figures

375

presented in this paper, it can be see that all the wounds were very similar. Furthermore,

376

in the present work fish were sampled following the healing progression time line

377

described in previous works [16,25,26].

378

In pigs and rats, it has been described that the healing progression of wounds can be

379

evaluated by means of computerized software and measuring the wound through

AC C

EP

TE D

M AN U

SC

RI PT

356

16

ACCEPTED MANUSCRIPT macroscopic images [38,40]. This method is a simple and fast way to determine the

381

wound area [40], and in the present study, we have adapted this method to fish skin

382

wounds. The initial stage of skin wound healing comprises the haemostasis/coagulation

383

and the inflammatory phase. These phases are closely related since inflammation is

384

activated during haemostasis/coagulation [41]. The inflammatory phase plays a central

385

role in wound healing, not only by encountering the invading microbes or new tissue

386

constituents, but also by participating in the tissue repair processes [12,25].

387

Inflammation prepares the wound for the subsequent phases of healing, and could be

388

divided into an early phase and a late phase [41]. In our study, the size of the wounded

389

areas increased immediately after wounding, probably due to an early inflammatory

390

response, which was also evident macroscopically [25,26]. The area of the wounds

391

started to fall by 4 days post-wounding, in accordance with the course of wound healing

392

course described for fish in general [16,42] and no statistically significant differences

393

were observed between wounds made above and below the lateral line. It has been

394

described that 5 or 6 days post-wounding wounds appear as vascular [16]. However, in

395

the seabream wounds studied in the present work the wounds were not vascularized at

396

any point up to the 7 days studied. This may have been related to the fact that in our

397

experimental wounds all the epithelia and some layers of muscle were removed, which

398

perhaps caused a delay in the healing process, which is faster if the lamina basal of the

399

epithelium is maintained [16].

400

In fish, dermal closure is initiated around 6 days post-wounding, concomitant with

401

granulation-tissue formation [2,25]. This stage is accompanied by attenuation of the

402

inflammatory response and the start of the proliferative stage. The objective of this stage

403

is to achieve protection of the wound surface and is characterized by the appearance of

404

red, fleshy granulation tissue, which ultimately fills the wound [41]. Our macroscopic

AC C

EP

TE D

M AN U

SC

RI PT

380

17

ACCEPTED MANUSCRIPT results agree with all these descriptions observed 7 days post-wounding, when the red

406

colour of some areas in wounds in fish from groups A and B was evident. Furthermore,

407

after 7 days of healing, no changes in skin pigmentation was observed in the wounded

408

area, since perhaps more days are needed to see hyper-pigmentation as a consequence of

409

melanocyte recruitment at the wound site. Besides this, melanocytes persist even after a

410

chronic wound has successfully healed [42]. Melanocytes are involved wherever a

411

recognizable sign of dark shadow on the skin and may be present in the epidermis, and

412

iridophores may be so densely packed, decreasing the space for other types of

413

chromatophores [41]. As mentioned above, no statistical differences were found

414

between the healing processes of A and B fish up to 7 days post-wounding probably due

415

to the high variability of the healing process among fish, as occurs in mammals [43].

416

In fish, an inflammatory response can be observed for 3 to 4 days and is evident 1 to 3

417

hours post injury [16]. In this phase, neutrophils act as a first line of defence in

418

contaminated wounds by destroying debris and bacteria through phagocytosis and

419

subsequent enzymatic and oxygen‐radical mechanisms [41]. Besides the cells, wound

420

healing comprises complicated processes with successive reactions [44] involving a

421

wide variety of innate immune molecules, including complement proteins, lysozyme,

422

proteases, lectins, esterases, immunoglobulins and AMPs, among others [5]. All of them

423

have been previously demonstrated in fish mucus and skin of non-wounded gilthead

424

seabream [7,8,33,45]. In teleosts, these molecules show a more diverse behaviour than

425

in mammals and the antimicrobial properties of epidermal mucus against infectious

426

pathogens has been demonstrated in several fish species [2]. Thus, the mucosal immune

427

response has a fundamental impact on the quality of the tissue response to an injury

428

[25]. For this reason the possible changes occurring in the skin mucus composition after

429

wounding above or below the lateral line were analysed to ascertain whether there were

AC C

EP

TE D

M AN U

SC

RI PT

405

18

ACCEPTED MANUSCRIPT differences in mucus composition that depended on the wound location in the fish body.

431

Regarding enzymatic activity, our previous data showed that several proteins were

432

involved in wound healing (proteases and antiproteases, peroxidase and esterase) [44].

433

Proteases are a group of enzymes responsible for the hydrolysis of peptide bonds

434

[45,46], and may be found in skin mucus, where they contribute to the natural resistance

435

of fish to infection [5] and play a key role in healing, especially in the case of the matrix

436

metalloproteinases (MMPs) and serine proteases [47,48]. In the normal course of the

437

healing process, there is a rapid initial increase in protease activity [47,49,50], reaching

438

a maximum at about 3 days , after which it begins to fall towards 5 days post-wound

439

[47]. No significant differences were recorded in the present study either in protease or

440

antiprotease activities in the mucus of fish from groups A and B withrespect to the

441

values recorded in non-wounded (control) fish and only a significant decrease in the

442

level of antiproteases was recorded in mucus from group B fish 7 days post-wounding.

443

Antiproteases have the capacity to inhibit the proteases which are present in skin mucus,

444

such inhibition perhaps being related to the correct modulation of the response after a

445

wound [46]. In fact, proteases and their inhibitors (antiproteases) contribute to the

446

balance between extracellular matrix (ECM) degradation and deposition [48], and so a

447

correct equilibrium between protease and antiprotease activities is needed for the

448

healing of skin wounds [45,48]. Moreover, peroxidase and esterase enzymes were

449

studied in this work because they act as important microbicidal agents [2,7]. Their

450

importance is maximum because their uncontrolled release might cause severe damage

451

to normal (unwounded) tissues of the host near the wounded zones [50]. The highest

452

values of these two enzymes were always found in gilthead seabream mucus from the

453

fish of group B. The results also point to high activity of these enzymes in skin mucus

454

of fish from groups A and B at 2 and 3 days post-wounding, compared to the values

AC C

EP

TE D

M AN U

SC

RI PT

430

19

ACCEPTED MANUSCRIPT recorded in the mucus of fish from control group, suggesting the importance of

456

secreting antibacterial substances in these early days of the skin healing process.

457

The principal immunoglobulin involved in teleost systemic immunity (the IgM) was

458

also analyzed in skin mucus of wounded fish. It is thought that the IgM antibodies

459

possess a limited antigen spectrum in fishes [5]. Furthermore, the distribution of these

460

antibodies is not uniform. Thus, Ig levels in channel catfish were found to be highest in

461

lateral skin, lower between the pectoral and anal fins, and lowest in the caudal fin and

462

ventral skin [5]. In our study, the decrease of these enzymatic activities and IgM level at

463

7 days post-wounding coincides with the onset of wound healing, the results agreeing

464

with those obtained in other animals and humans, where a higher level of some immune

465

activities in the initial stage of the wound (described as the inflammatory phase) has

466

been demonstrated [12,25,26,41,52]. In addition, ight gene was significantly up-

467

regulated 1 day post-wounding in skin from B fish. This Ig (IgT) represents the most

468

ancient specialized Ig in mucosal immunity. Besides, this Ig plays a key role in the

469

neutralization of bacterial microbiota and pathogens in the skin [53], which could

470

explain the up-regulation of this gene in skin of group B at 1 day post-wounding, that is,

471

when the wound is more susceptible to infection by pathogens and in the skin near the

472

vital organs. In the normal process of wound healing, immune cells and cytokines fall

473

within a few days after an injury [44] as found for all the immune parameters analyzed

474

in the present study.

475

Additionally, the expression of two genes involved in wound healing (grhl1 and krt1)

476

was studied and they were found to be differently regulated. More specifically, in the

477

case of grhl1 gene, significant differences were found at 3 days between the skin

478

samples from groups A and B. In group B fish this gene was significantly higher at day

AC C

EP

TE D

M AN U

SC

RI PT

455

20

ACCEPTED MANUSCRIPT 7 than at day 0. Grhl1 plays an important role in Drosophila organogenesis, epidermal

480

development and regeneration after wounding [51]. Some members of the grhl family,

481

like grhl3gene, are involved in mammalian skin regeneration [46]. Nevertheless, the

482

exact function of this gene in fish skin regeneration remains to be determined. The

483

presence of grhl1 gene has been reported in zebrafish non-keratinocyte epidermal cells

484

[54] and in seabream, during skin regeneration modulated by oestradiol-17β [48,55].

485

Based on these findings, our results could indicate an important role for grhl1 in the

486

wound healing process of gilthead seabream. Curiously, a gene which intervenes in

487

epidermis development [48], krt1 gene, was down regulated at all the experimental

488

times, although the deviations observed were only statistically significant in the skin of

489

fish from group A at 0, 1 and 3 days post-wounding. Our results agree with those found

490

for some keratins in gilthead seabream proteome [48]. Additional studies are needed

491

regarding the implication of this gene in fish skin regeneration.

492

It is known that when neutrophils and monocytes/macrophages arrive in the wound

493

area, they start to secrete large amounts of reactive oxygen species (ROS). The

494

generated ROS directly attack invading pathogens, and finally kill them to aid

495

phagocytosis. The excessive production of ROS is controlled by antioxidant substances

496

like superoxide dismutase (SOD) and catalase (CAT) [56]. The present results also

497

corroborate the involvement of such enzymes in wound healing because an increase in

498

sod and cat genes was observed 1 day post-wounding in skin from group A fish,

499

although the increase was not statistically significant. Furthermore, cat gene expression

500

showed significant differences between the skin of group A and B fish 2 days post-

501

wounding.

AC C

EP

TE D

M AN U

SC

RI PT

479

21

ACCEPTED MANUSCRIPT In conclusion, fish skin wound healing is a complex process that involves a wide variety

503

of substances. The present paper provides new insights into the wound healing process

504

and immunological properties of different skin zones in gilthead seabream. Our results

505

show that the healing process is faster for wounds below the lateral line above the line.

506

Apart from this, most of the parameters analyzed in the skin of group A fish were not

507

affected by the wound. By contrast, below the lateral line the immune parameters

508

analyzed showed variations that were more statistically significant, with respect to the

509

results recorded in skin from control fish. The results suggest that fish skin cells are

510

more sensitive to physical aggression in the area below the lateral line. However, the

511

gene expression of some immune-related genes increased to a greater extent in the skin

512

located above the lateral line. Future studies should focus on fish skin in both body

513

areas in order to understand further how and why the skin of different body locations

514

responds in different manners to the same injury.

515

TE D

M AN U

SC

RI PT

502

Acknowledgements

517

The authors thank A.I.S. and appreciate the services of SAI (Image Analysis Section)

518

from the University of Murcia for the technical support. D.C.F. is grateful to the

519

Spanish Ministry of Economy and Competitiveness (MINECO) for an F.P.I. fellowship

520

(grant no. BES-2015-074726). F.A.G. wishes to thank the Fundação para a Ciência e

521

Tecnologia (Portugal) for grant (SFRH/BPD/104497/2014). This work was supported

522

by the MINECO (grant no. AGL2014-51839-C5-1-R) co-funded by the European

523

Regional Development Funds (ERDF/FEDER) and Fundación Seneca de la Región de

524

Murcia (Grupo de Excelencia grant no. 19883/GERM/15).

AC C

EP

516

525 22

ACCEPTED MANUSCRIPT 526

References

527

[1]

528

Bargelloni, M. Clark, A.V. Canario, D.M. Power, Skin healing and scale regeneration in

529

fed and unfed sea bream, Sparus auratus, BMC Genomics. 12 (2011) 490.

530

[2]

531

Immunol. 2012 (2012) 1–29.

532

[3]

533

Delforges, B. Costes, F. Farnir, B. Leroy, R. Wattiez, C. Melard, J. Mast, F. Lieffrig, A.

534

Vanderplasschen, Skin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3

535

binding to epidermal cells, Vet. Res. 42 (2011) 92.

536

[4]

537

Kruse, R. Paus, “Fish matters”: The relevance of fish skin biology to investigative

538

dermatology, Exp. Dermatol. 19 (2010) 313–324.

539

[5]

540

Aquaculture (Beck BH, Peatman E, eds),( 2015) 67-93.

541

[6]

M. Stoskopf, Fish medicine, WB Saunder, Philadelphia, 1993.

542

[7]

F.A. Guardiola, A. Cuesta, E. Abellán, J. Meseguer, M.A. Esteban, Comparative

543

analysis of the humoral immunity of skin mucus from several marine teleost fish, Fish

544

Shellfish Immunol. 40 (2014) 24–31.

545

[8]

546

gilthead seabream (Sparus aurata L.) immune response under a natural lymphocystis

547

disease virus outbreak, J. Fish Dis. (2016) 1–10.

548

[9]

549

hydrolytic enzyme activities of naïve Atlantic salmon Salmo salar skin mucus due to

RI PT

F.A. Vieira, S.F. Gregório, S. Ferraresso, M. Thorne, R. Costa, M. Milan, L.

M.A. Esteban, An overview of the immunological defenses in fish skin, ISRN

M AN U

SC

V.S. Raj, G. Fournier, K. Rakus, M. Ronsmans, P. Ouyang, B. Michel, C.

TE D

S. Rakers, M. Gebert, S. Uppalapati, W. Meyer, P. Maderson, A.F. Sell, C.

AC C

EP

M.A. Esteban, R. Cerezuela, Fish mucosal immunity: skin.,Mucosal Health in

H. Cordero, A. Cuesta, J. Meseguer, M.A. Esteban, Characterization of the

N.W. Ross, K.J. Firth, A. Wang, J.F. Burka, S.C. Johnson, Changes in

23

ACCEPTED MANUSCRIPT 550

infection with the salmon louse Lepeophtheirus salmonis and cortisol implantation.,

551

Dis. Aquat. Organ. 41 (2000) 43–51.

552

[10]

553

Perspect. 109 (2001) 681–686.

554

[11]

555

for detecting skin ulceration in fish., Vet. Pathol. 39 (2002) 726–731.

556

[12]

557

Immunological aspects, Injury. 37 (2006).

558

[13]

559

ed., Elsevier Health Science. 2003.

560

[14]

561

(1997) 75–81.

562

[15]

563

738–746.

564

[16]

565

healing process, evaluation, and treatment, Vet. Clin. North Am. - Exot. Anim. Pract. 7

566

(2004) 57–86.

567

[17]

568

Ames (IA):, Iowa, 2000.

569

[18]

570

epidermis after wounding a cichlid fish, Hemichromis bimaculatus, Anat. Rec. 254

571

(1999) 435–451.

572

[19]

573

carp, J. Fish Biol. 36 (1990) 421–437.

574

[20]

M. Law, Differential diagnosis of ulcerative lesions in fish, Environ. Health

RI PT

E.J. Noga, P. Udomkusonsri, Fluorescein: a rapid, sensitive, nonlethal method

SC

A.K. Tsirogianni, N.M. Moutsopoulos, H.M. Moutsopoulos, Wound healing:

M AN U

Abbas A.K., A.H. Lichtman, S. Pillai, Cellular and molecular immunology, 5th

P. Martin, Wound healing-aiming for perfect skin regeneration., Science. 276

TE D

R.A. Clark, Singer A.J., Cutaneous wound healing., N. Engl. J. Med. (1999)

EP

D.K. Fontenot, D.L. Neiffer, Wound management in teleost fish: Biology of the

AC C

E.J. Noga, The clinical work-up. In: Fish disease: diagnosis and treatment.,

A. Quilhac, J.Y. Sire, Spreading, proliferation, and differentiation of the

Y. Iger, M. Abraham, The process of skin healing in experimentally wounded

J.M. Groff, Cutaneous biology and diseases of fish., Vet. Clin. North Am. Exot.

24

ACCEPTED MANUSCRIPT 575

Anim. Pract. 4 (2001) 321–411.

576

[21]

577

normal integument., Ocean. Mar. Biol. Ann. Rev. 13 (1974) 383–411.

578

[22]

579

Introduction and filament-containing cells, J. Ultrastruct. Res. 21 (1967) 194–212.

580

[23]

581

regulation of cutaneous immune responses in epidermal cells of Atlantic cod Gadus

582

morhua, Fish Shellfish Immunol. 36 (2014) 113–119.

583

[24]

584

cytokine profile revealed differences from dorsal and ventral skin susceptibility to

585

pathogen-probiotic interaction in gilthead seabream, Fish Shellfish Immunol. 56 (2016)

586

188–191.

587

[25]

588

into wound healing sequence of events., Toxicol. Pathol. 35 (2007) 767–79.

589

[26]

590

harmonic and fluorescence lifetime imaging. J. Biomed. Opt. 18 (2013) 61222.

591

[27]

592

Spauwen, J.A. Jansen, The influence of a PHI-5-loaded silicone membrane, on

593

cutaneous wound healing in vivo, J. Mater. Sci. Mater. Med. 18 (2007) 1449–1456.

594

[28]

595

immune components from the skin mucus of olive flounder (Paralichthys olivaceus),

596

Fish Shellfish Immunol. 24 (2008) 479–488.

597

[29]

598

quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72

599

(1976) 248–254.

Bullock A.M., Roberts R.J., The dermatology of marine teleost fish. I. The

RI PT

R.C. Henrikson, A.G. Matoltsy, The fine structure of teleost epidermis: I.

SC

C.C. Lazado, C.M. Caipang, Probiotics-pathogen interactions elicit differential

M AN U

H. Cordero, M. Mauro, A. Cuesta, M. Cammarata, M.Á. Esteban, In vitro

TE D

L. Braiman-Wiksman, I. Solomonik, R. Spira, T. Tennenbaum, Novel insights

G. Deka, W. Wu, F. Kao, In vivo wound healing diagnosis with second

AC C

EP

M. Van Rossum, D.P.P. Vooijs, X.F. Walboomers, M.J. Hoekstra, P.H.M.

K.J. Palaksha, G.W. Shin, Y.R. Kim, T.S. Jung, Evaluation of non-specific

M.M. Bradford, A rapid and sensitive method for the quantitation of microgram

25

ACCEPTED MANUSCRIPT 600

[30]

A. Hanif, V. Bakopoulos, G.J. Dimitriadis, Maternal transfer of humoral specific

601

and non-specific immune parameters to sea bream (Sparus aurata) larvae, Fish

602

Shellfish Immunol. 17 (2004) 411–435.

603

[31]

604

neutrophil primary granules, Vet. Immunol. Immunopathol. 58 (1997) 239–248.

605

[32]

606

are affected by immunomodulators in seabream (Sparus aurata L.), Vet. Immunol.

607

Immunopathol. 101 (2004) 203–210.

608

[33]

609

mucus proteome map of European sea bass (Dicentrarchus labrax), Proteomics. 15

610

(2015) 4007–4020.

611

[34]

612

real-time quantitative PCR and, Methods. 25 (2001) 402–408.

613

[35]

614

RT-PCR, Nucleic Acids Res. (2001) 29–45.

615

[36]

616

Toxicol. Pathol. 28 (2000) 807–823.

617

[37] H. Cordero, D. Ceballos-Francisco, A. Cuesta, M.A. Esteban, Dorso-ventral skin

618

characterization of the farmed fish gilthead seabream (Sparus aurata).PLoS

619

One. (2017) 12:e0180438.

620

[38]

621

cicatrizante de medicamentos, Rev. Cuba. Farm. 36 (2002) 189–196.

622

[39]

623

Abraham, A. Leask, Expression of integrin beta1 by fibroblasts is required for tissue

624

repair in vivo, J. Cell Sci. 123 (2010) 3674–3682.

RI PT

M.J. Quade, J.A. Roth, A rapid, direct assay to measure degranulation of bovine

SC

A. Cuesta, J. Meseguer, M.A. Esteban, Total serum immunoglobulin M levels

M AN U

H. Cordero, M.F. Brinchmann, A. Cuesta, J. Meseguer, M.A. Esteban, Skin

K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using

TE D

M.W. Pfaffl, A new mathematical model for relative quantification in realtime

AC C

EP

E.J. Noga, Review Article: Skin Ulcers in Fish: Pfiesteria and Other Etiologies,

R. González Escobar, Modelos experimentales para la evaluación de la acción

S. Liu, S. Xu, K. Blumbach, M. Eastwood, C.P. Denton, B. Eckes, T. Krieg, D.J.

26

ACCEPTED MANUSCRIPT [40]

W. Mekkes, J. R., & Westerhof, Image Processing in the Study of wound

626

healing, Clin. Dermatol. (1995) 13(4) 401-407.

627

[41]

E.D. G., Integumentary system, in: Microsc. Funct. Anat., 2000: pp. 271–306.

628

[42]

P. Martin, R. Nunan, Cellular and molecular mechanisms of repair in acute and

629

chronic wound healing, Br. J. Dermatol. 173 (2015) 370–378.

630

[43]

631

statistical analysis of murine incisional and excisional acute wound models, Wound

632

Repair Regen. 22 (2014) 281–287.

633

[44]

634

Biol. 3 (2015) 57–70.

635

[45]

636

humoral immune activities after storage of gilthead seabream (Sparus aurata) skin

637

mucus, Fish Shellfish Immunol. 58 (2016) 500–507.

638

[46]

639

neutrophilic lung disease - Relevance to drug discovery, Br. J. Pharmacol. 158 (2009)

640

1048–1058.

641

[47]

642

proteasas en el diagnóstico de heridas, Wounds Int. (2011) 16.

643

[48]

644

Wound Care. 2 (2013) 438–447.

645

[49]

646

inhibitors in intraperitoneal drainage fluid: Relationship to wound healing, Wound

647

Repair Regen. 11 (2003) 268–274.

648

[50]

649

and tissue repair or regeneration, Semin. Cell Dev. Biol. 20 (2009) 517-527.

RI PT

625

SC

D.M. Ansell, L. Campbell, H.A. Thomason, A. Brass, M.J. Hardman, A

M AN U

T. Kurahashi, J. Fujii, Roles of antioxidative enzymes in wound healing, J. Dev.

H. Cordero, A. Cuesta, J. Meseguer, M.A. Esteban, Changes in the levels of

TE D

C.M. Greene, N.G. McElvaney, Proteases and antiproteases in chronic

EP

S. Calne, K. Day, J. Beckford-Ball, Consenso internacional. Función de las

AC C

S.M. McCarty, S.L. Percival, Proteases and delayed wound healing., Adv.

E.A. Baker, D.J. Leaper, Profiles of matrix metalloproteinases and their tissue

S.A. Eming, M. Hammerschmidt, T. Krieg, A. Roers, Interrelation of immunity

27

ACCEPTED MANUSCRIPT 650

[51]

651

mediated by grainy head, Science. 308 (2005) 381–385.

652

[52]

653

freshwater fish., Curr. Eye Res. 2 (1982) 613–619.

654

[53]

655

R.D. Heinecke, K. Buchmann, S. LaPatra, J.O. Sunyer, Teleost skin, an ancient mucosal

656

surface that elicits gut-like immune responses., Proc. Natl. Acad. Sci. U. S. A. 110

657

(2013) 13097–13102.

658

[54]

659

of different non-keratinocyte epidermal cell lineages, which segregate from each other

660

in a Foxi3-dependent manner., Int. J. Dev. Biol. 54 (2010) 837.

661

[55] A. Ibarz, P.I.S. Pinto, D.M. Power, Proteomic approach to skin regeneration in a

662

marine teleost: modulation by oestradiol-17β, Mar. Biotechnol. 15 (2013) 629–646.

663

[56]

664

inflammatory skin conditions., Int J Mol Sci. 2013 Apr 26;14(5):9126-67.

J.L. Ubels, H.F. Edelhauser, Healing of corneal epithelial wounds in marine and

SC

RI PT

Z. Xu, D. Parra, D. Gomez, I. Salinas, Y.-A. Zhang, L. von Gersdorff Jørgensen,

M AN U

J.M. Chalovich, E. Eisenberg, Zebrafish grainy head-like1 is a common marker

EP

TE D

F.A. Wagener, C.E. Carels, D.M. Lundvig, Targeting the redox balance in

AC C

665

K.A. Mace, An epidermal barrier wound repair pathway in Drosophila is

28

ACCEPTED MANUSCRIPT 666

Figure legends

667 Fig. 1. Representative photographs showing wound healing progression from day 0 to

669

day 7 in skin of gilthead seabream. Control or non-wounded group (C), wounded above

670

the lateral line (A) and wounded below the lateral line (B) groups. Scale bar: 20 mm.

RI PT

668

671

Fig. 2. Experimental wounds (8 mm diameter) above (a, b) and below (c, d) the lateral

673

line at 0 (a, c) and 7 (b, d) days. Arrows indicate the wounds; LL=lateral line. Scale bar:

674

20 mm. e) Wound healing area (mm2). White bars (fish wounded above lateral line) and

675

black bars (below lateral line). Results are expressed as the mean ± SEM (n=3).

M AN U

SC

672

676

Fig. 3. Protease (a) and antiprotease (b) activities, expressed as percentage (%),

678

peroxidase (c) and esterase (d) activities, expressed as U mg -1, found in skin mucus

679

samples of gilthead seabream for each experimental day. White bar (control or non-

680

wounded group, C), grey bars (wound above lateral line, A) and black bars (wound

681

below lateral line, B). Results are expressed as mean ± SEM (n=3). Different letters

682

denote significant differences among groups when p<0.05.

EP

683

TE D

677

Fig. 4. Total immunoglobulin M (IgM) levels found in skin mucus samples of gilthead

685

seabream for each experimental day. White bar (control or no wounded group, C), grey

686

bars (wound above lateral line, A) and black bars (wound below lateral line, B). Results

687

are expressed as mean ± SEM (n=3). Different letters denote significant differences

688

among groups when p<0.05.

AC C

684

689

29

ACCEPTED MANUSCRIPT Fig. 5. Expression profile of pro-inflammatory genes [il1b (a), il6 (b) and tnfa (c)]

691

determined by qPCR in gilthead seabream skin during wound healing. Grey bars

692

(wound above lateral line, A) and black bars (wound below lateral line, B). Results are

693

expressed as mean ± SEM (n=3) fold increase relative to control. Asterisks denote

694

significant differences respect to control or NW group when p<0.05. Hash denotes

695

differences between wounded groups when p<0.05.

696

RI PT

690

Fig. 6. Expression profile of anti-inflammatory genes [tgfb (a) and il10 (b)] determined

698

by qPCR in gilthead seabream skin during wound healing. Grey bars (wound above

699

lateral line, A) and black bars (wound below lateral line, B). Results are expressed as

700

mean ± SEM (n=3) fold increase relative to control. Asterisks denote significant

701

differences with respect to control (group C) when p<0.05. Different capital letters

702

denote differences between experimental times in group A when p<0.05. Different

703

lowercase letters denote between experimental times in group B when p<0.05.

M AN U

TE D

704

SC

697

Fig. 7. Expression profile of immunoglobulins [ighm (a) and ight (b)] determined by

706

qPCR in gilthead seabream skin during wound healing. Grey bars (wound above lateral

707

line, A) and black bars (wound below lateral line, B). Results are expressed as mean ±

708

SEM (n=3) fold increase relative to control. Hash denotes significant differences

709

between wounded (A and B) groups when p<0.05.

AC C

710

EP

705

711

Fig. 8. Expression profile of genes [grhl1 (a) and krt1 (b)] determined by qPCR in

712

gilthead seabream skin during wound healing. Grey bars (wound above lateral line, A)

713

and black bars (wound below lateral line, B). Results are expressed as mean ± SEM

714

(n=3) fold increase relative to control. Asterisks denote significant differences with

30

ACCEPTED MANUSCRIPT 715

respect to the control (group C) when p<0.05. Hash denotes differences between

716

wounded groups when p<0.05. Different capital letters denote differences between

717

experimental times in group A when p<0.05. Different lowercase letters denote between

718

experimental times in group B when p<0.05.

RI PT

719

Fig. 9. Expression profile of oxidative stress genes [sod and cat] determined by qPCR

721

in gilthead seabream skin during wound healing. Grey bars (wound above lateral line,

722

A) and black bars (wound below lateral line, B). Results are expressed as mean ± SEM

723

(n=3) fold increase relative to control. Asterisks denote significant differences with

724

respect to control group when p<0.05.

M AN U

SC

720

725

AC C

EP

TE D

726

31

ACCEPTED MANUSCRIPT Table 1. Primers used in this study. Accession no.

elongation factor 1 alpha

ef1a

AF184170

ribosomal protein S18

rps18

AM490061

interleukin 1beta

il1b

AJ277166

interleukin 6

il6

AM749958

tumor necrosis factor alpha

tnfa

AJ413189

transforming growth factor beta

tgfb

AF424703

interleukin 10

il10

FG261948

grainyhead-liketranscription factor 1

grhl1

AM976768

keratin type 1

krt1

FJ744592

immunoglobulin m heavy chain

ighm

AM493677

immunoglobulin t heavy chain

ight

FM145138

Cu Zn-superoxide dismutase

sod

AJ937872

catalase

cat

FG264808

EP

TE D

Gene symbol according to zebrafish nomenclature (http://zfin.org/).

AC C

728

a

Primer sequence F: TGTCATCAAGGCTGTTGAGC R: GCACACTTCTTGTTGCTGGA F:CGAAAGCATTTGCCAAGAAT R: AGTTGGCACCGTTTATGGTC F:GGGCTGAACAACAGCACTCTC R: TTAACACTCTCCACCCTCCA F: AGGCAGGAGTTTGAAGCTGA R: ATGCTGAAGTTGGTGGAAGG F: TCGTTCAGAGTCTCCTGCAG R: TCGCGCTACTCAGAGTCCATG F: GCATGTGGCAGAGATGAAGA R: TTCAGCATGATACGGCAGAG F: AGGCAGGAGTTTGAAGCTGA R: ATGCTGAAGTTGGTGGAAGG F: GGTGCACCTCCAAACAAGAT R: ATAGCTTCCACCAGGCCTTT F: AGAGATCAATGACCTGCGGC R: CCCTCTGTGTCTGCCAATGT F: CAGCCTCGAGAAGTGGAAAC R: GAGGTTGACCAGGTTGGTGT F: TGGCAAATTGATGGACAAAA R: CCATCTCCCTTGTGGACAGT F:CCATGGTAAGAATCATGGCGG R:CGTGGATCACCATGGTTCTG F: TTCCCGTCCTTCATTCACTC R: CTCCAGAAGTCCCACACCAT

M AN U

Symbol

RI PT

a

Gene name

SC

727

32

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 1

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 2

e

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 3

AC C

EP

Fig. 4

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 5

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 6

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 7

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 8

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 9

ACCEPTED MANUSCRIPT Highlights •

Skin healing was studied in gilthead seabream wounded above or below the lateral line The healing process is faster for wounds below the lateral line

•

Fish skin cells are more sensitive to physical aggression in the area below the

RI PT

•

lateral line

The gene expression of some immune-related genes increased to a greater extent

AC C

EP

TE D

M AN U

in the skin located above the lateral line

SC

•