Local and systemic adaptive immune responses toward viral infection via gills in ginbuna crucian carp

Developmental and Comparative Immunology 52 (2015) 81–87

Contents lists available at ScienceDirect

Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i

Local and systemic adaptive immune responses toward viral infection via gills in ginbuna crucian carp Tomonori Somamoto a,*, Yuhei Miura a, Teruyuki Nakanishi b, Miki Nakao a a Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan b Department of Veterinary Medicine, Nihon University, Kanagawa, Fujisawa 252-8510, Japan

A R T I C L E

I N F O

Article history: Received 22 December 2014 Revised 23 April 2015 Accepted 24 April 2015 Available online 29 April 2015 Keywords: Gills Mucosal immunity Systemic immunity Ginbuna crucian carp Virus

A B S T R A C T

Recent studies on fish immunity highlighted the significance of gills as mucosal immune tissues. To understand potential of gills as vaccination sites for inducing adaptive systemic immunity, we investigated virus-specific cell-mediated and humoral immune responses following a “per-gill infection method”, which directly exposes virus only to gills. The viral load in crucian carp hematopoietic necrosis virus (CHNV)infected gills decreased after peaking at a particular time point. Furthermore, the viral titers in the gills following the secondary infection were lower than that after the primary infection, indicating that local adaptive immunity helped the elimination of virus. Gene expression analysis demonstrated that IFN-γ in gills and perforin in kidney were increased after the gill infection. CD8+ cells in kidney leukocytes increased after the secondary infection, whereas IgM+ cells decreased. These results suggest that IFN-γ and CTL contribute in controlling CHNV-replication in gills and kidney. Gill infection could induce specific cell-mediated cytotoxicity of peripheral blood leukocytes (PBL) and secretion of CHNV-specific IgM in serum, indicating that local priming of the gill site can generate adaptive systemic immunity. Thus, the gills could be prospective antigen-sensitization sites for mucosal vaccination. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction There are several anatomical and physiological differences between mammals and fishes. Contrast in their environmental habitat has created significant differences in mucosal organs; for example, the presence of gills and mucous skins and the absence of Peyer’s patches. The mucosal organs of fish prevent invasion of pathogens from their surrounding environment. Several recent reviews have suggested that fish have robust mucosal immune systems, and hence it is important to conduct further studies to improve our understanding of its functions (Gomez et al., 2013, 2014; Lazado and Caipang, 2014; Rombout et al., 2011, 2014; Salinas et al., 2011). The gills are one of the first organs exposed to pathogens, and thus, is thought to be an important organ in fish mucosal immunity (Andrews et al., 2010; Murray et al., 2007). Transcriptional analysis has demonstrated that many genes that are involved in adaptive immunity are abundantly expressed in gills (Abdelkhalek

* Corresponding author. Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan. Tel.: (81) 92 642 2895; fax: (81) 92 642 2897. E-mail address: [email protected] (T. Somamoto). http://dx.doi.org/10.1016/j.dci.2015.04.016 0145-305X/© 2015 Elsevier Ltd. All rights reserved.

et al., 2014; Aquilino et al., 2014; Koppang et al., 1998a,b; Rebl et al., 2014; Takizawa et al., 2011). In addition, antibody producing cells have been isolated from gills and IgM has been detected in gill mucus in several fish species (Salinas et al., 2011; von Gersdorff Jorgensen et al., 2011; Zilberg and Klesius, 1997). IgT, a unique immunoglobulin isotype in teleosts, appears to be specialized in mucosal immunity and is present in the gill mucosa (Aquilino et al., 2014; Fillatreau et al., 2013; Salinas et al., 2011). Castro et al (2014) have recently reported that the chemokine receptor CCR7, which represents an important determinant for circulating lymphocytes to enter lymph nodes in mammals, is mainly expressed in trout gills. Furthermore, a unique T-cell-rich intraepithelial structure in gills was identified in Atlantic salmon (Haugarvoll et al., 2008; Koppang et al., 2010; Aas et al., 2014; Weli et al., 2013). Thus, cellular, molecular, or histological analyses have provided evidence that the teleost gill is an important site to induce systemic adaptive immunity as a secondary lymphoid organ. Ginbuna crucian carp, a naturally occurring gynogenetic fish, is a useful model animal for studying adaptive immunity (Fischer et al., 2013; Nakanishi et al., 2011; Somamoto et al., 2014a). We have investigated the antiviral functions of cytotoxic T-lymphocytes (CTLs) and helper T cells (Th-cells) using this clonal fish and the crucian carp hepatopoietic necrosis virus (CHNV), suggesting that their functions are basically similar to those in mammals (Somamoto et al., 2009 2013 2014b). Because these studies demonstrated that

82

T. Somamoto et al./Developmental and Comparative Immunology 52 (2015) 81–87

intraperitoneal (i.p.) injection with virus can induce systemic CTL and Th-cell responses, it is unclear whether mucosal sensitization can also effectively activate adaptive anti-viral immunity. The “pergill infection method”, which directly exposes virus to only gills, has previously been developed as a procedure to infect fish with koi herpesvirus (Miyazaki et al., 2008). We believe that this method enables focusing on immune responses induced by sensitization through gills. Therefore, the present study has examined the systemic and mucosal immune responses following infection with CHNV via the gill. 2. Materials and methods

400 × g for 40 min at 4 °C. The cells at the top of the Histopaque were collected and washed twice with MEM-10. The cell number from each cell was adjusted to 0.5–1.0 × 106 cells/mL with MEM-10. The cell suspensions were equally divided into three parts (5 × 105 cells/ each suspension) and incubated with a 1:10,000 dilution of either rat anti-ginbuna CD8α mAb (mouse ascites), anti-ginbuna CD4 mAb, or anti-ginbuna IgM mAb for 30 min on ice (Somamoto et al., 2013; Toda et al., 2011a). The cells were then washed twice with medium, incubated for 30 min at 4 °C with a 1:100 dilution of anti-rat IgG goat antibody conjugated with phycoerythrin (PE; Abcam, Cambridge, MA, USA) and then washed additionally three times. The cell percentages were assessed by flow cytometry (EPICS XL; Beckman Coulter, Brea, CA, USA).

2.1. Fish and virus The S3n and OB1 strains of ginbuna crucian carp, Carassius auratus langsdorfii, were maintained at a temperature of 25 °C and were daily fed with commercial food pellets. CFS (Carassius fin from Lake Suwa) cells from the S3n strain of ginbuna crucian carp were used as syngeneic target cells and for propagation of the CHNV, as described by Somamoto et al. (2014a,b). The S3n strain was used in the cellmediated cytotoxicity assay, because the target cell line was available. The OB1 strain was used in experiments other than the cytotoxic assay. 2.2. Infection with CHNV through gills The method of infection was referred to as a “per-gill infection method”, which was previously reported (Miyazaki et al., 2008), and it was modified for CHNV and ginbuna crucian carp. Clonal ginbuna crucian carp (OB1 and S3n strain) were anesthetized in 25 mg/L quinaldine and inoculated with 0.5 mL/50 g fish weight of CHNV solution (107.8 TCID50/mL in PBS) into their gills (to both sides) in air. After the treatment, fish were wrapped with wet papers and kept in air for 5 min at 25 °C to allow the virus to adsorb into gill tissue. The fish were then returned to the tank and maintained at 25 °C. The control fish were treated with PBS instead of CHNV. Two weeks after the primary infection, the secondary infection was performed using the same procedure described earlier. 2.3. Virus titer from organs Eighteen ginbuna crucian carp (OB1 strain), weighing 18–23 g, was used in this experiment. The gills and trunk kidney were collected at 1, 12, and 24 h post-primary and -secondary infection. The respective organs were placed in MEM (1:10, weight: volume) containing 10% FBS, homogenized and then centrifuged at 1000 × g for 20 min. The supernatants were passed through a 0.45-μm membrane filter and the filtrate was then stored at −80 °C until further required. Virus titers were determined by a TCID50 endpoint titration in CFS cells incubated for 21 days at 25 °C. Three fish were sampled at each time point in both primary and secondary infections. The results were expressed as log10 TCID50 per gram of organ. 2.4. Leukocyte composition in the gill and kidney from infected fish The infection procedure described in the previous section was followed in this study too. Three fish were sampled at 24 h after the infection and PBS treatment. The trunk kidneys from control and infected fish were disaggregated by passing them through a 150gauge mesh stainless steel sieve in MEM supplemented with 10% heat-inactivated FBS (MEM-10) (Nissui Pharmaceutical Co., Tokyo, Japan). Whole gills including gill arch were excised and only the lamellae were disaggregated through the stainless steel sieve. The cells were washed with MEM-10, applied to a Histopaque density of 1.083 g/mL (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at

2.5. Real-time quantitative PCR Three fish from the control and infected groups sampled at 1, 12, and 24 h post-primary and post-secondary infections were employed for assessing the gene expression in the cells of interest that were collected as described in Section 2.4. Total RNA was extracted from 1.0 × 106 cells of the gill and kidney leukocytes using the NucleoSpin RNA II (Machery-Nagel. GmbH Co KG, Duren, Germany), according to the manufacturer’s protocol. First-strand cDNA was synthesized from total RNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA) with an oligo (dT) primer, according to the manufacturer’s instructions. Primers used for real-time PCR were designed to amplify cDNA fragments encoding the following cytokines and perforin: IFN-γ1 and IFN-γ2 (Yamasaki et al., 2014), IL-10 (accession HQ259106), and Perforin-1 (Toda et al., 2011b). An internal control for normalization was EF-1α (Toda et al., 2011b). The sequences or each primer sets of IFN-γ, perforin and EF-1α are indicated in the reference articles. The primer sequences for IL-10 were as follows: forward primer (5′-TTGGCACCATTACTCGATGA-3′) and reverse primer (5′TCCAAGTAGAAGCGCAGGAT-3′). Quantitative real-time PCR was performed in duplicate on a M × 3000P System (Stratagene, La Jolla, CA, USA) in 16 μL reaction mixtures containing 2 μL of template cDNA, 0.5 μM primers, and other reagent components from the Fast Start DNA Master SYBR_Green (Roche Applied Science, Mannheim, Germany). Thermal cycling was performed using a two-step thermal cycling mode composed of an initial denaturation for 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 or 40 s at 55 °C (EF-1α, IFN-γ1, and IFN-γ2) or 60 °C (IL-10 and perforin). The relative quantitative value of each gene was calculated according to the standard curve from a serial dilution of a reference cDNA in the same plates and normalized with the level of EF1α. 2.6. Cell-mediated cytotoxic assay The S3n strains of ginbuna crucain carp, weighing 63–80 g, were used in the cell-mediated cytotoxic assays. CHNV-infected fish (n = 3) were sampled 2 and 4 days post-secondary infection and were bled from the caudal vein into heparinized syringes. PBLs were isolated using a Percoll gradient method as previously described (Somamoto et al., 2013, 2014a,b). Blood were centrifuged (400 × g, 5 min, 4 °C), and the buffy coat was collected and diluted in OPTIMEM (Invitrogen, Carlsbad, CA). These cells were applied to a Percoll (Sigma Chemical Co., St. Louis, MO, USA) at a density gradient of 1.08 g/mL and centrifuged at 450 × g for 30 min at 4 °C. The top layer of cells in the Percoll was collected and the cells were washed twice with OPTI-MEM by centrifugation (450 × g, 5 min, 4 °C). Cells were then re-suspended in DMEM/F-12 medium (Invitrogen, Carlsbad, CA) without phenol red and with 1% FBS (DMEM/F12-1) and then used as effector cells for a cytotoxicity assay.

T. Somamoto et al./Developmental and Comparative Immunology 52 (2015) 81–87

2.7. Detection of CHNV-specific IgM by ELISA

8

Viral titer (Log 10 TCID50 / gram of the gill)

The cytotoxicity assay was previously described (Somamoto et al., 2013, 2014a,b). In brief, CFS cells, used as syngeneic target cells, were seeded in 96-well, flat-bottom microtiter plates (Nunc, Roskilde, Denmark) at 104 cells/well and allowed to settle for 6 h. Half of the wells with target cells were infected with CHNV at 25 °C for 3–4 h (MOI = 10). Effector cells were adjusted to 8.0 × 106 cells/mL and 4.0 × 106 cells/mL, and 100 μL of the effector suspension was added to each well. At least duplicate wells were analyzed. Cytotoxic activity was detected by lactate dehydrogenase (LDH) release using a Cytotoxicity Detection Kit-LDH (Takara Bio Inc, Shiga, Japan), according to the manufacturer’s protocol.

83

b

7

ab 6 5

a

4

a

a

a 3 2 1 0

CHNV-infected and control fish (OB1 strain of ginbuna crucian carp; weighting 74–94 g) were sampled at 7 days following the primary per-gill infection, and at 7, 14, and 21 days after the secondary infection. Less than 100 μL of blood was collected from caudal vessels, and it was allowed to clot at 4 °C for 2 h. The blood samples were collected from three fish in each group. The serum was separated by centrifugation at 2000 × g for 10 min at room temperature. The sera were stored at −80 °C until further required. Test sera diluted 1:80 in PBS were added to each well and then incubated for 2 h at room temperature. An ELISA to detect IgM directed against CHNV was performed as previously described (Somamoto et al., 2013). 2.8. Statistical analysis One-way ANOVA, followed by Tukey’s multiple comparison tests was used to understand the viral titer changes in gills, at different time points. A similar approach was employed in finding the differences in mRNA level fold changes in two tissue cell types, at a particular time point. To compare the lymphocyte subsets between the control group and infected group for a cell type, Welch’s t-test was performed. Welch’s t-test was also employed for comparing the CHNV-specific antibody production in control and infected fish. Cytotoxic activity of PBLs after secondary infection was analyzed using paired t-test. The reported significant differences are at P < 0.05. 3. Results 3.1. Viral replication in gills after per-gill infection The kinetics of virus replication in the gills is shown in Fig. 1. Viral loads in gills increased 1–12 h after both primary and secondary infections, confirming that CHNV replicated following pergill infected fish. The gills that were collected 12 h after the secondary infection had significantly lower virus titers than those after the primary infection (p < 0.05), indicating that adaptive immune responses were evoked in the gills by the infection. The virus titer was at undetectable levels in the kidney from all CHNV-infected fish (data not shown). 3.2. Expression of cytokines and perforin after per-gill infection Expression of cytokines and perforin in the gill and kidney of infected fish is shown in Fig. 2. IFN-γ1 mRNA levels in gill leukocytes were significantly upregulated 12 h post-primary infection and 1 h post-secondary infection (p < 0.05), whereas no significant difference was noted in kidney leukocytes. Perforin mRNA in kidney and gill leukocytes decreased 1 and 12 h post-secondary infection and then significantly increased at 24 h in gills only. In both the kidney and gills, expression of IL-10 and IFN-γ2 was not upregulated after the per-gill infection. Expressions of all genes were not

1

12

Primary infection

24

1

12

24

Secondary infection

Time after infection (hour) Fig. 1. Viral replication in the gills following per-gill infection. Viral titers in the gill after primary and secondary infections were determined by a TCID50 endpoint titration on CFS cells. Results are presented as the means of three individual fish, and error bars indicate the standard deviation (SD). Statistical comparisons in viral titers were made by one-way ANOVA and Tukey’s post hoc test. Means with different letters are significantly different (P < 0.05).

significantly different in control-treated templates (data not shown). Taken together, perforin, which is produced in cytotoxic cells such as NK-like cells, CTLs, and IFN-γ1, were elevated after gill infection, indicating that antiviral cell-mediated immunity was induced by the infection. 3.3. Composition of CD8α+, CD4+, and IgM+ cells in gills and kidney from per-gill infected fish The composition of lymphocyte subsets in the kidney and gills is shown in Fig. 3. The percentage of CD8α+ cells in the kidney leukocytes of infected fish was significantly higher than those of uninfected fish. In contrast, the percentage of IgM+ cells in infected fish was significantly lower than that in uninfected fish (p < 0.05). The percentage of CD4+ cells in infected fish was higher than that in uninfected fish, although no statistically significant difference between them was observed. Moreover, no differences were noted in the gills from infected and control fish for all the three cell types examined. 3.4. Systemic cell-mediated immune responses after gill infection The cytotoxic activity of PBL against virus-infected syngeneic cells is shown in Fig. 4. The effector cells from fish sampled at 2 days secondary post-infection (dpi) efficiently killed CHNV-infected syngeneic cells but not uninfected cells (p < 0.05). The activity disappeared at 4 days, suggesting that activity could have peaked at an earlier time point. No significant activity against CHNV-infected syngeneic cells was observed in the effector cells sampled 14 days after primary infection (day 0 post-secondary infection). The result indicated that sensitization of only the gill site could induce systemic antigenspecific cell-mediated immunity. 3.5. Systemic humoral immune responses after gill infection Antibody production of per-gill infected fish is shown in Fig. 5. In the primary infection, no significant difference of CHNV-specific IgM production between control and infected fish was observed. Antibody production became detectable 14 and 21 days after the

84

T. Somamoto et al./Developmental and Comparative Immunology 52 (2015) 81–87

Fold change in mRNA level

(A) Gill 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

18

Perforin

16

IFN-γ1

14 12 10

*

*

*

8

*

*

*

6 4 2 0

2.5

4.5

IL-10

4.0

2.0

IFN-γ2

3.5 3.0

1.5

2.5 2.0

1.0

1.5 0.5

1.0

0.0

0.0

0.5 0

1

12

24

1

12

0

24

1

Secondary

Primary

12

24

1

Primary

12

24

Secondary

Times after infection (hour)

(B) Kidney

Fold change in mRNA level

2.5

*

Perforin Perforin

2.5

IFN-γ1

2.0

2.0

1.5

1.5

*

1.0

*

*

1.0

0.5

0.5

0.0

0.0

3.5

1.8

3.0

1.6

IL-10

1.4 2.5

IFN-γ2

1.2

2.0

1.0 0.8

1.5

0.6

1.0

0.4

0.5

0.2 0.0

0.0 0

1

12 Primary

24

1

12 Secondary

24

0

1

12 Primary

24

1

12

24

Secondary

Times after infection (hour) Fig. 2. Quantitative expression profiles of IFN-γ1, IFN-γ2, perforin, and IL-10 in gill (A) and kidney (B) cells after primary and secondary per-gill infection. Data from the three individual experiments are shown as mean of fold change in mRNA expression relative to uninfected fish (time 0). No significant difference was observed in the expression of either IFN-γ2 or IL-10. Error bars indicate SD. In all quantitative real-time PCRs, melting curve analyses were performed and single specific melting peaks were observed, indicating amplification specificity. Statistical comparisons in each organ were made by one-way ANOVA and Tukey’s post hoc test. Asterisks indicate significant differences from uninfected controls (*P < 0.05).

Composition of lymphocyte subsets (%)

T. Somamoto et al./Developmental and Comparative Immunology 52 (2015) 81–87

25

85

20 15

CD4

*

CD8

*

IgM

20

15 10

15

10 10 5 5 5 0

0

0 Control

CHNV

Control

gill

Control

CHNV

CHNV

Control

gill

kidney

Control

CHNV

kidney

CHNV

Control

gill

CHNV

kidney

Fig. 3. Percentages of CD4+, CD8α+, and IgM+ cells from the gill and kidney leukocytes. Cells were collected at 24 h following the secondary infection and PBS administration. Results are presented as the means of three individual fish, and error bars indicate SD. The statistical significance compared with the control was determined by a Welch’s t-test (*P < 0.05).

secondary infection (p < 0.05). The results indicated that sensitization of only the gills could induce a systemic humoral immunity, with at least two sensitizations required to induce significant activity. 4. Discussion Recent studies relating to the development of a vaccine have demonstrated that strength and longevity of vaccine-induced immunity depend on the site of vaccination (Nizard et al., 2014; Shane and Klonowski, 2014). Mucosal immunization is considered to be an ideal strategy for vaccination due to advantages such as the ability to induce protective immunity in the mucosal area, a frontline of pathogen infection. The present study has shown that infection via gills can induce local as well as systemic antiviral immunity, indicating that gills could be a possible route for mucosal vaccination of teleost fish.

0.10

*

uninfected infected

30 25 20 15 10

*

*

21

28

44

(7)

(14)

(30)

Control fish 0.08

OD at 405 nm

35

Cytotoxicity (%)

Studies employing the clonal ginbuna crucian carp model have mainly demonstrated adaptive cell-mediated and humoral immunity by i.p. injection with CHNV (Somamoto et al., 2002, 2006, 2013). The present study has shown that infection via gills can induce significant cell-mediated cytotoxicity (CMC), upregulation of perforin mRNA, and an increased CD8α+ cell proportion in systemic tissue. In addition, the peak of CMC activity induced by gill infection was observed earlier than that by i.p. infection, as reported previously (Somamoto et al., 2002, 2013). Sato and Okamoto (2007, 2010) reported that antigen-specific cell-mediated immunity can be induced as a result of oral or anal administration with allo- or virusantigen, and that intestinal sensitization can elicit more efficient cell-mediated immunity than sensitization by i.p. injection. The present and previous studies indicate that mucosal vaccination is an effective way to induce systemic cell-mediated immunity in teleost fish. However, CHNV-specific IgM production in fish

Infected fish 0.06 0.04 0.02

5 0

0 -5 0

2

4

Days after secondary infection Fig. 4. Cytotoxic activity of PBLs against CHNV-infected syngeneic cells after secondary infection. The activity in the effector/target ratio of 100:1 is shown. Results are presented as the means of three individual fish, and error bars indicate SD. Black and white bars indicate the activities against infected and uninfected cells, respectively. The statistical significance compared with the activity against uninfected targets was determined by a paired t-test (*P < 0.05).

7

Days after primary infection Fig. 5. Detection of CHNV-specific antibody in sera by ELISA. Black and white bars indicate the antibody titer of infected and control fish, respectively. Arrowhead indicates the day of secondary infection. Numbers in parentheses indicate the days after the secondary infection. Results are presented as the means of three individual fish, and error bars indicate SD. The statistical significance compared with the control was determined by a Welch’s t-test (*P < 0.05).

86

T. Somamoto et al./Developmental and Comparative Immunology 52 (2015) 81–87

infected via gills are likely lower compared with those of i.p. infected fish which was reported in the previous study (Somamoto et al., 2002). Although the composition of IgM+ cells in the kidney from the infected fish was lower, the CD8α+ cell composition was higher in the infected fish. In addition, IFN-γ expression was upregulated in gills but not IL-10, suggesting that the infection with virus via gill induces a skewed Th-1 immunity in host. Therefore, sensitization with virus through the gills may not be capable of inducing an effective humoral immunity compared to cell-mediated immunity. CHNV in the gills was rapidly eliminated after the per-gill infection, indicating that local immunity may help to prevent the viral spread. The reduction of viral load following secondary infection showed induction of a secondary immune response in the gill site. The transcriptional analysis showed that IFN-γ mRNA was upregulated in the gill after infection. A previous study has shown that recombinant IFN-γ from the ginbuna crucian carp possesses antiCHNV activity (Yabu et al., 2011). Taken together, IFN-γ could possibly be an effective factor for clearing the CHNV in the gill. It has also been reported that the expression of immune-related genes such as chemokine receptors, immunoglobulin, IFN-relating genes, and MHC class II are upregulated in the gills following bath infection with virus in other teleost fishes (Aas et al., 2014; Aquilino et al., 2014; Austbo et al., 2014; Nuñez Ortiz et al., 2014). Perforin expression and CD8 + cell composition did not increase in gills, suggesting that CTLs do not provide efficient protection from the local viral infection. However, because CTLs increased in kidney, it is suggested that virus-specific CTLs are generated by gillsensitization, and play important roles in systemic organs. Histological analyses have also demonstrated that a T-cell-rich intraepithelial structure in the gills exists in Atlantic salmon, suggesting that this is a secondary lymphoid structure in teleost (Aas et al., 2014; Haugarvoll et al., 2008; Koppang et al., 2010). These findings provide an insight that the gill plays an important role as both a first line of defense and a secondary lymphoid organ. A secondary gill infection could induce significant cell-mediated and humoral immune response, suggesting that gill infection can generate immunological memory in host immunity. Antigen-specific memory T and B cells are key players for establishment of efficient and long-term sustainable immunity. Although the functions of memory B cell in teleost fishes have recently been reported (Ma et al., 2013; Piazzon et al., 2014; Ye et al., 2013), the role of memory cells in mucosal tissues still remains unknown. Recent studies relating to mammalian memory cells have focused on resident memory T cells (Trm), which are a non-recirculating subset located in non-lymphoid tissues to provide an early response to re-infection (Jiang et al., 2012; Schenkel et al., 2014a,b; Sheridan et al., 2014). The mechanisms for generation and maintenance of Trm vary depending on the route of infection and tissue type. Unlike terrestrial vertebrates, fish possess gills and mucosal skin and are then covered with mucus across their entire body, and thus, they may possess unique mucosal memory cells specialized for a water environment. Therefore, understanding the function of local memory cells would support the development of an effective vaccine for teleost fish. Acknowledgments This research was supported in part by a Grant-in-Aid for Scientific Research (C) (Grant Number 26450286) and a Grant-in-Aid for Scientific Research (B) (Grant Number 25292129) from the Japan Society for the Promotion of Science (JSPS). The costs of publication were supported, in part, by a Research Grant for Young Investigators of the Faculty of Agriculture, Kyushu University. The authors would like to thank Enago (www.enago.jp) for the English language review.

References Aas, I.B., Austbo, L., Konig, M., Syed, M., Falk, K., Hordvik, I., et al., 2014. Transcriptional characterization of the T cell population within the salmonid interbranchial lymphoid tissue. J. Immunol. 193, 3463–3469. Abdelkhalek, N.K., Komiya, A., Kato-Unoki, Y., Somamoto, T., Nakao, M., 2014. Identification of immune relevant genes using expressed sequence tags (ESTs) in common carp (Cyprinus carpio) gills and intestine. J. Fac. Agr. Kyushu U. 59, 109–116. Andrews, M., Battaglene, S., Cobcroft, J., Adams, M., Noga, E., Nowak, B., 2010. Host response to the chondracanthid copepod Chondracanthus goldsmidi, a gill parasite of the striped trumpeter, Latris lineata (Forster), in Tasmania. J. Fish Dis. 33, 211–220. Aquilino, C., Castro, R., Fischer, U., Tafalla, C., 2014. Transcriptomic responses in rainbow trout gills upon infection with viral hemorrhagic septicemia virus (VHSV). Dev. Comp. Immunol. 44, 12–20. Austbo, L., Aas, I.B., Konig, M., Weli, S.C., Syed, M., Falk, K., et al., 2014. Transcriptional response of immune genes in gills and the interbranchial lymphoid tissue of Atlantic salmon challenged with infectious salmon anemia virus. Dev. Comp. Immunol. 45, 107–114. Castro, R., Bromage, E., Abos, B., Pignatelli, J., Gonzalez Granja, A., Luque, A., et al., 2014. CCR7 is mainly expressed in teleost gills, where it defines an IgD+IgM- B lymphocyte subset. J. Immunol. 192, 1257–1266. Fillatreau, S., Six, A., Magadan, S., Castro, R., Sunyer, J.O., Boudinot, P., 2013. The astonishing diversity of Ig classes and B cell repertoires in teleost fish. Front. Immunol. 4, 28. Fischer, U., Koppang, E.O., Nakanishi, T., 2013. Teleost T and NK cell immunity. Fish Shellfish Immunol. 35, 197–206. Gomez, D., Sunyer, J.O., Salinas, I., 2013. The mucosal immune system of fish: the evolution of tolerating commensals while fighting pathogens. Fish Shellfish Immunol. 35, 1729–1739. Gomez, D., Bartholomew, J., Sunyer, J.O., 2014. Biology and mucosal immunity to myxozoans. Dev. Comp. Immunol. 43, 243–256. Haugarvoll, E., Bjerkas, I., Nowak, B.F., Hordvik, I., Koppang, E.O., 2008. Identification and characterization of a novel intraepithelial lymphoid tissue in the gills of Atlantic salmon. J. Anat. 213, 202–209. Jiang, X., Clark, R.A., Liu, L., Wagers, A.J., Fuhlbrigge, R.C., Kupper, T.S., 2012. Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity. Nature 483, 227–231. Koppang, E.O., Lundin, M., Press, C.M., Rønningen, K., Lie, Ø., 1998a. Differing levels of Mhc class II β chain expression in a range of tissues from vaccinated and non-vaccinated Atlantic salmon, Salmo salar L. Fish Shellfish Immunol. 8, 183–196. Koppang, E.O., Press, C.M., Rønningen, K., Lie, Ø., 1998b. Expression of Mhc class I mRNA in tissues from vaccinated and non-vaccinated Atlantic salmon Salmo salarL. Fish Shellfish Immunol. 8, 577–587. Koppang, E.O., Fischer, U., Moore, L., Tranulis, M.A., Dijkstra, J.M., Kollner, B., et al., 2010. Salmonid T cells assemble in the thymus, spleen and in novel interbranchial lymphoid tissue. J. Anat. 217, 728–739. Lazado, C.C., Caipang, C.M., 2014. Mucosal immunity and probiotics in fish. Fish Shellfish Immunol. 39, 78–89. Ma, C., Ye, J., Kaattari, S.L., 2013. Differential compartmentalization of memory B cells versus plasma cells in salmonid fish. Eur. J. Immunol. 43, 360–370. Miyazaki, T., Kuzuya, Y., Yasumoto, S., Yasuda, M., Kobayashi, T., 2008. Histopathological and ultrastructural features of Koi herpesvirus (KHV)-infected carp Cyprinus carpio, and the morphology and morphogenesis of KHV. Dis. Aquat. Organ. 80, 1–11. Murray, H.M., Leggiadro, C.T., Douglas, S.E., 2007. Immunocytochemical localization of pleurocidin to the cytoplasmic granules of eosinophilic granular cells from the winter flounder gill. J. Fish Biol. 70, 336–345. Nakanishi, T., Toda, H., Shibasaki, Y., Somamoto, T., 2011. Cytotoxic T cells in teleost fish. Dev. Comp. Immunol. 35, 1317–1323. Nizard, M., Diniz, M.O., Roussel, H., Tran, T., Ferreira, L.C., Badoual, C., et al., 2014. Mucosal vaccines: novel strategies and applications for the control of pathogens and tumors at mucosal sites. Hum. Vaccin. Immunother. 10, 2175–2187. Nuñez Ortiz, N., Gerdol, M., Stocchi, V., Marozzi, C., Randelli, E., Bernini, C., et al., 2014. T cell transcripts and T cell activities in the gills of the teleost fish sea bass (Dicentrarchus labrax). Dev. Comp. Immunol. 47, 309–318. Piazzon, M.C., Savelkoul, H.F., Pietretti, D., Wiegertjes, G.F., Forlenza, M., 2014. Carp Il10 has anti-inflammatory activities on phagocytes, promotes proliferation of memory T cells, and regulates B cell differentiation and antibody secretion. J. Immunol. 194, 187–199. Rebl, A., Korytar, T., Kobis, J.M., Verleih, M., Krasnov, A., Jaros, J., et al., 2014. Transcriptome profiling reveals insight into distinct immune responses to Aeromonas salmonicida in gill of two rainbow trout strains. Mar. Biotechnol. (NY) 16, 333–348. Rombout, J.H., Abelli, L., Picchietti, S., Scapigliati, G., Kiron, V., 2011. Teleost intestinal immunology. Fish Shellfish Immunol. 31, 616–626. Rombout, J.H., Yang, G., Kiron, V., 2014. Adaptive immune responses at mucosal surfaces of teleost fish. Fish Shellfish Immunol. 40, 634–643. Salinas, I., Zhang, Y.A., Sunyer, J.O., 2011. Mucosal immunoglobulins and B cells of teleost fish. Dev. Comp. Immunol. 35, 1346–1365. Sato, A., Okamoto, N., 2007. Oral and anal immunisation with alloantigen induces active cell-mediated cytotoxic responses in carp. Fish Shellfish Immunol. 23, 237–241.

T. Somamoto et al./Developmental and Comparative Immunology 52 (2015) 81–87

Sato, A., Okamoto, N., 2010. Induction of virus-specific cell-mediated cytotoxic responses of isogeneic ginbuna crucian carp, after oral immunization with inactivated virus. Fish Shellfish Immunol. 29, 414–421. Schenkel, J.M., Fraser, K.A., Beura, L.K., Pauken, K.E., Vezys, V., Masopust, D., 2014a. Resident memory CD8 T cells trigger protective innate and adaptive immune responses. Science 346, 98–101. Schenkel, J.M., Fraser, K.A., Masopust, D., 2014b. Cutting edge: resident memory CD8 T cells occupy frontline niches in secondary lymphoid organs. J. Immunol. 192, 2961–2964. Shane, H.L., Klonowski, K.D., 2014. Every breath you take: the impact of environment on resident memory CD8 T cells in the lung. Front. Immunol. 5, 320. Sheridan, B.S., Pham, Q.M., Lee, Y.T., Cauley, L.S., Puddington, L., Lefrancois, L., 2014. Oral infection drives a distinct population of intestinal resident memory CD8(+) T cells with enhanced protective function. Immunity 40, 747–757. Somamoto, T., Nakanishi, T., Okamoto, N., 2002. Role of specific cell-mediated cytotoxicity in protecting fish from viral infections. Virology 297, 120–127. Somamoto, T., Yoshiura, Y., Sato, A., Nakao, M., Nakanishi, T., Okamoto, N., et al., 2006. Expression profiles of TCRbeta and CD8alpha mRNA correlate with virus-specific cell-mediated cytotoxic activity in ginbuna crucian carp. Virology 348, 370–377. Somamoto, T., Okamoto, N., Nakanishi, T., Ototake, M., Nakao, M., 2009. In vitro generation of viral-antigen dependent cytotoxic T-cells from ginbuna crucian carp, Carassius auratus langsdorfii. Virology 389, 26–33. Somamoto, T., Nakanishi, T., Nakao, M., 2013. Identification of anti-viral cytotoxic effector cells in the ginbuna crucian carp, Carassius auratus langsdorfii. Dev. Comp. Immunol. 39, 370–377. Somamoto, T., Kondo, M., Nakanishi, T., Nakao, M., 2014a. Helper function of CD4(+) lymphocytes in antiviral immunity in ginbuna crucian carp, Carassius auratus langsdorfii. Dev. Comp. Immunol. 44, 111–115. Somamoto, T., Koppang, E.O., Fischer, U., 2014b. Antiviral functions of CD8(+) cytotoxic T cells in teleost fish. Dev. Comp. Immunol. 43, 197–204.

87

Takizawa, F., Koppang, E.O., Ohtani, M., Nakanishi, T., Hashimoto, K., Fischer, U., et al., 2011. Constitutive high expression of interleukin-4/13A and GATA-3 in gill and skin of salmonid fishes suggests that these tissues form Th2-skewed immune environments. Mol. Immunol. 48, 1360–1368. Toda, H., Araki, K., Moritomo, T., Nakanishi, T., 2011a. Perforin-dependent cytotoxic mechanism in killing by CD8 positive T cells in ginbuna crucian carp, Carassius auratus langsdorfii. Dev. Comp. Immunol. 35, 88–93. Toda, H., Saito, Y., Koike, T., Takizawa, F., Araki, K., Yabu, T., et al., 2011b. Conservation of characteristics and functions of CD4 positive lymphocytes in a teleost fish. Dev. Comp. Immunol. 35, 650–660. von Gersdorff Jorgensen, L., Heinecke, R.D., Skjodt, K., Rasmussen, K.J., Buchmann, K., 2011. Experimental evidence for direct in situ binding of IgM and IgT to early trophonts of Ichthyophthirius multifiliis (Fouquet) in the gills of rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis. 34, 749–755. Weli, S.C., Aamelfot, M., Dale, O.B., Koppang, E.O., Falk, K., 2013. Infectious salmon anemia virus infection of Atlantic salmon gill epithelial cells. Virol. J. 10, 5. Yabu, T., Toda, H., Shibasaki, Y., Araki, K., Yamashita, M., Anzai, H., et al., 2011. Antiviral protection mechanisms mediated by ginbuna crucian carp interferon gamma isoforms 1 and 2 through two distinct interferon gamma-receptors. J. Biochem. 150, 635–648. Yamasaki, M., Araki, K., Nakanishi, T., Nakayasu, C., Yamamoto, A., 2014. Role of CD4(+) and CD8alpha(+) T cells in protective immunity against Edwardsiella tarda infection of ginbuna crucian carp, Carassius auratus langsdorfii. Fish Shellfish Immunol. 36, 299–304. Ye, J., Kaattari, I.M., Ma, C., Kaattari, S., 2013. The teleost humoral immune response. Fish Shellfish Immunol. 35, 1719–1728. Zilberg, D., Klesius, P.H., 1997. Quantification of immunoglobulin in the serum and mucus of channel catfish at different ages and following infection with Edwardsiella ictaluri. Vet. Immunol. Immunopathol. 58, 171–180.