Fish & Shellﬁsh Immunology 36 (2014) 519e524
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Impairment of the immune system in GH-overexpressing transgenic zebraﬁsh (Danio rerio) Carolina R. Batista a, c, Márcio A. Figueiredo b, c, Daniela V. Almeida a, c, Luis A. Romano b, d, Luis F. Marins a, b, c, * a Programa de Pós-Graduação em Ciências Fisiológicas, Fisiologia Animal Comparada, Universidade Federal do Rio GrandedFURG, Avenida Itália, Km 8, CEP 96201-900 Rio Grande, RS, Brazil b Programa de Pós-Graduação em Aquicultura, Universidade Federal do Rio GrandedFURG, Avenida Itália, Km 8, CEP 96201-900 Rio Grande, RS, Brazil c Instituto de Ciências Biológicas, Universidade Federal do Rio GrandedFURG, Avenida Itália, Km 8, CEP 96201-900 Rio Grande, RS, Brazil d Instituto de Oceanograﬁa, Universidade Federal do Rio GrandedFURG, Avenida Itália, Km 8, CEP 96201-900 Rio Grande, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 August 2013 Received in revised form 20 December 2013 Accepted 23 December 2013 Available online 7 January 2014
Growth hormone (GH) is an important regulator of immune functions in vertebrates, and it has been intensively reported a series of stimulatory actions of this hormone over on the immune system. Within aquaculture, overexpression of GH has been considered a promising alternative for promoting higher growth rates in organisms of commercial interest. Considering the various pleiotropic effects of GH, there are still few studies that aim to understand the consequences of the excess of GH on the physiological systems. In this context, our goal was to present the effects of the overexpression of GH on immune parameters using a model of zebraﬁsh (Danio rerio) that overexpress this hormone. The results showed that GH transgenic zebraﬁsh had 100% of mortality when immunosuppressed with dexamethasone, revealing a prior weakening of the immune system in this lineage. Morphometric analysis of thymus and head kidney revealed a reduction in the area of these structures in transgenic zebraﬁsh. Moreover, the phenotypic expression of CD3 and CD4 thymocytes was also depreciated in transgenic zebraﬁsh. Furthermore, a decrease was noted in the expression of genes RAG-1 (60%), IKAROS (50%), IL-1b (55%), CD4 (60%) and CD247 (40%), indicating that development parameters, of innate and acquired immunity, are being harmed. Based on these results, it can be concluded that the excess of GH impairs the immune functions in GH transgenic zebraﬁsh, indicating that the maintenance of normal levels of this hormone is essential for the functioning of immunological activities. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Thymus Head kidney Immunity Gene expression Immunohistochemistry
1. Introduction The biotechnology applied to aquaculture has shown to be a promising alternative for raising animal growth rate through growth hormone (GH) transgenesis . Species with a commercial interest like the catﬁsh , Atlantic salmon , carp , tilapia [5,6], as well as species of salmon from the Paciﬁc , were already genetically modiﬁed for overexpression of this hormone. GH, a polypeptide synthesized and secreted through the adenohypophysis, is active in all organs and speciﬁc tissues that have the growth hormone receptor (GHR). Beyond its role in growth processes, this hormone has a number of pleiotropic effects on other physiological
* Corresponding author. Instituto de Ciências Biológicas, Universidade Federal do Rio GrandedFURG, Av. Itália, Km 8, CEP 96201-900 Rio Grande, RS, Brazil. Fax: þ55 53 32336848. E-mail address: [email protected]
(L.F. Marins). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.12.022
systems such as reproduction , behavior , osmoregulation  and on the immune system as well . Different organs and cells from the immune system have GH receptors. Thus, in mammals and in teleosts ﬁsh, both the endocrine secretion of GH and the paracrine and autocrine secretion by immunological cell types are able to modulate immunological functions [12,13]. Several studies have reported stimulatory actions of GH on various parameters of immunity. In mammals, for example, studies have demonstrated the inﬂuences exerted by a series of neuroendocrine hormones, including the GH, in the control of functions performed by the thymus . Increases in thymic cellularity, along with the production of cytokines, chemokines and hormones, are parameters which can be inﬂuenced by GH . Also, GH has been found to modulate the migration of thymocytes and peripheral T cells through increased production of proteins from the extracellular matrix . The interaction of GH with the immune system is not restricted to mammals. Teleost ﬁsh have an immune system similar to that of
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mammals, distinguished only by the hematopoietic site, which is located on the head kidney, and for not having lymph nodes . In this group, the GH plays a part in the promotion of immune functions such as phagocytosis, lysozyme activity, antibody production and the proliferation of lymphocytes . In corroboration with these studies, GH receptors have been characterized in the head kidney from Sparus aurata and there is evidence of the involvement of GH in hematopoiesis and as a differentiation factor . The GH-transgenesis, even when demonstrating its capacity to act as a viable alternative for promoting high growth rates, still needs further studies in order to verify what the possible additional effects caused in these systems are. With the aim of studying the physiological effects as a result of the transgenesis of the hormone in question, our group developed a GH-overexpressing model of transgenic zebraﬁsh (Danio rerio) . This lineage (named F0104) has proved to be an interesting model to study the relationship between high levels of GH and its function. Due to the correlation of GH with the immunological processes in mammals, widely described in the literature, the use of a transgenic ﬁsh model that produces GH in supraphysiological levels provides a tool for a better understanding of the effects of this hormone in the ﬁsh. In this study, the aim was to investigate the way in which the overexpression of GH could be inﬂuencing parameters of the innate and acquired immunity. For this, histological analyses were carried out on thymus and head kidney in order to determine the development of these organs in GH-transgenic animals. In addition, immunohistochemical analysis of the thymus, marked by CD3 and CD4 antibodies for quantiﬁcation of the total T lymphocyte population, and the T helper lymphocytes respectively, were performed. Furthermore, gene expression analyses for development factors and for components of innate and cellular immunity of the immune system were performed. Finally, transgenic and non transgenic ﬁsh were exposed to dexamethasone to assess the survival rates of the two groups through the chemical stressor. 2. Material and methods 2.1. GH-Transgenic ﬁsh and maintenance conditions The F0104 lineage used in this work was developed through the co-injection of two genetic constructs, both under the transcriptional control of b-Actin promoter from Cyprinus carpio carp. One of the constructs uses the GH cDNA from silverside ﬁsh Odontesthes argentinensis, and the other uses the green ﬂuorescent protein (GFP) cDNA . Transgenic and non transgenic ﬁsh were bred through the crossing of transgenic hemizygote males from the lineage F0104 and non transgenic females. Both types of ﬁsh were maintained with a controlled photoperiod and fed ad libitum twice a day with commercial ﬁsh food (Tetrabits, Tetra, Brazil). The chemical parameters of pH and nitrite concentration were measured weekly. The experiments were conducted in accordance to the Ethics Committee on Animal Use of the Federal University of Rio Grande (FURG). 2.2. Tolerance to immune system suppression In order to evaluate the tolerance between the transgenic and non transgenic individuals through a suppressant of the immune system, a test of resistance to exposure to dexamethasone (Sigmae Aldrich, Brazil) was conducted. Transgenic and non transgenic zebraﬁsh with 5 dph were exposed either to dexamethasone (100 mg/mL) or only to its dilution agent (0.4% ethanol) for ﬁve days. In each the four experimental groups, 20 ﬁsh with 5 dph were used. The dexamethasone concentration was chosen according to Langenau and colleagues (2004) , in which it was shown to be
an efﬁcient immunosuppressant in zebraﬁsh larvae. The exposure was performed in a 96-well plate, in which each individual was placed in a well with 200 ml of the dexamethasone solution or just with the control solution. After the ﬁfth day of exposure, the exposure solutions were replaced by water from the culture system, and the mortality rates were monitored for a further ﬁve days. Death was deﬁned using a magnifying glass, where it was found that cardiovascular movements had ceased. 2.3. Histological and morphometric analysis To perform the histological analysis and subsequent morphometric analysis, ten individuals of each strain aged 30 days were euthanized with tricaine (0.6 mg/L). The animals were ﬁxed in Bouin’s solution and processed according to Prophet and colleagues (1992) , for further immunohistochemical analysis. After that, the samples were dehydrated in graded ethanol series, incorporated with Paraplast Plus (Sigma Aldrich, Brazil) and cut in longitudinal sections (3e5 mm thick). The resulting sections were subsequently stained with hematoxylin and eosin. For morphometric analysis of the thymus and head kidney, longitudinal sections of about 0.5 mm in thickness were taken. The area of these organs was measured using the Weibel graticules, with lines, according to Weibel (1982) . 2.4. Analysis of gene expression For analysis of genes RAG-1, IKAROS, CD247, CD4, and IL-1b, seven samples of each group both non transgenic and transgenic were used, each sample consisting of a pool of three ﬁsh, all aged 30 days. Speciﬁc primers for the genes of interest were designed based on sequences available in GenBank (Table 1) using the Primer Express 3.0 software (Applied Biosystems, Brazil). For expression analysis of these genes, total RNA was extracted with Trizol Reagent (Invitrogen, Brazil) according to the manufacturer’s instructions. The conﬁrmation of the integrity of the RNA samples was done via gel electrophoresis in 1% agarose. cDNA synthesis was performed by reverse transcription of RNA using the High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems, Brazil). The gene expression analysis was performed by real-time quantitative PCR (qPCR). Preliminary tests with a serial dilution of cDNA showed that all primers used had efﬁciencies close to 100% (data not shown). For gene expression, each sample was analyzed in triplicate on ABI 7500 platform Real Time Systems (Applied Biosystems, Brazil) using the detection system Platinum SYBR Green qPCR SuperMix -
Table 1 Gene-speciﬁc primers used for quantitative PCR expression analyses. Gene
GenBank accession number
Recombination Activating Gene 1 (RAG-1) Ikaros Zinc Finger 1 (Ikaros) Cluster of Differentiation 247 (CD247) Cluster of Differentiation 4-2 (CD4) Interleukin 1 e beta (IL-1b) Elongation factor 1 e alpha (EF-1a)
F: 50 -AGCTGACGCCATGGAGAAAGGGA-30 NM_131389.1 R: 50 -ACAGTTCGTCAGGCATGGAGGC-3 F: 50 -GCCGACATGGTGGTCAGCCC-30 R: 50 -GTGCTCTGCGGCGCTGTCTT’3 F: 50 -GATCTGGAGGTCGCAGGCGTG-30 R: 50 -ACGGTACGTGTCGTCCCCCTT-30
F: 50 -GGTTCTGGTGCCACTGATCCTTGG-30 R: 50 -AGAGGCTGCCGCATGGATCTCA-30
F: 50 -CCACGTATGCGTCGCCCAGT-30 R: 50 -GGGCAACAGGCCAGGTACAGG-30 F: 50 -GGGCAAGGGCTCCTTCAA-30 R: 50 -CGCTCGGCCTTCAGTTTG-30
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UDG (Invitrogen, Brazil). The conditions for qPCR reactions were 50 C for 2 min, 95 C for 2 min, followed by 40 cycles of 95 C for 15 s and 60 C for 30 s. The gene of the elongation factor 1 alpha (EF-1a) was used as normalizer, which did not vary between experimental groups. 2.5. Immunohistochemical analyses In the immunohistochemistry of the thymus, the same animals processed for histological analyses were used. The ABC peroxidase method was applied (Vectastain Elite ABC Kit, Canada), as described by Hsu and colleagues (1981) . The sections were incubated with a human monoclonal antibody, anti CD3 and anti CD4 (Dako, Argentina) previously tested for ﬁsh in Xiphophorus helleri . Next, the sections were washed (0.1% diaminobenzidine solution), dehydrated and the slides were examined under an optical microscope. The evaluation of CD3 and CD4 receptors was performed by quantitative analysis of the phenotypic percentage by mm2 of tissue. The expression of these receptors was quantiﬁed using Bioscan OPTIMAS 6.1 software [23,26]. 2.6. Statistical analyses Tolerance to dexamethasone was analyzed using Chi square test. The Student’s t-test was used to verify differences in morphometric and immunohistochemical analysis. Signiﬁcant differences in gene expression data were veriﬁed using Relative Expression Software Tool e REST . For all tests, signiﬁcant differences were inferred when p < 0.05. 3. Results 3.1. Tolerance to depression of the immune system The results of the immunosuppressant dexamethasone exposure (Fig. 1) showed a mortality rate of 100% in the transgenic ﬁsh, while in the non transgenic ﬁsh the mortality rate was 70% (p 0.0001). Among the control-treatment ﬁsh (exposed only to the dilution agent of dexamethasone), no mortality was observed in any of the experimental groups. 3.2. Histological and morphometric analysis Morphometric analysis of the thymus and head kidney revealed a decrease in the area of these organs in transgenic ﬁsh when compared to non transgenic (Fig. 2). The area of non transgenic ﬁsh thymus was 159.2 mm2 0.2, whereas for the transgenic ﬁsh it was 123.5 mm2 0.3 (p < 0.0001) (Fig. 2(A)). Likewise, it was observed a
Fig. 1. Percentage of mortality of non transgenic (NT) and GH-transgenic (T) zebraﬁsh exposed to dexamethasone (100 mg/mL) for 5 days. Asterisk indicates signiﬁcant difference between NT and T zebraﬁsh (Chi square 53.78, df ¼ 1, p < 0.0001).
Fig. 2. Morphometric analysis of thymus (A) and head kidney (B), comparing non transgenic (NT) and GH-transgenic (T) zebraﬁsh. Asterisks indicate signiﬁcant difference between NT and T zebraﬁsh (t-test, p < 0.05).
decrease in the area of the anterior kidney of the transgenic subjects (208.9 mm2 0.2) compared to non transgenic (411.9 mm2 0.2) (p 0.0001) (Fig. 2(B)). 3.3. Analysis of gene expression Comparing transgenic and non transgenic individuals for the expression of genes related to immunity, the results showed a decrease (p 0.05) on the expression of genes CD4 (60%), CD247 (40%), RAG-1 (60%), IKAROS (50%) and IL-1b (55%) in transgenic ﬁsh (Fig. 3). 3.4. Analysis immunohistochemical The primary antibodies anti CD3 and anti CD4, produced to humans, showed cross-reactivity both to non transgenic and transgenic zebraﬁsh. The speciﬁc label of CD3 showed a decrease (p 0.0001) in the number of CD3 cells in the thymus of transgenic animals (25.5 mm2 1.1) compared to non transgenic (41.4 mm2 1.1) (Fig. 4(A)). The analysis of the CD4 receptor also showed a reduction (p 0.0001) in the number of CD4 cells in the thymus of transgenic ﬁsh (14.4 mm2 0.9) compared to non transgenic (27.2 mm2 0.8) (Fig. 4(B)).
Fig. 3. Gene expression analyses of immune system-related genes, comparing non transgenic (NT) and GH-transgenic (T) zebraﬁsh. The expression level of each gene was normalized by the expression of the elongation factor 1 alpha (EF-1a) gene. NT were considered controls, where gene expression ¼ 1. Data are expressed using a median SEM. Signiﬁcant differences (p < 0.05) between NT and T are denoted by asterisks.
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Fig. 4. Phenotypic expression of CD3 (A) and CD4 (B) receptors in the thymus of non transgenic (NT) and transgenic (T). Data are expressed as percentage of marked cells area (mm2). Asterisks indicate signiﬁcant difference between NT and T zebraﬁsh (t-test, p < 0.05).
4. Discussion It is well described in the literature that GH can exert stimulatory actions at various levels of control of immunological activities both in ﬁsh and in mammals. Unexpectedly, our results point to a loss of immune function in transgenic zebraﬁsh for the GH hormone. In our tests, the GH-transgenic ﬁsh showed a high mortality when immunosuppressed with dexamethasone. Furthermore, when the size of the head kidney and thymus was analyzed, areas were found to be smaller, and a decrease in the number of CD3 and CD4 thymocytes was also observed. Additionally, a decrease was seen in the expression of genes related to the immune system. Thus, our results suggest that supraphysiological levels of GH are responsible for damages caused in the immunity of these animals. Primarily, in order to verify changes in the response of the adaptive immune system of GH-transgenic ﬁsh, they were exposed to dexamethasone, a synthetic substance able to mimic the effects caused by glucocorticoids, such as cortisol. The immunosuppressive actions of dexamethasone are widely reported in ﬁsh  and mammals [29,30], as well as the inﬂuences of this compound in increasing the susceptibility to infections [31,32]. In the present study, mortality related to immunosuppression by dexamethasone was higher in GH-transgenic zebraﬁsh. However, we did not observe a straight relationship between mortality and infection diseases in dexamethasone-treated ﬁsh. It is already known that glucocorticoids could act in many other physiological aspects beyond the immune system including metabolism, osmoregulation and growth . Thus, we cannot conclude that mortality in this group has been originated only by previous GH immunosuppression action. It is possible that the dexamethasone-induced stress could not be neutralized due to the greater allocation of energy towards growth in GH-transgenic ﬁsh, leaving less energy available for stress response as observed in GH-transgenic Atlantic salmon . In order to verify how the head kidney and thymus could have been inﬂuenced by the overexpression of GH, a more rigorous investigation of these organs was set which evaluated the differences in the size of these structures due to transgenesis. Surprisingly, the total area of both organs was reduced in transgenic ﬁsh, and this decrease was even further aggravating in the head kidney, where the area measured was equivalent to 50% when compared to the area of the head kidney on non transgenic ﬁsh. Thus, the excess of circulating GH may be affecting the development of these organs when related to the migration of hematopoietic stem cells (HSCs)
for their colonization. It has been reported to zebraﬁsh that over embryogenesis, the head kidney is composed by cells of mesodermal origin that form the primordial organ, followed by the process of epithelialization of the pronephric duct . At this stage, the primary kidney is colonized by HSCs derived from the aorta-gonad-mesonephros (AGM) and will migrate along the pronephric tube to the pronephros in development . In turn, the thymus develops from the pharyngeal endoderm and subsequently is colonized by hematopoietic precursors. Therefore, the thymic epithelium differentiates into stromal cells, which secrete chemoattractants agents that lead to migration of precursors of T cells from the marrow . For the migration of HSCs to occur both in larval development and adult life, chemokines are released in order to attract the migration of HSCs to the releasing site. Impairments in this process of colonization, both in the early stages and during hematopoiesis in adults, could result in a reduced area of the head kidney and thymus. In this context, in mammals, stromal cell-derived factor-1 (SDF-1/CXCL-12) is involved in processes of migration of hematopoietic stem cells. In zebraﬁsh, the CXCL-12 expression has been found on kidney tubules, skin and gills, indicating that expression and function are conserved in teleost ﬁsh . Soriano and colleagues (2002) , veriﬁed the involvement of signaling between GH and CXCL-12 in GH-transgenic mice. In these mice, an over regulation of SOCS3 (Suppressors of Cytokine Signaling-3) was observed due to activation of the JAK/STAT pathway by GH. These authors reported that B cells and macrophages/granulocytes, when stimulated in vivo with CXCL-12, showed a decrease in migration associated with high levels of expression of SOCS3 in the spleen, lymph nodes and bone marrow. It was also proposed that SOCS3 is capable of interacting with the CXCR4 receptor, impairing the response generated by CXCL-12. In the strain F0104 it has been reported an increased expression of SOCS1 and SOCS3 in the liver, as a result of overexpression of exogenous GH . Furthermore, high levels of expression of SOCS1 and SOCS3 in the brain and skeletal muscle have been observed (data not published). Thus, it is possible that the migration of hematopoietic cells for the formation of the thymus and kidney of transgenic zebraﬁsh is affected by SOCS. Due to the importance of these organs for the operation of various aspects of immunity, the reduction in size of such structures could be causing damage to the amount of cells present in and generated by them. Taking into account the reduction in the thymus and kidney area in GH-transgenic ﬁsh, it is likely that there is a decrease in the expression of important genes for the function of these organs in the immune system. To this end, analyses were made on the expression of genes involved in the development of lymphocytes (RAG-1 and IKAROS), genes coding for membrane receptors of T cells (CD4 and CD247), and genes coding for the IL-1b cytokine secreted by cells from head kidney. All genes analyzed showed expression levels reduced by approximately 50% in transgenics, this decrease being possibly related to the reduction in the organ area and direct implications on the functionality of the immune system. The reduction of the head kidney may have a direct relation to the decrease in the expression of IL-1b, since cells such as monocytes and macrophages are produced in this organ and are considered as the main sites of release of this cytokine . Moreover, RAG-1 is involved in the maturation of T and B lymphocytes  and IKAROS is a transcription factor regulating the differentiation of T cells . Decreased expression of these genes indicates that there are fewer lymphocytes in the maturation process in transgenics. A decrease was also evident in the expression of CD247 and CD4 genes, demonstrating that besides a reduction in number of lymphocytes maturating, they are also reduced due to excess GH. Still, to investigate if indeed there was a decrease in the amount of CD3 and
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CD4 lymphocytes in the thymus as expected, immunohistochemistry of this organ for these receptors was performed. In fact, both the presence of CD3 and CD4 was decreased in transgenics (w40%). This result supports that seen in gene expression analyses, which also indicated small amounts of mature cell types as well as cells in the maturation process in the thymic microenvironment. Contrasting results have been reported about the effect of GHtransgenesis on ﬁsh immune system. As observed in our study, GH-transgenic coho salmon at smolt stage showed lower disease resistance when challenged with the bacterial pathogen Vibrio anguillarum . On the contrary, “all-ﬁsh” GH-transgenic common carp exhibited greater nonspeciﬁc immune function . A recent study with GH-transgenic Atlantic salmon demonstrated that transgenics have more difﬁculty in maintaining homeostasis after stress challenge . These controversial results demonstrate that the GH effect on immune system may be species-speciﬁc, and must be considered as an important factor to be individually evaluated for aquaculture practices. This study constitutes the ﬁrst report of a negative modulation exerted by GH on the immune functions in transgenic zebraﬁsh that overexpress this hormone. The decrease in area of the head kidney and thymus in transgenics resulted in a decrease in the number of CD3 and CD4 cells in the thymus as well as in the expression of genes related to these organs. These ﬁndings suggest that, as a result of exogenous GH, aspects of the development of these organs and the processes of colonization by HSCs may be harmed by pathways not yet fully studied. All this damage to the immune functions could contribute, together with others glucocorticoids physiological effects, to a higher mortality of transgenic ﬁsh when exposed to dexamethasone. Our study demonstrates that supraphysiological levels of GH end up impairing the development and the immune functions of GH transgenic zebraﬁsh, suggesting that maintaining adequate levels of this hormone is essential for the functioning of the immunological activities.
Acknowledgments This work was supported by Brazilian CNPq (Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico). L. F. Marins is a research fellow from CNPq (Proc. No. 304675/2011-3).
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