Full Length ArticleThe influence of 17β-oestradiol on lymphopoiesis and immune system ontogenesis in juvenile sea bass, Dicentrarchus labrax
Introduction
The vertebrate immune system consists of two major components: the adaptive and innate immune responses. The latter generally precedes, activates and determines the nature of the adaptive response, and co-operates in the maintenance of homeostasis (Fearon, 1997; Fearon and Locksley, 1996). The adaptive immune system –unique to jawed vertebrates– is highly specialized and based on two lineages of effector immune cells: the B and T lymphocytes (Kasahara et al., 2004), which are characterized by their respective T and B cell receptors (TCR, BCR). Recombination-activating genes (RAG) 1 and 2 encode nuclear proteins that directly mediate the recombination of TCR and BCR gene segments. This recombination is necessary to produce a diversity of receptors that are able to specifically recognize a large number of antigens (Randelli et al., 2009). The TCRs are formed by membrane heterodimers composed either of TCRγ and δ chains, or of TCRα and β chains, thus defining two fundamentally different T cell lineages. The γδ T cells have innate-like properties, including rapid effector function after activation and the capacity to bind antigens directly. The αβ T cells, on the other hand, are commonly referred to as ‘conventional T cells’ and recognize antigen peptides presented by the class I or II major histocompatibility complex (MHC), which are expressed by antigen presenting cells such as thymic epithelial cells (TEC) to select self-tolerant T cells (Klein et al., 2009; Mantegazza et al., 2013). Conventional T cells have a highly diverse TCR repertoire allowing a specific adaptive immune response, which follows the innate response.
The thymus plays a pivotal role in the ontogenesis of the adaptive immune system. It is the major site for T lymphocyte-education and -maturation, i.e., thymopoiesis. T cell-development, including T cell-commitment and -maturation, depends on the interaction with TECs, the function of which is regulated by the transcription factor forkhead-box n1 (FOXN1) (Bajoghli et al., 2009; Žuklys et al., 2016). Among the various conventional T cell subpopulations, the regulatory T cells (Tregs) are particular in that they develop either in the thymus, or in peripheral immune organs, such as the spleen or the liver, depending on their TCR specificity (Plitas and Rudensky, 2016). Their function is to regulate innate and adaptive immune cell activity and to maintain self- and non-self-tolerance (Quintana et al., 2010; Wing and Sakaguchi, 2010). The forkhead transcriptional regulator P3 (FOXP3) is essential for the genetic programming of Treg-differentiation and -function (Sakaguchi et al., 2001; Shevach, 2002). From the thymus, naïve and mature self-tolerant T cells emigrate to peripheral lymphoid organs, including the spleen and the liver (Bowden et al., 2005). Thymopoiesis and the regulation of the abundance of peripheral T cells is dependent on cell migration and on the expansion of proliferating cell nuclear antigen (PCNA)-positive T cells (Goronzy and Weyand, 2019). B and T lymphocytes colonize the spleen after they have been detected in the thymus and head kidney, proving that the spleen is a secondary lymphoid organ in jawed vertebrates (Boehm et al., 2012; Neely and Flajnik, 2016). The spleen plays an important role in antigen-presentation and in the initiation of the adaptive immune response, i.e., T and B cell-activation (Boehm et al., 2012; Mebius and Kraal, 2005). In mammals, the liver is also involved in T cell-education, although its innate immune functions appear to be more important. In fact, the liver promotes their dysfunction and mediates adaptive immune tolerance by acting as a ‘graveyard’ of activated T cells (Racanelli and Rehermann, 2006; Zheng and Tian, 2019). In fish, the role of the liver in immunity is far less documented. Nevertheless, intrahepatic immune cells, which are dominated by T cells, have been described by Möller et al. in rainbow trout (2014).
In mammals, early haematopoiesis already begins in the yolk sac and temporarily moves to the liver before definitive haematopoiesis is finally established in the bone marrow and thymus, where the full set of immune and blood cells will be generated (Jagannathan-Bogdan and Zon, 2013). In fish, however, the ontogeny of the haematopoiesis and the immune system is far less studied. It is believed that the protection of larvae and fry depends on maternally transferred components like lectins and antibodies and that a non-specific innate immunity is already present at early egg stages (Ferraresso et al., 2016). In several teleost species, the thymus is the first lymphoid organ in which lymphoid cells appear (Bowden et al., 2005). Nevertheless, the antibody response against T cell-dependent antigens, which highlights a mature adaptive immune response, occurs after the thymus becomes morphologically mature and is delayed in comparison to the maturation of humoral immunity (Lam et al., 2004). Accordingly, fish immunocompetence becomes enhanced as soon as thymic regionalization is completed (Seemann et al., 2017). Nevertheless, before thymus ontogenesis is completed, peripheral T cells are already detected (Huttenhuis et al., 2006; Romano et al., 2011; Scapigliati et al., 2018). Together with the expression of rag1 in peripheral T cells, this suggests that (i) innate-like T cells arise from both intra- and extrathymic origins in teleost fish, akin to mammals (Boschi et al., 2011; Cheroutre et al., 2011; Huttenhuis et al., 2006) and (ii) during ontogenesis, innate-like T cells probably represent a major component of the immune defence of fish, as has been shown previously for Xenopus tadpoles and mammals (Cheroutre et al., 2011; Edholm et al., 2016).
The oestrogenic regulation of the immune system, including the thymus, seems to be conserved across vertebrates (Paiola et al., 2018; Segner et al., 2013). Oestrogenic regulation in different tissues can result from either slow (via nuclear-initiated steroid signalization - NISS), or rapid (via membrane initiated steroid signalization - MISS) signalling through their binding to multiple oestrogen receptors (ERs). Most fish species have three intracellular nuclear receptors (ESRs), i.e., one Esr1 and two Esr2, while terrestrial vertebrates have only one Esr1 and one Esr2. In addition, fish may have two G Protein-Coupled Estrogen Receptors (GPERs), i.e., Gpera and Gperb, while mammals have only the GPER1 form. The functions of GPERs are less well understood, but it is now evident that both types of signalling and receptors may interact with each other (Amenyogbe et al., 2020; Lafont et al., 2016; Pinto et al., 2014; Thomas et al., 2010). The expression-pattern of the full set of nuclear and membrane receptors determines the global cellular response to oestrogens. According to the conserved immunomodulatory function of oestrogen, the various ER-isoforms have been detected in immune cells and immune organs, including the thymus, across numerous vertebrates from mammals to fish (Paiola et al., 2018; Segner et al., 2013). In addition to its modulatory role in thymic T cell-differentiation, as evidenced in mammals with a thymus atrophy during pregnancy or after puberty, oestrogen appears to be involved in thymus ontogenesis. In fact, aromatase-, ESR2-, GPER- and especially ESR1-knockout mice display defective thymus ontogenesis (Chunhe et al., 2008; Erlandsson et al., 2001; Li et al., 2002; Yellayi et al., 2000). Interestingly, studies from various jawed vertebrates, including the European sea bass (Dicentrarchus labrax), have shown that oestrogen can also increase thymus volume during critical windows of the thymus ontogenesis, during which immunocompetence is established (DeWitt et al., 2012; Kondo et al., 2004; Seemann et al., 2015). Therefore, oestrogen-exposure during thymus ontogenesis may have long-term consequences for the T cell-dependent immune response in mammals (Chapman et al., 2009; Forsberg, 2000) and in fish (Rehberger et al., 2020).
In the European sea bass, Seemann et al. (2015) observed that 17β-oestradiol (E2)-exposure triggers a change in thymus size, i.e., hypertrophy, in fish exposed for one month from 90 dph onward, but not from 60 dph or 120 dph. This E2-sensitive window of thymus ontogenesis seems to begin at 90 dph, which corresponds to the late stages of thymic regionalization. Therefore, in this study, we tested the hypothesis whether E2 is involved in immune system ontogeny by interfering with the thymic T cell development. To test this hypothesis, oestrogen responsiveness of the developing immune system was studied in European sea bass, at two developmental stages from 60 to 90 dph and from 93 to 122 dph. We investigated (i) the expression of the five fish oestrogen receptor isoforms in a central and a major secondary immune organ, thymus and spleen, respectively, and (ii) the thymic function, by studying the expression of thymic specific genes, as well as by measuring T cell-related genes in the periphery (spleen and liver). Potential effects on extrathymic T cell-differentiation were assessed by the measurement of rag2-expression in the spleen and liver.
Section snippets
Animal rearing
European sea bass fingerlings were obtained from the hatchery ‘Poissons du Soleil’ (Sète, France) at 40 dph, and then reared at the facilities of the Institute of Aquaculture Torre de la Sal (CSIC, Castellon, Spain) until they reached the appropriate age for the experimental assay (see Fig. 1 for details on the experimental design). During this time, fish were fed daily ad libidum with a diet manufactured by Life Bioencapsulation S. L. (Spin-Off Universidad de Almeria, Spain) and maintained in
Oestrogen concentrations and fish biometric measurements
The mean effective concentration of E2 in the treatment aquaria water over the 30 days of the exposure was of 20.22 ± 2.12 (S.E.) ng E2·L−1 and did not vary substantially, confirming the treatment nominal concentration in the two groups. The E2-concentration for the control aquaria was below the limit of detection (<1.39 ng·L−1) in all measurements. The liver samples from the E2-exposed fish presented a significantly higher concentration of E2 than the control group at 122 dph:
Discussion
In this study, we tested the hypothesis that oestrogens are directly involved in thymus maturation and associated T cell development in the European sea bass at two distinct developmental stages, previously identified as oestrogen-sensitive and non-sensitive windows of thymus ontogenesis, respectively (Seemann et al., 2015): a) between 60 and 90 dph, comprising the beginning of thymus regionalization (Romano et al., 2011), and b) between 93 and 122 dph, corresponding to the final phase of
Conclusion
To the best of our knowledge, this study is the first to investigate the regulatory role of E2, both in thymus and in secondary lymphoid organs, and the first to examine the dynamics of ESRs and GPERs gene expression in immune organs of a teleost fish during thymic development. We have confirmed the importance of oestrogen-signalling in the thymus development across different vertebrates. In fact, although the ER-isoforms have low or undetected levels of expression in the spleen of juvenile
Acknowledgments
This research was supported by ANR financed project ETaT (ANR-15-CE32-0014), by FCT (project UID/Multi/04326/2019, fellowship SFRH/BPD/84033/2012 and contract with the University of Algarve with reference DL57/2016/CP6321/CT0015 for P.I.S.P.) and by the EU H2020 Research and Innovation Programme AQUAEXCEL2020 (grant agreement 652831; TNA project AE050029) and ASSEMBLE PLUS (contract SR02022018976f).
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