1. Introduction
Fish are continuously exposed to a microbial-rich environment (freshwater or seawater) that circulates through and reaches every epithelial barrier of their body. Thus, compared to terrestrial animals, aquatic animals have a greater challenge coping with high microbial loads, which bombard their mucosal epithelial barriers.
The main mucosa-associated lymphoid tissues (MALT) of teleosts are the gut-associated lymphoid tissue (GALT), skin-associated lymphoid tissue (SALT), the gill-associated lymphoid tissue (GIALT) and the recently discovered nasopharynx-associated lymphoid tissue (NALT) (Figure 1).
Figure 1. Schematic representation of the four teleost main mucosa-associated lymphoid tissues (MALT) described so far and their anatomical localization. GALT: gut-associated lymphoid tissue; SALT: skin-associated lymphoid tissue; GIALT: gill-associated lymphoid tissue; NALT: nasopharynx-associated lymphoid tissue.
When any given mucosal barrier of an animal senses a danger signal, an immediate innate immune response is triggered. This initial cue is essential for the later establishment of specific adaptive immunity. Adaptive immunity based on B and T cells and recombinatorial rearranging receptors is a canonical feature of the immune system of jawed vertebrates [1].
This double-armed B/T cell system is present in both systemic and mucosal immune systems. At the mucosal barriers, B and T lymphocytes form a dynamic network for the induction and regulation of secretory antibodies and cytotoxic T lymphocyte (CTL) responses [2].
Mucosal B cells and T cells (and their respective receptors and signaling molecules) have specialized to meet the specific demands of the mucosal environment. Generally, the mucosal immune system favors a tolerogenic microenvironment that avoids constant immune responses against non-harmful antigens present for instance in the food or microbiota. In other words, immune tolerance to maintain homeostasis is a hallmark of the mucosal environment [3].
The presence of adaptive mucosal immune responses in teleost fish has been known for decades thanks to early oral and parenteral immunization studies conducted in rainbow trout (Oncorhyncus mykiss) and plaice (Pleuronectens platessa) [4,5].
Early biochemical analyses of antibodies revealed differences in mucosal and serum immunoglobulin (Ig) molecules of fish, suggesting the presence of specialized mucosal antibodies in this group [6–10]. Slow progress was made for some decades until the past six years or so, when a renaissance of teleost mucosal immunity studies took place with the discovery of IgT and its function in mucosal immunity.
Yet, the whole picture of how mucosal immune systems defend teleosts is far from clear. Thanks to substantial research efforts devoted to investigation of the evolution of Ig molecules in vertebrates including those present in teleosts [11,12], mucosal Igs and their function have been unraveled. However, in other realms the field of teleost mucosal adaptive immunity is clearly at its infancy. There are two major research areas that require further efforts: the biology of teleost mucosal T cells and the mechanisms by which memory is established and maintained at the mucosa.
The aim of this review is to describe the general aspects of teleost MALT anatomy as well as the adaptive immune cells, molecules and immune responses that occur at the mucosal barriers of fish including the skin, gut, gills and olfactory organ. Descriptions of the cell subsets known to be essential for mucosal adaptive immunity in mammals are included with a reference to whether or not they exist in teleosts, if known. The overview here provided should serve as a platform to encourage researchers to direct efforts towards unveiling the unique aspects of the teleost mucosal adaptive immune system. This, in turn, will lead to better mucosal vaccines for aquaculture and serve as a greater validation that teleosts are valuable models for the study of vertebrate mucosal immunity.
2. General Aspects of Teleost MALT Anatomy
Every vertebrate mucosal surface is armed with an associated lymphoid tissue also known as MALT. Depending on their localization in the body, MALT receive specific names. MALT appears to have first evolved as a network of diffuse leucocytes that are disseminated along the mucosal surfaces of all vertebrates. This is also known as diffuse MALT (D-MALT). On the other hand, organized lymphoid structures can be found within the mucosal epithelia of endotherms and are known as O-MALT. Some examples of O-MALT are the Peyer’s patches and tonsils.
These structures are believed to have provided the anatomical, physiological and immunological basis for the maturation of antibody responses, since they provide the niche where selection for high affinity B cells clones among the entire pool of B cells takes place. O-MALT structures do not exist in teleosts (Table 1). An exception may be the curious case of the interbranchial lymphoid tissue (ILT), identified in Atlantic salmon (Salmo salar) (Table 1).
This is a lymphocyte rich structure largely consisting of T cells embedded in a meshwork of epithelial cells, with no direct resemblance to previously described lymphoid tissues [13–15]. As discussed later, this structure plays a role in the immune response of salmon against viruses.
A total of four different MALTs have been described to date in teleosts (Figure 1 and Table 1). These are GALT, SALT, GIALT and NALT. The majority of what we know about fish MALT refers to studies from salmonids and cyprinids with an emphasis on the effects of mucosal vaccines. Teleost MALTs are composed of both innate and adaptive immune cells and molecules that work together to maintain homeostasis at the mucosa. It seems that all MALT in teleosts may operate under certain primordially conserved principles, although MALT-specific unique characteristics are likely to be unraveled and we study each of these tissues in depth. In mammals, mucosal immunologists have coined the terms “inductive mucosal site/tissue” and “effector mucosal site/tissue”.
Inductive sites are those where antigens sampled from mucosal surfaces stimulate cognate naive T and B lymphocytes. Effector sites, on the other hand, are those where the effector cells after extravasation, retention, and differentiation perform their action, for instance by contributing to the formation of secretory IgA antibodies [16].
Such distinction may not be easily made in teleosts, at least based on our current body of knowledge. Due to the lack of draining lymph nodes and O-MALT, we currently believe that each MALT in teleosts may function both as an inductive and effector tissue, at least with respect to IgT specific responses. Future studies on the migration, differentiation and function of mucosal B and T cells of fish may shed new light to this question.
Stimulation of one MALT often results in responses in other distant MALT. Whereas some level of inter-connectivity exists among teleost MALT, the molecular basis for a “common mucosal immune response” at multiple sites following stimulation or vaccination at one site remains to be studied [17]. It is also worth mentioning that the Society for Mucosal Immunology (SMI) does not support the use of the term “common mucosal immune system” due to fact that it is now clear that each MALT holds some degree of compartmentalization in mammals. This is still a point of debate in teleosts and it may be true that teleost MALT are not as compartmentalized as their mammalian counterparts. However, we recommend the use of this term with caution, as suggested by the SMI. With respect to ontogeny of adaptive immunity at mucosal barriers, it is clear that the first B and T cells appear at the mucosae much later than in primary lymphoid tissues. Additionally, studies in common carp (Cyprinus carpio) indicate that T cell appearance precedes that of B cells appearance in MALT [18].
3. Teleost Mucosal B Cells and Immunoglobulins
B cells, plasma cells and Igs have specialized to defend the complex environment that defines mucosal barriers. It appears that most vertebrates have an Ig isotype specialized in mucosal immunity [19,20]. The mucosal antibody repertoire in mammals is established by both T-dependent and T-independent mechanisms [21].
The second relies on the role of the microbiota to shape antibody production. Teleost fish have an associated microbiota in each of their mucosal barriers. How these microbial communities influence mucosal B cell biology of teleosts is largely unkown.
In the mucosal secretions of mammals, a wide diversity of different Ig isotypes is present including IgA, IgM and IgG, but IgA is the chief mucosal Ig playing a role in homeostasis, innate and adaptive immune responses [22]. Similarly, in teleost fish, both IgT and IgM are detectable at the protein level in a number of mucosal secretions using immunoblotting or ELISA (for a summary see [17]).
The biology and current knowledge on teleost mucosal B cells and Igs was recently reviewed [17,23]. Measuring the ratios of IgT to IgM in plasma and in mucosal secretions was the first indicator that IgT plays a major role in mucosal immunity.
Thus, in rainbow trout gut, skin and nasal mucus, the IgT/IgM ratio is much larger than in plasma in the absence of any antigenic stimulation [19,24,25]. IgT+ B cells are the preponderant B cell subset in GALT, SALT and NALT compared to the spleen or head kidney, where IgM+ B cells are the main subset [19,24,25].
The total percentage of B cells in the gut and skin of trout is ~4%–5% [19,24], whereas in the olfactory organ is ~40% [25] (Table 1).
Out of this total number, in all MALT approximately half of the B cells are IgT+ and the other half are IgM+ [19,24,25], although in the skin the proportion can be up to 60%/40% [24]. This is in agreement with results from other species, for instance carp, where the percentage of B cells was estimated to be 5%–10% in the intraepithelial lymphocyte (IEL) compartment and the same in the skin and the gills [26]. The role of IgD in vertebrate mucosal immunity continues to be in many ways an enigma. The presence of a V domain associated with the trout secretory IgD molecule indicates that this isotype may potentially be involved in specific antibody responses [27].
Total IgD levels range from 2 to 80 g/mL in the plasma of rainbow trout [27] but may be very low or below detection levels at mucosal secretions since no quantification of this Ig in mucus has been made. Total IgD secreting plasma cells were measured in systemic lymphoid tissues as well as the gills of rainbow trout and it was found that the IgD to IgM plasma cell ratio is about 1:1 in gills and approximately 4 fold lower in systemic lymphoid tissues [27]. This finding along with detection of secreted IgD transcripts in mucosally vaccinated trout may indicate a role for IgD in mucosal immunity. However, specific IgD plasma cells or specific secreted IgD in gill mucus in response to antigenic stimulation have not been measured to date. Functional experiments are critical for ascertaining the functional role of IgD in the mucosal adaptive immune response of fish. Overall, our knowledge on plasmablasts, plasma cells and memory B cells in teleost fish MALT is very scant [17]. Previously, hydroxyurea (HU) has been used to distinguish between HU-sensitive (plasmablast) and HU-insensitive (plasma cell) activities in rainbow trout [28].
However, we currently lack specific markers that define fish memory B cell populations. Using ELISPOTs, total numbers of plasma cells from MALT have been identified in a number of fish species and mucosal tissues (reviewed in [17]). However, only antigen specific IgM secreting plasma cells have been measured. IgM and IgZ-producing cells were detected by in situ hybridization in the gill of mandarin fish [29]. In the same study, no IgD-producing cells were detected in the gills, adding more controversy to the potential role of IgD in gill immunity. Generally speaking, it is unclear how naïve B cells become activated and how they mature into plasmablasts and plasma cells in the mucosal tissues of fish. Moreover, the maturation of mucosal B cells into plasma cells may be governed by distinct signals in the mucosa of teleosts compared to mammals; a question that needs to be resolved in fish. It has been proposed that teleost gut has a limited number of classical plasma cells and that they are not easily detectable in the mucosal tissues [10]. Whereas long-lived plasma cells have been identified in the main lymphoid organs of teleosts, whether or not these exist in MALT is unknown.
4. Teleost Mucosal T Cells
Generally speaking, teleost fish have T cell populations with similar characteristics to those found in mammals. Two major T cell receptors (TCR), TCRαβ and TCRγδ have been described in teleosts.
Additionally the CD4 and CD8 co-stimulatory molecules have been cloned and some antibodies against these molecules have been produced. These two molecules define the CD8+ and CD4+ T cell subsets which appear to have conserved functions in vertebrates: cytotoxic versus helper T lymphocytes [30]. The description of several key T cell markers including CD4, CD8, CD3, CD28, CTLA4, as well as important cytokines suggest that, similar to mammals, different T helper (Th) subtypes (Th1, Th2 and Th17) exist in teleost fish [31].
Additionally, the availability of monoclonal antibodies against the T cell markers, CD8 and CD3ε , in rainbow trout [32,33] and CD3ε in Atlantic salmon [14] has helped the study of mucosal T cells. Finally, the specific T cell monoclonal antibody DLT15 detects T cells in European seabass (Dicentrarchus labrax) [34] whereas the WCL38 antibody detects T cells in the common carp [26] and those two tools have been very valuable for the study of mucosal T cells in both species. In every vertebrate, mucosal T cells possess unique features that make them particularly suitable for the mucosal microenvironment, where millions of food antigens and symbionts are present. Intestinal T cell subset development is controlled by the microbiota according to murine studies [35].
T cells appear to be very abundant in GALT, SALT and GIALT of common carp, accounting to 50%–70% of all lymphoid cells [26] and T cell markers have been found in GALT, SALT, GIALT and NALT of teleosts [10,25,26,34,36–39]. Additionally, studies in carp using WCL38 suggested that T cells from skin, gut and gills represent a distinct subset from those present in systemic lymphoid tissues [26]. Gut mucosal T cells are divided into two populations, the IEL subset and the lamina propria leucocyte (LPL) subset. In mammals, IELs are predominantly CD8+ T cells, whereas CD4+ T cells dominate in the lamina propria (LP). In teleost, IELs are also predominantly CD8+ T cells (reviewed by [10]). Interestingly, studies on rainbow trout showed that the gut IEL T cell repertoire is not distinct from its systemic counterpart. TCR transcripts of rainbow trout IELs are highly diverse and polyclonal in adult naive individuals, in sharp contrast with the restricted diversity of IEL oligoclonal repertoires described in birds and mammals [38]. Teleost IELs share similar features to systemic T cells and therefore may not represent a distinct compartment such as that present in mammals. In GIALT, T cells may represent around 10%–20% of all lymphoid cells [33]. Additionally, the discovery of the interbranchial lymphoid tissue (ILT) in Atlantic salmon as a CD3ε rich lymphoid tissue makes this species somewhat unique among other vertebrates and supports the importance of T cells in teleost gill adaptive immunity [14,15]. In carp skin, T cell numbers are also very abundant [26] and transcription levels of T cell markers point to a generally high presence of T cells in the teleost skin. Thus far, NALT T cells have not been studied (Table 1) but it is likely that T cells are abundant in the teleost NALT. It is clear that further studies on specific T cells and T cell functions will lead to further expansion and understanding of teleost mucosal immunity. Below, the most important T cell subsets known to play a role in the mucosal immune system of mammals are discussed. In those instances where information is available within the teleost fish literature, teleost-specific information is included.