2.5.1 Pathogen recognition
Once a pathogen succeeds in breaching the innate physical barriers, it is recognized by pattern recognition receptors on immune cells, this step being the first key factor in the triggering of all subsequent immune responses.
These receptors are mainly present in DCs and macrophages, but also in other cell types, such as B lymphocytes or endothelial cells, and are activated in response to conserved motifs in pathogens, designated as pathogen-associated molecular patterns (PAMPs), or in response to damage-associated molecular patterns (DAMPs), cell components released during cell damage.
Once these receptors are activated, several intracellular activation routes are triggered. All of them essentially lead to the activation of pro-inflammatory or antimicrobial genes. TLRs are the best-known receptors in this group that also includes C-type lectin receptors, NOD-like receptors, RIG-I-like receptors, and peptidoglycan recognition proteins (PGRPs). All five types of receptors have been reported in teleost fish (Boltana et al., 2011).
C-type lectin receptors exist as both soluble and transmembrane proteins. In fish, only seven transmembrane C-type lectin receptors have been described to date, and, although they are expressed in immune tissues and modulated upon infection, their immune role has yet to be elucidated (Chen et al., 2010a; Goetz et al., 2004; Soanes et al., 2004).
NOD-like receptors are cytosolic proteins involved in autoimmunity, bacterial and viral responses, as well as apoptosis (Martinon and Tschopp, 2005).
In fish, these receptors form a large family with up to 70 members in species such as zebrafish (Laing et al., 2008).
RIG-I-like receptors are a type of intracellular pattern recognition receptors involved in the recognition of viruses that activate host IFN upon recognition of viral RNA. The RLH family contains three members, RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2).
Fish also possess RIG-I-like receptors, among which RIG-I has been shown to activate the IFN response via a mitochondrion-associated signaling pathway (Biacchesi et al., 2009; Lauksund et al., 2009). Finally, recent studies performed in trout revealed that MDA5 and LGP2 act as independent positive regulators of the IFN response, while an LGP2 variant identified in this species antagonizes with LGP2 function (Chang et al., 2011). The PGRPs recognize peptidoglycans from both gram-positive and gram-negative bacteria and have a well-conserved structure between invertebrates and vertebrates.
In fish, 10 different PGRPs have been identified to date (Boltana et al., 2011).
Finally, the TLR group is a well-characterized family that comprises membrane receptors and endolysosomal receptors. In fish, they have been reported in large numbers in more than a dozen different fish species that include modern fish, such as the sea bream, or ancient species such as the lamprey (Boltana et al., 2011; Pietretti and Wiegertjes, 2014; Rebl et al., 2010).
Several reviews provide detailed descriptions of the down stream activating routes triggered by TLRs in fish (Collet and Secombes, 2002; Rebl et al., 2010). Most of the studies dealing with TLRs in fish report only transcriptional changes, but of course consistent changes in TLR transcription in the same species or across different fish species should be considered as an indirect indication of functional relevance of this TLR in a defined situation.
Thus, it has been mainly through these types of studies that we have seen a high degree of conservation in TLR recognition and functionality in fish compared to their mammalian counterparts. In mammals, TLR3, 7, 8, and 9 are mainly involved in viral detection, whereas TLR1, 2, 4, 5, 6, 7, 8, and 9 recognize highly conserved structures of bacterial origin. Nevertheless, remarkable distinct features of teleostean TLR cascades have been discovered and have been reviewed elsewhere (Pietretti and Wiegertjes, 2014; Rebl et al., 2010).
For example, it has been shown that TLR3 in fish can detect bacterial molecular patterns, in addition to viral patterns in contrast to mammalian TLR3. Regarding TLR4, responsible for sensing LPS in mammals, it seems that this receptor is missing from the genome of several fish species (Pietretti and Wiegertjes, 2014), whereas it does not recognize LPS in some others (Sepulcre et al., 2009). Finally, while the orthologs of some human TLRs are missing in teleosts (TLR6 and TLR10), some piscine TLRs are encoded by duplicated genes, such as salmonid TLR22, found in several fish species, but only as a nonfunctional pseudogene in humans (Rebl et al., 2010).
2.5.2 Phagocytosis
The cells that primarily sense the pathogens through receptors, such as DCs and macrophages, are also characterized by a strong phagocytic capacity. This phagocytosis contributes, on one hand, to pathogen clearance, while, at the same time, phagocytosis constitutes a necessary step for antigen presentation by these cells.
Since the latter will be addressed in the next section, in this section we will focus on the capacity that cells of the immune system have to clear pathogens through phagocytosis. This mechanism mainly relies on the activities of the two so-called “professional phagocytes,” macrophages and neutrophils.
Even though the differential features of these two cell types have been extensively studied in mammals, it has been through the use of transgenic zebrafish larvae that it has been revealed that macrophages efficiently engulf bacteria from blood or fluid-filled body cavities; neutrophils barely do so. By contrast, neutrophils are very efficient at sweeping up surface-associated, but not fluid-borne, bacteria (Colucci-Guyon et al., 2011).
Furthermore, it is especially the phagocytic ability of neutrophils that is essential for the removal of microbes and damaged tissue needed for the resolution of inflammation. For this, subsequent reduction in neutrophil numbers at a site of tissue injury occurs by several different processes: by neutrophil apoptosis, by removal in inflammatory exudates, or by reverse migration, all of them demonstrated in the zebrafish model (Henry et al., 2013).
Once the pathogen is inside a phagocyte, intracellular killing mechanisms are triggered. It is interesting to note that these intracellular killing mechanisms are triggered, not only when the pathogen has been internalized in a phagosome as a consequence of active phagocytosis, but also when the pathogen itself penetrates the cells (Rieger and Barreda, 2011).
In case the pathogen has been phagocytized, lysosome granules migrate towards and fuse with the phagosome, forming a phagolysosome structure, resulting in the release of acidic and enzymatic lysosomal contents into the phagosomal lumen and subsequent degradation of phagolysosome contents. Phagolysosome fusion has been shown to occur in goldfish monocytes and mature macrophages (Rieger et al., 2010).
Upon pathogen internalization, phagocytes also trigger the production of oxygen and nitrogen radicals as one of the main intracellular killing mechanisms. The production of oxygen radicals or respiratory burst is a process catalyzed by NADPH-oxidase, a multicomponent enzyme assembled on the inner surface of the plasma membrane following appropriate activation. This activation leads to the production of superoxide anion.
NADPH-oxidase has been identified in diverse teleost fish (reviewed in Rieger and Barreda, 2011) and the stimulation of respiratory burst, especially in macrophages, has been widely studied in diverse species, (reviewed in Rieger and Barreda (2011) and Forlenza et al. (2011)). Furthermore, it is known that several fish cytokines can augment this respiratory burst.
These include TNF-a (Grayfer et al., 2008), IFN-g (Arts et al., 2010; Grayfer and Belosevic, 2009; Grayfer et al., 2010; Zou et al., 2005), CSF-1 (Grayfer et al., 2009), and IL-8 (Harun et al., 2008). Interestingly, in some species, TNF-a has no direct effect on phagocytes, but instead it activates endothelial cells, that then lead to an indirect activation of phagocytes (Forlenza et al., 2009; Roca et al., 2008).
It is worth noting that in 2006, the active phagocytic capacity of teleost B cells was revealed as a breakthrough discovery in immunology (Li et al., 2006), posteriorly reported for some subsets of mammalian B cells (Nakashima et al., 2012; Parra et al., 2012). Both IgM+ (Li et al., 2006) and IgT+ (Zhang et al., 2010) cells were shown to actively internalize beads or bacteria, leading to phagolysosome fusion. Although the actual contribution of teleost B cell to pathogen clearance in comparison to other professional phagocytes may be low, the phagocytic activity suggests a higher capacity of B cells in fish to present antigens, a hypothesis also supported by the reported high levels of expression of DC-like markers in teleost B cells (Johansson et al., 2012; Zhang et al., 2009).
2.5.3 Antigen presentation
MHC class I molecules are found on nearly every nucleated cell of the body. They bind peptides mainly generated from degradation of cytosolic proteins by the proteasome. The MHC I:peptide complex is then exposed on the cell surface. Thus, the function of the class I MHC is to display intracellular proteins produced during an intracellular infection or through a cell cycle alteration (tumor cells) to Tc lymphocytes.
Recognition of antigenic peptides through MHC class I by Tc lymphocytes leads to the killing of the target cell, which is either infected by virus or by intracellular bacteria, or is otherwise damaged or dysfunctional.
MHC class II molecules on the other hand are found only on specific cell types, which in the case of fish could include macrophages, DCs, B cells, thrombocytes, and acidophilic granulocytes (in gilthead sea bream), all of them assumed to have antigenpresenting properties (Cuesta et al., 2006; Forlenza et al., 2011; Kollner et al., 2004; Li et al., 2006; Lugo-Villarino et al., 2010).
The antigens presented by class II peptides are derived from extracellular proteins that have been taken up through phagocytosis by these cells. The protein is then processed into the phagolysosome that breaks into smaller peptides, which are then exposed on the cell surface in the context of class II MHC molecules. This MHC:antigen complex is then recognized by T cells, usually CD4+ Th cells that become activated and secrete cytokines to modulate the adaptive immune response. In mammals, this presentation takes place in the spleen or in the lymph nodes.
In fish, since there are no lymph nodes, antigen presentation seems to take place locally where the antigen is present (Castro et al., 2014b). Despite the fact that fish DCs have not been clearly characterized, the antigen presenting capacity of the DC-like cells described in fish has been established (Bassity and Clark, 2012; Lugo-Villarino et al., 2010).
The up-regulation of MHC-I and MHC-II levels in response to different pathogens or immunostimulants has been reported in various fish species (Cuesta and Tafalla, 2009; Dixon et al., 1995; Goetz et al., 2004; Ingerslev et al., 2009; Miller et al., 2004; Olsen et al., 2011; Rakus et al., 2009; Stet and Egberts, 1991); however, many details of its regulation remain unsolved. One of the limiting factors in understanding teleost major histocompatibility receptor function is the lack of knowledge about antigen presentation accessory molecules. Some of these molecules have been identified in some fish species and seem to have an expression regulation different from that of tetrapods (Fujiki et al., 2003).
2.5.4 Cytotoxic responses
In the process of recognition of self from non-self, immune cells can detect nonspecifically most pathogens and altered cells via cell surface receptors that recognize expressed pathogen-associated molecular patterns, non-self cells (with heterologous MHC class I molecules in their surface) and foreign molecules presented in the context of MHC class I (intracellular antigens) or MHC class II (extracellular antigens). In this sense, both NK-like cells and Tc lymphocytes are responsible for identifying and killing these cells.
NK cells sense the down-regulation or altered expression of MHC-I to identify altered cells, whereas Tc cells identify a certain antigenic peptide exposed in a self MHC-I complex through an interaction with the TCR complex. Furthermore, in comparison to NK cells, Tc cells require priming and clonal expansion for an adequate response. However, their mode of killing is very similar and is based on two different types of mechanisms. On the one hand, granule-exocytosis releases pore-forming molecules, such as perforin, granulysin, and NK-lysin, that disrupt the membrane of the target cells that allow entry of granzymes that provoke cell apoptosis.
Perforin and granzyme has been identified in CD8+ Tc lymphocytes and NK cells in various fish species (reviewed in Fischer et al., 2013) and their expression is up-regulated in response to virus infection (Aquilino et al., 2013), suggesting an equivalent role as to that of mammals. On the other hand, the second killing mechanism is mediated through FasL/Fas interaction. FasL proteins produced by cytotoxic cells provoke apoptotic cell death through its interaction with Fas receptor (FasR) on the surface of the target cell. In mammals, FasL can be expressed in a membrane-bound form and as a cytosolic soluble form, whereas in fish only cytosolic forms have been identified (Fischer et al., 2013). This killing mechanism has been demonstrated in catfish and zebrafish.
2.5.5 Antibody production
B cells express Ig either on the cell surface as antigen receptors (BCR) or secrete them as soluble Igs or antibodies. Igs are composed of two identical heavy (H) chains and two identical light (L) chains that are encoded by the IgH locus and IgL locus, respectively. Both the H and L chains are composed of a variable (V) and a constant (C) domain. The V domain is composed of one of each V, D, and J gene segments and is responsible for antigen-binding, whereas the C domain is responsible for binding to effector molecules, triggering complex signaling pathways that lead to the elimination of the antibody-coated foreign material.
Two different types of genomic arrangement of the Ig loci have evolved. Mammalian V, D (only in the H chain), J, and C gene segments encoding Ig H or L chains are organized in one type of locus containing multiple consecutive V segments, D segments, and J segments upstream in the constant region in an organization known as translocon arrangement. In cartilaginous fish, successive individual clusters of V-(D)-J-C gene segments are repeated 100–200 times in what has been termed as multicluster arrangement (VDJ-C)n. IgH genes in teleost are organized in a translocon configuration as in mammals, whereas the Ig L-chain gene organization is of multicluster type (Hsu et al., 2006; Malecek et al., 2008; Warr, 1995). Cartilaginous fish possess three Ig heavy chain isotypes, namely, m (IgM), d (IgW), and IgNAR, and four light chain isotypes, namely, σ-cart, σ, k, and l. For teleost, three heavy chain isotypes – m (IgM), d (IgD), and τ (IgT) – and three light chain isotypes – σ, k, and l – have been described (Das et al., 2012). Igs are expressed in the membrane of B cells, defining the different B cell subtypes (see Section 2.3.1.1). Upon the interaction of a pathogen with the variable region of these Igs, the B cell becomes activated and part of its progeny initiates Ig secretion as part of the process that will lead the cell to become an antigen-secreting cell (ASC). The production of specific IgMs in response to antigens has been reported against several types of pathogens, including virus, bacteria, and parasites and in some occasions this antibody production seems to be directly related to protection (Raida et al., 2011; Romstad et al., 2013; Ye et al., 2013). In those cases when protection is mediated by antibodies, they can protect fish against a reinfection with the same pathogen and can last for a long time (Ma et al., 2013a; Ye et al., 2013). In most teleosts, serum IgM is expressed as a tetramer in different redox states, variability suggested as important for their function, although not clearly elucidated yet (Kaattari et al., 1998). IgM can be present in serum and secretions, including mucus, and the antibody response intensity varies among fish species, from salmonids that produce a large amount of specific antibodies to a variety of antigens, to cod that has very low antibody responses, being mainly natural, nonspecific antibodies (Solem and Stenvik, 2006). The secreted antigen bind to the pathogen and in some cases directly interferes with their replication capacity (neutralizing antibodies). Furthermore, antibody-pathogen complexes attract phagocytes through receptors (Fc receptors) that recognize the Fab portion of the antibody bound to the antigen, facilitating phagocytosis. The Fc receptor-antibody complex can also create by-products, such as C3b and C4b, that activate the complement system (Stafford et al., 2006). Recently, the production of specific IgDs and IgTs have also been described, suggesting that all three Igs cooperate in pathogen clearance even though their specific function is not clear yet (Bromage et al., 2004; Xu et al., 2013; Zhang et al., 2010). Before the identification of an IgD homolog in catfish, IgD was believed to represent a recently evolved Ig described only in primates and rodents (Wilson et al., 1997). Since then, IgD was identified in all vertebrates except birds, indicating that this Ig represents an ancient Ig class. A secreted IgD was identified in serum of catfish as a monomer, although this protein may not contain a functional V-region (Bengten et al., 2002). On the other hand, rainbow trout possess a secretory IgD product that is associated with a Cm1 exon and a V-region, and its secretory arrangement has been inferred in other teleost species analyzing the IGH locus expressed sequence transcripts (ESTs) (Ramirez-Gomez et al., 2012). This analysis includes Atlantic salmon, Atlantic cod (Gadus macrocephalus), and grass carp (Ctenopharyngodon idella). A single positive IgD population was recently described in trout. This B cell population, mainly present in the gills, has still an unknown function, although it is regulated upon viral infection. In mammals, a memIgD+ memIgM− population present mainly in the upper aerodigestive mucosa arises in humans after active IgM-to-IgD class switch. These plasmablast-like cells that retain IgD in the membrane secrete highly mutated mono- and polyreactive IgD, providing a layer of mucosal protection by interacting with pathogens, and are either retained locally or are circulated in the blood, where they can account for up to 0.5–1% of circulating B cells (Chen and Cerutti, 2010; Chen et al., 2009). IgT is expressed as a monomer in rainbow trout serum and as a tetramer in gut mucus (Zhang et al., 2010). This Ig has been described as a specialized Ig in mucosal immunity in rainbow trout. So far, IgT positive populations have been described as the prevalent B cell subset in the gut and skin. Although fish do not produce IgA, IgM produced at mucosal sites can be secreted and so does IgT/Z (Rombout et al., 2011). In trout, a strong gut local IgT response was elicited by a gut protozoan parasite, while the IgT levels in serum remained at low levels. In contrast a strong IgM response was restricted to serum (Xu et al., 2013; Zhang et al., 2010). In the case of the skin, IgT responses against a skin parasite were mainly limited to the skin, whereas IgM responses were almost exclusively detected in the serum (Xu et al., 2013). These results suggested that IgT could be an equivalent of IgA; however, systemic IgT responses to either virus (Castro et al., 2013) or DNA vaccination (Castro et al., 2014b) suggest that IgT also plays an important role outside mucosal tissues. The repertoire of the BCR was inferred in zebrafish and rainbow trout using spectratyping and high-throughput sequencing methods. The expressed variable domain (VDJ region) of the IgHm, IgHd, and IgHτ was studied in naïve fish and these repertoires are polyclonal and highly diversified (Castro et al., 2013; Weinstein et al., 2009). The B cell response against a rhabdovirus, viral hemorrhagic septicemia virus (VHSV), was also studied, vaccinating fish with an attenuated strain and challenging them 3 weeks later with the same strain. The study of the BCR repertoire in the spleen of these fish showed a strong IgM response with clonal expansion of several VDJ sequences, a more moderate IgT response, and an IgD repertoire that remained essentially unaltered (Castro et al., 2013).
It is worth noting that what have been designated as natural antibodies are present in the serum of vertebrates at any time without apparent antigenic stimulation. In mammalian species they are mostly of the IgM type, secreted by the long-lived, selfrenewing B1 subset of B cells, which are generated during fetal or neonatal development. These antibodies are polyreactive and have low-binding affinity, binding various autoantigens like thyroglobulin, single stranded DNA and heat shock proteins, or haptens like 2,4,6-trinitrophenyl (TNP) or 2,4-dinitrophenyl (DNP) (Boes, 2000; Casali and Schettino, 1996). Their role is mainly to provide local confinement of infection, to enhance IgG response, to take part in homeostasis and clearing of cell debris, and to link innate and adaptive immunity (Boes, 2000; Lutz, 2007; Ochsenbein et al., 1999). Natural antibodies have been described in several species of chondrichthyes and teleosts (Gonzalez et al., 1988; Sinyakov et al., 2002; Vilain et al., 1984). In a comparative study of the natural antibody repertoire of different fish species, it has been shown that teleost natural antibodies have specificity primarily for haptenated protein (TNPBSA), while those of elasmobranchs and sturgeon show specificity for various self and non-self-antigens but a lower anti-TNP-BSA titre (Gonzalez et al., 1988). The contribution of these natural antibodies in the immune defense against both bacterial and viral diseases has been studied in cod, goldfish, and rainbow trout (Gonzalez et al., 1989; Magnadottir et al., 2009; Sinyakov et al., 2002). In cod, special attention has been paid to natural antibodies because of the peculiarities of the antibody response in this species. When fighting an infection, cod relies more on the quantity than the specificity of the antibody repertoire. Thus, immunization and challenge experiments have resulted in poor acquired antibody response, even if the IgM concentration in serum is relatively high compared with other fish species. In contrast, the activity of natural antibodies against TNP-BSA is commonly high, and these natural antibodies have a broad specificity and relatively strong affinity (Magnadottir et al., 2009).