2.4 Soluble mediators of immunity
An important array of regulatory mediators, highly toxic soluble molecules, degradative enzymes, and antimicrobial peptides provide strong immune humoral protection against a wide range of pathogens, orchestrate the majority of the immune mechanisms, and contribute to maintain the host homeostasis. These mediators, constitutively expressed or secreted upon induction by different immune cell types, can be found in all the extracellular body fluids, mainly serum and mucus, and at interstitial spaces.
2.4.1 Cytokines
Within these soluble factors, cytokines include a broad category of small proteins (∼5–20 kDa) that mediate cell signaling within the immune system. Cytokines are released by cells (mainly leukocytes) and regulate immune functions through the interaction with a specific receptor on the surface of other cells (paracrine) or the same cell that produced it (autocrine). In some cases, systemic effects can also be produced through their release (endocrine). Each cytokine can be produced by different cell types, but in the same way; its receptor can be expressed on the surface of many different leukocyte types. Finally, several cytokines may exert very similar roles and thus there is a high degree of duplication.
2.4.1.1 Chemokines
Chemokines are a family of cytokines that regulate immune cell migration, maturation, and functionality of the recruited cells in response to inflammation. Constitutively expressed chemokines also play a role in physiological leukocyte transit and microenvironmental segregation within lymphoid organs (Cyster et al., 1999; Warnock et al., 2000).
They are produced by different cell types and act on leukocytes through interaction with G-protein-linked chemokine receptors on the cell surface. Additionally, many chemokines also have a role outside the immune system in processes such as angiogenesis (Arenberg et al., 1997; Keane et al., 1998), neurological development and function (Belmadani et al., 2006; Gordon et al., 2009), or organogenesis and germ cell migration (DeVries et al., 2006; Doitsidou et al., 2002; Knaut et al., 2003).
They are defined by the presence of four conserved cysteine residues and are divided into four subfamilies depending on the arrangement of the first two conserved cysteines in their sequence, into CXC, CC, C, and CX3C classes. The CXC and the CC families constitute the two largest chemokine families, both in mammals and fish. CXC chemokines can contain an ELR (Glu-Leu-Arg) motif at the N-terminus of their sequence responsible for receptor binding and activation of neutrophils, whereas CXC chemokines that lack this motif do not attract neutrophils and act on monocytes and lymphocytes (Clark-Lewis et al., 1991, 1993).
There are seven human ELR+ CXC chemokine genes (CXCL1–5, 5–8) located on the same chromosome and known to act through receptors CXCR1 and CXCR2 and nine ELR− CXC chemokines that interact with receptors CXCR3–6 (Mackay, 1997; Zlotnik et al., 1999). In fish, this ELR motif is usually replaced by a defective DLR motif (Asp-Leu-Arg) thought, at first, to be active due to the fact that mammalian ELR motifs mutated to DLR retained the capacity to attract neutrophils (Hebert et al., 1991). However, it has been recently demonstrated that this DLR motif is not essential for the attraction of neutrophils by fish CXC chemokines, and, therefore, DLR+ fish chemokines attract neutrophils even when this motif is eliminated (Cai et al., 2009).
Phylogenetic analyses of teleost CXC chemokine sequences have identified six different teleost CXC chemokine clades: CXCa, CXCb, CXCc, CXCd, CXCL12, and CXCL14 (reviewed in Huising et al., 2003). However, chemokines from each clade have not been identified in every species. A wide review of the CXC chemokines identified in fish to date was published in 2011 (Alejo and Tafalla, 2011), and, recently, a unified nomenclature for CXC chemokines in fish, amphibians, and reptiles has been proposed based on an extensive phylogenetic study (Chen et al., 2013). The CC chemokine family, distinguished by adjacent cysteine residues in a conserved position, has suffered a large increase in some fish species, evidencing extensive, species-specific intrachromosomal duplications.
So far, 18 different genes have been identified in rainbow trout (Dixon et al., 1998; Laing and Secombes, 2004b; Liu et al., 2002), 30 in Atlantic salmon (Peatman and Liu, 2007), 26 in channel catfish (Ictalurus punctatus) (Bao et al., 2006; Peatman and Liu, 2007), and 81 in zebrafish (Nomiyama et al., 2008).
CC chemokines were first divided into “inflammatory” (or “inducible”) CC chemokines, which are expressed only after immune stimulation, and “homeostatic” (or “constitutive”) CC chemokines, which are produced under normal physiological conditions (Laing and Secombes, 2004a; Zlotnik, 2006). However, as more information becomes available, many chemokines appear to have a dual role.
Therefore, seven large groups of fish CC chemokines were established after an extensive phylogenetic analysis: the CCL19/21/25 group, the CCL20 group, the CCL27/28 group, the CCL17/22 group, the macrophage inflammatory protein (MIP) group, the monocyte chemotactic protein (MCP) group, and a fish-specific group (Peatman and Liu, 2007). It was suggested that this “fish CC chemokine group” may represent a subset of ancestral chemokines that carry on important functional roles common to all teleost fish, not yet identified.
Concerning the other two mammalian chemokine families, C and CX3C, so far in teleost fish, C chemokines have only been identified in zebrafish (Nomiyama et al., 2008), whereas CX3C chemokines have never been reported. Moreover, a novel family of chemokines named CX, which includes five different members, has been established in zebrafish (Nomiyama et al., 2008).
These chemokines differ from the C family in that they lack one of the two N-terminus conserved cysteine residues but retain the third and fourth one, while the C family only retains the second and fourth signature cysteine residues. There is no information available yet on the bioactivity of these chemokines. Despite the great number of chemokine genes identified in diverse fish species, only very few studies have demonstrated the chemotactic capacity of these molecules (Alejo and Tafalla, 2011). Furthermore, although in some species, such as zebrafish, pufferfish, or rainbow trout (Daniels et al., 1999; DeVries et al., 2006; Dixon et al., 2013; Nomiyama et al., 2008; Zhang et al., 2002), the number of chemokine receptor genes identified also begins to grow, there are only a few of receptor:chemokine pairs revealed so far (Knaut et al., 2003; Perlin and Talbot, 2007). Until this information is available, it will be very difficult to understand the functionality of these molecules.
2.4.1.2 IFNs
Interferons (IFN) are virus-inducible cytokines with antiviral activity that, in mammals, can be classified into three subfamilies (I, II, and III), depending on their genomic structure, the cell types that produce them, and the receptors through which they signal (Zou and Secombes, 2011). In mammals, different type I and III IFNs exist, whilst there is only one single type II IFN gene. In fish, type I and II IFNs have been reported and at least two type II IFN genes are known to be present in some species. Type I IFNs can be produced by any cell type in response to a viral infection and constitute the main antiviral mechanism in vertebrates.
Although type I IFN-like activities were reported many years ago in fish, it was only until 2003 that it was cloned and reported in three independent studies in zebrafish (Altmann et al., 2003), Atlantic salmon (Robertsen et al., 2003), and pufferfish (Takifugu rubripes) (Lutfalla et al., 2003). After those reports, type I IFN genes have been identified in multiple fish species (reviewed in Zou and Secombes, 2011). The number of IFN present in one species varies from one to another, but, in general, salmonids and cyprinids appear to have more copies than species of the Acanthopterygian superorder such as pufferfish (Zou and Secombes, 2011).
Furthermore, fish IFNs can be divided according to the number of cysteines in their sequence. These two groups are referred to as group I type I IFN (2C) and group II type I IFN (4C), but, so far, group II IFNs have been found only in salmonids and cyprinids where their expression seems to be restricted to specific cell types such as leukocytes (Zou and Secombes, 2011; Zou et al., 2007). Bioactivity studies have mostly focused on group I type I fish IFNs for which antiviral activity, as well as capacity to induce downstream molecules, has been demonstrated (reviewed in Zou and Secombes, 2011). Although the bioactivity of group II type I IFNs has not been studied as extensively, recent evidence suggests they have complementary antiviral effects (Aggad et al., 2009; Levraud et al., 2007; Lopez-Munoz et al., 2009; Zou et al., 2007).
The direct antiviral effects of type I IFN in infected and surrounding cells are mediated by the induction of a set of IFN-stimulated genes such as Mx, oligo2’,5’-adenylate synthetase, or protein kinase R (PKR). The direct antiviral effects of these different proteins are not completely clear even in mammals, and, moreover, different viruses exert different sensibilities to their actions (Staeheli, 1990). Mx is the IFN-induced protein most widely studied in teleost fish, and up to three isoforms of Mx proteins have been reported in multiple species (Abollo et al., 2005; Altmann et al., 2004; Chen et al., 2006; Jensen and Robertsen, 2000; Lee et al., 2000; Plant and Thune, 2004; Robertsen et al., 1997; Tafalla et al., 2004; Trobridge et al., 1997; Trobridge and Leong, 1995; Zhang et al., 2004).
However, its antiviral activity has been demonstrated only in a few cases (Caipang et al., 2003; Fernandez-Trujillo et al., 2011, 2013; Larsen et al., 2004). Other IFN-induced antiviral proteins reported in fish include the viperin, protein kinases, ISG15, galectins, and finTRIM proteins (reviewed in Verrier et al., 2011). In mammals, type II IFN (IFN-g) is produced by Th1 cells and NK cells to promote Th1 responses mainly directed against intracellular pathogens, such as viruses and intracellular bacteria, through the activation of Tc lymphocytes and macrophages (Boehm et al., 1997). Orthologs to IFN-g have been identified in many teleost species (reviewed in Zou and Secombes, 2011), and, in some species, an additional IFN-g gene cataloged as IFN-grel has also been reported (Chen et al., 2010b; Grayfer and Belosevic, 2009; Igawa et al., 2006; Milev-Milovanovic et al., 2006; Stolte et al., 2008; Zou and Secombes, 2011).
It is generally accepted that this gene is a second member of the type II IFN family that appeared in teleost after duplication of the IFN-g gene (Zou and Secombes, 2011). Several IFN-g have been recombinantly produced and their bioactivity tested, mainly on macrophages.
In these cells, fish IFN-g has been shown to increase the expression of MHC-I and MHC-II (Martin et al., 2007; Zou et al., 2005), the production of nitric oxide (Arts et al., 2010; Grayfer and Belosevic, 2009), the respiratory burst (Grayfer and Belosevic, 2009; Zou et al., 2005), and the phagocytic capacity (Grayfer and Belosevic, 2009). Furthermore, some of the antiviral genes usually induced by type I IFN, such as Mx, can also be induced by type II IFN (Jorgensen et al., 2007), even though the antiviral capacity of this IFN is much weaker than that of type I IFN (Xu et al., 2010).
2.4.1.3 Interleukins
The term “interleukin” (IL) was first coined in 1979 to refer to molecules that signal between different leukocyte types, and, although nowadays it is known that these ILs are not only synthesized nor have effects exclusively on leukocytes, the term has been maintained (Secombes et al., 2011). In mammals, 35 ILs have been identified so far; however, some of these numbers refer to subfamilies of molecules rather than to single molecules. Secombes et al. (2011) excellently reviewed the ILs described to that current date in fish, classifying them into different families; therefore, in this chapter, we will mention only the main roles and members of these IL groups.
The IL-1 family groups 11 members in mammals, namely, IL-1a (IL-1F1), IL-1b (IL-1F2), IL-1 receptor antagonist (IL-1ra/IL-1F3), IL-18 (IL-1F4), IL-1F5-10, and IL-33 (IL-1F11). All these molecules are either pro-inflammatory or agonists of other pro-inflammatory family members. IL-1b was first discovered in fish (rainbow trout) in 1999 (Zou et al., 1999), and it has been reported in many different teleost species since then (reviewed in Secombes et al., 2011). IL-18 has been identified in pufferfish and trout, and, although no functional studies have yet been performed, its transcription is modulated in response to different immune stimuli (Huising et al., 2004; Zou et al., 2004).
Although no clear homologs of other family members are present in the locus of IL-1b as occurs in mammals, other family members with high relatedness to IL-1b exist in some fish species in different locus, for example, the molecule named novel IL-1F (nIL-1F), thought to be an IL-1b agonist (Wang et al., 2009a). There are other pro-inflammatory cytokines outside the IL-1 family such as IL-6. The IL-6 family includes IL-6, IL-11, IL-31, and other mammalian cytokines such as CNTF, LIF, OSM, CT-1, and CT-2 (Secombes et al., 2011).
Although not clear in relation to other family members, there seem to be clear homologs of IL-6 and IL-11 in several fish species, which can be modulated in response to diverse stimulation (reviewed in Secombes et al., 2011). The IL-2 subfamily of cytokines include those that signal via the common gamma chain (gC or CD132), a member of the type I cytokine receptor family expressed on most leukocytes.
In mammals, the IL-2 subfamily is formed by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. All these molecules are implicated in T cell memory or correspond to molecules released by different Th subsets upon stimulation. All of them, except IL-9, have been found in fish (Secombes et al., 2011). The IL-10 subfamily groups IL-10, an anti-inflammatory cytokine, and other class II cytokine family members such as IL-19, IL-20, IL-22, IL-24, IL-26, and the IFNs (Lutfalla et al., 2003). The induced transcription of IL-10 in response to LPS or bacterial infection has been demonstrated in several fish species (Inoue et al., 2005; Lutfalla et al., 2003; Pinto et al., 2007; Savan et al., 2003; Seppola et al., 2008; Zhang et al., 2005; Zou et al., 2003a); however, its regulatory or anti-inflammatory effects have not been characterized yet in fish. The IL-17 cytokine subfamily in mammals is composed of 6 members, IL-17A to IL-17F, with IL-17E also known as IL-25.
This gene family is considered very ancient and is the only IL family to have been found in invertebrates as well as in agnatha (Secombes et al., 2011). Several of these family members have been found in fish, even though their homology to mammalian counterparts has been difficult to establish. Several ILs with very diverse functions, such as IL-12, IL-23, IL-27, and IL-35, can be grouped because they are formed as heterodimers and share some of the peptide chains that form them. These chains include p19, p28 (also called IL-30), p35, p40, and Epstein-Barr virus induced gene 3 (EBI3); consequently, IL-12 is formed by p35 and p40, IL-23 by p19 and p40, IL-27 by p28 and EBI3, and IL-35 by p35 and EBI3 (Secombes et al., 2011). Additionally, the p40 homodimer can function as an inhibitor of IL-12 (Jana and Pahan, 2009). To date no functional studies have been performed with any of these cytokines; however, the p19, p35, p40, and EBI3 chains have been identified in several fish species (reviewed in Secombes et al., 2011).
2.4.1.4 Other cytokines
Other important cytokine families include TNFs, TGFs, and colony-stimulating factors (CSFs). In mammals, the TNF superfamily groups 19 members with a wide range of functions, including key roles in inflammation, host defense, autoimmunity, organogenesis, cellular apoptosis, and differentiation (Ware, 2003).
TNF-a, the best-known family member, has been identified in several fish species, often finding two molecules per species (Kinoshita et al., 2014; Zou et al., 2003b). Zebrafish TNF-a has been shown to mediate virus-induced apoptosis in fish cells, suggesting that the function of this molecule is conserved (Wang et al., 2011).
Using two different fish models, it has been shown that TNF-a mediates pro-inflammatory responses through the promotion of Eselectin and chemokine expression in endothelial cells and a subsequent recruitment and activation of phagocytes (Roca et al., 2008).
Recently, the inclusion of TNF-a in an oral Vibrio vaccine in sea bass produced an extended protective response and higher local IgT transcription levels (Galindo-Villegas et al., 2013).
TGF-b is the best-known cytokine belonging to a family of multifunctional cytokines involved in cell growth, migration, differentiation, and apoptosis (ten Dijke and Hill, 2004).
TGF-b also acts as an important regulator in the proliferation of T and B cells, having effects on macrophages, DCs, and NK cells (Kee et al., 2001; Li and Flavell, 2006; Strobl and Knapp, 1999). Although dose-dependent, often these effects on immune cells are antiinflammatory, restoring lymphocyte homeostasis. Three isoforms of the TGF-b family (e.g., TGF-b1, TGF-b2, and TGF-b3) have been identified in mammals.
These isoforms are similar in structure and biological function, with TGF-b1 as the predominant form in the immune system. All isoforms of the TGF-b family have been identified in teleosts, including zebrafish (Kohli et al., 2003), rainbow trout (Hardie et al., 1998), sea bream (Tafalla et al., 2003), grass carp (Ctenopharyngodon idella) (Yang and Zhou, 2008), and striped bass (Morone saxatilis) (Harms et al., 2000).
However, information on biological functions of these molecules in the fish immune system remains limited. In grass carp, it has been demonstrated that TGF-b1 inhibits in vitro peripheral blood leukocyte proliferation induced by LPS and PHA, but could up-regulate MHC class I expression and proliferation in nonstimulated peripheral blood leukocytes (Yang and Zhou, 2008). CSFs are cytokines with a central role in hematopoiesis, activating intracellular signaling pathways that can cause the cells to proliferate and differentiate into a specific kind of blood cell. Additionally, they can influence specific mature cells to modulate immune functions and maintain homeostasis and immune competence (Barreda et al., 2004). Belonging to this group, macrophage-CSF (M-CSF) and granulocyteCSF (G-CSF) are relatively lineage-specific, having a role in the proliferation, differentiation, and survival of macrophages, neutrophils, and their precursors. On the other hand, granulocyte/macrophage-CSF (GM-CSF) and multi-CSF (IL-3) function at earlier stages of lineage commitment regulating the expansion and maturation of primitive hematopoietic progenitors.