Innate Immunity

Abstract

Research on the innate immunity has accelerated over the last decades. The main reason for this has been the discovery of receptors recognizing danger molecules from pathogens. This has been facilitated through genome and transcriptome sequencing of different fish species. Also, endogenous host molecules from sterile physiological insults may also bind to certain receptors and induce immunological processes. The magnitude and quality of adaptive immunity are known to be dependent on the instructions the innate response gives. This chapter gives an overview of selected innate immune organs/tissues, factors, and processes that have been suggested to possess important roles during innate immune response in fish.

2.1 Introduction

2.1.1 Innate Immunity: The Concept

Innate immune defense is important for protecting the host from infection, not only in naïve fish but also in fish that have previously been infected. “Innate immunity has shed its older, disparaging title of ‘non-specific immunity’ and now stands as a proud partner with the adaptive immune system in protecting human hosts from infectious insults. For any who doubt the impressive protective capacity of the innate immune system, it is instructive to consider that only vertebrates boast the added benefits of an adaptive immune system, leaving most organisms on our planet to survive on innate immunity alone” (Turvey and Broide 2010). Indeed, this applies also to fish. The immune system of teleost fish is composed of two kinds of receptor types: The germline-encoded pattern recognition receptors (PRRs) and the antigen-specific receptors are made from gene arrangement after, e.g., pathogen infection. The latter consist of, e.g., antibodies, MHC I and MHC II, and T-cell receptors. In addition, numerous other receptors/molecules can take part in the innate immunity. The innate mechanisms can be divided into constitutive and inducible. The former represents rapid ongoing ligand binding to receptors and a quick response, while the inducible (e.g., many PPRs) acts slower—but with a higher magnitude (Paludan et al. 2020) (Figs. 2.1 and 2.2).

Fig. 2.1

Simplified illustration shows how inducible innate immunity changes over time, whereas the constitutive is stable vs. time. To get complete sterilization and resolution, both inducible and constitutive innate immune responses plus antibodies are often needed. Epigenetic changes may contribute to better fitness/increased protection when fish are exposed to a second infection. Targeted gene expression surveys or transcriptomics has focused primarily on describing or identifying inducible genes (e.g., DEGs), while in contrast, factors contributing to the constitutive arm have been poorly described

Fig. 2.2

Hypothetical time-course study of a gene (qPCR) expression. The orange line represents a gene with constitutive expression upon treatment with stimulant 1, and the black line demonstrates an early expression of the same gene after the fish were treated with stimulant 2, whereas the dotted red line represents a delayed expression of this gene after the fish were treated by stimulant 3. A specific gene may be induced by a certain stimulant and not by others, or there may be a stable, rapid, or delayed induction. The magnitude of induction may likely be dependent on number of specific receptors on cells or/and the number of cells that harbor specific receptors

2.1.2 Innate Receptors

Innate and adaptive immunity can cooperate to clear the infections. Central receptors in the early innate responses are so-called Toll-like receptors (TLRs) and are vital for the communication between the innate and adaptive branches (Rivera et al. 2016). The germline-encoded pattern recognition receptors (PRRs) are central in the recognition of microbial components and for the activation of innate immunity, which may induce inflammatory response to eliminate pathogens. The PRRs, expressed in innate immune cells, include receptors such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs). Upon recognition of microbial components known as pathogen-associated molecular patterns (PAMPs), PRRs induce intracellular signaling networks to activate transcription factors that regulate genes involved in inflammatory responses. Importantly, these innate immune signals also trigger dynamic chromatin changes. Such changes may in turn induce modulated gene-specific expression patterns resulting in even more pathogen elimination.

2.2 Cells in the Innate Immune Response

The traditional view that the adaptive and innate immune defense is divided into two compartments is now more or less history. The fish innate immune cells comprise not only the “traditional” innate cells such as macrophages (Kordon et al. 2018; Rieger and Barreda 2011; Grayfer et al. 2014, 2018) and granulocytes (Pijanowski et al. 2013; Schmidt 1905) but also red blood cells (Puente-Marin et al. 2018, 2019b; Dahle et al. 2015; Wessel et al. 2015), thrombocytes (Stosik et al. 2019), B cells (Wu et al. 2020), and subtypes of T cells (Scapigliati et al. 2018).

2.2.1 Monocytes/Macrophages

Monocytes are large mononuclear circulating leukocytes, which become macrophages when they settle tissues and organs. The nucleus may display different oval-, kidney-, or bean-shaped conformations, while the cytoplasm is usually pale and agranular, with varying amounts of vesicles and lysosomes. Macrophages are one of the main immune cells performing phagocytosis, the other presumably being the neutrophilic granulocytes. Phagocytosis is a multistage process for removal and cellular ingestion and destruction by intracellular enzymes and other substances (Grayfer et al. 2014; Hodgkinson et al. 2015). In addition to being professional phagocytes, macrophages can also function as professional APCs by presenting antigen to T cells on MHC class II. Such functions have also been suggested from studies on fish monocytes/macrophages (Sugamata et al. 2009; Wittamer et al. 2011). It has been suggested that fish display at least three different phenotypes of macrophages, based on their activation processes: innately, classically, and alternatively activated macrophages (Wentzel et al. 2020; Hodgkinson et al. 2015). Innate activation occurs when a macrophage receives a stimulus from the recognition of a microbial substance (e.g., PAMP) through cell receptor(s) without any need for any co-stimulation. Classical activation, however, occurs with the combination of such a stimulus and the cytokine interferon-gamma (IFN-gamma). Both innate activation and classical activation typically lead to increased pro-inflammatory response as opposed to alternative activation. A presence of cytokines (interleukin 4 (IL4) and/or interleukin 13 (IL13)) induces a macrophage phenotype with a resolving function (wound healing and tissue repair). There is also a suggestion for a fourth type of macrophages, namely regulatory macrophage. Regulatory activation is associated with the cytokine interleukin 10 (IL10) and important for downregulation of the inflammatory process (Wiegertjes et al. 2016). However, macrophages are apparently able to change between different phenotypes, and there is still some uncertainty whether all these activation pathways perform the same way in fish, as in mammals (Forlenza et al. 2011). Please see Chap. 6 for a more thorough overview of macrophages in fish.

2.2.2 Dendritic Cells

Dendritic cells (DCs) are categorized as a professional APCs found within several different tissues and are very effective at initiating both innate and adaptive immune responses in mammals (Banchereau et al. 2000; Worbs et al. 2017). DCs are typically small cells, with several elongated, cytoplasmic processes (dendrites) that increase the cell surface area (Collin and Bigley 2018). Cell populations with DC-like morphology and functions have been reported from teleost fish (Shao et al. 2015; Bassity and Clark 2012; Haugland et al. 2012), but due to lack of specific markers it is currently unknown whether they are true homologs of the mammalian cell type.

2.2.3 Granulocytes

Granulocytes are leukocytes with cytoplasmic granules and often nucleus with varying shapes (lobes) (Flerova and Balabanova 2013). They are central pool of the innate immune cells (Lieschke and Trede 2009). The granulocytes have traditionally been grouped into neutrophilic granulocytes (neutrophils), eosinophilic granulocytes (eosinophils), and basophilic granulocytes (basophils), based on their staining characteristics with different dyes. However, this classification was originally developed for use in mammalian hematology and does not appear to always correlate well with characteristics of fish granulocytes (Kelenyi and Nemeth 1969; Drzewina 1909; Rombout et al. 2005).

2.2.3.1 Neutrophils

Neutrophils typically possess nucleus with varying degrees of lobulation and contain granules that usually do not display marked affinity for staining with basic or acid dyes (such as hematoxylin and eosin). Neutrophils are generally most abundant between the granulocytes. In mammals, neutrophils are very mobile and are usually among the first cells to infiltrate tissue during onset and early phases of inflammation (Rosales 2018). Similar cellular recruitment speed has also been reported from teleosts (Lamas and Ellis 1994; Katzenback and Belosevic 2009; Havixbeck and Barreda 2015). Neutrophils are armed with a diverse arsenal of cellular weapons, making them effective combatants against invading pathogens (Havixbeck and Barreda 2015). Like macrophages, they are able to degrade ingested microbes and particles through production and release of reactive oxygen species (ROS) and proteases (Katzenback and Belosevic 2009; Rieger and Barreda 2011). They can also release different granules, upon degranulation, containing antimicrobial proteins and enzymes such as myeloperoxidase (MPO) (Lieschke and Trede 2009). In addition, neutrophils are able to form extracellular traps, which contain antimicrobial factors (Palic et al. 2007; Pijanowski et al. 2013; Chi and Sun 2016; Zhao et al. 2017; Van et al. 2020).

Eosinophils, or acidophils, are described to contain cytoplasmic granules that stain bright red with the acidic dye (eosin). However, cellular identification based solely on cytochemical and/or histochemical staining characteristics may lead to misinterpretation as basophils, eosinophilic granule cells, mast cells, some neutrophils (also called heterophils), and rodlet cells also are capable to be dyed to various degrees. Consequently, it has been suggested that mammalian terminology should be used whenever possible for describing these cell types (Watanabe et al. 1997; Suljevic et al. 2017). In mammals, the eosinophils have immunological roles regarding both immune regulation, defense against parasitic infections, and allergic inflammatory reactions (Hogan et al. 2008). Teleost eosinophils have been reported to be phagocytic (Watson et al. 1963), and they increase in cell numbers and increase the degranulation activity as a response to infection (Balla et al. 2010).

2.2.3.2 Basophils

Basophils are large granulocytes with staining of their cytoplasmic granules with a basic dye (hematoxylin). Basophils are rarely observed in teleost species (Tavares-Dias 2006). Their granules contain histamine, an inflammatory mediator, and basophils are associated with anaphylaxis, allergy, and hypersensitivity reactions (Chirumbolo 2012). As such, they are similar to the mast cells. Although not fully established, these granulocytes might also have other functions within the fish immune system (Odaka et al. 2018).

2.2.3.3 Eosinophilic Granule Cells

Eosinophilic granule cells (EGCs) and rodlet cells have been observed in fish (Reite and Evensen 2006). Such cells resemble the classical mast cells (Reite 1998). Teleost EGCs have been identified in several species, as part of the host inflammatory response to injected vaccines, bacterial infection, parasite infestations, or other types of noxious stimuli (Rombout et al. 2011).

2.2.4 Thrombocytes

Thrombocytes are oval-shaped, nucleated, and agranular cells located in fish. In some fish species, thrombocytes have been shown to be phagocytic and it has been discussed whether thrombocytes can function as APCs and/or is coupled to the innate immunity (Stosik et al. 2019; Passantino et al. 2005).

2.2.5 Red Blood Cells

From a RNAseq study on trout red blood cells exposed to either poly I:C, it was found that the cells expressed numerous transcripts of immune molecules—such as ifna, tlr3, tlr9, mx, and ccl4 (Morera et al. 2011). Thus, the authors suggested that red blood cells indeed participate in innate immune response.

2.3 Epigenetic Control of Innate Immunity

2.3.1 Epigenetics: The Concept

Epigenetics involves heritable factors that regulate spatiotemporal genome expression, which may induce different phenotypes. Two of the molecular mechanisms, histone modifications and DNA methylation, regulate gene expression at the chromatin level. In contrast, microRNAs are molecules that affect gene expression at the posttranslational level. Epigenetic histone modification involves acetylation/deacetylation, methylation/demethylation, and phosphorylation/dephosphorylation of specific histone amino acids. Pathogens have evolved a variety of strategies to modify host epigenetics. For example, they can (1) directly modify host proteins and chromatin, (2) attenuate PRR binding and signaling pathways, and (3) modulate the expression of activators and repressors of innate immunity. Hosts can abrogate pathogen-induced epigenetic changes to maintain their innate defense characters (Zhang and Cao 2019). Analysis of posttranslational processes on immunity has not generally been well studied in fish. However, in one study, the impact of histone modification after infectious necrosis virus infection (IPNV) and temperature control has been shown (Boltana et al. 2018). In this study, IPNV-infected fish that preferred a given temperature showed histone modification, which could explain modulated expression of il1, il2, ifng, and ifnrg receptor. The pattern of histone modification was different from IPNV-infected fish kept at constant temperature. In another study, spring viremia of carp virus (SVCV) infection induced histone modification in zebrafish (Danio rerio). The authors indicated that the ifn, tlr, and C-reactive protein promoters were methylated postinfection; thus, these genes were upregulated compared to controls (Medina-Gali et al. 2018). Since epigenetic modification of the genome is a heritable trait, epigenetic programming of brood stockfish, by, e.g., immunostimulants, may be a viable approach to produce offspring with higher innate disease resistance (Zhang et al. 2019).

2.3.2 Micro RNA

MicroRNAs (miRNAs) are a family of small noncoding RNAs that play vital roles in modulating host immune response. Accumulating evidence demonstrates that host miRNAs are involved as mediators in regulating viral replication and host antiviral immunity in mammals. In a miiuy croaker macrophages, miR-3570 that was upregulated after rhabdovirus infection interfered and led to downregulation of type I interferon in the cells. In turn, this downregulation caused increased virus replication in cells (Xu et al. 2018a). Binding to Toll-like receptors (TLRs) and subsequent intracellular signaling may also bring about production of microRNA. This may result in a positive or negative feedback loop system regulating immune response. More on this complex issue is described in a review authored by Zhou et al. (2018).

2.3.3 Long Noncoding RNA

lncRNAs have been demonstrated to play pivotal roles in various biological processes, especially gene expression regulations, including transcriptional regulation, posttranscriptional control, and epigenetic processes. The functional significance of lncRNA lags far behind what is the status on mammals. However, a novel lncRNA (SETD3-OT) in turbot (Scophthalmus maximus) has been identified. From the annotation of neighboring adjacent genes, SETD3-OT might be involved in the regulation of cell apoptosis and cycle, the immune cell development, and the immune response against infection. The expression pattern of SETD3-OT was similar to the majority of the neighboring genes following Aeromonas salmonicida challenge. The SETD3-OT expression was high levels in mucosal surfaces in controls fish (intestine, gill, and skin), but was downregulated following Vibrio anguillarum infection (Yang et al. 2020). In another study, Nodavirus infected European sea bass (Dicentrarchus labrax) displayed many putative lncRNA, suggested to possibly be involved in immune responses (Pereiro et al. 2020). Other studies have also suggested lncRNA to be involved in the regulation of immune responses (Boltana et al. 2016; Valenzuela-Miranda and Gallardo-Escarate 2016; Paneru et al. 2016; Valenzuela-Munoz et al. 2018, 2019).

2.3.4 Small Interfering RNA and Circular RNA

In addition to microRNA and lncRNA, the methylation of mRNA, occurrence of small interfering RNAs, and circular RNAs may all contribute to epigenetic modulation of gene expression in vertebrates, including fish (Wang et al. 2018a). Olive flounder (Paralichthys olivaceus) experimentally infected with Edwardsiella tarda showed differentially expressed circRNA. The authors suggested that these belonged to the circRNA-miRNA-mRNA network, where KEGG analysis indicated that they were part of the Herpes simplex infection and intestinal immune network for IgA production (Xiu et al. 2019). Another study showed that circRNAs are involved in mammalian antiviral immunity (Wang et al. 2017). KEGG (Kyoto Encyclopedia of Genes and Genomes; www.genome.jp), a huge database integrating genomic, chemical, and systemic functional information, is often used to find what cellular networks/pathways the DEGs belong to. It refers to what is described in and annotated from human/mice systems.

2.4 Mucosal Innate Defense

2.4.1 Innate Immune Molecules of the Fish Skin

The skin of fishes protects fish from external pathogens. The outermost layer is mainly composed of epithelial cells, termed keratocytes. These cells cover scales and are highly phagocytic toward certain particles. They are also motile. The motility of fish keratocytes is studied in a number of fish species (Asbakk and Dalmo 1998; Tsuchida and Theriot 2013; Galbraith and Sheetz 1998; Jurado et al. 2005; Okimura et al. 2018; Ream et al. 2003). There has been limited research on the production of innate defense factors, but this topic deserves more attention. Whether the cells possess phagocytic receptors is not known, it seems that the cells are able to discriminate the uptake dependent on the kind of bacteria (Karlsen et al. 2012). The skin mucus contains an array of molecules enabling protection from pathogens. In a study on yellow catfish (Pelteobagrus fulvidraco), 133 differentially expressed proteins were found after bath infection with Edwardsiella ictaluri. A minority of these differentially expressed proteins were directly immune-related. Examples of the upregulated genes were complement component c3, MAP kinase 1, and interferon-induced 35 kDa protein (Xiong et al. 2020).

Among the antibacterial enzymes, the best studied in the fish skin is lysozyme. Lysozyme is a glycoside hydrolase that catalyzes the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan, which is the major component of gram-positive bacterial cell wall. However, it seems that lysozyme-like enzymes have activity also against gram-negatives, parasites, and virus, as reviewed by Dash et al. (2018). This review also contains detailed description of other skin-related innate immune factors (Dash et al. 2018).

How the mucus is obtained, for concomitant analysis of factors, will inevitably decide which substances will be found during a screening process. As an example: If the mucus sample contains cells or scales, it is clear that the samples also contain cellular factors and most probably also immune factors normally localized in deeper layers (e.g., connective tissue and muscle). The most gentle and sensible protocol is to adsorb the mucus using a tissue paper. While a wiping method using tissue paper also gives a good protein yield, this method comes with some degradations. If the research requires a high mucus yield together with substances from the epithelial layer, the wiping method is preferable (Faeste et al. 2020). It is quite difficult to discriminate between substances normally found in mucus compared to what is intracellularly or extracellularly localized in epidermis, subdermis, and connective tissue. Thus, many reports describe the presence of substances not (only) found in the mucus itself but also found in the underlaying tissue. As an example of the latter, the transcriptomic analysis of a skin sample (3 × 1 cm) from large yellow croaker (Larimichthys crocea) followed by Cryptocaryon irritans challenge revealed up to 1055 DEGs (differentially expressed genes) (96 h postinfection). Since many of the DEGs were clearly innate immune-related, it would have been interesting to see how many and which transcripts were from epithelial cells, connective tissue cells, blood cells, and muscle cells, respectively. Probably, a similar sampling protocol was followed by Liu et al. (2020a, b) where zebrafish were challenged with spring viremia of carp virus (SVCV)—causing skin lesions. This study revealed 320 DEPs (differentially expressed proteins) (48 h postinfection) and 181 DEPs (96 h postinfection). Sixteen of these were confirmed by means of QPCR analysis (Bai et al. 2020). DEPs often found are complement factors, and chemokines, heat shock proteins, MHC, cell adhesion molecules, TNF-induced protein, and many more were regulated (Liu et al. 2020b). In conclusion, analysis of skin innate defense mechanisms should discriminate between mucus itself, epidermis, subdermis, and connective tissue.

The epidermis consists of keratocytes, which are highly mobile cells and also possess (mostly overlooked) phagocytic activity (Asbakk and Dalmo 1998; Sveen et al. 2020). The immunological significance of their phagocytic ability is not yet fully understood. One theory is that they engulf as many particles they can before going into a cell death pathway and are sloughed off from the epidermis (Asbakk and Dalmo 1998). It is speculated that these cells possess some innate defense mechanisms (e.g., receptors) (Lindell et al. 2012). The epithelial layer of the fisheye cornea consists of cells that highly resemble skin keratocytes. These cells are not studied with respect to their innate defense abilities. We have preliminary results showing that these cells also engulf foreign particles (Fig. 2.3). For more details on mucosal immunity in fish, see Chap. 12.

Fig. 2.3

Corneal epithelial cells of Atlantic salmon (Salmo salar) possess phagocytic ability, as illustrated by the intracellular presence of (cyan) microbeads. Lysosome is stained by pink color. Courtesy: Dalmo, Wolfson, Kjølstad, Svartaas (UiT)

2.4.2 Nasopharynx-Associated Lymphoid Tissue (NALT)

NALT has been discovered to harbor lymphocytes, but also genes central in induction of innate immunity. These include mx1, tlr3, il1r, il8, tnfr, myD88, c3, c4, c7–1, cxc9, cxcl9, cathl1, ccl19, and il6 (Tacchi et al. 2014; Yu et al. 2018). The significance of NALT-mediated innate response, compared to, e.g., skin or intestine, is not clear. Another assemblance of lymphoid cells can be found in the buccal cavity of rainbow trout (Oncorhynchus mykiss) that have been infected by Flavobacterium columnare. After infection, this buccal cavity lymphoid tissue was found to express innate factors such as il8, il1b, chemokine like 19, cathl1 and cathl2, rig1, among other adaptive immune genes (Xu et al. 2020).

2.4.3 Gills in Innate Immunity

Gill-associated lymphoid tissue (GIALT) has been characterized in Atlantic salmon and different fish species (Resseguier et al. 2020; Haugarvoll et al. 2008). This tissue was, in Atlantic salmon, identified to express upregulated genes such as complement component c3, il18, mx3, il20, ifn type II, viperin, rig1, and ifna after ISAV challenge (Austbo et al. 2014; Valenzuela-Miranda et al. 2015). Pro-inflammatory (il6, il17c1) and anti-inflammatory (il10, tgfb) genes have been found, in rainbow trout gills, after Ich (Ichthyophthirius multifiliis) infection (Syahputra et al. 2019). Another study aimed at doing a transcriptomic survey of Atlantic salmon gills suffering from multifactorial pathologies. Genes that were differentially expressed were depicted to be involved in pathways such as cellular immune response (IL-17 signaling, IL-6 signaling, granzyme A signaling, crosstalk between dendritic cells and natural killer cells, granulocyte adhesion and diapedesis, and HMGB1 signaling), cytokine signaling (IL-17 signaling, IL-6 signaling, acute-phase response signaling, role of JAK family kinases in IL-6 type cytokine signaling, TNFR2 signaling, and HMGB1 signaling), and tissue damage and repair (Krol et al. 2020). Some of these genes possess central functions in innate immunity. More details on the gills’ function in the immune response, please see Chap. 1.

2.4.4 Intestine in Innate Immunity

During the recent years, many excellent review articles describing the fish’s intestinal immunity have been published (Dawood 2020; Nadal et al. 2020; Sitja-Bobadilla et al. 2016; Brugman 2016; Dezfuli et al. 2016; Scapigliati et al. 2018; Brinchmann et al. 2018). Recently, there have been many innovative approaches to better understand intestinal immunity. In one of these studies, proteomic and transcriptomic examination of the intestinal mucus in Tilapia infected with Streptococcus agalactiae showed that innate factors such as c1r-like EGF domain, c1q-binding protein, hsp1, hsp90b, galectin, and membrane attack complex component/perforin domain, conserved site, complement factor D, C-type lectin fold, il1, il1r, and foxp3 (Wu et al. 2016). Another study, in grass carp, did a transciptomic and proteomic examination of the intestine after oral DNA vaccination (Li et al. 2020). The study revealed 250 and 50 immune-related DEGs and DEPs, respectively, after the oral vaccination. KEGG enrichment analysis showed genes and proteins participating in the Toll-like receptor signaling pathway, MAPK signaling pathway, NOD-like receptor signaling pathway, and the complement cascade were present both in the mucous and tissue homogenates. It is obvious that the intestinal innate mechanisms are quite diverse. More research using modern omic technologies will inevitably give us more information about the significance of the various intestinal innate factors that have on disease resistance.

Recently, a lymphoid structure in the cloacal region was discovered in Atlantic salmon (Loken et al. 2020). This may be the same as Inami et al. (2009) described in Atlantic cod—although the work on cod did not specify the anatomical localization properly (Inami et al. 2009). Whether this collection of lymphatic cells has any function in innate defense is not clear. However, through gene expression studies, genes encoding il1b, il8, il10, hepcidin, and ccl19 were found. These genes likely play roles in the innate immunity.

Future studies of intestinal mucus must be carefully planned and executed to avoid contaminant cells and blood. This may give false assumptions with regard to the actual presence of innate factors.

2.5 Innate Defense Mechanisms in Muscle Tissues

Skeletal muscles have been implicated in several atypical physiological processes including immune response, especially after pathogen challenge. When zebrafish were intramuscularly challenged by Salmonella enterica, pro-inflammatory il1b and tnfa were highly expressed in their skeletal muscle. Likewise, hep (hepcidin) and il10 were also expressed (Chatterjee et al. 2016). The authors did not examine any presence of leukocytes in muscle tissue samples after the fish were challenged with Salmonella. Thus, it is likely that inflammatory leukocytes did contribute to the expression of the pro-inflammatory cytokines. The contribution of incoming leukocytes to the inflammatory event is discussed by Valenzuela et al. (2017) and Kaitetzidou et al. (2012). Similar to the induction of innate immune genes in skeletal muscle, Atlantic salmon infected with salmonid alphavirus and piscine reovirus showed altered gene expression in the heart tissue (Johansen et al. 2015). In this study, several innate genes were expressed in the heart muscle. The authors did not elaborate whether this contribution was caused by inflammatory cells or not.

2.6 Innate Defense Mechanisms in Kidney and Spleen

The kidney and spleen are hematopoietic organs capable of inducing and exerting innate immune responses (Uribe et al. 2011; Svingerud et al. 2012; Kumar et al. 2018). The head kidney is the principal immune organ responsible for phagocytosis, antigen processing, and formation of Igm and immune memory (Page et al. 2013; Stosik et al. 2018; Rauta et al. 2012; Rombout et al. 2005; Kim et al. 2017). Kidneys in fish are paired and have a Y shape along the body axis. The immune relevant part, the head kidney, is located anteriorly. The posterior is mostly the renal system. The form of the head kidney varies between species. In some species, there are two separate extensions in the most anterior part of the organ, while in salmonid species the kidney is present as a single organ (Press and Evensen 1999). It is acknowledged that the head kidneys’ main function is hematopoiesis of lymphocytes, phagocytosis, antigen presentation, and maturation of lymphocytes. Its significance in innate immunity is not very well researched yet, although the head kidney leukocytes are armed with innate factors (Aballai et al. 2017; Gerdol et al. 2015; Cao et al. 2020; Hwang et al. 2017; Rozas-Serri et al. 2019; Zhou et al. 2019). It should be clear that cells in the posterior part of the kidney also have capability to express immune genes after pathogen challenge, as reported by Sudhagar et al. (2019).

2.7 Innate Defense Mechanisms in the Spleen

It is acknowledged that the main functions of the spleen are in hematopoiesis of lymphocytes, antigen trapping, and destruction of red blood cells (Press and Evensen 1999). However, from RNAseq analysis it is evident that the spleen cells contain and express numerous innate immune genes such as those involved in chemokine signaling, Toll-like receptor signaling, RIG-1, and NOD-mediated signaling and complement cascade (Ali et al. 2014).

2.8 Innate Defense Mechanisms in the Liver

The liver is acknowledged to produce acute-phase proteins, including complement components following infection or physiological insult. An array of innate defense factors has been found following a transcriptomic study of rainbow trout. This study revealed transcripts coding for genes important in acute-phase response, inflammatory response, genes coding for PAMP-binding receptors, and molecules central in chemotaxis (Martin et al. 2010). This finding suggests that the liver also has capacity to mount innate responses.

2.9 Receptors and Molecules of the Innate Immune Defense

Innate immunity is orchestrated by numerous molecules such as cytokines, complement factor, and receptors. Many molecules participate in both innate immunity and adaptive immunity. The following chapters describe the roles of selected innate molecules that have been ascribed to innate immunity—as central components.

2.9.1 Toll-Like Receptors (TLRs) as Pattern Recognition Receptors (PPRs)

The number of TLRs adds to other pattern recognition receptors (Tribouley et al. 1978) (including splice variants) such as different C-type lectin receptors, NOD-like (nucleotide-binding oligomerization domain-like) receptors (NLRs), RIG-1-like receptors, and scavenger receptors (Brubaker et al. 2015), and suggests that fish may very well be equipped with innate receptors that may likely be targets for innate immune training. TLRs are a family of pattern recognition receptors that bind pathogen-associated molecular patterns (PAMPs) (Pietretti & Wiegertjes, 2014). In addition, several TLRs are able to bind certain endogenous molecules called damage-associated molecular patterns (DAMPs) (following, e.g., trauma). TLRs are highly important since they represent a considerable diversity in their ligand-binding properties and thus facilitate responses against a wide array of pathogens. Genome duplication events in fishes during evolution have been attributed to the diversity of TLRs; therefore, differences with respect to the number of TLR loci exist between mammalian species and many fish species (Palti 2011). As an example: The genome of a mudskipper species (Periophthalmodon schlosseri) contains 11 copies of tlr13 (You et al. 2014). Most vertebrate genomes are recognized to have at least one gene representing each of the seven major tlr1, tlr2, tlr3, tlr4, tlr5, tlr7 and tlr11 families (Roach et al. 2005). Within Osteichthyes, the large tlr1 subfamily members include tlr1, tlr2, tlr14, tlr18, tlr25, tlr27, and tlr28 (Nie et al. 2018). The tlr3, tlr4, and tlr5 subfamilies recognize dsRNA, LPS, and bacterial flagellin. The tlr7 subfamily ligands are nucleic acid motifs, whereas the tlr11 family TLRs recognize an array of different molecules—from proteins to nucleic acids—reviewed by Nie et al. (2018). The ligand specificities for each TLR have not been very well studied in fish, though flagellin, synthetic triacetylated lipopeptide (Pam3CSK4), lipopeptides from gram-positive bacteria, and short double-stranded RNA (dsRNA) have been shown to interact with/bind to tlr1/2, tlr5, and tlr22, respectively (Nie et al. 2018). This means that fish immunologists assume that tlr localization and ligand specificities of fish tlrs are similar to mammalian counterparts. This is reviewed by Pietretti et al. (2014) and Kanwal et al. (2014). Tlrs are, in mice and humans, localized in the cell membrane and in the endoplasmic reticulum (ER), endosomes, and lysosomes (Fink et al. 2016). Tlr receptors 1, 2, 6, and 10 have, in human or mice models, been found to recognize a broad range of peptidoglycans and lipoproteins from, e.g., bacteria and parasites. These are located on the cell surface and, following engagement, there is intracellular signaling ending in NF-kB-dependent gene expression. NF-kB promotes expression of pro-inflammatory cytokines. Viral recognition may be brought about by tlr3, tlr7, tlr8, and tlr9 where they potentially can bind dsRNA, single-stranded RNA (ssRNA), and CpG DNA. As found in zebrafish, the tlr22 may also bind dsRNA or poly I:C (a dsRNA mimic) (Li et al. 2017b) [Fitzgerald, 2020 #954]. These “antiviral receptors,” upon ligand binding, confer (via TRIF or/and MyD88) activation of interferon regulatory factors 3 and 7 (transcription factors), which in turn facilitates transcription of interferon type I expression.

Taken together, the common interpretation is that TLR activation results in the production of pro-inflammatory cytokines (e.g., tnfa and IL-1ß) and/or in the expression and synthesis of transcription factors involved in protection against viruses, bacteria, and parasites (Sahoo 2020; Kanwal et al. 2014; Rauta et al. 2014; Zhang and Gui 2012; Palti 2011).

Tables 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, and 2.22 give an updated overview of tlrs 1–5, 7–9, 12–14, and 18–28 found in different fish species. This list is continuously growing as genome, and transcriptome sequences from new species are completed and analyzed. This will be done during the “Fish10K” project where the aim is to genome sequence 10,000 fish species during a ten-year period (Fan et al. 2020), and through the ongoing “Fish1K” project (Sun et al. 2016b) and Earth Biogenome Project (Lewin et al. 2018).

Table 2.1 Updated list of tlr1 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr3 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. Tlr1 can associate with tlr2 and tlr6 to expand their ligand-binding specificities. In general, tlr1 may bind bacterial peptidoglycan and triacyl and diacyl peptides. ND not determined

Table 2.2 Updated list of tlr2 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. ND not determined

Table 2.3 Updated list of tlr3 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr3 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. Tlr3 is able to bind double-stranded RNA, which is a molecular signature of certain viruses. ND not determined

Table 2.4 A list of tlr4 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr4 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. TLR4 binds the lipid moiety of bacterial lipopolysaccharide. ND not determined

Table 2.5 List of tlr5 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr5 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. Bacterial flagellin is the ligand for TLR5. ND not determined, S soluble, m membrane

Table 2.6 Updated list of tlr7 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr7 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. TLR7 recognizes internalized single-stranded RNA found in viruses. ND not determined

Table 2.7 List of tlr8 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr8 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. TLR8 may recognize guanosine and uridine-rich single-stranded RNA found in viruses. Tlr8 associates with tlr7. ND not determined

Table 2.8 A list of tlr9 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr9 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. TLR9 binds unmethylated CpG sequences in DNA—often found in prokaryotes. ND not determined

Table 2.9 Tlr12 found in Yellow River scaleless carp (common name). See citation for corresponding Latin names. TLR12 recognizes profilin found in certain parasites. ND not determined

Table 2.10 Updated list of tlr13 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr13 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. It has been suggested that tlr12 binds 23S ribosomal RNA (rRNA) found in bacteria. ND not determined

Table 2.11 A list of tlr14 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr14 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. The natural ligand for TLR14 is not known. Since it is a member of tlr1 subfamily one can speculate that this receptor may bind similar ligands as TLR1/2/6—which are ligands from bacteria. ND not determined

Table 2.12 List of tlr18 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr18 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. TLR18 is also a member of the TLR1 subfamily and may bind molecules from bacteria. ND not determined

Table 2.13 Updated list of tlr19 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr19 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. Teleost-specific TLR19 is localized in the endosomes and recognizes dsRNA analogs. ND not determined

Table 2.14 A list of tlr20 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr20 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. TLR20 is proposed to play a role in the immune response of carp to protozoan parasites. ND not determined

Table 2.15 Updated list of tlr21 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr21 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. It has been suggested, similar to TLR9, that TLR21 may bind unmethylated CpG sequences in DNA. ND not determined

Table 2.16 List of tlr22 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr22 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. TLR22 is a typical fish-specific TLR and is suggested to bind double-stranded RNA. ND not determined

Table 2.17 Updated list of tlr23 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr23 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. The ligand for TLR23 is unknown, ND not determined

Table 2.18 List of tlr24 found in different fish species (common names). See citation for corresponding Latin name. The main tissue distribution of tlr24 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. The ligand for TLR23 is unknown

Table 2.19 Updated list of tlr25 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr25 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. The ligand for TLR25 is unknown, ND not determined

Table 2.20 Updated list of tlr26 found in different fish species (common names). See citations for corresponding Latin names. The main tissue distribution of tlr26 mRNA is also given, where the highest expression is found in the first organ/tissue listed, and so on. TLR26 is not yet found in any other fish species. The ligand is unknown

Table 2.21 Tlr27 is found in spotted gar, coelacanth, and elephant shark. See citation for corresponding Latin name. ND Not determined. Its ligand is unknown

Table 2.22 Tlr28 is found in miiuy croaker. See citation for corresponding Latin name. Like tlr27, the tlr28 belongs to the tlr1 subfamily. Its ligand is unknown

2.9.2 Interferon Type I

Interferons (IFNs) are a group of cytokines with important roles in defense against viral pathogens (cf. Chaps. 13 and 14). They are divided into two families, type I and type II, based on structural properties and functions. Both the type I and II IFN systems are essential to antiviral defense in innate and adaptive immunity (Zou and Secombes 2011) (Tables 2.23 and 2.24). In contrast to type I IFNs, which are more important in innate immunity, IFN-γ (type II IFN) is exclusively produced in immune-related cells and is more important later in the immune response. In innate immune responses, IFN-γ is produced by natural killer cells (Jung et al. 2012). During adaptive cell-mediated immune responses, IFN-γ is produced by CD4-positive Th1 cells and CD8-positive cytotoxic T lymphocytes. IFNs induce the expression of a broad array of IFN-stimulated genes (isgs), which encode for proteins with direct antiviral activity, including inhibition of viral transcription, degradation of viral RNA, inhibition of translation, or modification of protein function. Several reviews of the interferon system of teleost fish have been presented over the years (Robertsen 2006; Workenhe et al. 2010; Zou and Secombes 2011; Secombes and Zou 2017). Chaves-Pozo and coworkers investigated the interferon response in the ovary of rainbow trout (O. mykiss). They found that the VHS virus strongly upregulated all the ifn genes studied, while the IPN virus either had no effect or strongly suppressed ifn gene expression (Chaves-Pozo et al. 2010). Valero and coworkers investigated ifns in the gonads of gilthead sea bream (Sparus aurata) and European sea bass (D. labrax). They evaluated the expression after infection with the disease viral nervous necrosis (VNN) in the brain (Valero et al. 2015). The orange-spotted grouper, Epinephelus coioides, is a commercially important fish that is widely farmed in tropical waters, e.g., in Taiwan, Japan, Australia, and also Europe. Chen et al. characterized a type I ifn from this fish and determined the expression during nodavirus infection. Groupers infected with nodavirus had elevated levels of ifn and administration of recombinant IFN type I, which led to upregulated antiviral activity (Chen et al. 2014). In large yellow croaker (L. crocea), a type I group II interferon was identified by Ding and coworkers. The ifn was constitutively expressed in all examined tissues, spleen, liver, skin, head kidney, gills, blood, muscle, heart, brain, and intestine. The expression was rapidly upregulated in spleen and head kidney by poly I:C and Aeromonas hydrophila (Ding et al. 2019). A type I interferon gene was identified in Japanese eel (Anguilla japonica). The ifn was expressed constitutively in liver, spleen, intestine, gills, skin, kidney, heart, and muscle. After injection with LPS, poly I:C, and live A. hydrophila, expression levels increased in both liver, spleen, and kidney (Feng et al. 2017). A transgenic cell line for the detection of salmon interferons has been established. It is based on a CHSE-214 cell line containing a reporter construct expressing firefly luciferase under the control of a rainbow trout promoter for the IFN-induced mx1 gene. The mx promoter was shown to respond to both salmon IFN type I and trout IFN type II in a dose-dependent manner, while there was no response to recombinant tnfa and ilb (Jorgensen et al. 2007). Three distinct members of type I interferons were identified in the mandarin fish (Siniperca chuatsi) by Laghari et al. Fish injected intraperitoneally with poly I:C resulted in an enhanced expression of all three genes in the head kidney. The disease infectious spleen and kidney necrosis virus (ISKNV) caused an increased but delayed response of ifns (Laghari et al. 2018). Liu and coworkers studied ifn subgroups of salmonid species like rainbow trout (O. mykiss), chinook salmon (Oncorhynchus tshawytscha), coho salmon (Oncorhynchus kisutch), Atlantis salmon (Salmo salar), and Arctic charr (Salvelinus alpinus) and compared them with other species. The analysis confirmed that salmonids have a complex (in terms of ifn subgroups present) and (large number of genes) type I ifn repertoire relative to other teleost fish (Liu et al. 2020a). Milne et al. studied three distinct type I interferons in meagre (Argyrosomus regius), namely ifnc, ifnd, and ifnh. Constitutive expression was analyzed during larval development and in adult tissues (gills, midgut, head kidney, spleen). The spleen had high transcript levels of all three ifns. Ifnd and ifnh were also highly expressed in gills. The expression of each subgroup increased significantly across all four tissues following injection of poly I:C (Milne et al. 2018). In Atlantic salmon, Sun et al. identified an ifn multigene cluster encoding three ifn subtypes (ifna, ifnb, and ifnc). Each ifn subtype was constitutively expressed in head kidney. The three subtypes showed a striking difference in expression properties in response to stimulation with poly I:C. Both ifna and ifnc transcripts increased, while ifnb was only slightly induced by poly I:C (Sun et al. 2009). Type I interferon genes were cloned and characterized in the rock bream (Oplegnathus fasciatus). Their expression was upregulated in blood cells and head kidney by LPS, poly I:C, E. tarda, Streptococcus iniae, and iridovirus, and recombinant ifn I protein induced a rapid and transient expression of the mx gene in head kidney cells (Wan et al. 2012). An overview of type I ifns in different fish species with their activities is found in Table 2.23.

Table 2.23 Innate immunity: interferon type I genes in fish

Table 2.24 Innate immunity: Presence of interferon type II (ifng) genes in fish

2.9.3 Interferon Type II

Interferon-gamma (ifng), the only type II interferon, is a pleiotropic pro-inflammatory and antiviral cytokine. In mammals, it is constitutively produced by NK cells, whereas T lymphocytes produce IFNG after activation or differentiation. IFNG is a key cytokine for innate and adaptive immunity against viral and intracellular bacterial infections and is involved in tumor control. An updated teleost interferon-gamma review has recently been published (Pereiro et al. 2019). Arts and coworkers made recombinant proteins of the carp (Cyprinus carpio) IFN-γ sequences of both clusters (ifng1 and ifng2) and tested their effects on expression of pro-inflammatory mediators (Arts et al. 2010). An interferon-responsive stable cell line RTG-3F7 has been developed for rainbow trout by modifying the RTG-2 cell line through transfection with a plasmid construct containing a promoter element from the IFN-γ responsive gene TAP2 linked to a luciferase reporter gene and a hygromycin resistance gene. The results indicate that the stable cell line RTG-3F7 is an excellent tool for monitoring the presence of trout ifng in biological samples (Castro et al. 2010). The large yellow croaker (L. crocea) is an important mariculture fish species in China, and the bacterium Vibrio harveyi and the ciliate protozoan C. irritans are the two major pathogens of this species. The nucleotide sequence of ifng was obtained, and expression studies were performed. Fish were challenged with V. harveyi and C. irritans, respectively. One day after injection with V. harvey, all 10 tissues investigated had a higher expression of ifng, while only spleen, muscle, intestine, heart, and skin had higher expression after infection with C. irritants (Chen et al. 2015). Jung and coworkers produced a recombinant ifng (rifng) from the olive flounder (P. olivaceus). Stimulation of kidney leukocytes in vitro with rinfg induced the gene expression of il1b, signal transducer and activator of transcription 1 (stat1), CXCL-13-like chemokine (cxcl13), and ifng. Intraperitoneal injection of a mixture of rifng and E. tarda into olive flounder resulted in a survival rate of 60% compared to 0% in the group treated with E. tarda only (Jung et al. 2012).

The extensive use of paraquat (PQ) in agricultural practice throughout the world may compromise the integrity of biological systems in fish. PQ toxicity has been found to be mediated by the production of free radicals, which cause oxidative damage to cells. In a study by Ma et al. (2014), the acute toxicity of PQ in common carp (C. carpio) was determined. The results suggest that PQ exposure may result in suppression or excessive activation of the immune system that leads to immune dysfunction and reduced immunity (Ma et al. 2014). In the report of Pereiro et al. (2016), an antiviral turbot (S. maximus) interferon-gamma gene was characterized, and its expression pattern under basal conditions, after type I ifn administration and viral and bacterial infections, was evaluated (Pereiro et al. 2016). The intramuscular injection of an expression plasmid encoding turbot ifn gene was not able to affect the transcription of numerous immune genes directly related to the activity of ifng. It was neither able to reduce the mortality caused by a VHSV nor A. salmonicida challenge. Shibasaki and coworkers cloned and characterized two ginbuna crucian carp (Carassius auratus langsdorfii)-specific isoforms of ifng called ifng rel1 and ifng rel2. Recombinant ifng rel1 and ifng rel2 showed high antiviral activities against the lethal crucian carp hematopoietic necrosis virus (Shibasaki et al. 2014).

The antiviral activity of ifn gamma against IPNV and salmonid alphavirus (SAV) was studied by Sun et al. (2011). The studies were performed in Atlantic salmon TO cells and Chinook salmon embryo cells (CHSE-214). Ifn-γ induced antiviral activity against both IPNV and SAV3 in salmon cells (Sun et al. 2011). The marine flatfish Atlantic halibut (Hippoglossus hippoglossus) is of great commercial interest. However, due to poorly developed larva at hatching and a long live-feed stage, aquacultural use of this species is limited. Øvergård and coworkers cloned and characterized the gene encoding the ifng. A constitutive expression was found in both lymphoid and non-lymphoid organs with relatively high expression in the thymus and gills (Overgard et al. 2012). An overview of type II ifng in different fish species is presented in Table 2.24.

2.9.4 Tnfa

Tumor necrosis factor-alpha (tnfa) is a cytokine involved in systemic inflammation, apoptosis, cell proliferation, and regulation of immune cells (Wiens and Glenney 2011). It is produced mainly by activated macrophages as a membrane or secreted form. The main pro-inflammatory effects are mediated through the activation of endothelial cells (Roca et al. 2008). In bony fish, tnfa was first discovered in Japanese Flounder (Hirono et al. 2000) rainbow trout (Laing et al. 2001) and has since been characterized in a number of species. Fish have 14 tumor necrosis family genes. Their genomic existence and location have been investigated in the Japanese pufferfish (fugu) (Takifugu rubripes) (Biswas et al. 2015). Fugu was found to possess nine tnf superfamily genes including seven newly identified and two that had been previously reported. Poly I:C caused an elevated expression of three fugu tnf superfamily 10 genes in head kidney cells. Tnfa is an important factor for bacterial pathogen killing. A. salmonicida subsp. salmonicida is highly pathogenic for turbot, an economically important cultured flatfish in Europe, China, and Chile. In A. salmonicida-infected fish, the number of tnfa immunopositive cells was significantly increased in the kidney and spleen (Coscelli et al. 2016). Immunoreactive cells were also present in the digestive tract, liver, heart, gills, and skin (Ronza et al. 2015).

The striped trumpeter (Latris lineata Forster) is a new species in Tasmanian waters. The tnfa was cloned, and the expression was analyzed in response to an ectoparasite Chondracanthus goldsmidi. A significant upregulation was found in the gills, which are the site of parasite attachment. Head kidney cells showed a significant upregulation of tnfa, but spleen cells did not (Covello et al. 2009). The European sea bass (D. labrax) is intensely aquacultured in the Mediterranean area. The bacterial pathogen V. anguillarum provokes the highest mortality among several pathogens of this species. Available vaccines do not achieve the desired protection. In a recent study, recombinant tnfa was used as adjuvant in a commercial sea bass oral vaccine against V. anguillarum. Tnfa significantly enhanced disease resistance and induced recruitment of gut intraepithelial lymphocytes (Galindo-Villegas et al. 2013). In rainbow trout, two tnfa genes have been described. Recently, a third tnfa (tnfa3) that has low identities to known trout molecules was reported. The constitutive expression of tnfa3 was generally lower than the other two genes in tissues and cell lines. Expression of all three tnfa isoforms could be modulated by crude LPS, peptidoglycan, poly I:C and recombinant Ifng in cell lines and primary macrophages, and bacterial and viral infections (Hong et al. 2013). The genomic location of the two tnfa genes in zebrafish (D. rerio) and medaka (Oryzias latipes) was recently determined. Zebrafish tnfa1 and tnfa2 were found on chromosomes 19 and 15, and medaka tnfa1 and tnfa2 on chromosomes 11 and 16, respectively. There was a constitutive expression of the genes in different tissues. An increased expression of both was induced in head kidney cells by LPS in vitro (Kinoshita et al. 2014). Li and Zhang studied a tnfa homologue from the Tongue sole (Cynoglossus semilaevis) named CsTNF1. Expression of CsTNF1 was detected in liver, spleen, kidney, blood, gill, brain, muscle, heart, and intestine, and was upregulated by experimental challenge with bacterial and viral pathogens (Li and Zhang 2016).

Meagre (Argyrosomus regius) is an emerging aquaculture species found in the Mediterranean area and Black Sea due to its large size, fast growth, low feed conversion ratio, and high processing yield. Two types of tnfa were expressed in meagre (type 1 and type 2). Tnfa1 was more highly expressed in head kidney and gills. Both isoforms increased in expression in head kidney following injection with LPS (Milne et al. 2017). Atlantic bluefin tuna (Thunnus thynnus) was introduced into Mediterranean aquaculture in the early nineties, and has become the most valuable finfish aquaculture, representing more than half of the world’s total production (Pleic et al. 2014). Tuna aquaculture is a capture-based activity, where wild-caught tuna is cultured in marine cages for a period of time in order to increase their protein and fat content. The full-length cDNA and gene sequences of Bluefin tuna tnfa1 and tnfa2 were determined, and expression studies showed that they were constitutively expressed in liver and head kidney at similar levels. Expression of both cytokines was examined in acute and chronic natural infection of the parasites Pseudocycnus appendiculatus and Didymosulcus katsuwonicola (Pleic et al. 2015). D. katsuwonicola-infected gills showed significantly higher expression of tnfa2, while tnfa1 showed no difference in expression with either Pseudocycnus appendiculatus- or Didymosulcus katsuwonicola-infected gills.

Rainbow trout red blood cells (RBCs) are able to endocytose nanostructured tnfa in vitro despite not being phagocytic cells, and in response to nanostructured tnfa, the expression of different immune genes could be modulated (Puente-Marin et al. 2019a).

Tnfa was cloned in large yellow croaker (Pseudosciaena crocea), mainly distributed in coastal regions of East Asia, and is one of the most important cultured marine fish in China. Vibrio parahemolyticus challenge demonstrated enhanced expression of tnfa in head kidney and blood (Xie et al. 2008). Tnfa was also identified in grass carp (Ctenopharyngodon idella), and its role in signaling was defined (Zhang et al. 2012). Additionally, tnfa is involved in the control of ovulation (Crespo et al. 2010, 2015) (Table 2.25). For more details, please see Chap. 10 (“cytokines”) by Dr. C. Secombes.

Table 2.25 Innate immunity: tnfa genes in different fish species

2.9.5 The Complement System

The mammalian complement system is composed of about 35 plasma and membrane-associated proteins. The main functions of the complement system are opsonization, inflammation, and formation of the cytolytic membrane attack complex. The proteins are mostly produced by liver hepatocytes and secreted to the blood, except for some like factor D and c1q. Several components of the teleost complement contain isoforms like c3, c4, c5, c7, factor B, factor I, and MBL. Most homologs of mammalian complement components are present in teleosts and have been shown to be expressed in a variety of tissues like the kidney, skin, and intestine. Several reviews on the complement system of teleosts are available (Zhang et al. 2013b; Nakao et al. 2011; Uribe et al. 2011; Boshra et al. 2004, 2006). Additional information of the complement system in fish is given in Chap. 9.

2.9.5.1 c3

c3 is the central complement component and has been isolated, purified, and characterized in many teleost species. Recently, four c3 isoforms were purified from the Nile tilapia (Oreochromis niloticus) serum and were shown to possess an intrachain thioester bond. All named c3-1, c3-2, c3-3, and c3-4 showed the two-chain polypeptide structure typical of c3 (Abdel-Salam et al. 2014). Forn-Cuni et al. confirmed the presence of three c3 genes and in addition identified five more c3 genes in the zebrafish (D. rerio) genome (Forn-Cuni et al. 2014). Maternal immunization of female zebrafish with formalin-killed A. hydrophila caused a significant increase in c3 and factor b contents in the mother, a corresponding rise in the offspring, and induced a remarkable increase in the hemolytic activities in both the mother and offspring (Wang et al. 2009). The dojo loach (Misgurnus anguillicaudatus) is one of the most commercially cultured fish species in Eastern Asian countries including China, Japan, and Korea. Three isoforms of c3 were discovered in M. anguillicaudatus named c3-1, c3-2, and c3-3, respectively. The expression of c3–1 and c3–3 was mainly detected in liver followed by spleen and gonad. The mRNA levels were upregulated in the gill, skin, liver, and spleen after bath infection with A. hydrophila (Xu et al. 2018b). Furthermore, the complete nucleotide sequence of c3 from two Antarctic teleosts Trematomus bernacchii (two isoforms) and Chionodraco hamatus (a single isoform) was determined (Melillo et al. 2015).

Rainbow trout c3a and c5a receptors were cloned and functionally characterized. Both anaphylatoxin receptors were expressed at considerable levels by B cells. Treatment by lipopolysaccharide led to a significant upregulation of both receptors, suggesting that B cells play a role in the development of an inflammatory response (Li et al. 2007).

2.9.5.2 Classical Pathway

The classical complement pathway involves c1, c2, and c4. The first complement component (c1) is activated by recognition of antigen-bound immunoglobulins, and proteolytically activates c4 and c2 into c4b and c2a, respectively. Rock bream (Oplegnathus fasciatus) is one of the most economically important marine fish species in South Korea, which geographically distribute in the coastal water, especially in coral beds of the Pacific and Indian Ocean. Rock bream complement components c1r and c1s were characterized, and homology analysis showed 73.4% and 58% amino acid identity with orthologs of Pundamilia nyererei of Lake Victoria and the Japanese rice fish, Oryzias latipes, respectively. c1r was highest expressed in blood and c1s in the liver. The transcription of both components was found to be upregulated in response to pathogenic bacteria E. tarda and S. iniae and virus (rock bream iridovirus) (Godahewa et al. 2015).

Grass carp (C. idella) is susceptible to A. hydrophila infections. In the study of Dang et al., grass carps were given intraperitoneal injections of live A. hydrophila and 4, 8, 12, 24, 48, and 72 h after RNA sequencing of spleen tissue was performed. Four to 72 h after infection, the complement system, represented by c2, c3, c4, c5, c8a, c1q, and mbl, was upregulated with a transitory downregulation at 12 h (Dang et al. 2016).

Tissue of C. idella was infected with A. hydrophila. Two cDNA sequences of c4 from the common carp (C. carpio) were isolated sharing only 32% identity of amino acid level and having distinct binding specificities (Mutsuro et al. 2005).

2.9.5.3 Alternative Pathway

Factor B and factor D are both components of the alternative pathway of complement. Following activation of the alternative pathway, factor B is cleaved into Ba and Bb fragments. In rainbow trout, factor B is known to act as a c3 convertase, but the function of the Ba fragment is unknown. The expression patterns of tongue sole (Cynoglossus semilaevis) factor B and the biological activity of the Ba fragment were studied by Li and Sun (2017). Expression of factor B was high in liver, muscle, and heart and low in intestine, blood, and kidney. Bacterial infection (E. tarda, Pseudomonas fluorescens, and V. harveyi) induced an expression in kidney, spleen, and liver in a time-dependent manner. For the first time, it was found that overexpression of Ba significantly reduced bacterial dissemination in fish tissues, indicating that Ba possesses antimicrobial activity and may inhibit bacterial infection in fish (Li and Sun 2017).

Rock bream (Oplegnathus fasciatus) complement factor D (Cfd) was characterized and expression was analyzed. Factor D encodes 277 amino acids for a 30 kDa polypeptide and was most highly expressed in the liver and spleen. Transcription of factor D was upregulated in the spleen by lipopolysaccharide, S. iniae, rock bream iridovirus, and poly I:C (Godahewa et al. 2016). Rainbow trout liver seems not to be an important transcription site of the genes c1q, factor B (cfb), and c7–2. The novel characterized factor D of rainbow trout had 253 amino acids with a molecular weight of 27.2 kDa and shared a sequence identity with its human ortholog of 45% (Kobis et al. 2015).

2.9.5.4 Lectin Pathway

The central components of the lectin pathway are MBL and MASPs. Teleost fish often possess several genes encoding different subtypes. Kania et al. (2010) characterized three homologs of mannan-binding lectin (named MBL H-1, MBL H-2, MBL H-3) (Gene: mbl and variants) in the rainbow trout. They were expressed in the spleen, anterior intestine, and liver. MBL H-1 and H-3 were also found in the vascular system. MBL H-1 had the highest expression level in the anterior intestine followed by gill, thymus, and skin, while the highest expression level of MBL H-2 and MBL H-3 occurred in the anterior intestine (Kania et al. 2010).

C. semilaevis mannan-binding lectin (Mbl)-associated protein 34 (MAP34) and Mbl-associated serine protease 1 (MASP1) are key factors involved in complement activation through MAPs’ ability to bind to M1 and MBL. Remarkably, in contrast to a negative regulatory role of mammalian MAP, the teleost C. semilaevis Map 34 exerts a positive effect on the activation of the lectin pathway (Li et al. 2016).

2.9.5.5 Terminal Pathway

c5, c6, c7, c8, and c9 are the components engaged in building the membrane attack complex. Native c5a of rainbow trout and recombinant infectious hematopoietic necrosis virus glycoprotein (G) fusion protein was constructed to test the adjuvant activity of rainbow trout c5a. At four to sixteen weeks postinjection, the serum Igm antibody levels were higher than those injected with G-protein alone, suggesting that c5a acts as molecular adjuvant in rainbow trout (Wu et al. 2014).

Grass carp (C. idella) is an economically important species, and its global production is more than 4.5 million tons per year making it the most highly consumed freshwater fish species in the world. A. hydrophila is the causative pathogen of intestinal hemorrhage, which has caused great economic loss in grass carp aquaculture. Fish were intraperitoneally injected with A. hydrophila, and the transcriptomic response was tested in the spleen. A total of 1591 genes were upregulated, and 530 were downregulated. c1, c7, and c8b were upregulated indicating activation of the classical pathway (Yang et al. 2016). c7 was isolated and characterized from grass carp. The predicted amino acid sequence of c7 cDNA exhibited 55.4% and 48.3% homology with rainbow trout c7–1 and zebrafish c7, respectively. c7 gene expression was detected in trunk kidney, liver, head kidney, skin, spleen, heart, and intestine. Significant changes in c7 transcript expression were detected following A. hydrophila infection, especially in head kidney and spleen (Shen et al. 2012).

Full-length c8a and c8b sequences from a cDNA library of rock bream (Oplegnathus fasciatus) and their genomic sequences were obtained. Quantitative real-time PCR analysis showed that both components were expressed in all examined tissues, with highest expression in the liver. Pathogen challenge, including E. tarda, S. iniae, and rock bream iridovirus, led to upregulation of both (Wickramaarachchi et al. 2013).

Complement component c9 is the last component that is involved in the formation of the membrane attack complex on the surface of target cells. The full-length c9 cDNA sequence was found in the southern catfish (Silurus meridionalis) and showed similarity with other teleost fish. The mRNA expression was highest in the liver and observed also in the spleen, head kidney, stomach, and intestine. Intraperitoneal injection of A. hydrophila gave upregulation of c9 in the liver, spleen, and intestine (Fu et al. 2019).

The large yellow croaker Larimichthys crocea is one of the most important marine fish in China and East Asian countries. Complement components c7 and c9 were characterized by Guo et al. (2016). c7 and c9 were mainly expressed in liver, but low levels were also constitutively expressed in most tissues. Fish challenged with Vibrio alginolyticus showed a rapidly upregulated response in the liver and head kidney (Guo et al. 2016). Miiuy croaker, Miichthys miiuy, belongs to the family Sciaenidae of the order Perciformes and mainly distributes from the western Japan Sea to the East China Sea. In China, it has been widely cultured since the late 1990s for its good taste and high nutritive and medicinal value. A truncated c9 cDNA sequence encoding 461 amino acids was cloned and characterized in the miiuy croaker (M. miiuy). The c9 of miiuy croaker shows the highest amino acid identity score with fugu c9 (61%) and the lowest with zebrafish c9 (36%). The highest levels of transcripts were detected in liver of both healthy and V. anguillarum-infected fish (Meng et al. 2012). Full-length c9 sequence was identified from a cDNA library of rock bream (O. fasciatus), and its genomic sequence was obtained. Quantitative real-time RT-PCR analysis confirmed that c9 was constitutively expressed in all the examined tissues, with highest expression occurring in the liver. Pathogen challenge including E. tarda, S. iniae, lipopolysaccharide endotoxin, and rock bream iridovirus led to upregulation of c9 in liver but resulted in no change in the peripheral blood cells (Wickramaarachchi et al. 2012). The transcriptional expression of central complement components during the ontogeny of the common sole (Solea solea) was studied by Ferraresso et al. (2016). The c2, c3, and fb showed a gradual increase in expression between 4 and 33 days post-hatch (dph). c4 and masp1 showed no differences in expression during the development, while c1qb showed a very high level of expression. Terminal components, c5, c6, c7, c8, and c9, showed an increase in expression until the onset of metamorphosis and a second increase after metamorphosis (Ferraresso et al. 2016).

2.9.5.6 Complement Regulation

Complement activation is controlled by both fluid phase and membrane inhibitors. Factor I regulates complement by proteolytic cleavage of components c3b and c4b. Factor H, the main cofactor of factor I, regulates the alternative pathway by acting in the breakdown of c3b to ic3b. Factor I (cfi) and factor H (cfh) of rainbow trout were cloned and characterized. The deduced amino acid sequences of factor I and factor H exhibited 42% and 32% identity with human orthologs, respectively (Anastasiou et al. 2011). The deduced amino acid sequence of factor H from large yellow croaker (Larimichthys crocea) showed 28% and 34% identity with human and rainbow trout orthologs, respectively. The highest expression levels were found in liver, kidney, and spleen. After injection with V. alginolyticus, the expression levels were upregulated in all three tested tissues (Qi et al. 2018a). Black rockfish (Sebastes schlegelii) is an important aquaculture species in the Republic of Korea. A c1 inhibitor gene from black rockfish was cloned and characterized by Nilojan et al. The c1 inhibitor was most highly expressed in the liver followed by the gills (Nilojan et al. 2018) (Table 2.26).

Table 2.26 Table of complement components found in the listed fish species

2.9.6 Acute-phase Component

During infection, stimulation with strong danger signals or stress, the fish may respond to produce acute-phase proteins (APPs). Especially, IL-1, IL-6, and tnfa are able to induce acute-phase response, as observed in higher vertebrates. The most common APPs are pentraxins such as serum amyloid A (SAA) and C-reactive protein (CRP). Dissimilar to many mammalian species, the fish show a modest acute-phase response when it comes to concentration of pentraxins in serum. CRP may be able to bind to (opsonize) certain bacteria, fungi, and parasites, activate the complement system, agglutinating particles, and may infer production of cytokines. There are two forms of SAA, one of them being acute-phase SAA. SAA may neutralize pathogen activity, reduce tissue damage, and restore homeostasis. Transferrin, haptoglobin, ceruloplasmin, alpha-2-macroglobulin, lectins, and complement component c3 are all considered to be AAPs. Most of these have regulatory activities limiting infection and restoring the physiological balance. Several reviews covering this topic are recommended (Roy et al. 2017; Bayne and Gerwick 2001; de Magalhaes et al. 2020; Magnadottir 2014; Nakao et al. 2011).

2.9.7 Chemokines and Their Receptors

The major function of chemokines is to guide the migration of cells. An example is chemokine-guided migration of leukocytes to inflammatory foci. Other functions involve immune surveillance where chemokines direct homing of leukocytes to lymphatic tissues. Some chemokines have function in growth of new blood vessels and wound healing. Chemokines are classified into four main subfamilies (CXC, CC, CX3C, and XC) dependent on the amino acid sequences (first two cysteine amino acid residues). Most of the chemokines bind to specific chemokine receptors on cells. Fish display numerous genes for different chemokines and chemokine receptors, suggested due to gene duplication events. Functional and significance studies of chemokine expression are generally not very well examined in fish. However, exceptions exist. We list the most recent findings within functional chemokine research. It has been shown that grass carp cxcl20b possessed antibacterial activity by attaching to the bacterial membrane (Xiao et al. 2020). In another study, it was shown that common carp Cxcb1 stimulated neutrophil extracellular trap formation—which was suggested to be an antipathogenic event (Pijanowski et al. 2020). Moreover, an ayu (Plecoglossus altivelis) CC-like chemokine was found to possess chemotactic activity against monocytes and neutrophils in vivo and in vitro (Yu et al. 2019). Chemotactic activity of Ccl4 has been shown in the golden pompano (Trachinotus blochii). This recombinant chemokine had also antimicrobial activity against E. tarda and Escherichia coli (Sun et al. 2019). Furthermore, a rainbow trout CC chemokine (Ck11) also displayed antimicrobial activity against different gram-positive and gram-negative bacteria by attaching to and disrupting their cell membranes (Munoz-Atienza et al. 2019).

2.9.8 Antibacterial Peptides (AMPs)

AMPs are a diverse class of highly conserved molecules that are produced as a first line of defense in all multicellular organisms, including fish. These small peptides (12–50 amino acids) are essential components of innate immunity capable of antimicrobial activity against a broad range of microbial pathogens (Semple and Dixon 2020; Zhang and Gallo 2016). Functionally, they can be described as either membrane disruptive AMPs, which induce membrane permeabilization, or they can be non-membrane disruptive where they can be internalized in cells and act on intracellular targets (Semple and Dixon 2020). In general, fish AMPs may be categorized into five different classes based on their structure: β-defensins, cathelicidins, hepcidins, histone-derived peptides, and piscidins (Brunner et al. 2020). As for chemokines and TLRs, fish possess numerous genes for antimicrobial peptides. Recently, reviews on the significance of AMPs are published (Brunner et al. 2020; Chaturvedi et al. 2020; Valero et al. 2020; Chen et al. 2020; Shabir et al. 2018).

2.10 Conclusion and Future Research

It is clear that fish are indeed equipped with an arsenal of defense mechanisms to prevent infection. An earlier report has used Rag knockout mutants (rag1^−/− zebrafish), which possess no serum Igm, to assess the significance of the innate immune system in comparison with control fish with a rag1^+/− genotype. An experimental challenge experiment revealed that the rag ^−/− zebrafish displayed similar protection as the controls (Tokunaga et al. 2017). This underscores the notion that the innate immune system alone may likely be as effective as a fully immune-equipped fish. However, immunized fish will normally acquire higher disease resistance than naïve fish. The concept of trained innate memory should be addressed as a trained innate immune system likely would add higher protection level during infection. Trained innate immunity involves activation of innate defense factors that in turn confer increased disease resistance to infection by homologous or heterologous pathogens. Trained immunity can be transferred to offspring as training induces heritable epigenetic changes.

The future will bring a vast more knowledge of innate immune factors through fish genomic and transcriptomic studies, and it is likely that many more innate immune factors will be revealed. To find their significance in the innate immune defense, these must be functionally examined.