Cytokines Studied in Carp (Cyprinus carpio L.) in Response to Important Diseases

Abstract

Cytokines belong to the most widely studied group of intracellular molecules involved in the function of the immune system. Their secretion is induced by various infectious stimuli. Cytokine release by host cells has been extensively used as a powerful tool for studying immune reactions in the early stages of viral and bacterial infections. Recently, research attention has shifted to the investigation of cytokine responses using mRNA expression, an essential mechanism related to pathogenic and nonpathogenic-immune stimulants in fish. This review represents the current knowledge of cytokine responses to infectious diseases in the common carp (Cyprinus carpio L.). Given the paucity of literature on cytokine responses to many infections in carp, only select viral diseases, such as koi herpesvirus disease (KHVD), spring viremia of carp (SVC), and carp edema virus disease (CEVD), are discussed. Aeromonas hydrophila is one of the most studied bacterial pathogens associated with cytokine responses in common carp. Therefore, the cytokine-based immunoreactivity raised by this specific bacterial pathogen is also highlighted in this review.

1. Introduction

The common carp (Cyprinus carpio L.) is one of the most important cultivated fish species contributing to the global economy. Carp are currently cultured worldwide at a rate of over 3 million metric tons/year, which represents around 10% of the global production of freshwater fish in aquaculture [1,2]. Historically, the common carp was cultured only for human consumption, beginning around 3000 years ago in China (FAO, 2006). Currently, it is a widely cultured fish species in over 100 countries, and not only used for human consumption but also as an ornamental fish and for recreational fishing purposes [2]. In recent decades, the common carp has been subjected to intensive rearing patterns, which may induce stress and lead to a weakened immune system, increasing susceptibility to various pathogens [3,4].

Thanks to more advanced approaches and different, newer technologies, we now have a deeper understanding of the immune processes protecting fish against the effects of infectious agents, which can be tracked to help prevent diseases.

Great progress has been made in isolating and characterizing cytokines in fish in recent years. However, immunoreactive responses vary due to the presence of different pathogenic stimuli, which is a focal issue for research. Evaluating cytokine responses, which develop in the early stages of infections, is essential in determining the behavior of both host and pathogen during and/or after the infection. Consequently, preventive measures, such as the development of vaccines and protective approaches related to the fishes’ environment, can be carried out. In this review, particular attention has been paid to discussions of the cytokine responses provoked in common carp due to cyprinid herpesvirus 3 (CyHV-3), spring viremia of carp virus, carp edema virus, and Aeromonas hydrophila infections. The selected disease-causing agents represent important pathogens that occasionally infect farmed carp worldwide, resulting in substantial economic losses due to high mortality rates and/or officially ordered culling of fish stocks, as in the case of KHV.

Koi herpesvirus disease (KHVD) was first diagnosed in the late 1990s in Israel, and it has since spread across the world, primarily due to the ornamental fish trading industry [1]. Due to its infectivity, lethality, and economic impact, it was listed as a non-exotic disease in a European Council directive (2006/88/EC), and more recently in Annex II of Animal Health Law (Regulation (EU) 2016/429). It is also an OIE (World Organization for Animal Health) listed disease [2,3].

Carp edema virus disease (CEVD) first occurred in European fish farms about 10 years ago. Clinical signs are very similar to KHV (gill necroses, sunken eyes, irregular skin-mucus production, mortality). The disease often appears in lower water temperatures than KHVD [4]. The causative agent is probably the same virus responsible for “sleepy koi disease”, diagnosed in the 1970s in koi farms in Japan [5]. However, several different genogroups of this viral pathogen have recently been distinguished by molecular biology tools [6]. Although the spread and economic impact of CEVD are similar to KHVD, to date, it remains a non-notifiable disease, and its prevalence is relatively unknown. In the OIE, it is listed as an emerging disease.

Unlike KHV and CEV, which are host-specific only for carp and koi, SVCV has been diagnosed in most members of the Cyprinidae family, as well as in several other fish species [7]. Although the disease has not caused significant economic losses in carp farming in recent decades, it is still one of the diseases monitored by the OIE. The virus is present in most European countries, North America, South America, and Asia (OIE, 2017); however, owing to the absence of systematic monitoring, information about the true spread of SVCV worldwide is not available.

Aeromonas hydrophila belongs to the motile aeromonads group, which are the most abundant bacteria in aquatic environments. It is responsible for causing carp erythrodertmatitis; sometimes, the disease is recognized as “motile Aeromonas septicemia” (MAS) [8]. An opportunistic pathogen, A. hydrophila usually causes disease in fish under stress in various fish species [9]. The conditions caused by A. hydrophila infection in fish farms are accompanied by high death rates and, subsequently, large economic losses [10].

2. Fish Immune System

The immune system of vertebrates consists of several immune organs, which can be divided into two categories, primary and secondary. Within these organs, immune cells, including differentiated and non-differentiated leukocytes, represent an essential part of the lymphoid system. In addition, they can be disseminated in blood or other tissues, along with various substances produced by these cells. The immune organs of teleost fish are somewhat different from those of other vertebrates, with fewer consistent lymphatic compartments distinguishing the lymphoid system from the circulatory system. However, the tissues of lymphatic organs are equally responsible for the production and storage of lymphocytes and providing sites for their interactions with antigens [11]. The primary lymphoid organs in fish include the head kidney and thymus, while the secondary lymphoid organs consist of the spleen, trunk kidney, and mucosa-associated lymphoid tissue (MALT), such as gills, skin, intestine, oral and nasal mucosa, and urogenital tract [12,13]. According to anatomical location, MALT can be further subdivided into skin-associated lymphoid tissue (SALT), gut-associated lymphoid tissue (GALT), and gill-associated lymphoid tissue (GIALT) [14,15].

The teleost immune system is similar to that of higher vertebrates, comprising both specific (adaptive) and non-specific (innate) responses to overcome invasion by various pathogens, such as viruses, bacteria, and parasites [16]. The non-specific immune response is considered the very first defense against pathogens. Unlike other vertebrates, fish primarily depend on the non-specific immune system for survival during early embryonic development [17]. Not only that, but due to fish being poikilothermic, they lack some of the conventional characteristics of adaptive immunity represented by limitation in antibody defenses and slow proliferation, maturation, and memory possessiveness of lymphocytes. Therefore, in fish, innate immune responses are considered fundamentally important defenses and are mainly classified into three basic mechanisms: physical (epithelial/mucosal) barrier, cellular component, and humoral responses [18]. The mucosal barrier of the skin, gills, and gut comes into direct contact with causative agents of diseases found in the environment. Hence, it constrains the invading pathogens through a series of mechanisms involving the secretion of antimicrobial humoral factors by immune cells or tissues. These factors may include specific peptides that are able to identify certain pathogen-associated molecular patterns (PAMPs), such as microbial endotoxins such as lipopolysaccharides (LPS), and polysaccharides, or viral DNA and RNA, bacterial DNA, or other molecules that are naturally found on the surface of different microorganisms [19]. Such antimicrobial agents are known as immunological identifiers due to their ability to recognize molecules on pathogens’ surfaces and aid in eliminating them either through direct destruction by phagocytosis or via regulation of cellular immune responses. Examples of these identifiers include members of the major histocompatibility complex (MHC), lytic enzymes, growth inhibitors, antibodies, antibacterial peptides, chemokines, and cytokines.

3. Cytokines

Cytokines are proteins produced and secreted by a variety of immune cells (T and B lymphocytes, mast cells, and endothelial cells) with development, differentiation, and activation capabilities that influence the nature of immune responses [20]. Cytokines are engaged in various immune reactions, from induction of non-specific immune response to the generation of the primary effector cells of the innate immune system known as cytotoxic T cells, up to the production of antibodies by B cells [21]. The cytokines can be grouped into distinct families based on their structural characteristics, such as interferons (IFNs), interleukins (ILs), tumor necrosis factors (TNFs), transforming growth factors (TGFs), and chemokines [22].

Interferons (IFN) play a crucial role in inhibiting viral replication in different viral infections in vertebrates [23]. The underlying mechanism involves recognizing and inhibiting viral nucleic acid via binding to specific cell receptors and initiation of gene expression cascade responsible for encoding antiviral proteins [24]. For instance, an experimental study on infectious pancreatic necrosis virus in Atlantic salmon evidenced that AS-IFN can specifically inhibit virus activity following the induction of interferon-stimulated genes [25]. However, unlike mammals, where interferons are divided into three classes (type I, II or IFNγ, and III), in fish, only classes I and II have been discovered [26,27]. Nevertheless, fish seem to have a collection of cytokines akin to those of mammals [28]. Various homologs of the cytokines and their suppressors reported in mammals were successfully cloned in several fish species, either purified and identified as native fish proteins or theoretically predicted according to a similarity in structure or sequence [29].

In addition to interferons, interleukins (IL) are also important cytokines in the fish immune system, produced by different types of leucocytes [30]. The most important interleukins consist of pro-inflammatory (IL-1, IL-2, IL-6, IL-11, IL-17, IL-18, and IL-33) and anti-inflammatory (IL-4, IL-6, IL-10, and IL-13) cytokines. The mechanistic effects of these molecules tend to either suppress the activities of individual members or induce pro-inflammatory effects [31]. For instance, IL-10 can be expressed by all immune system cells, including dendritic cells (DC), macrophages, mast cells, natural killer cells (NK), eosinophils, neutrophils, B cells, and CD8⁺ T cells. Despite being an anti-inflammatory cytokine, IL-10 produced by these cells seems to inhibit the production of proinflammatory cytokines in most cases. Hence, it might be viewed as a pleiotropic cytokine with both robust immunosuppressive and anti-inflammatory capabilities [32]. Among several cytokines, IL-1 was found to be the earliest cytokine identified in fish [33]. In mammals, genes encoding IL-1α and IL-1β are recognized, whereas, in fish, only genes encoding IL-1β have been discovered as of yet, which is homologous to that of mammals [29]. Furthermore, in fish, the role of interleukins represented mainly by IL-1β is proposed as analogous to that in mammals through regulation of immune responses via the early activation of T cells [34].

Tumor necrosis factor (TNF) is a critical cytokine that plays an essential role in physiological and pathological processes. It consists of various family members, including TNF alpha (TNF-α) and TNF beta (TNF-β). Both are expressed in macrophages, T, B, and natural killer (NK) cells, and in the TNF-related apoptosis-inducing ligand and CD40 ligand (primarily expressed on the surface of T cells). Using different mechanisms of action than interferons and interleukins, tumor necrosis factors α and β are confirmed to promote phagocytosis and nitric oxide production in teleost during viral and bacterial infections [35,36]. Both TNF family members (TNF-α and TNF-β) are produced from macrophages and T lymphocytes, respectively, while also being expressed by other immune cells at low levels. In fish, TNF-α has been cloned in several species, including common carp (C. carpio) [37], gilthead seabream (Sparus auratus) [38], catfish (Ictalurus punctatus) [39], grass carp (Ctenopharyngodon idella) [40], zebrafish (Danio rerio) [41], and sea bass (Dicentrarchus labrax) [42]. In light of its importance for the mammal immune system, it is surprising that no TNF-β gene has been identified in fish. However, the missing function of TNF-β may be performed by alternative proteins. Analyses of phylogenetic trees and genetic co-localization in certain loci of fish chromosomes in some teleost indicate the existence of two types of TNF-α, named I and II. Type-II TNF-α, which is reported in catfish (type-II catfish TNF-α) [39] and trout (type-II trout TNF-α3) [38], proposedly have a similar function to that of TNF-β in mammalian species.

Transforming growth factor β (TGF-β) is a key inhibitory and inflammatory cytokine responsible for tissue repair during pathophysiological reactions [43]. TGF-β regulates cell development, differentiation, and proliferation and modulates different leukocytes and their lineages, including lymphocytes, macrophages, and NK cells [44]. In mammals, TGF-β maintains immune tolerance and is a well-known immunosuppressive cytokine [45]. The immunosuppressive effect is primarily due to the stimulation of T lymphocytes. For instance, during in-vitro study, the activation of resting T cells limits the lymphocytes’ clonal expansion, resulting from increased levels of TGF-β receptors and their mRNA expression [46]. In teleost immunity, the role of TGF-β appears to be parallel to that in mammals. Although limited investigations of TGF-β immune activity in fish have been carried out, a few studies have examined its role in some fish species. For instance, in a study on the top mouth culter (Culter alburnus), TGF-β1 was found to express highly during lipopolysaccharides (LPS) stimulation, where it blocked the mRNA expression of pro-inflammation cytokines, such as IL-β and TNF-α [47]. A similar immune suppressive function of TGF-β was noticed in LPS treated common carp and goldfish when the nitric oxide response of TNF-α activated macrophages were significantly reduced, and considerable downregulation of TNF-α was noticed [48,49].

Chemokines, also known as chemoattractant cytokines, regulate the immune cells and control their migration to the site of inflammation. In mammals, chemokines are divided into four subfamilies according to their peptide structures: CXC (α), CC (β), C, and CX3C classes [22]. Fish chemokines (CXCα, CXCβ1, CCβ, and C) and their receptors are described in some teleost, including zebrafish, rainbow trout, carp, catfish, gilthead seabream, and Japanese flounder [50]. Limited progress has been made in studying their role in teleost immunity. Nevertheless, their contribution to immunity in several vertebrates through localizing and regulating the function of immune cells calls for more investigation about their importance in fish [50].

3.1. Cytokine Studied in Response to Cyprinid Herpesvirus 3 Infection

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpes virus (KHV), is the causative agent of koi herpesvirus disease (KHVD). This DNA virus belongs to the family Alloherpesviridae [51]. The disease affects both common carp (C. carpio) and its ornamental variety, koi (Cyprinus rubrofuscus). External clinical signs include lethargy and anorexia followed by excessive mucous production on the gills and skin and necrosis of gill tissue. Petechial bleeding spots can also be seen in the final stages of infection on the trunk, vent, and around the mouth [52,53]. KHV is categorized as an emerging disease that causes massive mortality resulting in substantial economic losses to the aquaculture industry (FAO, 2010).

The coevolution of fish herpesviruses and their hosts is believed to have occurred about 400 to 450 million years ago [54,55]. Over time, hosts have adapted a more developed and divergent immune system to safeguard themselves in the face of viral or other pathogenic infections. In different vertebrates, including humans, it has been proven that the non-specific immune reaction against herpesviruses generally incorporates an activation of (NK) cells and the production of interferons and different types of interleukins [56].

Interferon (IFN) plays a crucial role in innate immunity to guard the body against viral invasion. This cytokine significantly contributes to the early containment of herpesvirus infections because of its immunoreactive properties. Several type 1 IFN reactions, including interferon regulatory genes (ISGs), are characterized during viral infections [57,58]. Multiple studies have also shown that recombinant interferon or the stimulation of type I IFN production has a protective effect against numerous fish viruses, whether performed in vitro or in vivo. The magnitude of type I IFN responses are essential for increased resistance to virus-induced mortality in fish infected with viruses with more complex genomes, like alloherpesviruses. However, in the case of CyHV-3 infection, skin is considered a major entry organ and the primary site for infection in carp [59,60]. Fish skin serves as a good protection barrier from different types of pathogens [61]. Few studies have been carried out to gain insight into the type I IFN response during CyHV-3 infection exclusively. For instance, a relationship between the upregulation of type 1 IFN response and CyHV-3 infection was observed in the skin of infected common carp [57] (

Table 1. List of cytokines studied during cyprinid herpesvirus 3 (CyHV-3), spring viremia of carp virus (SVCV), carp edema virus (CEV), and Aeromonas hydrophila (A. hydrophila) infections in common carp (C. carpio).

CyHV-3
Assay Condition	Tissue/Cell Type	Studied Cytokine	Observed Effect	Reference
in vivo	kidney	IFNαβ, IL-12p35	↑	[63]
	gill	type 1 IFN, Vip, PKR	↑	[64]
	gill, kidney, spleen	IL-10	↓	[65]
	spleen	IL-1β, IL-10, IL-12p35, IL-6 and IFNαβ	↑	[66]
	skin, head kidney	Type 1 IFNs	↑	[67]
	anterior kidney	IL-10	↑	[68]
	spleen	Type 1 IFN, Vip2 and IL-8	↑	[69]
	spleen	IFNγ-1, IFNγ-2, IL-1β, IL-10, and IL-12	↑	[70]
	spleen, kidney, intestine	IL-10	↑	[71]
	spleen	IFNγ-2, IL-6, Mx and IL-8	↑	[72]
	gills	IL-10 and TNF	—	[73]
	muscles	IL-1β, TNF-α, IFNγ2ab, ISGs (Mx1, Vip2 and PKR3)	↑	[74]
	serum	IL-1β	—	[75]
in vitro	head kidney leucocytes	type 1 IFNs	↑	[62]
in vitro/in vivo	gill, kidney, head kidney, skin	type 1 IFN	↑	[76]
	plasma	TNF-α	↑	[77]
	spleen	IL-10	↓	[78]
svcv
in vivo	spleen, head kidney, intestine, thymus, blood	IRFs	↑	[79]
in vitro	head kidney leucocytes (HKLs)	type 1 IFNs	↑	[62]
in vitro/in vivo	kidney and spleen	type 1 IFN, Mx1, Rig1	↑	[80]
	skin, kidney, head kidney	IFNα2 and Vig1	↑	[76]
	muscles and blood	IL-1β, IL-6, TNF-α, IFNγ2a, IFNγ2b, IFNø1, IFNø2, Mx1, Mx2, Vip 2, PKR3	↑	[81]
CEV
in vivo	gill	Type 1 IFN (Vip and PKR)	↑	[64]
A. hydrophila
in vivo	spleen	IL-1β, IL-10, IL-12, IL-6, IL-8, IRFs (1, 4, 7 and 8)	↑	[82]
	head kidney	IL-1β, IL-10, TNF-α, Cc and Cxc-chemokines	↑	[83]
	spleen and head kidney	IL-1β, TNF-α	—	[84]
	spleen and liver	IL-1β, IL-10	↑	[85]
	spleen, kidney, and liver	IL-17	↑	[86]
	head kidney and pituitary	IL-1β	↑	[87]
	head kidney, spleen, foregut, hindgut, and endothelial progenitor cells (EPCs)	IRF9, Type 1 IFN, PKR, ISG15, TNF-α	↑	[88]
in vitro	spleen, head kidney, foregut, hindgut, peripheral blood leucocytes (PBLs) and head kidney leucocytes (HKLs)	IRF10	↑	[89]
in vitro/in vivo	head kidney leucocytes (HKLs)	IL-1β, IL-10	↑	[90]
	head kidney and trunk kidney	IL-17, IL-1β	↑	[91]

IFN—interferon; IL—interleukin; TNF—tumor necrosis factor; TGF—tumor growth factor; Mx—myxovirus resistance protein; Vip—viperin; PKR—protein kinase RNA-activated; IRF—interferon regulatory factor; Vig-1—VHSV-induced gene; Rig-1—retinoic acid-inducible gene-1; ↑—overall upregulation; ↓—overall downregulation; ——no changes.

). However, the localized intervention of IFN-1 on the skin interprets the absence of interferon type 1 response during in-vitro testing on C. carpio brain (CCB) cells infected with CyHV-3 [62]. Moreover, lack of type 1 interferon response on other sites could be extrapolated as antiviral capabilities adopted by koi herpesvirus against IFN-1.

During infection with herpesviruses, activation of adaptive immune response occurs in the later stages. The response is carried out by stimulation of the natural killer T cells and B lymphocytes to induce interleukins, in addition to TNF-β and IFN-γ, to initiate and prolong the responses by enhancing the production of antibodies [92]. However, the role of these specific cytokines is not fully elucidated in CyHV-3 infection. Rather, infected carp produce specific immunoglobulins (Ig) and mount cell-mediated immune responses [93]. Fish that survive a CyHV-3 infection acquire resistance, leading to a remarkable reduction in mortality [94,95]. Despite that, the capacity to build up a long-lasting latent infection is the hallmark of all known herpesviruses, including fish herpesviruses. Those latent infections are either controlled through the virus mimicking host immunoreactions or by encoding their own antiviral-like proteins such as IL-10 [96,97,98].

In order to replicate more effectively, several viruses encode their own viral IL-10 homolog (vIL-10), which exert an immunosuppressive effect in the host at the beginning of the infection [96,97]. CyHV-3 vIL-10 was initially described in common carp and the European eel [98]. Later on, Sunarto et al. [65] also reported that CyHV-3 captures an IL-10 gene from the host and modulates its features to tackle the host immune response. However, an in-vitro study conducted by Ouyang et al. [78] demonstrated that CyHV-3 ORF134 (the gene encoding an IL-10 homolog) was neither essential for viral replication nor virulence in common carp.

Nevertheless, CyHV-3 appears to manipulate host antiviral mechanisms more prominently via interaction with the TNF-α pathway. A study conducted by Rakus et al. [77] revealed a significant connection between CyHV-3 infection and the development of inflammatory responses controlled by TNF-α. Based on the findings of the study, infected carp demonstrated a delay in fever manifestation, which is an essential infection phase that promotes viral replication. This modulation in disease development was associated with viral CyHV-3 ORF12 encoding the TNF-α soluble decoy receptor, which is known to block cytokine activity [77].

The research of gene expression in carp has yielded an insightful perception of the CyHV-3 pathogenesis associated with cytokine secretion. Specific groups of cytokines including IFNγ-1, IFNγ-2, IL-12, IL-10, IL-1β, TNF-α1, and genes of major histocompatibility complex (MHC-II), are found to be over- or under-expressed, respectively, to the ongoing infection stage [70]. For instance, fish tested during acute phases of infection show a higher expression rate in all interleukin and interferon genes, while class II MHC and TNF-α1 genes are downregulated [70]. Furthermore, a survey conducted by Rakus et al. [66] on two carp lines (R3 and K) following CyHV-3 infection has revealed a disparity in the kinetics of cytokine genes expression on different days post-infection. The R3 line exhibited significant upregulation of IL-1β, IL-10, IL-12, and MHC class I genes and more disease resistance compared to the K line, whereas no significant differences in IFN expression were detected in both lines during the infection. It can be speculated that differential expression of those cytokine genes in carp lines could be due to host-related genetic factors incorporating the progress of the infection phase and shifting to adaptive immunity.

The use of cytokine or cytokine inducing cells (i.e., macrophages, B cells, and dendritic cells) as an adjuvant to improve the immunogenicity of DNA vaccines in fish is another growing research interest [99,100]. A potential protective effect of IL-β in combination with the CyHV-3 ORF25 DNA vaccine has been observed in common carp [75]. Likewise, the mammalian granulocyte–macrophage colony-stimulating factor, which in mammals is described as a regulator cytokine of immune cells proliferation, differentiation, and maturation [101], has also improved the immunogenicity of DNA and subunit vaccines in fish [102]. Moreover, the effect of the CyHV-3 ORF25 DNA vaccine on IL-β, cxca, cxcb1 chemokines, and interferon-stimulated genes (Mx1, vip2, pkr3, and isg15) have been examined in muscles of common carp by Embregts et al. [74]. In their study, they used ORF25-based DNA vaccine in different vaccination routes, i.e., intramuscular injection (i.m.) or oral gavage, in one or multiple doses. A strong immunostimulant effect was observed through the repeated i.m. injection, which resulted in cytokine expression and interferon-stimulated genes.

3.2. Cytokine Studied in Response to Spring Viremia of Carp Infections

Spring viremia of carp (SVC) is a highly contagious viral disease affecting carp (C. carpio) and other cyprinid species. The disease is caused by the Carp sprivivirus (originally SVC virus), a single-stranded RNA virus belonging to the family Rhabdovividae [7,103]. The condition is associated with edematous symptoms such as exophthalmia, edema of the underlying tissues, and abdominal distention [7]. Gross signs include petechial hemorrhages on the eyes, skin, gills, and internal organs, specifically on the swim bladder [104].

The SVCV genome encodes five different viral proteins, including matrix protein (M), nucleoprotein (N), glycoprotein (G), phosphoprotein (P), and viral RNA-dependent polymerase, important for viral transcription and replication, endocytosis, and infectivity [105]. The accessibility of the entire genomic information of SVCV, and its homology to mammalian rhabdoviruses, has allowed us to understand the function of these proteins more accurately [7,106].

Rhabdovirus infections, in fish, are controlled through the responses of group I and II IFNs. The recombinant IFNs group I appears to inhibit virus replication at any of the replicative stages [107,108,109,110]. The mechanism of rhabdovirus containment by group II IFNs varies intrinsically [110]. Recombinant IFNs-II was found to induce a protective effect against SVCV infection in zebrafish through upregulation of interferon regulatory genes such as myxovirus resistance protein (Mx) and viperin. On the contrary, group 1 IFNs upregulate both ISGs and pro-inflammatory cytokines (IL-10β and TNF-α) more persistently [111]. Conclusions from in-vitro and in-vivo research in carp indicate alternative responses of IFNs-I during SVCV and CyHV-3 infections. For instance, in a study on CCB cells infected with either SVCV or CyHV-3, a higher IFN-I upregulating response was detected through SVCV compared to CyHV-3, which oppositely diminished IFN-I production [62]. A similar trend of IFN response was noticed when different common carp strains (Amur wild carp, Amur sasan, AS; Ropsha scaly carp, Rop; Prerov scaly carp, PS; and koi) were challenged with the same viruses [76]. Rop was found to be more resistant to SVCV infection than the PS strain. During CyHV-3 infection, both Rop and AS displayed an increased survival rate compared with the PS strain. Interestingly, these observations were associated with increased activity of IFN-I among the SVCV resistant carp more than that in the CyHV-3 resistant group. The contribution of type 1 interferon to prevent SVCV, but not CyHV-3, infection appears to be a host–pathogen-specific interaction with the SVC virus.

Type 1 interferon, in addition to IL-12, are collectively known as signal-3 cytokines, which have been shown to activate clusters of differentiation 8 (CD8) T cells [112]. The cluster comprises CD8a and CD8b; both are dimeric co-receptors recognizing peptides presented by MHC class I molecules. They have a crucial role in immune defenses against various pathogens, including viruses. Both type 1 IFN and IL-12 were found to support the expansion and differentiation of CD8 T cells in vitro [113,114]. CD8 T cells also display an equivalent gene expression profile in the presence of either type 1 interferon or IL-12 and also upon in vitro stimulation. A survey conducted by Forlenza et al. [93] evaluated transcription of signal-3 cytokines, evident a concomitant of IL-12 and type 1 interferon, with CD8ab during SVCV infection in common carp. Precisely, signal-3 cytokines coincided with CD8ab upregulation at day 4 post SVCV infection. Initiation of CD8ab via signal-3 cytokines during SVCV infection indicates that these pro-inflammatory molecules play a regulatory and reciprocal role via the MHC-I pathway, which involves activating antigen-directed adaptive immunity.

Earlier research revealed the presence of active IFNs in sera of fish infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and SVCV [115]. More recent follow-up research has confirmed the presence of these similar interferons via transcriptional upregulation of genes after experimental infection with the viruses mentioned above [111,116].

IFN-γ, in fish, generally evokes an immune response that features overriding virus-replicative activity. For instance, recombinant IFN-γ upregulates some interferon regulatory genes, including ISG12, ISG15, and Mx [117], and shows antiviral effects, such as the inhibition of viral structural proteins synthesis and the reduction of virus titer in different cell lines of Atlantic salmon [118]. In cyprinids, i.e., zebrafish, the biological activities of IFN-γ, in addition to groups 1 and 11 of IFNs that are classified as type I interferons in fish, have been evaluated in vivo during SVCV infection. Strikingly, and unlike groups 1 and 11 IFN-I, zebrafish interferon (zfIFNγ) failed to produce pro-inflammatory genes and antiviral effects when administered [111].

As previously mentioned, IFNs have a significant role in establishing an antiviral response against Carp sprivivirus involving initialization of the adaptive immunity. Despite that, Carp sprivivirus was reported to have the ability to evade host immune response [119]. Studies conducted by Li et al. [120] and Lu et al. [121] demonstrated that the overexpression of SVCV phosphoprotein N and glycoprotein G inhibits IFN synthesis in carp and zebrafish by reducing IFN transcription in host cells.

Interferons are not the only cytokines involved in the immune response during Carp sprivivirus infection. Some studies also observed an interleukin activity in response to this pathogen. For instance, carp IL-10 paralogues IL-10a and IL-10b possess similar protein sequences and phagocytes-provoking bioactivity were tested on carp head kidney cells. IL-10b had a low tissue constitutive expression in the liver, gut, gills, spleen, thymus, peripheral blood leucocytes, head, and trunk kidney when assessed in the absence of pathogens. However, it was upregulated during SVCV infection in the head and trunk kidney, followed by other tissues. Contrary to this, IL-10a gene expression did not change throughout the infection period despite its tissue constitutive expression being higher in healthy tissue [122].

3.3. Cytokine Studied in Response to Carp Edema Virus Infection

Carp edema virus disease (CEVD) or sleepy koi disease (KSD) is a rising threat to koi and carp aquaculture. The disease was first reported in koi carp farms in Japan in 1974, where it caused substantial mortalities [123,124,125] and economic losses. The nomenclature of sleepy koi disease is attributed to the behavioral abnormalities showed by affected carp, including lethargy and unresponsiveness. Consequently, the disease is called sleepy koi, and fish can be seen lying in the bottom of tanks for extended periods [126]. Gross lesions incorporating spreading hemorrhagic skin lesions with edema, particularly in the abdomen, pale gills, sunken eyes, and ulcerative inflammation on the anus may also be seen [127,128,129]. Overproduction of mucus on the skin and gills is also observed [130,131].

Carp edema virus (CEV), belonging to the Poxviridae family, consists of double-stranded DNA about 250–280 nm in diameter, with a mulberry-like structure [128]. Gills seem to be the primary site for the infection. In diseased fish, poxvirus-like structures were confirmed by electron microscopy in morphologically altered gill tissue [125,126,132]. Three different genogroups of CEV have been so far characterized: I, IIa, and IIb [5,6]. Genogroup I, which has been detected in most European waters, is mainly infective to common carp. Genogroup IIa is almost entirely, but not exclusively, reported in koi, while genogroup IIb has been detected in both carp and koi samples.

Poxviruses are contained through innate immunity undertaken by the inflammatory and NK cells [133]. These responses control viral replication and mount an antigenic adaptive response [34]. Similar to other viral infections, interferons (α, β, and γ) play an important role during poxvirus infections. Interferons produced by NK cells, fibroblasts, lymphocytes, and leucocytes conclusively control poxvirus replication. Nevertheless, poxviruses also modulate the host immune responses to guarantee their survival [134]. The implicated mechanism incorporates interruption of ISGs synthesis resulting in IFN inhibition. Furthermore, poxviruses possess immunomodulating genes able to competitively antagonize the IFN-α via encoding a homolog of the ligand-binding receptors chain for this interferon [135].

However, during CEV infection in carp, the host–virus interaction is reciprocally impacted by host genetic competitiveness and virus genomic characteristics. In other words, the carp edema virus, although it still remains unclassified, can probably vary in its genotype, as several emerging genogroups have so far been identified among carp populations worldwide. On the other hand, these genogroups are seemingly selective mutations targeted towards the numerous variants within the common carp species. It is reported that the underlying strains of carp highly influence the expression profile of cytokines during infections, specifically the IFN and ISGs [69].

The only study based on IFN and ISGs concerning CEVD was conducted by Adamek et al. [64]. In their study, different carp strains, including AS, PS, Rop, and koi carp, were exposed to CEV, genogroups I and II, to investigate their susceptibility to infection. Their analysis comprised the expression of type I interferon, ISGs encoding IFNa2 in gills. Amur wild carp was found to be more resistant to the infections than the other three strains/species. The significant finding of this study was the downregulation of IFNa2 and ISGs in response to CEV infection in AS, known to be a highly resistant species compared to other strains/species. Such an effect indicates an activation of the adaptive immunity at the proposed time of infection marked by suppression of cellular immunity through a shutdown of interferon expression. Potential development of humoral immunity and antibody production might have been the reason for resistance to CEV infection displayed by the AS compared to the other strains/species. Likewise, a lack or delay in initiating an antigenic-based adaptive immunity in susceptible strains could be responsible for the high mortalities. A similar cytokine cellular immune-responsiveness trend is reported in association with CyHV-3 and SVCV. Previous transcriptomic analysis of differentially expressed immune genes during CyHV-3 infection elucidated a rapid upregulation of IFNs and ISGs in susceptible strains versus resistant ones [69]. These observations were supported by other findings reporting an influence of carp strain on the interferon expression rate in fish infected with CyHV-3 or SVCV [76]. In contrast to CyHV-3 and SVCV infections, where the mechanism of IFN response has been studied quite thoroughly, there remains a paucity of research on IFN response during CEV infection in common carp. Overall, the response of IFNs in different carp strains/species to CEV infections is consistent with previous studies [69,76]. However, more research is required to support the IFNs response to CEV infection in carp strains.

3.4. Cytokine Studied in Response to Aeromonas hydrophila Infections

Aeromonas hydrophila is a rod-shaped Gram-negative bacterium and one of the most common bacterial pathogens infecting fish in freshwater [136]. Aeromonas hydrophila naturally inhabits the gastrointestinal tract of fish [83]. This bacterium predominantly affects teleosts that are highly susceptible to the infection, such as common carp, catfish, goldfish, and other tropical and ornamental fish [137,138]. Infected fish manifest different clinical signs, including swimming abnormalities, generalized edema, pale gills, and deep dermal ulcers [139]. In common carp, a distended abdomen and scale blisters on the skin are the prominent signs of this disease [140].

Eliminating bacterial pathogens in the host is carried out through an immediate and non-specific response. Upon the stimuli of the immune system with bacterial agents, the recruitment of several phagocytes involved in cytokine production like neutrophils, macrophages, and dendritic cells takes place [141]. During A. hydrophila infections, the secretion of cytokines such as ILs, IFNs, and TNFs hold great importance. Interleukins seem to be a focal point for immunological research interest, followed by tumor necrosis factors and interferons.

It is believed that the expression of IL1-β, IL-10, and IFN in carp during A. hydrophila invasion is triggered primarily by the toll-like receptor 18 (Tlr18), as indicated by a study on the epithelioma papulosum cyprini cell lines (EPC) [90]. This important pattern recognition molecule usually exists on macrophages and dendritic cells and has a fish-specific expansion that plays a crucial role in innate immunity against pathogens via cytokines activation.

In response to bacterial infections, IL-1β induces adhesion molecules (selectins, integrins, and cadherins), which assist in recruiting neutrophils to the site of inflammation [142]. Several studies have been conducted to evaluate the response of IL-1β during A. hydrophila infections. For instance, carp subjected to high temperature (30 °C) for 30 days and subsequently challenged with A. hydrophila had significantly elevated IL-1β and TNFα expression in the spleen and head kidney. Other immune or stress-related genes, such as inducible nitric oxide synthase (iNOS), glucocorticoid receptor (GR), and superoxide dismutase (SOD), were also suppressed [84].

The emergence of A. hydrophila in the carp population is common around the globe, but using antibiotics as a preventive measure has been questioned due to bacterial resistance and potential risk to consumer health [143]. Additionally, A. hydrophila has a significant strain diversity with plentiful pervasiveness in aquatic environments [144]. Therefore, developing an effective vaccine to control this pathogen remains an issue of interest. At present, various A. hydrophila vaccines have been developed, such as inoculations comprising killed whole bacteria, components of the pathogen macromolecules, biofilms, or non-replicated protein isolates [34,145,146,147,148,149]. Most of these vaccines, however, exhibit nonfunctional immunogenicity. Considering this fact, multiple research attempts have aimed to evaluate the efficiency of live, attenuated (XX1LA) or killed (by formalin) A. hydrophila to induce an early and adequate immune stimulation in carp. In both cases, upregulation of the IL-1β- and IL-10-related genes occurs in the spleen and liver specifically, as indicated in a study conducted by Jiang et al. [85]. Although other parameters, such as survival rate, antibody titer, and serum lysozyme were vaccine-dependent, it can be demonstrated that the IL-1β and IL-10 are major game changers in the innate immune defense against A. hydrophila with association to tissue-specific gene regulation.

Interleukin IL-17 is another nominated key pro-inflammatory cytokine involved in immunoreactions against bacterial infections in carp. In fact, IL-17 with pro-inflammatory characteristics plays an essential role against a wide range of viral, bacterial, and fungal infections [150]. Providing mucosal immunity at the first entry sites, i.e., gills and skin, against various pathogens is the hallmark of this cytokine, which exists abundantly in mucosal and immune tissues [151]. In mammals, interleukin comprises seven ligands, from IL-17A to IL-17F. IL-17A and IL-17F are pivotal cytokines that play a significant role in autoimmune disorders and immunological responses to pathogens [152]. These cytokines regulate gut microbiota and act as the first line of defense in mucosal tissue against invading pathogens.

In fish, all seven ligands have been identified, and transcriptional studies have shown the tissue-specific ligand role of IL-17 in all tissues [151]. Unsurprisingly, these ligands’ expression was abundant in mucosal tissues, signifying their essential role in the first entry sites (gills, skin, and intestine) for pathogens. Fish IL-17A and IL-17F show the same genomic organization as that of mammals, therefore named as IL-17A/F. Later on, IL-17N fish-specific ligands were described in the fish genome and known as a teleost-specific ligand [152]. The expression of genes from the IL-17 family was actively observed in the gills of common carp in response to A. hydrophila [86]. In the appointed observations, upregulation of most IL17 genes occurred at early stages of microbial challenging (4 h post infection) in gills and then declined slowly. The only genes that were substantially upregulated for a prolonged time (12 h post-infection) were IL17A/F2 and IL17N. In the most recent experiment, common carp interleukins (IL-17Na and b) were identified and confirmed through real-time polymerase chain reaction (real-time PCR) [153]. When carp were challenged with A. hydrophila infection, IL-17Ns were highly expressed in the brain tissue at 6 h post-infection; at day 1 post infection, the expression was upregulated in other tissues (head kidney, spleen, liver, and muscle) as well. In addition to that, the upregulation of the cytokines IL1β, IFN-γ, and IL-6 and chemokine CCL were also noticed post infection. In conclusion, the varying pattern of expression for different genes in the IL-17 family during A. hydrophila infection in gills and other tissue of carp indicates a pivotal role performed by these genes in regulating early immune responses.

Upon IFNs discovery, their biological function was first attributed to antiviral mechanisms, but later on, IFNs acquisition of antibacterial activity was also recognized [154]. Interferons inhibit bacterial infection by recognizing Toll-like receptors (TLR) and/or pattern recognition receptors (PRRs) present on infected cells [19]. These receptors have been found to bind molecules from a range of bacterial cellular components such as lipopolysaccharides, polysaccharides, and bacterial DNA to initiate the production of IFN genes controlling the infection [155]. Following these discoveries, a revolution of genetic identification and characterization for various IFNs regulatory genes aided in promoting the study of their gene expression during diseases’ development.

Interferon regulatory factors (IRFs) play an important role in regulating both type 1 IFN and IFN-stimulated genes [89]. They are crucial in the innate immune response that regulates the expression of IFNs and initiate an antiviral and antibacterial response in teleost. For instance, carp challenged with A. hydrophila and polyinosinic:polycytidylic acid (poly I:C) stimulation upregulated IRF9 expression in the spleen, head kidney, foregut, and hindgut at different time intervals. Furthermore, transfection of IRF9 with poly I:C and LPS stimulation upregulated the expression of cytokines, including interferon-stimulatory genes (ISG)15, type 1 IFN, and TNFα in epitelioma papulosum cyprini (EPC) cells [88]. This finding suggests the potential antiviral and antibacterial role of IRF9 in fish. Furthermore, the so-called carp interferon regulatory gene (CcIRF10) apparently participates in stabilizing the bacterial infection via negative regulation of IFN response [89]. This inhibitory effect of IRF10 on IFN has been similarly described in a study on zebrafish infected with SVCV [156]. The authors of the underlying study linked their findings to the possible strategy of the immune system to avoid excessive immunopathological reactions resulting in response to the viral infection. However, documenting the same effect in the absence of viral intrusion, as in the study conducted by Zhu 2020 et al. [89], may further explain an intrinsic role of IRF10 in carp following the onset of bacterial infection. The implicated mechanism of interferon inhibition by IRF10 might have taken place to suppress the development of severe inflammation or as a proactive tactic to reduce the implying of potential secondary viral infection. In this regard, examining the specific role of IRF10 in carp co-infected with A. hydrophila and SVCV might be an issue of interest for future research orientation. Upon review, these findings suggest that interferon regulatory factors play a significant role in regulating type 1 IFN and ISG genes in common carp to A. hydrophila infection. Further understanding the levels and activity of IRF genes in common could be the future interest.

4. Conclusions

Cytokines are essential components in the innate immune system, which play a crucial role in maintaining fish health. Their immunoreactive function in common carp to studied pathogens gives us an insight into what may have been the minimal or even the absolute cytokine network needed to initiate innate and adaptive immune responses. The information presented in this review article indicates that cytokines, including interferons, interleukins, and tumor necrosis factors, play an important role in the clearance of pathogens and the enhancement of antimicrobial activities in carp. Hence, analyzing and assessing their expression profile in common carp to the diseases mentioned above could be an advantage for developing vaccines for aquaculture. The type of cytokine in common carp and its induced response can depend on the nature of infectious agents. Variation of cytokine responses depends on the disease, as demonstrated by the differences between interferons and interleukins produced when infected with cyprinid herpesvirus 3, rhabdovirus, and A. hydrophila infections, respectively. However, the inconsistent expression of cytokines could also be due to the different genetic backgrounds of carp variants. Therefore, the genetic diversity of carp strains and their cytokine response to pathogenic stimuli is a domain where much remains to be discovered. In conclusion, a deeper understanding of the cytokine profile in common carp associated with various infectious agents is a continuing research demand.