Teleost NOD-like receptors and their downstream signaling pathways: A brief review

HIGHLIGHTS

• •Homologs of mammalian NOD1, NOD2, NLRC3, NLRC5 and NLRX1 present in fishes.
• •The NLR-C/NLRC3-like/NLR-B30.2 subfamily appears unique to teleosts.
• •Nodosome signaling cascade is well conserved in fishes and mammals.
• •Signaling related to other NLRs is still obscure in fishes.
• •Recently discovered mammalian NLRP1/3 homolog indicate conserved inflammasome pathway.

Abstract

Nucleotide-binding oligomerization domain-like receptors (NOD-like receptors or NLRs) are key members of the immune system that act as intracellular sentinels. These pathogen recognition receptors are essentially characterized by a central nucleotide binding domain and a C-terminal leucine rich repeat domain responsible for recognition of pathogens. Over the past decade, our understanding of teleosts' NLRs has enhanced significantly although the signaling pathways remain to be elucidated. In this brief review, we have tried to decipher the structural and functional aspects of NLRs in teleost. The review also engages in illustrating the various downstream signaling pathways/molecules reported so far in fishes that enable the NLRs to act as important players in immune responses and defense mechanisms against pathogens. Importantly, we try to explore the lacunae in structural and mechanistic details of NLRs in the teleost that would help in identifying key areas in which research is needed to complete our understanding of NLRs and their structural and functional evolution.

1. Outline of pathogen recognition receptors

Pathogen recognition receptors (PRRs) are germ-line encoded receptor proteins aimed to recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) resulting in mobilization of downstream signaling responses leading to inflammation, microbial destruction and eventually activation of adaptive immune responses. PAMPs are conserved molecular structures expressed on similar pathogenic microorganisms, and include peptidoglycans, lipids, bacterial DNA, viral RNA, nucleic acids. DAMPs on the other hand are non-pathogenic endogenous host-derived molecules, such as heat shock proteins (HSPs), high mobility group box 1 (HMGB1), adenosine triphosphate (ATP) and histones released by damaged or necrotic cells [1]. In addition to directly binding and activating the PRRs, some DAMPs are known to have different targets that in turn affect the signaling by PRRs (Figure 1). For example, uric acid indirectly activates NLRP3 via reactive oxygen species (ROS) induction [2] whereas ATP activates NLRP3 via P2X7R signaling. HMGB1 binds to DNA and augments activation of TLR9 via crosstalk with receptor for advanced glycation end products (RAGE) [1]. Hyaluronan also does not recognize TLR4 alone but a unique complex of TLR4 with molecules such as MD-2 and CD44 [3]. In addition to PAMPs and DAMPs, molecular patterns involved in the activation of PRRs are MAMPs (microbial-associated molecular patterns). MAMPs include virulence factors in addition to essential components of both commensals and pathogens, e.g., lipopolysaccharide (LPS), peptidoglycan, or nucleic acids [4]. There are several families of PRRs that are either membrane-bound or located intracellularly. Transmembrane PRRs in plasma membrane include toll-like receptors (TLRs) [5] and C-type lectin receptors (CLRs) [6]. Intracellular PRRs include retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), cytosolic DNA sensors (CDSs), and the newly identified cytosolic absent in melanoma (AIM)-like receptors [7], [8], [9], [10]. The current review is focused on NOD-like receptors or NLRs that act in tandem with other transmembrane and intracellular receptors to orchestrate an efficient host immune response against microbial invasions and endogenous danger signals. Members of the NLR family possess a C-terminal LRR (leucine-rich repeats) domain and a central NACHT domain (named after NAIP, CIITA, HET-E, and TP1). In addition, many NLRs in metazoans also have a N-terminal effector domain.

Figure 1.

Schematic representation of NOD-like receptors and their signaling pathways in mammals and teleost. MAMPs, PAMPs and DAMPs are recognized by various NLRs. This leads to the activation of downstream signaling cascades resulting in inflammatory responses. (PAMPs- pathogen-associated molecular patterns, DAMPs- damage-associated molecular patterns, MAMPs-microbial-associated molecular patterns, dsRNA-double stranded RNA, LRR- leucine rich repeats, CARD-caspase activation and recruitment domains, NACHT-NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), FISNA- fish-specific NACHT associated, STING- stimulator of interferon genes, NFkB- nuclear factor kappa-light-chain-enhancer of activated B cells, P13K- phosphatidylinositol-3-kinase, AKT- Protein kinase B, mTOR- mammalian target of rapamycin, TLR- Toll-like receptor, IFN- interferon, MDA5- melanoma differentiation-associated protein 5, RIG-1- retinoid acid -inducible gene I, JNK- janus kinase, STAT- signal transducers and activators of transcription, VEGF- Vascular endothelial growth factor, MHC- major histocompatibility complex).

In the 1990s, a large family of conserved genes that encode proteins characterized by nucleotide binding domain and leucine rich repeats (NBD-LRRs) was discovered in the animal kingdom. In human, NLR genes were named variously such as CATERPILLER [11,12], NODs [13], NOD-LRR [14,15], NACHT-LRR, [16] and NOD-like receptor [17,18], until the introduction of the standard nomenclature “NLR'' in 2008 after the approval of the Human Genome Organisation (HUGO) Gene Nomenclature Committee and the Mouse Genomic Nomenclature Committee [19]. In teleosts, NLRs were first reported in 2008 by Laing et al. in zebrafish [20]. The next few sections describe the structural and functional aspects of NLRs in teleosts and compare it with information already available in mammals.

2. Diversity of NLRs

The members of the NLR family are reported to vary in different species across vertebrates. In mammals, the number of NLRs range from 23 in humans to more than 30 in mice [21,22]. Invertebrate NLRs are much higher in number, extending from 135 in sponges [23] to 200 in sea urchins [24]. Similarly, in teleosts, different NLR proteins have been shown with nearly 400 NLRs being identified in the zebrafish genome [25].

Structure of mammalian NLRs has been determined employing X-ray diffraction, solution nuclear magnetic resonance and electron microscopy [26]. They have a tri-domain architecture comprising of an amino-terminal effector-binding domain, a central nucleotide-binding domain termed NOD domain and a carboxy-terminal leucine-rich repeat (LRR) domain. NOD domain also referred as NACHT domain has three associated helical domains namely, helical domain 1 (HD1), winged helix domain (WHD), and helical domain 2 (HD2). The central NACHT domain is unique to NLRs and is critical for their oligomerization, while the C-terminal LRR domain serves to recognize PAMPs and associated danger signals. Ligand binding to LRR causes oligomerization of NACHT and leads to conformational changes in receptor such that N-terminal effector domains become accessible to interact with downstream signaling molecules or molecular adaptors [10].

Mammalian NLRs have been broadly classified according to their effector domain, phylogenetic relations and physiological functions. According to the HUGO classification depending upon their effector binding domain (EBD) at N-terminal, NLRs are categorized into NLRA (MHCIIACTVATR domain (AD) or CARD-like domain prior to the AD domain in splice variants), NLRB (Baculoviral IAP repeats, BIR), NLRC (CARD domain), NLRP (pyrin domain, PYD) and NLRX (yet to be identified domain). Based on phylogenetic relations, NLR family is divided into NODs (NOD1, NOD2, NOD3/NLRC3, NOD4/NLRC5, NOD5/NLRX1, CIITA), NLRPs (NLRP1-NLRP14) and IPAF (ICE-protease-activating factor e.g., IPAF/NLRC4 and NAIP) [27]. According to their physiological functions, Coutermarsh-Ott et al. [28] introduced another system of grouping. First group includes inflammasome-forming NLRs (NLRP1,3,4,6,7,10,12) associated with production of proinflammatory cytokines IL-1β and IL-18 as well as cell death known as pyroptosis [28,29]. Other groups are reproductive NLRs (NLRP2,4,5,7-9,11,13,14) involved in reproduction and embryogenesis [30,31], and regulatory NLRs having either positive (NOD1, NOD2) or negative (NLRP12, NLRX1, NLRC3) role in regulating diverse immune signaling pathways.

In non-mammalian vertebrates, studies are mostly confined to teleosts and meagrely explored in birds, amphibians and reptiles wherein the reports lack functional studies/signaling pathways. The last decade has seen a steep increase of reports in fish NLRs but a clear picture with respect to nomenclature and signaling is still lacking. Initially it was believed that NLRs first appeared in teleosts via domain shuffling as presence of their orthologs could not be demonstrated following in silico analysis in classical invertebrate model organisms such as Drosophila melanogaster, Ciona intestinalis and Caenorhabditis elegans [11]. However, the discovery of NLRs in many classes of invertebrates indicated the evolutionary conservation of these cytosolic PRRs.

In cyclostomes, the most primitive vertebrate group, presence of 34 NLR genes has been reported in the genome of the sea lamprey [32]. In addition, recently, 9 NLR genes identified in the Far Eastern brook lamprey genome have been grouped into two subfamilies (NLRA and NLRC) based on comparative genomics and phylogenetic analysis [33]. The same study has also shown high expression of these genes in immune tissues and their significant response to bacterial/viral ligands. The presence of NLRs has been predicted in chondrichthyes such as grey bamboo shark [34], whale shark [35] and major ray-finned fishes like bichir, American paddlefish, alligator gar, spotted gar and bowfin [36] following in silico analysis. In teleosts, whole genome sequencing along with advanced bioinformatics tools has facilitated the identification of several NLR transcripts over the years (Table 1) having similar domain architecture to mammals. However, NLR classification in this extremely phenotypically diverse group that comprises more than half of extant vertebrates is at times confusing. In 2008, Laing et al. [20] provided a basic framework for classification wherein NLRs in zebrafish were categorized into three subfamilies NLR-A, -B and -C (Figure 2). The NLR-A subfamily (NLR-A1 to A5) resembles mammalian NODs, NLR-B (NLR-B1 to B6) are structurally similar to mammalian NALPs, and NLR-C subfamily (NLR-C1 to C62) with unusual C-terminal domain that is unique to the bony fishes phylogenetically relates to mammalian NOD3 [20]. In the following year, Sha and group [37] used the HUGO nomenclature [19] to name NLRs in channel catfish as NOD1, NOD2, NLRC3, NLRC5 and NLRX1 and suggested that these were orthologous to their mammalian counterparts. Thereafter, Rajendran et al. [38] re-classified the NLRs in channel catfish employing subfamily nomenclature used in zebrafish. They reported 22 NLRs, 6 belonging to subfamily A (renamed as per HUGO nomenclature), 2 belonging to subfamily B, 11 belonging to NLR-C subfamily, and 3 NLRs that do not belong to any of these subfamilies. It is to be noted that except NLR-B2 in zebrafish (CARD-NACHT) and NLR-B1 in miiuy croaker (CARD-NACHT-LRR) all other NLR-B genes lack N-terminal as well as C-terminal domains [20,38,39]. In zebrafish, 6 genes belonging to the NLR-B subfamily were identified which clade with mammalian NALPs following phylogenetic analysis based on similarity in the region of NACHT domain. Despite the suggestion that NLR-B subfamily have structural similarity to mammalian NALPs/NLRPs, NLR-B genes do not contain pyrin domain distinctive of NALP genes thus the idea of these genes being functional equivalents to NALPs subfamily is debatable [38]. In addition to NLR-A, -B and -C, another subfamily NLRX1 with unknown effector domain has been identified in fugu [40] although Li et al. [41] suggested NLRX1 to be a part of the NLR-A subfamily. After that, HUGO nomenclature has been used invariably to classify NLRs in teleosts. However, it is to be noted that NOD1, NOD2, NLRC3, and NLRC5 belonging to subfamily C in mammals have been grouped under subfamily A in teleosts. The recent discovery of mammalian homologues of NLRP1 and NLRP3 in zebrafish [42,43] suggests the presence of a conserved inflammasome pathway. In the following subsections, we will focus on the NLRs that are actively involved in providing robust immune responses.

Table 1.

:

Fish species	Members/reported NLRs	Mammalian orthologs/ novel molecules	References
Zebrafish	NLRA1	NOD1	[20]
	NLRA2	NOD2
	NLRA3	NOD3
	NLRA4	NOD4
	NLRA5	NOD5
	NLRB1- NLRB6	NALPs/NLRPs	[20]
	NLRC1- NLRC62	Novel to teleosts	[20]
	NLRP1	NLRP1	[42]
	NLRP3	NLRP3	[43]
Channel Catfish	NOD1	NOD1	[37]
	NOD2	NOD2
	NLRC3	NLRC3
	NLRC5	NLRC5
	NLRX1	NLRX1
	NOD1	NOD1	[38]
	NOD2	NOD2
	NOD3a	NLRC3
	NOD3b	Novel to teleosts
	NOD4	NLRC5
	NOD5	NLRX1
	NLRB1	Novel to teleosts	[38]
	NLRB2	NALPs/NLRPs but lacks pyrin and LRR domain
	NLRC1-NLRC11	Novel to teleosts	[38]
Common Carp	CcNLRC	Novel to teleosts	[187]
Gold fish	gfNod1	NOD1	[74]
	gfNod2	NOD2
	gfNlrx1	NLRX1
	Nlrc3L (NLRC3-like molecule)	Novel to teleosts	[101]
Japanese flounder/ Olive flounder	NOD1	NOD1	[68]
	NLRC5	NLRC5	[93]
	JfNLRC	NLRC3	[90]
	NLRC3	NLRC3	[39]
Rohu	NOD1	NOD1	[69, 70]
	NOD2	NOD2
Grass Carp	NOD1	NOD1	[71]
	NOD2	NOD2
	NLRC1-NLRC48	Novel to teleosts	[100]
Miiuy croaker	NOD1	NOD1	[41, 88]
	NOD2	NOD2
	NLRC3	NLRC3
	NLRC5	NLRC5
	NLRX1	NLRX1
	NLRB1 and NLRB2	NALPs/NLRPs	[97]
	NLRC1-NLRC48	Novel to teleosts	[97]
Mrigal	NOD1	NOD1	[73]
	NOD2	NOD2
Orange-spotted grouper	NOD1	NOD1	[77]
	NOD2	NOD2
Nile tilapia	NOD1	NOD1	[76]
	NOD2	NOD2
	NLRC3	NLRC3
Catla	NOD1	NOD1	[188]
Atlantic salmon	NLRC5	NLRC5	[92]
	NLRC3	Novel to teleosts
Rainbow trout	NOD2a	NOD2	[67]
	NOD2b	NOD2	[189]
	NOD1	NOD1
			[88]
	NLRC3	NLRC3
	NLRC5	NLRC5
	NLRX1	NLRX1
Asian seabass	NLRC3	NLRC3	[87]
Blunt snout bream	NLRC3	Novel to teleosts	[98]
Turbot	NLRC3a	NLRC3	[85]
	NLRCb	Novel to teleosts
Japanese pufferfish	NOD2	NOD2	[190]
	NLRX1	NLRX1
	NHLRC1-NLRC13	Novel to teleosts	[36]
Spotted snakehead	NOD1	NOD1	[191]
Ya-fish	NOD2	NOD2	[80]

Figure 2.

Domain architecture of NLRs in teleosts. NLRs are broadly divided into three subfamilies namely NLR-A, NLR-B and NLR-C. Members of NLR-A subfamily have a varied number of CARD domains at the N-terminal. NLR-B family has been characterized in three fish species (zebrafish, channel catfish, miiuy croaker) so far. ‘#’ denotes domain organization depending on fish species. NLR-B1 in zebrafish contains CARD, NACHT and LRR domains whereas in channel catfish and miiuy croaker, the LRR domain is reported to be absent. ‘*’ denotes that differential domain organization in NLR-B2 wherein both CARD and NACHT domains are present in zebrafish while only NACHT domain in channel catfish and CARD domain in miiuy croaker. NLR-C subfamily is unique to teleosts with different domain organization across species and within the same species. Till date, the N-terminal domain of NLRX1 remains unidentified. (LRR- leucine rich repeats, BIR- baculovirus IAP repeat, CARD-caspase activation and recruitment domains, NACHT-NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein), FISNA- fish-specific NACHT associated).

2.1. NLRs involved in inflammatory responses

Structural and functional aspects of NLRs implicated in mediating inflammatory responses are well characterized in mammals. NOD1 and NOD2 sharing one or two CARD domains are known to trigger immune defense in response to bacterial peptidoglycan via detecting muropeptides released from bacterial peptidoglycan (PG) [44,13,[45], [46], [47]]. The mutation in NOD2 has been associated with serious inflammatory diseases such as Crohn's disease and Blau syndrome in humans [48], [49], [50], emphasizing its importance in immune responses. In addition, NOD1 and NOD2 transcripts have been detected in rat's Sertoli cells and germ cells implying their role in reproduction [51]. NLRC3 and NLRC5 having a single N-terminal CARD domain share high structural similarity. They are believed to be negative regulators of inflammatory immune responses [52], [53], [54], [55], [56] and are also reported to regulate adaptive immunity. NLRC4 reported only in mammals so far is not seen involved in sensing pathogens. Rather, it acts downstream of NAIPs (NLR family, apoptosis inhibitory proteins) to form inflammasome [57] that promotes activation of caspase-1 [58,59].

Unlike these receptors in which CARD domain is present at N-terminal, NALPs (NACHT-LRR-PYD-containing proteins) in mammals are characterized by the presence of PYD effector domain [60]. These receptors are activated by a variety of damage-associated molecular patterns (DAMPs) and microbial products including double-stranded RNA from bacteria and viruses [61], toxins [62], muramyl dipeptide (MDP) [63] and ATP [58]. Also, host-derived danger signals, such as uric acid crystals released by necrotic cells activate NALP receptors [64]. It is interesting to note that some of the NALPs play a pivotal role in inflammasome formation. Another member of the NLR family that could be envisioned to be involved in inflammatory responses is NLRX1. N-terminal of this receptor is yet to be characterized although the crystallographic data of human NLRX1 C-terminal fragment revealed presence of N-terminal helical domain (LRRNt), central leucine-rich repeat modules (LRRM) and a C-terminal three-helix bundle (LRRCT) [65]. This receptor is unique because it is the only NLR that is localized in mitochondria. Loss of function of NLRX1 has been linked to a variety of autoimmune diseases, including inflammation in CNS and bowel, colitis and cancer in mammals [66].

In teleosts, NOD1 and NOD2 have been extensively studied and their ubiquitous expression has been seen [67], [68], [69], [70]. Moreover, infection studies involving bacterial ligands [71,[68], [69], [70],[72], [73], [74], [75], [76]] and virus [71,67,77,[68], [69], [70],75] have reported change in basal NOD1 and NOD2 expression suggesting their critical role in host defense against bacteria and viruses [74]. Although the existence of multiple splicing variants of NOD1 have been reported in human databases, only one form of NOD1 has been reported in zebrafish via Western blotting [78]. It is noteworthy to mention that NOD2 splice variants similar to human NOD2 have been reported in zebrafish [79], rainbow trout [67] and Ya-fish [80]. In rainbow trout, the shorter transcript NOD2a encodes the normal form whereas NOD2b has a longer 5’ untranslated region (UTR) with incomplete first CARD domain. Splice variants are suggested to have a role in a harmonized defense response and variants with deletion might be involved in competitive inhibition of downstream signaling underlying the normal form [80]. Despite its importance in immune responses, NOD2 has been reported as a pseudogene in pangolins [81], and absent in birds [82], amphibians [83] and reptiles [84] suggesting its functional replacement by other PRRs. For example, in contrast to mammalian NOD2 which recognizes MDP, in chicken the function has been replaced by NLRP3 [63]. It has been proposed that different environmental conditions resulting in unique selection pressures induce differentiation in structure and function among NLR family members [82].

Mammalian ortholog of NLRC3 termed as NOD3 in channel catfish [38] and NLRC3 in turbot [85], Nile Tilapia [76], Asian seabass [86], miiuy croaker [87] and rainbow trout [88] belonging to the NLR-A subfamily, is shown to contain one N-terminal CARD domain. In these species, the constitutive and ubiquitous expression of NLRC3 in immune tissues suggests its role in innate immunity. NLRC3 expression is reported to be upregulated in response to poly I:C [86], [87], [88] and bacterial ligands such as LPS and PGN [86,88,89] as well as during bacterial and viral infection in teleosts [89,90,87,39,85,76]. NLRC3 has been suggested to positively [88] as well as negatively influence inflammatory responses [86,89]. Interestingly, unlike mammals where no splice variants for NLRC3 are reported [91], two splice variants NLC3a and NLRC3b in turbot [85], and two splice variants NOD3a and NOD3b [37,38] have been described.

Mammalian NLRC5 ortholog in teleosts contains either one CARD domain as seen in catfish [37] or no CARD domain as in Atlantic salmon [92]. The constitutive expression of NLRC5 has been seen in Atlantic salmon [92] and Japanese flounder [93]. Its expression was shown to be upregulated in response to bacterial or viral ligands in channel catfish [90], miiuy croaker [87], Japanese flounder [93], rainbow trout [88] and zebrafish [94]. Similar to humans, five transcript variants in zebrafish are reported having conserved 5′ region with variation in length of the LRRs. The diverse functions of these isoforms have been speculated in zebrafish during viral infection. Overexpression of isoform zfNLRC5d had a pronounced effect against viral infection whereas zfNLRC5a overexpression had no significant effect [95]. In addition, NLRC3 and NLRC5 novel to teleosts which might belong to the NLR-C subfamily have been reported [39,40,[96], [97]]. NLRC3-like (NLRC3L) or NLR-B30.2 characterized by a conserved NACHT domain with an unusual C-terminal domain is unique in teleosts. This receptor is thought to have evolved from NLRC3/NOD3 due to high similarity of their NACHT domain [20]. Members of this subfamily possess an additional domain that includes a PYRIN/ RING/ FISNA and uncharacterized (X) domain at N-terminus, and a B30.2 (PRY/SPRY) domain at C-terminus [98]. The B30.2 domain consisting of Serine–Proline-Arginine-Tyrosine (SPRY) and Proline-Arginine-Tyrosine (PRY) has an important role in innate immunity. Additional category of NLRC3-like proteins consisting of a distinctive PYD-FISNA-NACHT structure was cloned in zebrafish and blunt snout bream [94,99]. Recently, NLRC3-like proteins in grass carp have been explicitly classified into four categories according to their domain architecture [100]: (i) having PYD, FISNA, NACHT and LRR domains, (ii) possessing FISNA-NACHT-LRR structure, (iii) containing FISNA, NACHT, LRR and SPRY-PRY-SNTX domains and (iv) consisting PYD, FISNA and NACHT domains, no LRRs. The ubiquitous expressions of NLRC3-like genes have been reported in various teleosts, namely zebrafish [20], Japanese flounder [39,89], blunt snout bream [98], goldfish [101] and Atlantic salmon [92]. Moreover, studies have shown the regulation of piscine NLRC3-like genes by bacterial and viral infection [38,39,99,102]. Some of these genes in miiuy croaker [39] and grass carp [100] have been reported to exhibit more sensitivity to virus than bacteria although the mechanism for mediating host defense is unclear [103].

A study based on phylogenetic analysis and synteny has reported duplication of NLRX1 gene into NLRX1a and NLRX1b before the divergence of sarcopterygii and actinopterygii. The study revealed loss of NLRX1a in actinopterygii and selection of NLRX1b during evolution which is referred to as NLRX1 [87]. In teleosts, NLRX1 was first identified in channel catfish [37]. The domain(s) present at the N-terminus of NLRX1 has not been identified so far. Bioinformatics analysis of NLRX1 in rainbow trout has demonstrated the presence of a mitochondrial-targeting sequence (MTS) and two potential transmembrane domains [88]. An upregulation in NLRX1 mRNA levels in response to bacterial infection was shown in channel catfish [37], goldfish [74] and turbot [104]. Also, bacteria and Poly I:C are shown effective in inducing NLRX1 expression in miiuy croaker [87].

3. Downstream signaling mechanism of NLRs

The downstream signaling pathway of NLRs has been mostly unraveled by studies in mammals. In case of teleosts other than the nodosomes, the adaptor molecules of different NLRs and their signaling cascades are still obscure. In addition, unlike mammals in which ligand-specificity of different NLRs are well defined, ligands recognized by NLRs in fishes vary depending upon species.

3.1. Nodosome signaling

In response to bacterial infection, proinflammatory function of nod proteins via NF-κB signaling is reported in mammals [105], [106], [107], [108]. After recognition, NOD1/2 undergoes oligomerization that leads to exposure of its CARD domain for interaction with CARD domain of RIP2 (Receptor-interacting protein 2) adaptor protein, also known as RICK (RIP-like interacting clarp kinase) [15]. Active RIP2 kinase leads to ubiquitination of NEMO (NF-κB essential modulator) and subsequent activation of NF-κB pathway. NF-κB induces the expression of several proinflammatory cytokines and growth factors [109], [110], [111] (Figure 3). In addition to inducing classical NF-kB pathway via RIP kinase, NOD2 is reported to modulate NF-kB signaling by interacting with TAK1 [TGFb (transforming growth factor b)-activated kinase 1] and GRIM-19 (gene associated with retinoid-IFN-induced mortality 19) [112,113]. The involvement of both, NOD1 and NOD2, have been shown in synthesis of anti-microbial peptides in response to bacterial stimuli [106,108,114]. Further, processing of pro-inflammatory cytokines through interaction with caspase-1 [115] and regulation of apoptotic pathway via COP9 signalosome [116] and RIP2K mediated JNK pathway [117] have been attributed to NOD1 (Figure 3). Also, RIP2 has been shown to interact with ASC to activate caspase-1 under inflammatory conditions [118], [119], [120] and hence, NOD1/2 may be considered as a modulator of caspase-1 mediated apoptotic pathway.

Figure 3.

Nodosome signaling pathways. NOD1 and NOD2 after sensing PAMPs interact with RIPK leading to induction of pro-inflammatory cytokines via NF-kB pathway. In mammals, NOD2 is also reported to interact with TAK1 and GRIM-19 to induce NF-kB pathway. NOD1 and NOD2 are involved in synthesis of anti-microbial peptides via ERK and NOD1 interacts with COP9 signalosome to bring about apoptosis. RIPK also interacts with ASC and activate caspase-1 involved in processing of pro-inflammatory cytokines. In addition, NOD2 is also seen to interact with MAVS either via RIG-1, MDA5 or directly in response to ssRNA virus and might induce IFN gene expression in case of teleosts (denoted by red/dotted lines). (PAMPs- pathogen-associated molecular patterns, PGN-peptidoglycan, MDP- muramyl dipeptide, ERK- extracellular signal-regulated kinase, RIPK- receptor interacting protein kinase, ASC- apoptosis-associated speck-like protein containing a CARD, AP-1- activator protein-1, NEMO- NF-kB essential modulator, NF-kB- nuclear factor kappa-light-chain-enhancer of activated B cells, TAK1- TGFb-activated kinase 1, GRIM-19- gene associated with retinoid-IFN-induced mortality, MAVS-mitochondrial antiviral signaling protein, MDA5- melanoma differentiation-associated protein 5, RIG-1- retinoid acid -inducible gene I, IRF- interferon regulatory factor, IFN- interferon).

As in case of mammals, the versatile immune regulator RIP2 is considered as a downstream adaptor molecule for NOD1/2 signaling in teleosts [77,69,70,73]. In zebrafish [121], mrigal [73], muiiy croaker [122,123] and goldfish [124], nod proteins after recognizing appropriate ligands are shown to participate in activation of NF-κB pathway via interaction with RIPK2. Additionally, NOD1 [125] and NOD2 [75] induces other PRRs involved in anti-viral responses such as RIG-1 and MDA5 (Figure 3). This interaction between NOD2, RIG-I and MDA5 allows NOD2 to take part in RLR-mediated signaling pathways and hence induce IFN promoter activity [75]. Moreover, the induction of IFN-γrel (IFN-gamma related gene) expression after bacterial and viral infections as reported in teleosts such as zebrafish [126], channel catfish [127], grass carp [71] and rohu [128] is contemplated to be regulated via NOD2 [129,71] (Figure 3). It is interesting to note that unlike mammals where NOD1 and NOD2 signaling is primarily implicated in mounting inflammation in response to bacterial ligands, NOD1 and NOD2 in teleost is reported to be involved during viral infection via induction of other PRRs. As far as involvement of the nodosome in processing of proinflammatory cytokines is considered, there is no evidence to suggest the activation of caspase 1 by nodosome. Nonetheless, the involvement of caspase 1 in processing of IL-1β is shown in zebrafish [130] and seabass [131] though it is still unclear whether the mature IL-1β after processing has a biologically active function [132].

3.2. NLRC3, NLRC4, NLRC5

NLRC3, C4 and C5 are known to regulate immune responses by interacting with various signaling molecules/adaptors. Mammalian NLRC3 is implicated in viral sensing and considered as a negative regulator of innate immunity as it is shown to inhibit type I/II IFN response. The receptor also regulates various inflammatory signaling pathways viz. STING [56,133], NF-κB and ERK [134,135], PI3K/AKT/mTOR [136], [137], [138], TCR [139] pathway (Figure 4). Further, NLRC3 is reported to inhibit toll-like signaling pathway by interfering with K63-linked ubiquitination of TRAF6 (TNF receptor-associated factor 6) which is a TLR signaling adapter [55]. NLRC3 also competes with ASC for caspase-1 binding to inhibit inflammation [140,141] (Figure 4).

Figure 4.

Signaling pathways of NLRC3, NLRC5. In mammals, NLC3 inhibit TLR signaling via TRAF6, type I/II/III IFN response and immune pathways viz. STING, NF-kB, ERK, P13K/AKT/mTOR, TCR. It also competes with ASC to bind with pro-caspase-1 and interferes with the assembly of NLRP3 inflammasome. In addition to generally inducing MHC class I genes, NLRC5 inhibit RIG-1, MDA5 and type I IFN as well modulate immune pathways such as NF-kB, JAK2/STAT3, AKT/VEGF-A, PI3/AKT and IFN-1 pathway. In teleosts, NLRC3/C3L modulate cytokine expression while (possibly) interacting with RIPK (denoted by red/dotted lines). NLRC3L is reported to inhibit NOD1-mediated downstream signaling pathways (denoted by red line). NLRC3 is also reported to interact with ASC, however the downstream signaling remains elusive. (STING- stimulator of interferon genes, NF-kB- nuclear factor kappa-light-chain-enhancer of activated B cells, ERK- extracellular signal- regulated kinase, P13K- phosphatidylinositol-3-kinase, AKT- Protein kinase B, mTOR- mammalian target of rapamycin, TCR- T cell receptor, ASC- apoptosis-associated speck-like protein containing a CARD, IFN- interferon, MDA5- melanoma differentiation-associated protein 5, RIG-1- retinoid acid -inducible gene I, JAK/STAT- janus kinase/signal transducers and activators of transcription, VEGF-A- Vascular endothelial growth factor A, MHC- major histocompatibility complex, RIPK- receptor interacting protein kinase).

NLRC4 acts as an adaptor protein for NAIP and forms inflammasome in response to intracellular bacterial flagellin [142], [143], [144], [145] as well as other virulence factors such as PrJ-like proteins of Type III bacterial secretion system [146]. Thereafter, procaspase-1 is recruited to induce the process of pyroptosis [147,148] (Figure 5). Also, CARD-CARD interaction of NLRC4 with procaspase-1 leads to processing of pro-inflammatory cytokines resulting in neutrophil recruitment [149,150]. Interestingly, in case of Casp1^−/− mice, Naip5/NLRC4/ASC inflammasome activates caspase-8 and induces cell death in response to flagellin-positive bacteria, indicating the existence of an additional pathway which is NLRC4 dependent but caspase-1 independent [151]. NLRC5 induces transcription of MHC class I genes by regulation of chromatin remodeling in lymphocytes and several phagocytic cells. In addition, it inhibits activation of RIG-I, MDA5 and type I IFN production thus, affects viral infection [152,153] (Figure 4). The role of NLRC5 in antiviral signaling is further validated in primary human fibroblasts and several cell lines in which NLRC5 gene knockout showed significant decreased levels of IFN-a/b in response to virus [154,155]. Besides, NLRC5 has been shown to regulate NF-κB and IFN-I signaling pathways both positively [156] as well as negatively [53,157]. It also modulates other signaling pathways such as JAK2/STAT3 [158], AKT/VEGF-A [159], PI3K/AKT [160], and inflammasome activity [161] (Figure 4). Surprisingly, Kumar et al. [162] did not observe any role of NLRC5 in cytokine production in virus-infected macrophages and dendritic cells. The influence of NLRC5 in inflammatory pathways has also been seen in birds [163,164].

Figure 5.

Signaling pathways of NLRX1. In response to PAMPs, NLRX1 interacts with UQCRC2 leading to ROS production and induction of NF-kB and JNK pathways. It also negatively regulates MAVS-mediated RLR signaling and TRAF6-NF-kB signaling. In teleosts, NLRX1 inhibit gene expression of IRFs and IFNs (denoted by red lines). (UQCRC2- Ubiquinol-Cytochrome C Reductase Core Protein 2, RLH-RIG-1-like helicases, MAVS- mitochondrial antiviral signaling protein, TRAF-6-Tumor necrosis factor receptor (TNFR)-associated factor 6, FN- interferon).

In teleosts, role of NLRC3 or NLRC3-like genes in viral infection has not been explored though systemic inflammation with higher expression of proinflammatory cytokines has been observed in NLRC3-like homozygous mutant zebrafish [94]. In another study, overexpression of zebrafish NLRC3-like 1 upon bacterial infection has shown a decrease in expression of proinflammatory cytokines [102]. These studies indicated a suppressive role of NLRC3 in inflammation in zebrafish. In contrast, NLRC3 or NLRC3-like protein in Japanese flounder positively regulates cytokine expression [90]. The presence of CARD domain in NLRC3 raises the possibility of its interaction with other downstream signaling components such as RIPK2 via CARD domain in a manner similar to NOD1 and NOD2 (Figure 4). This assumption is supported by a study in zebrafish in which interaction is reported between NLRC3 and apoptosis-associated speck-like protein (ASC) containing a CARD (caspase activation and recruitment domain) (Figure 4). Similarly, zebrafish NLRC3-like 1 has been shown to interact with RIPK2 which in turn inhibit NOD1-mediated downstream signaling pathways and proinflammatory cytokine production [102]. With regard to NLRC4, in spite of the fact that it is involved in host defense, particularly against enteric pathogens in human with possible association with human auto inflammatory diseases such as infantile enterocolitis and macrophage activation syndrome [165], the presence of its orthologs has not been reported in fishes, herptiles and birds so far.

As far as role of NLRC5 in immune defense is concerned, overexpression of NLRC5 [93] or its isoform, zfNLRC5d [95] in zebrafish resulted in significant inhibition of SVCV (spring viraemia of carp virus) infection though it did not transactivate IFN1 and IFN3 in response to Poly (I:C) and LPS. This suggests that unlike mammals, NLRC5 in fishes is involved in an IFN-independent antiviral response possibly through transcriptional regulation of TLRs and NF-κB signaling (Figure 4). On the other hand, overexpression of zfNLRC5 isoforms contributed to negative regulation of antibacterial immune response, with the decreased expression of IκBα whose function has evolved from being NF-κB inhibitors to transcriptional coactivators, positively regulating gene transcription [95]. Thus, on the basis of preliminary evidence, we speculate that unlike mammals, NLRC3 and NLRC5 acts as positive regulators of immune responses in teleosts.

3.3. NLRX1

The mitochondrially located protein NLRX1 has atypical features and plays a crucial role in dampening inflammatory responses. NLRX1 has been shown as a negative regulator of TRAF6-NF-kB signaling [166,167], and RLH- as well as MAVS-mediated antiviral signaling [168]. Recent evidence indicates that NLRX1 causes degradation of MAVS leading to the negative regulation of IFN signaling pathway and promoting HCV infection [169]. However, NLRX1 is also shown to potentiate TNF-α/bacterial/double stranded RNA-induced ROS production in HeLa cells [170]. The direct interaction between C-terminal fragment of NLRX1 and RNA ligands (polyI:C and single stranded RNA) is reported crucial for NLRX1-mediated ROS activation. The synergistic role of NLRX1 in ROS production could be seen in light of the fact that NLRX1 interacts with a matrix-facing protein UQCRC2 of the respiratory chain complex III. Increased ROS generation has been reported to activate pro-inflammatory pathways such as NF-kB and JNK. Although the increased ROS production may seem contradictory to the anti-inflammatory role of NLRX1, it has been implicated that this actually leads to apoptosis which is a typical host response to viral infection and tumorigenesis [171]. Interestingly, unaltered antiviral and inflammatory gene expression in response to influenza A virus or intraperitoneal injection of poly (I:C) in NLRX1-deficient mice [172] raises question on pivotal role of this mitochondrial protein in immune responses.

In teleosts, NLRX1 functions as a suppressor of inflammatory response. In grass carp, similar to mammals, NLRX1 attenuates innate immunity by interacting with TRAF6. In the same study, enhanced gene expression of IRF3, IRF7, and IFN-I following poly (I:C) stimulation has been observed in NLRX1 deficient grass carp kidney cell line [173]. In black carp, direct association of the NACHT domain of NLRX1 with MAVS has been seen as critical for negative regulation of antiviral signaling [174]. In a recent study in this fish, like mammals, a complex formation between NLRX1 and Tu translation elongation factor (TUFM) has been speculated to negatively regulate MAVS-mediated RLR/IFN signaling [175] (Figure 5).

3.4. NLRP

Depending on the signaling pathway, NLRPs are broadly divided into two categories. In canonical NLRPs such as NLRP1, formation of inflammasome is followed by the activation of caspase-1, whereas NLRP6 and NLRP12 follow a non-canonical pathway which is inflammasome-independent or may involve alternate caspases such as caspase-4, -5 and -11 [176]. NLRP3, the most widely studied member of this group, can follow either canonical or non-canonical signaling pathways depending upon the stimulus and type of tissue. In the canonical pathway, activation of NLRP by PAMPs/DAMPs results in recruitment and polymerisation of apoptosis-associated speck-like (ASC) adaptor molecules via homotypic interaction. In humans, the CARD-CARD interaction between oligomerised prion-like ASC and pro-caspase-1 leads to formation of multi-protein complex known as inflammasome responsible for the processing and maturation of the proinflammatory cytokines, IL-1β and IL-18 [60,177] (Figure 6). ASC is critical for caspase-1 activation although human NLRP1 can directly recruit caspase-1 with/ without interacting with ASC due to presence of C-terminal CARD domain [176]. Mouse NLRP1 has been well characterized and has been shown to be essential in regulating host immune response [178]. In case of non-canonical pathways involving caspase-1 independent activation of pyroptosis and/or secretion of IL-1β and IL-18 occurs that have wide effects on both, innate and adaptive immunity. Other than these two pathways, some of the NLRPs modulate MAPK and NF-κB signaling pathways in mice [176].

Figure 6.

Signaling pathways of inflammasome forming NLRs. PAMPs interact with inflammasome forming NLRs (NAIP-NLRC4, NLRP1, NLRP3) as well as non-inflammasome forming NLRs such as NLRP6 which then activates procaspase-1 leading to inflammatory response. In non-canonical pathway, NAIP-NLRC4 interact with caspase-8 whereas NLRP6 and NLRP12 interact with caspase-4,5,11 to induce pyroptosis. In addition, in teleost, NLRP3rel has also been shown to follow similar inflammasome signaling pathways (denoted in red lines). (LTA- lipoteichoic acid, ATP- adenosine triphosphate, S. aureus- Staphylococcus aureus NAIP- neuronal apoptosis inhibitory protein, ASC- apoptosis-associated speck-like protein containing a CARD).

Unlike NLRP1 and NLRP3, less information is available about the other NLRP subfamily members such as NLRP6 and NLRP12. NLRP6 is a key molecule that links inflammation with gut microbiota by activating both canonical and non-canonical pathways, thereby influencing gastrointestinal inflammatory, infectious and neoplastic diseases in the case of mice and serves as a protective agent in liver in humans [179]. In addition, mice NLRP12 has been implicated in suppressing and acting as regulator of colonic microbiota [180]. Despite having a crucial role in immunity, the ligands sensed by NLRP6 and NLRP12 are yet to be identified [181,182]. Moreover, in addition to the reproductive role of human NLRP7 [183], contradictory roles in immunity have been reported, having both positive and negative effects on inflammasome responses [184].

In teleosts, Laing et al. [20] had shown that the NLR-B family clade with mammalian NALPs (NLRPs) though they do not take part in inflammation, probably due to missing domains. Later, Li et al. [42,43] has described NLRP1 and NLRP3 in zebrafish which are structurally homologous to mammalian NLRP1 and NLRP3. As far as signaling is concerned, NLRP1 and NLRP3 in zebrafish [42,43], and NLRP3 in Japanese flounder [185] are shown to initiate ASC dependent caspase activation leading to IL-1 beta processing. In addition, NLRP3rel along with adaptor protein ASC have been functionally characterized in goldfish and is suggested to have similar inflammasome signaling pathway as in mammals [186] (Figure 6). These studies in teleosts point towards the evolutionary conservation of inflammation signaling pathways.

4. Conclusion

NLRs have emerged as pivotal players that act in tandem with other PRRs to orchestrate an effective immune response. The evolutionary conservation of NLRs throughout the animal kingdom highlights their significance in host defense mechanisms. Nevertheless, depending upon the pathogenic challenges faced by organisms in their particular niche, these NLRs show structural and functional diversity. NLRs, in general, are involved in translating pro-inflammatory responses but there are certain NLRs that act as negative regulators dampening the strong stimulatory responses, thus maintaining tissue homeostasis crucial for cell survival. Interestingly, NLRs in teleosts show a vast spectrum of variation in structural and functional aspect, and ligand recognition depending on species. Despite the close structural and functional similarities of some teleost NLRs with their mammalian counterparts, there are others such as NLR C-like receptor that do not have any mammalian homologs and are absent in all tetrapods. There is scarcity of literature on downstream signaling pathways and adaptor molecules of NLRs in teleost. To develop a thorough understanding of these receptors in regulation of immune responses, extensive studies in teleosts are required taking into account the signaling cascades and crosstalk with other PRRs and inflammatory pathways. The understanding of functional and structural aspects of NLRs in teleost is not only essential from an evolutionary point of view, but also because teleosts are an important economic resource. Extensive aquaculture predisposes fish to a variety of infectious diseases and deciphering signaling mechanisms underlying the activation of these receptors would provide promising opportunities for therapeutics.

Funding Statement

This work was supported by the Department of Biotechnology, Ministry of Science and Technology, India (Ref No. BT/PR19619/BIC/101/57/2016); Institute of Eminence, University of Delhi, India (IoE/FRP/LS/2020/27) and 7University Grants Commission, India (Ref. No. 19/06/2016(i)EU-V).

Conflict of interest

The authors declare no conflict of interest.