OPEN
REVIEW
Pattern recognition receptors in zebrafish provide
functional and evolutionary insight into innate immune
signaling pathways
Yajuan Li, Yuelong Li, Xiaocong Cao, Xiangyu Jin and Tengchuan Jin
Pattern recognition receptors (PRRs) and their signaling pathways have essential roles in recognizing various
components of pathogens as well as damaged cells and triggering inflammatory responses that eliminate invading
microorganisms and damaged cells. The zebrafish relies heavily on these primary defense mechanisms against
pathogens. Here, we review the major PRR signaling pathways in the zebrafish innate immune system and compare
these signaling pathways in zebrafish and humans to reveal their evolutionary relationship and better understand
their innate immune defense mechanisms.
Cellular & Molecular Immunology (2017) 14, 80–89; doi:10.1038/cmi.2016.50; published online 10 October 2016
Keywords: caspases; innate immune system; NOD-like receptors; pattern recognition receptor; RIG-I-like receptors;
Toll-like receptors; zebrafish
INTRODUCTION
Zebrafish (Danio rerio) is extensively used as a model organism
in many fields, including developmental biology, cancer and
immunology.
1–3
There is a clear temporal separation between
the innate and adaptive immune responses of this organism.
Zebrafish relies more on the innate immune system than
mammals because it does not have a morphologically and
functionally mature adaptive immune system until 4 weeks
after fertilization. As a result, the zebrafish model can provide
new insights into the function and evolution of innate immune
responses.
As the first line of host defense, the innate immune system
relies on a large family of pattern recognition receptors (PRRs)
to recognize pathogen-associated molecular patterns (PAMPs)
derived from various microbial pathogens, including viruses,
bacteria, fungi, parasites and protozoa,
4
and danger-associated
molecular patterns (DAMPs) that are present in aberrant
locations or abnormal molecular complexes as the consequence
of infection, inflammation or cellular stress.
5,6
Currently, the
four best characterized groups of PRRs include the Toll-like
receptors (TLRs), the nucleotide-binding oligomerization
domain (NOD)-like receptors (NLRs), retinoic acid-inducible
gene-I (RIG-I)-like receptors (RLRs) and the absent in
melanoma-2 (AIM-2)-likereceptors (ALRs). PRRs can be
localized at the cell surface (TLRs, CLRs), within the cytoplasm
(NLRs, RLRs and ALRs) or in endosomes (TLRs). The cell
surface PRRs are responsible for surveying the extracellular
environment, whereas the cytoplasmic PRRs sense intracellular
pathogens or danger signals. Endosomal PRRs are used to
detect microbes that have entered the phagolysosome. On
PAMP or DAMP recognition, PRRs activate signaling cascades
leading to the NF-κB and interferon (IFN) response factor
(IRF) transcription factors, thus leading to the induction of
proinflammatory cytokines, chemotactic cytokines and anti-
microbial responses.
Zebrafish rely on cytokine and IFN production, complement
activation, innate immune cell activation and cellular cytotoxic
stimulation as host defense mechanisms against pathogens.
1
Several classical receptor families that are involved in primary
immune responses have been identified in the zebrafish genome.
Counterparts of the majority of vertebrate PRRs and downstream
signaling components have been identified in zebrafish, and some
of these have been functionally characterized (Table 1). In this
review, we focus on the pattern recognition receptors and their
Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Science,
School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027, China
Correspondence: Dr T Jin, Laboratory of Structural Immunology, CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in
Molecular Cell Science, School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027, China.
E-mail: jint@ustc.edu.cn
Received: 1 April 2016; Revised: 4 August 2016; Accepted: 4 August 2016
Cellular & Molecular Immunology (2017) 14, 80– 89
&
2017 CSI and USTC All rights reserved 2042-0226/17
www.nature.com/cmi
signaling pathways in zebrafish and compare them with their
counterparts in mammals, including humans, to reveal their
evolutionary relationship and provide new insight into innate
immune defense mechanisms.
PATTERN RECOGNITION RECEPTORS
Toll-like receptors
The mammalian TLR family consists of 12 members that are
integral glycoproteins possessing an extracellular (or intra-
endosomal) ligand-binding domain with a leucine-rich repeat
(LRR) motif and a cytoplasmic signaling Toll/Interleukin-1
(IL-1) receptor homology (TIR) domain.
7,8
Some TLRs (TLR1,
-2, -4, -5, -6 and -10) are expressed at the cell surface, whereas
others (TLR3, -7, -8, -9, -11 and -13) are located almost
exclusively in intracellular compartments, including endosomes
and lysosomes. TLRs are principally responsible for the
recognition and response to pathogen ligands such as lipopo-
lysaccharide (LPS) from Gram-negative bacteria (TLR4
ligand),
9
lipoteichoic acid from Gram-positive bacteria (TLR2
ligand)
10
and the flagellin protein (TLR5 ligand).
11
Nucleic
acids such as dsRNA, ssRNA and single-stranded unmethylated
CpG motif-containing DNA are recognized by TLR3, TLR7
and TLR9, respectively, in antiviral or antibacterial
pathways.
12–16
In a typical mammalian TLR signaling cascade,
ligand binding to the extracellular leucine-rich repeats region
causes TLRs to dimerize and undergo conformational changes
and/or oligomerization of their intracellular TIR domains,
which in turn recruit and activate cytosolic TIR domain-
containing adaptor molecules such as MyD88, MyD88 adaptor-
like protein (MAL/TIRAP), TRIF/TICAM1 and TRIF-related
adaptor molecule (TRAM/TICAM2) to transduce signals from
the membrane surface to the cytosol and then to the nucleus
via the activation of downstream transcription factors such as
ATF, NF-κB, AP-1, IRF and the STAT family. The MyD88-
dependent signaling pathway recruits downstream IRAKs and
activates TRAF6 and the I κB kinase (IKK), leading to the
translocation of NF-κB to the nucleus and the secretion of anti-
infection molecules and inflammatory cytokines and type-I IFN
production in dendritic cells (DCs). In the MyD88-independent
signaling pathways, the non-typical IKKs IKKi/IKKε and TBK1
mediate the activation of IRF3 downstream of TRIF,
17
which is
responsible for type-I IFN responses in non-DCs.
The TLR protein family is conserved from insects to
mammals, but TLR signaling pathways in fish exhibit different
features than those in mammals.
18
TLRs are highly expressed in
the skin of zebrafish, which suggests a prominent role in the
defense against pathogens. Zebrafish has an almost complete
set of 20 putative TLR variants.
19,20
Among them, 10 are
orthologs of human TLR family members, and TLR22 belongs
to a fish-specific subfamily that is closely related to the
Drosophila melanogaster toll-9 gene. TLR21 is common to
birds, amphibians and fish. Zebrafish TLR9 and TLR21 were
found to have similar expression profiles and antimicrobial
activities toward CpG-ODNs.
21
Because the genome of
teleost fish was duplicated during evolution, zebrafish have
two counterparts of some mammalian TLRs, including tlr4ba/
tlr4bb for the LPS-specific TLR4 and tlr5a/tlr5b for TLR5, and
tlr8a/tlr8b. Homologs of mammalian TLR6 and TLR10 are
absent from fish, but TLR14 and TLR18 are non-mammalian.
It has been reported that Japanese flounder TLR14 shares some
features with TLR1, TLR6 and TLR10, suggesting that TLR14
Table 1 PRRs and downstream signaling components have
been identified in zebrafish
Zebrafish Mammal
TLR
TLR1 TLR1
TLR2 TLR2
TLR3
TLR4b.a/b TLR4
TLR5a/b TLR5
TLR6
TLR7 TLR7
TLR8a/b TLR8
TLR9 TLR9
TLR10
TLR14
TLR18
TLR19
TLR20a
TLR21
TLR22 TLR3
MyD88 MyD88
TICAM1/TRIF TICAM1/TRIF
Lost TICAM2/TRAM
IRF10, IRF11
Divergent TRAF1
TRAF2a/b TRAF2
TRAF4a/b TRAF4
TIRAP/MAL TIRAP/MAL
NLR
NOD1 NOD1
NOD2 NOD2
NLRC3 NOD3
NLR-B NALPs
NLRP3
NLR-C
ASC ASC
Caspy Caspase-1
Caspy 2 Caspase-4/5/11
RLR
RIG-Ia/b RIG-I
MDA5 a/b MDA5
Two LGP2 isoforms LGP2
MAVS MAVS
STING STING
TBK1 TBK1
IRF3 IRF3
IRF7 IRF7
Abbreviations: IRF, interferon response factor; NLR, NOD-like receptor; NOD,
nucleotide-binding oligomerization domain; PRR, pattern recognition receptor;
RLR, RIG-like helicase receptor; TLR, Toll-like receptor.
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might be a functional substitute for mammalian TLR6 and
TLR10.
22
TLR18 in zebrafish and channel catfish is the
homolog of human TLR1
23,24
and may correspond to TLR14
in other fish.
18
In addition, Peitretti et al.
25
identified TLR20 in
zebrafish and common carp, finding that this protein is similar
to TLR11 and TLR12 in mice. It is not clear when such gene
duplications or deletions occurred during evolution, suggesting
that the innate immune system of fish may be more complex
than that of mammals. The ancient jawless vertebrate Japanese
lamprey has no more than 16 TLR genes,
26
whereas there are
up to 20 putative TLR variants in zebrafish. Ohno proposed
that two rounds of whole-genome duplication have occurred
during early vertebrate evolution, with a third fish-specific
genome duplication occurring later in a basal teleost.
27
A
fourth whole-genome duplication even occurred in some
cyprinids 8–21 million years ago,
28,29
resulting in the appear-
ance of paralogs of ancestral genes and the development of
neofunctionalization.
30
With respect to the TLR repertoire, it is
of interest to note that, except for a single tlr21 gene, two tlr23
genes and tlr22-related genes, most TLR genes seem absent
from the Atlantic cod,
31
which may be the result of positive
selection to generate neofunctionalization.
32
However, whether
the increase in the number of TLRs in fish is associated with
diversification in ligand recognition is still unknown.
The ligands of some zebrafish TLR receptors have been
identified (Table 2). For example, TLR2 can form heterodimers
that are responsible for the recognition of bacterial lipopro-
teins/lipopeptides or Pam3CSk4.
20,33
TLR3, -5 and -9 recognize
dsRNA, flagellin and unmethylated CpG DNA, respec-
tively.
11,13,16,34,35
However, zebrafish TLR4 is not responsive
to LPS stimulation, despite the fact that MD1 and Rp105,
which mediate ligand delivery and/or recognition, have been
identified. Biochemical and functional studies indicate that
MD1 binds both Rp105 and TLR4 in zebrafish,
36
but accessory
molecules such as LBP, CD14 and MD2 have not been isolated
from fish,
30,37,38
suggesting that TLR4 ligand specificity is not
conserved in zebrafish.
39
The knockdown of tlr4a, tlr4b and
myd88 did not disrupt the zebrafish immune response to LPS
exposure,
40
suggesting other unidentified roles of TLR4 signal-
ing in PAMP responses. In addition, zebrafish lacks a clear
ortholog of caspase-11, which serves as an intracellular LPS
receptor in mice. On one hand, Zebrafish Caspy 2 shows the
highest homology to human caspase-5 and a preference for
caspase-5-like substrates, and Caspy 2 also induced apoptosis in
mammalian cells that was inhibited by general caspase inhibi-
tors;
41
on the other hand, the Caspy 2 does not contain a
CARD domain as found at the N-terminal of caspases. Actually
the N-terminal domain of Caspy 2 is most homologous to the
N-terminal domain of human NLRP3 (46% similarity),
a pyrin-domain-containing NOD-family protein,
41
thus it
remains to be seen whether fish Caspy 2, which is similar to
human caspase-4/5 (caspase-11), can interact with LPS directly
and activate the inflammasome. The above discussion suggests
that the mechanism of LPS recognition in fish could be
different from that in mammals, and it is possible that ancestral
or other genes are involved in sensing LPS. In addition, the
fish-specific TLR22 recognizes dsRNA viruses or PolyI:C; this is
followed by the recruitment of TRIF to induce IFN expression.
Thus, TLR22 could be a functional homolog of mammalian
TLR3.
35
In fact, zebrafish also has TLR3, and TLR3 and TLR22
recruit a common adaptor TRIF to augment the local IFN
response to viral infection.
35
The innate immune signaling molecules downstream of
TLRs are conserved in zebrafish as well, and include ortholo-
gous MyD88, SARM1, Tollip, IKAP (IKK complex associated
protein), NEMO (NF-κB essential modulator), TIRAP, TRIF
and the central intermediator IRFs, the signal transducers and
activators of transcription (StatS), AP-1,
42
and all of the TRAF
family members (traf1 to traf7).
43
MyD88, the most-studied TLR adaptor in zebrafish, has
important roles in host defense against microbial infections.
44
In fact, all adaptor molecules except for TICAM2 (TRAM)
have been identified in zebrafish. Although mammals have two
copies of TICAM, TICAM2/TRAM was lost specifically from
teleost fish, and the only homologous gene is distantly related
to TICAM1 or TICAM2, indicating that an ancestral gene
subsequently diverged to two copies. Zebrafish TICAM1
localizes to the Golgi apparatus and lacks the N-terminal and
C-terminal proline-rich domains found in the mammalian
protein,
45
despite being able to activate NF-κBpromotersand
IRF3- and IRF7-mediated pathways.
46,47
The partial function
attenuation of TIRAP in zebrafish, which may weaken its
ability to recruit MyD88,
18,48
leads to low sensitivity to LPS.
Overexpression of the adaptor TRIF induces IFN production in
zebrafish, suggesting that zebrafish trif is a true homolog of the
mammalian gene.
45
In conclusion, TLR adaptors are highly
conserved between mammals and teleosts, suggesting that the
signaling downstream of TLRs is highly conserved between the
innate immune responses of mammals and fish.
The zebrafish has unique features in the components of its
TLR downstream pathways, including some gene duplications
or losses. With regard to IFN response factors, all of the
nine IRF orthologs of mammals have been identified in fish.
There are two additional IRFs in zebrafish, namely IRF10 and
IRF11, which are absent in mammals.
49
Furthermore, zebrafish
traf1 differs from mammalian traf1 in that it contains a single
zinc finger motif. In addition, traf4 and traf2 are duplicated in
zebrafish.
50
Table 2 Known ligands of zebrafish TLRs
TLR Ligands References
TLR1 TLR1–TLR2 heterodimer
19,20,94
TLR2 Lipopeptides; Pam3CSk4
20,33
TLR3 dsRNA; polyI:C
20,95,96
TLR5a/b Flagellin
20,33
TLR9 CpG-ODNs
20,97
TLR21 CpG-ODNs
20,97
TLR22 dsRNA; polyI:C
19,20,35,95,96
Abbreviation: TLR, Toll-like receptor.
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The zebrafish is now being utilized as a model for infectious
disease because of the ease of using particular stages to examine
immune responses to infection as well as host–microbe
interactions. TLRs are the best understood innate immune
receptors that respond to infection in fish. In 2002, Neely
et al.
51
established a zebrafish model of Streptococcus infection.
Their studies suggested that the principles underlying host–
pathogen relationships in fish are very similar to those in
humans. TLR3 mRNA was upregulated in zebrafish upon
infection with Gram-negative bacteria.
52
Mycobacterial infec-
tion of zebrafish results in elevated TLR1, TLR2, TLR5a/5b and
TLR18 expression and also induces the expression of fish-
specific TLR20a and TLR22. These studies suggest that the
microbial PAMP recognition mechanism is already established
in the common vertebrate ancestor and is conserved in
mammals and teleosts.
The zebrafish MAL orthologs that are downstream of TLRs
also show increased expression, whereas the expression levels of
other TIR domain-containing adaptor genes such as MyD88,
TRIF and SARM are not responsive to Mycobacterial
infection,
24
suggesting that the signaling pathways downstream
of fish TLRs are different from those in mammals. Of course,
infecting the entire animal with a live pathogen induces
alterable changes in gene expression due to the presence of
multiple PAMPs. As a result, it is difficult to distinguish the
correlation between the upregulation of TLR signaling genes
and the recognition of specific pathogen-derived ligands.
NOD-like receptors and inflammasome pathways
Intracellular monitoring is performed by several families of
receptors to detect those pathogens that evade extracellular and
endosomal surveillance. These include the NLRs for different
PAMPs and DAMPs,
53
RIG-like helicase receptors (RLRs) for
viral RNA,
54
and the ALRs for cytosolic DNA.
55–58
The innate
immune signaling pathways associated with the NLRs and
RLRs are largely conserved in mammals and teleost fish, but
the signaling pathways for the ALRs are not. The expression of
ALRs is restricted to and conserved among mammalian species,
and the loss of ALRs in fish suggests they have evolved alternate
mechanisms to cope with DNA viruses and intracellular
bacteria.
59
Owing to the importance of inflamma somes in the innate
immune defense system, numerous studies were extended to
vertebrates to uncover their functions. At present, the function
of NLRs in lower vertebrates and invertebrates is less well
understood than that in mammals. With nearly 421 NLR family
members in zebrafish,
60
it is predicted that at least one prototype
gene exists in lower organisms. With events such as gene loss,
duplication and acquisition in various species, this unique gene
family is likely to have formed gradually in vertebrates during
evolution. By analyzing the molecular phylogeny and expression
of NLR subfamilies in zebrafish, three NLR subfamilies have
been identified, with the first subfamily (NLR-A) containing
eight genes that resemble mammalian NODs, the second
subfamily (NLR-B) containing nine genes that resemble mam-
malian NACHT-, LRR- and PYD-containing proteins (NLRP),
and the third subfamily (NLR-C) containing 405 NLR genes that
are unique to teleost fish.
60,61
Recent reports have indicated that
several members of the mammalian NLR family, namely NOD1,
NOD2 and NLRC3, are conserved in zebr afish.
62
NOD2
cooperates with the dual oxidase (DUOX) enzyme to produce
bactericidal reactive oxygen species in epithelial cells in
mammals.
63
Similarly, the morpholino-mediated depletion of
zebrafish NOD1 or NOD2 significantly decreases zebrafish
DUOX expression, which reduces the ability of embryos to
control systemic infection in a Salmonella infection model
64
and
suggests that NOD-like receptors are also important for innate
antibacterial immunity in teleost fish.
In contrast, NLRP3, the most extensively studied mamma-
lian NLR in the inflammasome, is not conserved in
zebrafish,
61,65,66
and there are no direct NLRP3 orthologs in
fish. Although Boyle identified a gene consisting of an NTPase
domain, leucine-rich repeats and a C-terminal PRY-SPRY
domain on zebrafish chromosome 17 using human NLRP3
in a BLASTP search, the lack of an N-terminal effector domain
distinguished this gene from mammalian NLRP3.
66
In addi-
tion, there are not any other putative NLRP3 orthologs in the
genomes of other fish. Stein et al.
65
has suggested that these
NLRPs in lower vertebrates are not similar to the NLRs in
mammals and are not the origin of inflammasome compo-
nents, as these genes provide diverse responses to infection and
injury in different species. Such differences between fish and
mammals are interesting and may encourage scientists to
reconsider the mechanism and evolution of the inflammasome.
In brief summary, NLRPs are not functionally conserved in
fish, and whether other zebrafish-specificNLRsmayfunction
through inflammasome-like molecular complexes awaits future
investigation.
Despite these differences, some components of innate
inflammatory pathways are conserved in zebrafish. As a key
adaptor molecule in the inflammasome pathways, apoptosis-
associated speck-like protein containing a CARD (ASC) links
upstream receptors (such as NLRs/PYHINs) and downstream
signaling caspases through homotypic or heterotypic protein–
protein interactions to form the inflammasome complex,
a granular structure formed in the perinuclear region of the
cytosol upon inflammasome activation that ultimately leads to
the processing of pro-IL-1β and pro-IL-18.
67,68
ASC is highly
conserved in vertebrates but is not present in invertebrates.
Zebrafish has a single ortholog of ASC (Figure 1, accession
number NM_131495) that is also composed of an N-terminal
PYD domain and a C-terminal CARD domain. zASC has been
observed to form SPECKs in vitro and in the embryo (Li and
Jin, unpublished data), suggesting a conserved function of ASC
in inflammasome assembly.
In addition to the canonical inflammasomes that activate
caspase-1, a non-canonical caspase-11-dependent inflamma-
some pathway was recently discovered.
69–71
The N-terminal
CARD domain of caspase-11 serves as a direct receptor for
intracellular LPS. LPS binding induces conformational changes
and the oligomerization of caspase-11, activating caspase-11.
Activated caspase-11 cleaves gasdermin D to induce
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pyroptosis.
72–74
In zebrafish, there are two caspase genes,
namely Caspy and Caspy 2. Interestingly, their N-terminal
regions share higher sequence similarity with the PYD than the
CARD domain of mammalian caspases, both of which belong
to the death-fold superfamily. These zebrafish caspases
function as inflammatory rather than apoptotic mediators.
41
The sequence of the zebrafish Caspy PYD domain is similar to
the N-terminal domains of caspases from non-mammals such
as tetrapod, suggesting their functional similarity. Comparative
alignment of the PYD or CARD domains of zebrafish shows
that the N-terminal domain of Caspy has sequence similarity to
the zebrafish ASC PYD domain, and the sequence of the
zebrafish ASC CARD domain is similar to the CARD domain
from zebrafish NLP3 (NACHT, LRR and PYD domains-
containing protein 3-like isoform X2; Figure 2). Caspy
performs the function of human caspase-1, and Caspy 2 shows
the highest sequence similarity to human caspase-4/5, both of
which have substrate specificity.
41
Only Caspy can be activated
through interaction with zASC. Although the function of these
fish caspases is poorly defined, they appear to be essential for
the morphogenesis of the jaw and gill-bearing arches. It is not
yet clear whether fish Caspy 2, which is similar to human
caspase-4/5 (mouse caspase-11), can directly interact with LPS
and activate the inflammasome or cleave the relevant substrates
and induce, pyroptosis as its orthologs do in mammals.
Interestingly, the zebrafish dfna5 gene (AY603655) also con-
tains a gasdermin family domain, a recently identified caspase-
4/5 substrate and the cleavage of which induce pyroptosis in
mammals. But the role of zebrafish dfna5 in pyroptosis awaits
further studies.
Zebrafish also express IL-1, which is similar to IL-1β in
mammals.
75
Zebrafish IL-1 represents a single ancestral gene
for the IL-1 superfamily in mammals and functions similarly to
IL-1β in mammals. Zebrafish IL-1 also requires proteolytic
cleavage for maturation into an activated form.
76
However, the
processing and cleavage mechanism of pro-IL-1β is less clear in
fish because fish IL-1β lacks the conserved caspase-1 cleavage
site.
75
With respect to function, IL-1β behaves the same in
teleost and mammalian immunity. A study demonstrated the
caspase-1-mediated processing of IL-1β at an aspartic residue
distinct from the cleavage site of mammalian IL-1β in the
European sea bass,
77
suggesting that fish have a more sophis-
ticated inflammasome activation mechanism than mammals.
Thus, more research is needed to illuminate the innate
immune mechanisms in fish.
Figure 1 Evolutionary relationships of ASC. ASC sequences were
obtained from the NCBI database (http://www.ncbi.nlm.nih.gov).
Sequences were aligned with Clustal-Omega, and the molecular
phylogeny tree was inferred using the Neighbor-Joining method in
MEG6.0. The scale bar reflects 0.1-nt substitutions per site. ASC,
apoptosis-associated speck-like protein containing a CARD.
Figure 2 Comparative sequence alignment of PYD or CARD domains in zebrafish. The conserved residues are indicated by red vertical
lines between the sequences. High (:) and low (.) sequence similarities are marked. ASC, apoptosis-associated speck-like protein
containing a CARD.
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