Along the Axis between Type 1 and Type 2 Immunity; Principles Conserved in Evolution from Fish to Mammals

Abstract

A phenomenon already discovered more than 25 years ago is the possibility of naïve helper T cells to polarize into TH1 or TH2 populations. In a simplified model, these polarizations occur at opposite ends of an “immune 1-2 axis” (i1-i2 axis) of possible conditions. Additional polarizations of helper/regulatory T cells were discovered later, such as for example TH17 and Treg phenotypes; although these polarizations are not selected by the axis-end conditions, they are affected by i1-i2 axis factors, and may retain more potential for change than the relatively stable TH1 and TH2 phenotypes. I1-i2 axis conditions are also relevant for polarizations of other types of leukocytes, such as for example macrophages. Tissue milieus with “type 1 immunity” (“i1”) are biased towards cell-mediated cytotoxicity, while the term “type 2 immunity” (“i2”) is used for a variety of conditions which have in common that they inhibit type 1 immunity. The immune milieus of some tissues, like the gills in fish and the uterus in pregnant mammals, probably are skewed towards type 2 immunity. An i2-skewed milieu is also created by many tumors, which allows them to escape eradication by type 1 immunity. In this review we compare a number of i1-i2 axis factors between fish and mammals, and conclude that several principles of the i1-i2 axis system seem to be ancient and shared between all classes of jawed vertebrates. Furthermore, the present study is the first to identify a canonical TH2 cytokine locus in a bony fish, namely spotted gar, in the sense that it includes RAD50 and bona fide genes of both IL-4/13 and IL-3/ IL-5/GM-CSF families.

Full text

Biology 2015, 4, 814-859; doi:10.3390/biology4040814
biology
ISSN 2079-7737
www.mdpi.com/journal/biology
Review
Along the Axis between Type 1 and Type 2 Immunity;
Principles Conserved in Evolution from Fish to Mammals
Takuya Yamaguchi 1,†, Fumio Takizawa 2,†, Uwe Fischer 1 and Johannes M. Dijkstra 3,*
1 Laboratory of Fish Immunology, Institute of Infectology, Friedrich-Loeffler-Institut, Südufer 10,
Greifswald-Insel Riems 17493, Germany; E-Mails: Takuya.Yamaguchi@fli.bund.de (T.Y.);
Uwe.Fischer@fli.bund.de (U.F.)
2 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania,
Philadelphia, PA 19104, USA; E-Mail: fam64tak@hotmail.com
3 Institute for Comprehensive Medical Science, Fujita Health University, Dengakugakubo 1-98,
Toyoake, Aichi 470-1192, Japan
† These authors contributed equally to this works.
* Author to whom correspondence should be addressed; E-Mail: Dijkstra@fujita-hu.ac.jp;
Tel.: +81-562939381.
Academic Editor: Brian Dixon
Received: 8 September 2015 / Accepted: 19 October 2015 / Published: 17 November 2015
Abstract: A phenomenon already discovered more than 25 years ago is the possibility of
naïve helper T cells to polarize into TH1 or TH2 populations. In a simplified model, these
polarizations occur at opposite ends of an “immune 1-2 axis” (i1-i2 axis) of possible
conditions. Additional polarizations of helper/regulatory T cells were discovered later, such
as for example TH17 and Treg phenotypes; although these polarizations are not selected by the
axis-end conditions, they are affected by i1-i2 axis factors, and may retain more potential for
change than the relatively stable TH1 and TH2 phenotypes. I1-i2 axis conditions are also
relevant for polarizations of other types of leukocytes, such as for example macrophages.
Tissue milieus with “type 1 immunity” (“i1”) are biased towards cell-mediated cytotoxicity,
while the term “type 2 immunity” (“i2”) is used for a variety of conditions which have in
common that they inhibit type 1 immunity. The immune milieus of some tissues, like the
gills in fish and the uterus in pregnant mammals, probably are skewed towards type 2 immunity.
An i2-skewed milieu is also created by many tumors, which allows them to escape
eradication by type 1 immunity. In this review we compare a number of i1-i2 axis factors
OPEN ACCESS

Biology 2015, 4 815
between fish and mammals, and conclude that several principles of the i1-i2 axis system
seem to be ancient and shared between all classes of jawed vertebrates. Furthermore, the
present study is the first to identify a canonical TH2 cytokine locus in a bony fish, namely
spotted gar, in the sense that it includes RAD50 and bona fide genes of both IL-4/13 and IL-3/
IL-5/GM-CSF families.
Keywords: immunology; evolution; fish; TH1; TH17; Treg; TH2; i1-i2 axis; cytokines; IL-5
1. Introduction
General Principles of the i1-i2 Axis as Exemplified by Major Polarizations of Mammalian Helper and
Regulatory T Cells
Depending on the stimuli, largely in primary immune organs, hematopoietic stem cells can develop
into a large array of morphologically and functionally different leukocyte populations [1–3]. At the sites
of activation, these mature but “naïve” immune cells can then further polarize towards phenotypically
distinct cell populations depending on the conditions. The polarized phenotypes can be more or less fixed
by epigenetic changes, including chromatin folding, DNA methylation and histone modification [4–6]. Very
important for polarization of immune cells is a loosely defined “axis” of conditions that favor type 1 or
type 2 immunity, and which we call here the “i1-i2 axis”. In helper T (TH) cells the i1-i2 axis end
conditions induce pronounced polarizations, TH1 and TH2 respectively, which are rather stably imprinted
in the cell clones by heritable epigenetic changes [5,7–11]. The pronounced and stable character of TH1
and TH2 polarizations allowed their discovery already more than 25 years ago [7–9,12]. The type 1 end
of the i1-i2 axis is represented by conditions which stimulate expression of interferon γ (IFNγ) and are
enhanced by this cytokine, while for the type 2 end of the i1-i2 axis a self-stimulatory marker cytokine
is interleukin 4 (IL-4; [5]). Important transcription factors for TH1 cells are T-bet and STAT4 [5,13,14],
and important transcription factors for TH2 cells are GATA-3 and STAT6 [5,15–19]. The i1-i2 axis
affects polarization of various types of leukocytes, and shifts along the axis are not only determined by
IFNγ and IL-4 concentrations, but are also affected by other cytokines, pathogen-associated molecular
patterns (PAMPs), danger-associated molecular patterns (DAMPs), the strength and nature of cell-cell
interactions, and physiochemical variables such as the concentrations of nucleotides and their
derivatives, glucocorticoids, and oxygen [5,20–24]. In this review we will only discuss a few relevant
factors, mainly concentrating on several important cytokines and transcription factors.
The term “type 1 immunity” relates to a milieu skewed towards cytotoxic functions including
enhanced natural killer (NK), TH1, and CD8+ T cell activities. The major function of type 1 immunity is
to kill cancer cells or cells with intracellular pathogens. Cell killing processes can be expected to be in
relative disregard of damaging host tissue, but many tissue damaging inflammations for which originally
TH1 cells were blamed are actually mediated by TH17 cells [25]. TH17 cells are only partially shifted
towards the i1-end of the i1-i2 axis (Figure 1A), and are representative for what can be called “type 3
immunity” (“i3”) [26]. Characteristic for type 3 immunity is the involvement of transcription factors
RORα and/or RORγt, secretion of the cytokines IL-17A, IL-17F and IL-22, and the activation of

Biology 2015, 4 816
neutrophils [26–28]. Type 3 immunity has an important function in protection against extracellular
bacteria and some fungi. In the healthy intestine, TH17 cells form an important role in a complex network
of interactions between commensal bacteria and immune cells and help to maintain tissue homeostasis and
barrier integrity [29–31]. Although phenotypically TH17 polarizations are often considered to be more
plastic than TH1 and TH2 polarizations, some epigenetic modifications acquired during TH17 polarization
are rather stable [32].
The use of the term “type 2 immunity” can somewhat differ between researchers and research fields,
but tends to encompass both milieus with dominant immunosuppressive functions, for which TGF-β and
IL-10 are marker molecules, and inflammatory milieus with dominant functions of cytokines IL-4, IL-5
and/or IL-13. Characteristic for type 2 inflammation are anti-parasite activities involving the activation
of mast cells and eosinophils, and the secretion of IgE by B cells. In allergy diseases, these types of
reactions are triggered by allergens. Generally, i2-skewed immune milieus may be more protective of
tissues than i1-skewed milieus [33,34], which also agrees with type 2 immunity being important in wound
healing [23,35]. However, also type 2 immune reactions can cause considerable tissue damages (e.g., [36]),
amongst which tissue fibrosis [37]. The stimulation of TH2 polarization by the alarmin IL-33, which is
released from damaged tissue, can be understood from the importance of type 2 immunity in tissue
regeneration and wound healing [23,35,38]. Similarly, the expression of chitinase-like proteins (CLPs),
which is enhanced by helminth infection or injury, also induces TH2 responses, although CLPs can also
stimulate IL-17 release [39–42].
Figure 1 is our attempt to summarize some principles in cell polarization as they have been described
for mammals. The horizontal axis relates to the concentrations of some important factors, whereas the
vertical depiction of “energy valleys” relates to the relative stability of a cell polarization; the depiction
with energy valleys only serves to explain a model, and the depicted valley depths have no absolute
meanings. Some of the molecules characteristically expressed by the respective polarized cells are listed
within those valleys, with blue arrows highlighting the cytokines that help fixing the cell phenotype as
part of self-stimulatory loops [5,13,43–46]. White arrows refer to studies that described how some
already polarized phenotypes are plastic in that they can be modified towards other polarizations; our
figure is a simplification in the sense that the cell types produced by this type of route can be somewhat
different from those directly produced from naïve T cells [47–53]. Our choice of the white arrows in
Figure 1, with numbers for references described in the figure legend, represents our attempt to summarize
major literature, and these possible conversions highlight similarities between polarizations in the order
that they are depicted as “neighbors” in Figure 1. Readers should, however, realize that also conversions
between “non-neighboring” (defined by Figure 1) polarizations have been reported possible (not shown
in Figure 1; e.g., [54]), underlining that the Figure 1 depiction is only a model which explains some but
not all principles of immune polarization. Importantly, though, the continuous axis-nature of the
polarizations as depicted in Figure 1 is also supported by shared expressions of some marker molecules
between “neighbors”, with typically in one of the populations the expression being considerably lower
or restricted to subpopulations (indicated by italic font in Figure 1). Naturally, Figure 1 is an enormous
simplification, describing only a few major factors and categorizing only a few major cell populations.
Especially T lymphocyte types with regulatory functions are a complex set of cells widely distributed
along the i1-i2 axis [55], and the single regulatory T cell valley in Figure 1A is only representative for
major sets of Treg cells. The stability of Treg polarizations is believed to differ between subtypes [56].

Biology 2015, 4 817
Figure 1. Schematic depiction of the i1-i2 axis affecting the polarizations of mammalian
TH/Treg cells (A) and macrophages (B). Only some relevant factors, and not all known
polarizations, are summarized. The figure organization and purpose is explained in the main
text. Italic font relates to molecules that have been described as molecules especially
expressed by that respective polarized cell population, but are present in lesser amounts than
in other polarized populations for which they are more characteristic. Our depictions of
factors relevant to TH/Treg cell polarizations are the summaries of mainstream ideas, with
most references given in the main text. For GATA-3 expression in Treg cells see [57].
For RORγt expression in early Treg, and FOXP3 expression in early TH17, see review [58]. For
STAT4 expression in TH17 cells see [59]. The macrophage polarization figure (B) is importantly
based on a figure by Mantovani et al. [60], while modifications were made based on additional
literature as referenced in the main text. The names between brackets are alternative designations
that have been used for the respective macrophage polarizations. The blue arrows relate to

Biology 2015, 4 818
self-amplifying loops as described by: For interferon γ (IFNγ) in TH1 cells, see [5,13]; for IL-21
in TH17 cells see [45]; for TGF-β in Treg see [44]; for IL-10 in Treg see [43,46]; for IL-4 in TH2
see [5]; for TNF-α in M1 macrophages see [61,62]; for IL-10 and TGF-β in M2c macrophages
see [63] and [64], respectively. The white arrows relate to experiments that described how
addition (+) or repression (−) of factors could push already polarized TH/Treg cells into
another polarization state, with superscript numbers indicating the respective literature: 1, [49];
2, [50]; 3, [53]; 4, [48]; 5, [47]; 6, [51].
In Figure 1, the actual biological situation of polarizations would probably be better represented by
a three-dimensional energy landscape with many hills, ridges and valleys [65,66], where the 1-2 axis
might be something like the East-West axis and with multiple possible routes between the East and West
sides, and further addition of dimensions would further improve the figure; however, that would need
a lot more information than currently available.
In Figure 1A we did not include TH9 and TFH cells because currently it is impossible to discuss their
possible presence in the context of fish because relevant genes (e.g., IL-9) or tissues (e.g., functional
equivalents of lymph nodes) have not been found/clarified in fish yet. It is very likely that fish do not
have the exact same immune cell polarizations as found in mammals, although the major principles of
the i1-i2 axis appear to be the same as we argue in this article. If we somewhat freely interpret the work
by Kaplan and co-workers [67], those authors arranged mammalian TH9 and TFH cells in the order
TH1-TFH-TH17-Treg-TH9-TH2 along the i1-i2 axis. However, others have found similarities between TFH
and TH2 cells [68] or stressed the heterogeneity and the existence of subpopulations among TFH cells [69];
probably the adaptation of cells to TFH function should not be understood as a unique polarization along
the i1-i2 axis.
TGF-β limits how far cells polarize along the i1-i2 axis in either direction, and it is an important
cytokine for the development of TH17 and Treg cells. A higher concentration of TGF-β favors development
of Treg over that of TH17 [70,71]. Whereas STAT3 activation in TH17 cells is especially enhanced by
IL-6, IL-21 and IL-23, the STAT3 activation in Treg cells is especially enhanced by IL-10 [46]. It is tempting
to speculate that the relatively common transcription factor STAT3 [72] blocks development of the “more
extreme” axis-end phenotypes TH1 and TH2. However, although STAT3 is known to suppress expression
of TH1 marker genes [73], it was reported necessary for TH2 development [74]. Because the dependency
of TH2 cells on STAT3 has not been studied intensively, we used a dashed line in Figure 1A for the yet
better to be clarified main factors that stimulate STAT3 in TH2 cell development.
Although STAT5 activity can stimulate survival and proliferation of different sets of lymphocytes [75],
it represses TH17 differentiation and shifts the development of common TH17/Treg precursor cells
towards Treg [76]; the important inducer of STAT5 activity in Treg is IL-2, a cytokine which can also
stimulate other lymphocyte populations [77]. The Figure 1 model does not include the STAT5 activity
enhancers IL-9 and thymic stromal lymphopoietin (TSLP), which both stimulate type 2 immunity [38],
because these two genes have not been found in fish (yet). STAT5 activities in NK cells and CD8+ T
cells (not shown in Figure 1), important for type 1 immunity, can be induced by IL-15 that is expressed by
dendritic cells or monocytes/macrophages [77].
Although most researchers will agree that polarizations of immune cells depend both on various
gradients of factors as well as on more discrete sets of conditions, to try to catch that in a figure with

Biology 2015, 4 819
only a single axis as in Figure 1 could be righteously considered presumptuous, overly simplified, and
misleading. However, we argue that in such it doesn’t stand out negatively from more popular figures
trying to summarize leukocyte polarizations. Furthermore, we argue that if we wish to compare immune
polarizations of different cells and tissues, of healthy vs. diseased conditions, and among species as
diverged as mammals and fish, we need a kind of articulated bird-view of the i1-i2 axis as attempted in
Figure 1. In the current study we use the Figure 1 model for analyzing published data in fish, and
conclude that the immune systems of mammals and teleost fish seem to obey to at least some similar i1-i2
axis principles.
2. Polarizations along the i1-i2 Axis of Mammalian Leukocytes Other than Helper and
Regulatory T Cells
Polarizations towards type 1, type 3 and type 2 immunity, which are very reminiscent of the ones
found for TH cells, have been described for innate lymphoid cells (ILCs) (reviews [26,78,79]). Marker
molecules expressed by ILC1 cells are transcription factor T-bet and cytokine IFNγ, marker molecules
expressed by ILC3 cells are transcription factor RORγt and cytokines IL-17 and IL-22, and marker
molecules for ILC2 cells are transcription factor GATA-3 and cytokines IL-5 and IL-13. The intermediate
position of ILC3 along the i1-i2 axis, similar to as found for TH17 cells, is supported by sharing of some
marker transcription factors and cytokines with either ILC1 or ILC2 cells, while ILC1 and ILC2 cells
appear to lack unique overlaps with each other [26]. ILC3 cells can be converted into ILC1 cells by stimulation
with IL-12, resulting in downregulation of RORγt and upregulation of T-bet [80]. Some difficulties in
classification of ILCs are caused by the existence of multiple ILC1-type populations, and by differences
in their regulation between human and mouse [78]. Most researchers do not distinguish a separate “ILCreg”
population, but besides aiding type 2 inflammation, ILC2 cells are known to have important functions
in tissue homeostasis and tissue repair [81,82]. Very interestingly, recently also ILC3 subsets were found
to have Treg-like functions in the sense that they could negatively select antigen-specific T cells [83]. Thus, like
found among T cells, among ILCs there is an overlap between type 1 and type 3 immunity, between type 3
immunity and regulatory functions, and between regulatory functions and i2 inflammation.
Except for regulatory/helper T and ILC populations, i1-i2 polarizations similar to the ones listed
above because involving at least several of the same marker molecules have been reported for CD8+ T
cells [84], B cells [85], neutrophils [86] and dendritic cells [87]. However, it is beyond the scope of this
article to discuss those polarizations. Macrophage populations, on the other hand, will be discussed here,
because macrophage polarizations have been studied relatively intensively and are of major importance
in the creation of immune milieus and in tissue modeling. Furthermore, there are some functional data
on macrophage polarizations in teleost fish (see further below).
In Figure 1B we made an attempt to characterize major polarizations of mammalian macrophages
along the i1-i2 axis. The figure is a modified version from a distribution figure by Mantovani et al. [60],
and as in Figure 1A, the depths of the “energy valleys” only serve to explain a model and have no
absolute meanings. Very importantly, what emerges as a general impression from literature is that
macrophage populations have less pronounced self-amplifying loops (although for an autocrine TNF-loop
see [61] and [62], for an autocrine IL-10 loop see [63], and for an autocrine TGF-β loop see [64]) than
known for TH/Treg polarizations, and that macrophage polarizations appear to be rather unstable and

Biology 2015, 4 820
hence a rather direct reflection of their immune environment [88,89]. This makes sense since in
contrast to T cells which are antigen-specific and whose epigenetic modifications contribute to immune
memory [19,90], macrophages interact with a large number of antigens. Their plasticity probably is an
important reason why macrophage polarizations were discovered later and remain poorer characterized
than TH/Treg polarizations. Many researchers only distinguish between M1 and M2 macrophages, without
further subdivisions.
Macrophages are sensitive to DAMPS and PAMPs. LPS is an important PAMP for shifting macrophage
polarizations towards the i1-end of the i1-i2 axis, and can stimulate the development of both M1 and
M2b macrophages [60,89]. Viral dsRNA mimic poly(I:C) also induces M1 polarization [91,92].
M1-skewed macrophages express IL-12 which is important for the initiation of TH1 polarization [89],
and they also express IL-15 [93,94] which is especially important for the stimulation of NK and CD8+
T cells [77,95]. Both M1 and M2b macrophages express inducible nitric oxide synthase (iNOS) and are
active in clearance of bacteria through NO production [94,96]; the big difference of M1 vs. M2b cells is
the abundant production of IL-12 vs. IL-10, so that M1 cells support type 1 immunity and M2b cells are
able to support type 2 immunity [96–98]. The expression (-pathways) of iNOS and arginase affect each
other negatively [99–101]. Expression of arginase in M2c and M2a cells leads to production of ornithine,
a precursor of extracellular matrix components that contributes to wound healing [60,102]. M2a and
M2c macrophages appear to participate in tissue regeneration following tissue injury [103], which
explains why M2a polarization can be enhanced by alarmin IL-33 ([104,105]. We did not include alarmin
IL-25 (alias IL-17E) in Figure 1, although it also supports type 2 immunity, because of difficulties to find
an orthologue in fish [106,107]. M2a polarization involving i2 cytokines induces macrophages to express
CLPs [40,108,109]. M2c macrophages have anti-inflammatory properties and are stimulated by
glucocorticoids, IL-10 and TGF-β [110,111]. It is of note that in human, different from the mouse
situation, some M2 polarizations may not be typified by high levels of arginase expression [112,113].
In many tumors the tumor cells attract monocytes/macrophages and skew their development towards
type 2 immunity; the M2 macrophages then in reciprocal interaction with the tumor cells remodel the
tumor microenvironment, which aids the tumor cells and protects them from type 1 immunity [114,115].
The current successes in cancer immunotherapies are largely based on shifting the tumor milieu from
type 2 towards type 1 immunity, and one of the focuses of investigation concerns macrophage
polarizations (e.g., [116,117]).
3. Fish Orthologues of Mammalian genes for i1-i2 Axis Functions
For all the mammalian genes encoding the proteins shown in Figure 1, homologues could be found
in ray-finned and/or in cartilaginous fish, in most cases including probable orthologues. Examples are
shown in Figure 2 plus supplementary file 1.
For teleost fish now a relatively large number of “whole genome” sequences have been published.
However, for cartilaginous fish, the only species for which the sequence of a large part of the genome
has been published is the chimaera elephant shark (Callorhinchus milii; [107]); hence, gaps in the
published elephant shark genome can’t be “filled in” with information from other cartilaginous fish.

Biology 2015, 4 821
Figure 2. Schematic depiction of the conservation of the IFNG and TH2 cytokine loci in fish
and mammals. The pentagon orientations correspond with gene directions. Depicted gene
organizations are based on analysis of genomic sequence information available for elephant
shark (Callorhinchus milii) provided by the Elephant Shark Genome Project ([118] and
GenBank accession number AAVX02000000) and for the other species in the following
datasets of the Ensembl database [119]: human (Homo sapiens), GRCh38.p2; spotted gar
(Lepisosteus oculatus), LepOcu1; zebrafish (Danio rerio), GRCz10. Between human IL-5 and
GM-CSF lays a 465 kb stretch with a number of genes which are not shown in this figure.
Most of the depicted gene organizations have been described before [107,120–125]. The
deduced elephant shark IL-26L amino acid sequence is MRCAAACLLVSLGVCVVRTSTA
TCKPKVSDRLIQDFIRCVGNVMNASQHYWGSSWSDGKGYRFLPKPVKMTKHGKC
TVVKKALEFYLIFLKQYRPMPDGFKQDLIKVKHYLEEMYAKTRCDECKSSKDLNAE
RAIKRLEKEICKARCSKHTSVTKKSIIFQLYILRNLITNMA. For the deduced encoded
elephant shark TH2 locus cytokine sequences see Table S3 (except for IL-4/13D these are also
described in [124]). For the spotted gar IL-4/13A sequence we refer to [125]. The deduced
spotted gar IL-5famA sequence is MSMYLVLLILGVHYSGVRTQHYHFISEIISHIENAK
QGVVHTILLTPQNVLNANCTASYSKIFLKGIKHLSVHSEHGSQEELKLIIHNMERMD
VICPNLKHQVPDCEVQDTSTFQFLRQFTKFLQKIKRSDCFRLRSEYPFSA, which is compared
with other sequences in Figure 3.

Biology 2015, 4 822
Nevertheless, for most of the molecules depicted in Figure 1 probable gene orthologues can be found
in the published elephant shark genomic sequences, and for the exceptions the incomplete nature of the
published sequences might be to blame. It has been argued that elephant shark does not have an
RAR-related orphan receptor (ROR) gamma gene [107]. That may be true, although such conclusion
would need full genome sequence information, and in our preliminary phylogenetic tree analyses
(data not shown) the molecule encoded by the elephant shark “RORA-like” (RORAL) gene at scaffold
2358 (supplementary file 1) clusters with RORγ sequences. A serious analysis of the evolution of the
ROR family of transcription factors would need a more serious effort than feasible within the scope of
this article. Regardless, because in mammals not only RORC but also RORA can contribute to TH17
development [28], the question on C or A identity might not be so relevant across these wide species
borders when addressing the possibility of TH17 polarization.
An early ancestor of all extant teleost fishes experienced a whole genome duplication event [126],
and several teleost fish lineages experienced an additional genome duplication event (e.g., [127]). This
was frequently followed by gene losses, lineage specific gene duplications and/or translocations, causing
a tendency for the analysis of orthologous relationships between mammals and teleost fish to be more
complicated than between tetrapods, cartilaginous fish and non-teleost primitive bony fish. However,
overall and in principle, many gene organizations in teleost fish resemble those of mammals and elephant
shark [107,126], and it was because of conserved syntenies that we and others could identify teleost fish
genes for small cytokine genes despite their poorly conserved sequences (e.g., [120–122,128,129]).
A number of the gene syntenies between fish and human depicted in Figure 2 and supplementary file 1
were already described earlier (e.g., [107,130]), but we feel it is convenient for the readers to have the
data presented together.
Figures 1 and 2, and supplementary file 1 do not provide information regarding the relevant receptors
and neither regarding many of the pathway molecules, because we argue that the currently depicted
molecules are sufficiently representative for their functional pathways. However, it is important to
realize that also the relevant cytokine receptor and pathway molecules tend to be rather well conserved
between fish and mammals (e.g., [107,131–133]). Furthermore, regarding the molecules depicted in
Figure 1, we did not analyze the genomic locations of fish genes involved in glucocorticoid pathways and of
fish CLP genes; for these genes we refer interested readers to references [134] and [135], respectively.
As a negative exception among the proteins depicted in Figure 1, for IL-33, which is a highly diverged
member of the IL-1 family with poor sequence conservation even between mammals and birds (see
Ensembl accession ENSGALG00000020558), we could not find a likely gene candidate in any of the
investigated fish species. However, in teleost fish multiple IL-1 family members have been found [136,137],
and teleost genes have been annotated as IL1RL1 (alias ST2 or ST2L; [136,138,139]) which in mammals
encodes the receptor for IL-33. Since IL1RL1 maps to a locus with multiple similar genes of the IL-1
receptor family [140], this IL1RL1 designation in fish probably would need a more intensive analysis
than has been published to date or is feasible within the scope for our present study. In short, fish may
have IL-33 (-receptor) function, but there is no real evidence to support that.
From the early days that we started to identify i1-i2 axis cytokine genes in fish despite of their very
poorly conserved sequences with the help of gene syntenies (e.g., [120,122,129]), we have been
fascinated by the high conservation of loci between fish and mammals, often even in simple 1:1
orthologies. The high level of evolutionary conservation of the genomic organization of many of the i1-i2

Biology 2015, 4 823
axis gene loci, as shown in Figure 2 and supplementary file 1, contrasts with the abundant locus turnovers
and copy number differences found for other genes of the immune system, like for example genes
encoding MHC molecules [141,142], chemokines [143], and type I interferons [144]. For several NK cell
receptor families there may not be close relatives in fish at all [145]. That many of the gene loci
important for the i1-i2 axis are so well conserved between jawed fish and mammals strengthens the
idea that in all jawed vertebrates major principles of the i1-i2 axis system have been conserved as core
mechanics of their immune system. Our attempts to find i1-i2 axis genes in published sequences of
jawless fish (lampreys, hagfish) and invertebrates proved to be difficult/impossible (data not shown),
and future careful analyses should determine if to any extent some principles of the i1-i2 axis might
be present in those species. For reviews on the immune systems of jawless fish, which are fundamentally
different from those in jawed vertebrates, we refer to [146–148].
4. Conservation of the IFNG and TH2 Cytokine Loci
Very important in the TH1 and TH2 polarizations are their divergent epigenetic modifications of the
IFNG and TH2 cytokine loci [5,149]. Especially the pronounced modifications of the TH2 cytokine locus,
including chromatin refolding, have received a lot of attention [4,18,150–153]. Binding of transcription
factor GATA-3 and STAT6 induces chromatin refolding by inducing interactions between (inter-) gene
regions of IL-5, RAD50, IL-4 and IL-13 [18,19]. It is fascinating to see how well the IFNG and TH2
cytokine loci have been conserved between fish and mammals (Figure 2). The name IL-4/13 is used for
genes related to tetrapod IL-4 and IL-13, because it can’t be decided to which of the two tetrapod genes
the fish genes are closer related, and the gene duplication leading to IL-4 vs. IL-13 may have occurred
after the separation between the ancestors of tetrapods and ray-finned fish [122].
Most of the gene organizations shown in Figure 2 have been reported before [107,120–125], but different
from previous publications [107,124,125] we found (i) IL-26-like (IL-26L; for the encoded sequence see
the Figure 2 legend) in the elephant shark IFNG locus, (ii) an extra IL-4/13 copy, IL-4/13D, that we had
missed [124] but which was properly annotated as an IL-4/13 gene by automated database gene prediction
(XM_007902044) in the elephant shark TH2 cytokine locus, and (iii) an IL-5 family (IL-5fam) member
in the spotted gar TH2 cytokine locus (for the encoded sequence see the Figure 2 legend). Our analysis
of the spotted gar TH2 locus constitutes the first identification of a bony fish TH2 cytokine locus that
includes RAD50 and seemingly bona fide genes of both IL-4/13 and IL-3/IL-5/GM-CSF families.
Expression of elephant shark IL-26L was confirmed by sequence read archive (SRA) database reports
(data not shown), and the gene has an intron-exon organization typical of the IL-10 family (to which also
IL-22 and IL-26 belong). Phyre2 software [154] predicts that elephant shark IL-26L protein has multiple
α-helices, and that its structure is similar to IL-10 (confidence 52%). Although the deduced molecule
does not have a typical IL-10 signature motif in the carboxy-terminal α-helix, it shares some specific
cysteines with IL-26 (data not shown), and despite the minimal similarity we considered “IL-26L” to be
the best possible name.
In a previous paper we depicted the elephant shark IL-5famA and IL-5famB genes as “IL-5A” and
“IL-5B”, upon request of the respective journal who considered the “fam” indication (for family) to be
confusing for a general audience ([124]; the “fam” designations were only given as optional in the
supplement of that publication). But, although the genes are related to IL-5, and are situated at the

Biology 2015, 4 824
expected IL-5 location, we have no strong arguments for their closer relation to IL-5 than to IL-3 or
GM-CSF (for discussion of the evolution of the TH2 cytokine locus see also [122]). So in the current
article we like to use the nomenclature including “fam”, although both nomenclatures are defendable.
The cytokine family including IL-3, IL-5 and GM-CSF is characterized by extremely poorly conserved
sequences, especially among the IL-3 molecules [155], with only a few typical and conserved sequence
motifs (Figure 3). Whereas hitherto for bony fish no convincing IL-5-family candidates were reported,
we now found such gene at the expected IL-5 site in the spotted gar TH2 cytokine locus which we
designated IL-5famA (Figures 2 and 3). It has the family-typical intron-exon organization, and in contrast
to the other detected IL-5fam molecules in fish, gar IL-5famA has a cytokine as top-match upon blastp
comparison with the NCBI database (Genbank accession KFO26617); the relevant unknown cytokine
gene appears to be correctly predicted for cattle (GenBank accession XP_010796657), maps directly
downstream of mammalian IL-3 and GM-CSF, is predicted to encode multiple α-helices according to
Phyre2 software, and appears to have pseudogene identity in humans (data not shown). We may discuss
this hitherto unknown mammalian cytokine in more detail in a future publication, and only mention it here
as additional evidence that the fish IL-5fam molecules truly belong to the IL-3/IL-5/GM-CSF family.
In teleost fish gene candidates for the common β receptor chain (alias IL-3Rβ) have been known for
a long time [131], and accordingly the finding of IL-3/IL-5/GM-CSF family genes has been anticipated.
However, the best that we could do so far was a zebrafish gene with unclear signature which we
designated “IL-5?” [122] and which we now in Figure 2 designate as “IL-5fam?A”. We actually are still
insecure whether this somewhat peculiar zebrafish gene is an intact gene, as it may not have a normal
exon1 sequence (data not shown), and therefore it is indicated by a dashed line in Figure 2. However,
we are now confident that the zebrafish gene is at least related to intact cytokine genes, as orthologous
and apparently bona fide cytokine genes can be found in carps and goldfish. Like in zebrafish, in common
carp the gene is also linked with KIF3A (GenBank accession LN591230). Figure 3 shows four cyprinid
IL-5fam? sequences, namely the LN591230 encoded common carp sequence, a goldfish sequence
encoded by GenBank TSA accession GBZM01010380, and two very similar golden mahseer sequences
assembled from SRA reads (data not shown; we don’t show the individual SRA accessions). The
question mark in the “IL-5fam?” nomenclature expresses our insecurity about the molecule identities,
because whereas in our opinion the gar and elephant shark molecules have a convincing IL-3/IL-5/
GM-CSF family signature, the only partially conserved signature in cyprinid sequences fails to convince
us (Figure 3). Nevertheless, because of genomic location and lack of better matching candidates, the most
likely hypothesis appears to us that these cyprinid sequences are highly diverged members of the
IL-3/IL-5/GM-CSF family. To our frustration, even with the knowledge of the gar and cyprinid
IL-5fam(?) sequences, we have been unable so far to find any IL-3/IL-5/GM-CSF family gene
candidates in non-cyprinid teleosts. Although negative findings for gene members of these small
cytokine families with poorly conserved sequences shouldn’t be overvalued (see [107] and [124]), it might
be speculated based on the lack of convincing gene candidates that the importance of the IL-3/IL-5/GM-
CSF family was reduced in teleost fish compared to other classes of vertebrates. Functional analyses of
the fish molecules, including their possible interaction with the common β receptor chain, should clarify
these matters.
As a general statement based on our many years of experience in identifying genes of the immune
system, we feel that at the genetic level the immune systems of elephant shark and gar have more

Biology 2015, 4 825
similarities with the mammalian immune system than found between teleost fish and mammals. Slow
evolution towards the elephant shark genome and rapid evolution towards the extant teleost fish genomes
have been noted before [107,126,156]. When fish research is performed with the aim to deduce the
ancestral features of the human immune system, it might be worth considering to move research away
from teleosts to for example gar (Lepisosteus oculatus).
Figure 3. Cont.

Biology 2015, 4 826
Figure 3. Alignment of (deduced) IL-3/IL-5/GM-CSF family member amino acid sequences.
(Predicted) leader peptides are indicated with gray shading; for predictions SignalP software
was used (http://www.cbs.dtu.dk/services/SignalP/). The alignment is organized according
to the matching exons, and brackets relate to intron positions with the number indicating the
intron phase. The α-helices αA-to-αD, of human IL-5, GM-CSF and IL-3 are indicated by
underlining following [157], [158], and [159], respectively. Sequences were aligned by hand,
based on considerations regarding structure and evolution (as in [122], although we made some
different choices now). For the alignment among the core regions of human IL-3, IL-5 and
GM-CSF we mostly followed the structural alignments by [159] and [160], with as notable
exception the α-helix B sequences of IL-5 in which we introduced a gap for a better match of
sequence identities with the other cytokines. Readers should realize that alignments of such
highly differentiated sequences remain discussable. Conserved motifs are highlighted by
different color shading in a somewhat instinctive and random manner. Some of the
highlighted motifs can also be found in cytokines not belonging to the IL-3/IL-5/GM-CSF
family, but not in this combination (compare with [122,160,161]. The yellow shaded glutamic
acid in α-helix A is important for function [162–164], and, at least for GM-CSF, for binding
the common β receptor chain [165]. Aligned, in that order, are the following sequences:
Human (Homo sapiens) IL-5, GenBank accession NP_000870; mouse (Mus musculus) IL-5,
NP_034688; nine-banded armadillo (Dasypus novemcinctus), XP_004456511; Tasmanian
devil (Sarcophilus harrisii) IL-5, XP_003756529; gray short-tailed opossum (Monodelphis
domestica) IL-5, XP_001371840; chicken (Gallus gallus) IL-5, ADL28818; white-throated
sparrow (Zonotrichia albicollis) IL-5, XP_005483812; rock pigeon (Columba livia) IL-5,
EMC79983; elephant shark (Callorhinchus milii) IL-5famA and IL-5famB, see supplementary
file 2 and [124]; spotted gar (Lepisosteus oculatus) IL-5famA, see the Figure 2 legend; goldfish
(Carassius auratus) IL-5fam?A, GBZM01010380; golden mahseer (Tor putitora) IL-5fam?A
and IL-5fam?B, see supplementary file 2; common carp (Cyprinus carpio) IL-5fam?A, compare
LN591230 or LHQP01003280 with the goldfish or golden mahseer sequences; human
(Homo sapiens) GM-CSF, NP_000749; mouse (Mus musculus) GM-CSF, CAA26192;
chicken (Gallus gallus) GM-CSF, NP_001007079; human (Homo sapiens) IL-3, AAH66275.

Biology 2015, 4 827
5. Investigation of Tissue-Specific Co-Expressions of TH1, TH17, Treg and TH2 Signature Genes in
Fish; Gills Consistently Express High Levels of TH2 Signature Genes
Previously we showed that in salmonid fishes the expression of GATA-3 and IL-4/13A are high in
gill, skin and thymus, also in relation to other genes of the immune system, and we assumed that these
tissues are skewed towards type 2 immunity [166]. Similar findings for high GATA-3 and IL-4/13A
expression in teleost gill were also made by others [121,167,168]. In the current study we extended this
type of investigation with database mining, in which we blasted sequences against tissue-specific single
SRA datasets available for healthy individuals of several fish species (http://www.ncbi.nlm.nih.gov/sra)
(data not shown) as summarized in Table 1. We found our previous observations of high IL-4/13A and
GATA-3 expression in rainbow trout and Atlantic salmon gills confirmed by transcriptome analyses for
these two species, but also for pike (like salmonids member of Protacanthopterygii) and golden mahseer
(a cyprinid fish) (Table 1). Excitingly, even in elephant shark the GATA-3 and IL-4/13 expressions appear to
be particularly high in gill, coinciding with high IL-5fam gene expression, which suggests that the
elephant shark IL-5fam/RAD50/IL-4/13 locus is a similar GATA-3 driven TH2 cytokine locus as present
in mammals, and that the i2-skewage of the gill immune milieu is ancient. More analysis would be
necessary to determine how the i2-skewage is distributed over the gill, and to what extent it maps to
interbranchial lymphoid tissues (ILT; [169]). Interestingly, in Golden mahseer, the expressions of IL-4/13B
and IL-5fam? genes are not tightly associated with those of IL-4/13A and GATA-3, and are not consistently
although often high in gill (Table 1 and data not shown). This suggests that only one of the two teleost
copies of the TH2 cytokine locus resulting from the teleost ancestral whole genome duplication, namely
the RAD50 + IL-4/13A locus (Figure 2; see also [122]), retained the expression mode of the ancestral TH2
cytokine locus. In accordance, in the promoter regions of teleost IL-4/13A genes and not in those of teleost
IL-4/13B genes we found a rather well conserved GATA-3 binding motif [122]. In elephant shark STAT6
expression is highest in gill, as expected from an i2-skewed tissue, but for unknown reason in teleost fish
it does not tightly associate with the high GATA-3 and IL-4/13A expression found in gill (Table 1).
Besides a clustering of high expressions of TH2 signature genes in gill, the investigated elephant
shark individual displays such clustering for TH1 signature genes in its spleen and for TH17-signature
genes in its intestine (Table 1). Although this appears very interesting, and may be indicative of ancient
tissue-specific immune biases, these data do need confirmation in other cartilaginous fish individuals
before allowing conclusions.
In the investigated teleost fish individuals, besides the consistent link between GATA-3 and
IL-4/13A expression, we also found a consistent link between high STAT1 and STAT4 expression. The
highest STAT1 and STAT4 expressions correlated relatively well with the highest T-bet (alias TBX21)
expression, but the tissue of highest expression differed among the investigated teleosts and there was
no clear correlation with IFNG expression (Table 1). Whether the lack of consistencies seen in Table 1
represent genuine differences between species or are due to random differences between fish individuals
or between sampling techniques, can’t be decided without further investigation. However, it is of note
that, for example, in another study comparing among healthy trout and salmon individuals we also found
considerable variation regarding the tissue of highest IFNG expression [166].
In the teleost fish turbot the highest expression of both IL-17A/F and IL-22 was found in the
intestine [170], which would agree with the findings in elephant shark shown in Table 1. However,

Biology 2015, 4 828
before concluding that the fish intestinal immune milieu—or part of it—tends to be i3-skewed, more
research is needed, and the data of the teleost fish individuals summarized in Table 1 argue against it.
For signature genes of regulatory functions such as FOXP3, IL-10 and TGF-β, we could not distinguish
any notable expression patterns among healthy tissues of either elephant shark or teleost fish (Table 1).
Table 1. Expression levels of immune signature genes in various tissues determined by
BLAST analysis against cartilaginous and teleost fish sequence read archive (SRA) datasets.
Read numbers per 5 × 107 reads of various immune signature genes of cartilaginous and
teleost fish species were determined by similarity searches against tissue-specific SRA
datasets (from http://www.ncbi.nlm.nih.gov/Traces/sra/ (data not shown); see Table 1C)
using the BLAST search function at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). ORF
sequences (see supplementary file 2) were subjected to “Megablast” analysis (blastn) using
default software settings except that the “max target sequences” number was changed to
20,000 and that the “word size” number was changed to 64. To ensure specificity of the
Megablast analysis, only matches with “score” values ≥128 for elephant shark, ≥251 for
golden mahseer, ≥168 for northern pike and Atlantic salmon, and ≥169 for rainbow trout,
were counted. Colored backgrounds highlight the tissues with the highest relative expression
of the respective gene, and the red frames highlight the consistent high expression of IL-4/13A
and GATA-3 in teleost gills.
A. Cartilaginous fish
Elephant Shark (Callorhinchus milii)
Gill
Kidney
Spleen
Intestine
TH1-signature
T-bet
4
1
121
3
STAT1
6006
701
5263
2168
STAT4
726
131
3873
738
IFNγ
17
1
78
6
TH17-signature
IL17A/F1
1
0
0
7
IL17A/F2
21
0
2
66
IL-21L
0
0
3
2
IL-22
4
0
0
15
Treg-signature
Foxp3
10
1
51
3
IL-10
7
4
185
13
TGFβ1
72
36
66
3
TH2-signature
GATA3
2287
92
575
95
STAT6
290
41
199
102
IL-4/13A
3
0
0
0
IL-4/13B
8
0
0
1
IL-4/13C
0
1
0
0
IL-4/13D
0
0
0
0
IL-5A
0
0
0
0
IL-5B
3
0
0
0

Biology 2015, 4 829
Table 1. Cont.
B. Teleost fish (bony fish)
Golden mahseer (Tor putitora)
Northern pike (Esox lucius)
Rainbow trout (Oncorhynchus mykiss)
Atlantic salmon (Salmo salar)
Gill
Kidney
Spleen
Gill
Kidney
Head
Kidney
Spleen
Intestine
Gill
Kidney
Spleen
Intestine
Gill
Kidney
Spleen
Intestine
TH1-
signature
T-bet
4
47
54
91
114
663
899
28
12
20
22
11
134
320
1434
66
STAT1_1
523
1174
42
2589
1555
3200
2579
930
2041
2219
5794
2422
5608
5386
10317
2530
STAT1_2
1990
1870
3563
1878
STAT4_1
1525
1714
686
1866
1375
3702
2708
785
822
964
1122
679
1611
729
2273
1916
STAT4_2
484
37
62
88
IFNγ_1
85
47
31
8
0
4
11
1
19
0
189
84
6
6
32
2
IFNγ_2
17
0
211
99
TH17-
signature
IL-17A/F1a
14
21
13
13
15
2
0
2
14
5
1
9
0
5
0
0
IL-17A/F1b
1
6
0
4
5
11
0
0
IL-17A/F2a
11
6
4
10
0
0
0
0
4
5
0
2
8
39
0
0
IL-17A/F2b
3
0
0
2
1
0
0
2
IL-17A/F3
6
10
6
75
7
7
4
1
3
0
2
0
2
1
2
1
IL-21
8
28
19
53
35
26
14
123
1
0
0
6
0
0
0
0
IL-22
2
4
1
15
2
0
5
2
13
0
11
5
15
5
7
4
Treg-
signature
Foxp3_1
11
12
9
100
62
257
138
32
61
11
28
35
230
143
666
149
Foxp3_2
115
25
43
63
IL-10
32
179
130
23
127
111
109
8
0
2
7
1
1
2
2
0
TGFβ1a
98
279
59
1374
891
1549
1330
270
177
659
888
211
66
122
125
7
TGFβ1b
220
410
910
500
TH2-
signature
GATA3
>20590
472
113
10801
409
365
202
141
2669
98
13
24
16724
219
334
84
STAT6
460
870
17
2354
2183
2650
2781
875
872
827
1274
827
1719
1313
1688
788
IL-4/13A
1147
56
36
25
7
4
3
4
29
9
5
5
112
42
33
15
IL-4/13B1
13
3
2
3
0
1
0
2
6
0
1
1
31
7
8
14
IL-4/13B2
1
6
5
3
8
5
2
2
7
7
3
0
7
IL-5fam?A
29
13
9
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
IL-5fam?B
6
29
12
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.

Biology 2015, 4 830
Table 1. Cont.
C. Accession numbers of the SRA datasets and their number of total reads
Elephant shark (Callorhinchus milii)
SRA
dataset
SRX154852
SRX154856
SRX154860
SRX154855
Tissue
Gill
Kidney
Spleen
Intestine
No. of
reads
71430454
118965654
83369382
147745918
Golden mahseer(Tor
putitora)
Northern pike (Esox lucius)
Rainbow trout (Oncorhynchus mykiss)
Atlantic salmon (Salmo salar)
SRA dataset
SRX768559
SRX768561
SRX767362
SRX514237
SRX514263
SRX514240
SRX514270
SRX514238
ERX297522
ERX297511
ERX297523
ERX297509
SRX608399
SRX608574
SRX608599
SRX608567
Tissue
Gill
Kidney
Spleen
Gill
Kidney
Head
Kidney
Spleen
Intestine
Gill
Kidney
Spleen
Intestine
Gill
Kidney
Spleen
Intestine
No. of reads
41751362
34023336
51857480
58499888
60694314
61054936
61731442
60466858
39064840
32103778
41714660
40271788
59793962
61054936
60203316
59806348

Biology 2015, 4 831
6. Evidence Supporting the Existence of TH Cells in Fish
In this and the following chapters with “fish” we refer to teleost fish if not mentioned otherwise.
Readers should realize though that the relatively little information available for sharks suggests that in
essence they have immune systems similar to those found in other jawed vertebrates [147,171]. It is of
note, however, that despite the overall similarities, there are also some aspects of the fish immune system
that importantly differ from the mammalian situation, such as the poikilothermic conditions and the
absences of lymph nodes, of mammalian-type haematopoietic bone marrow, and of antibody class
switching [147,171]. It is also of note that the general pattern of basic similarity does not involve all
jawed fish species, like for example gadoid fish do not have an MHC class II presentation system [172].
In the below we only try to summarize the (teleost) fish consensus situation.
Formally, helper T cell function in fish probably cannot be considered proven. However, multiple
lines of evidence indicate that fish TH cells similar to their human counterparts do exist. Fish have B
cells and macrophages, which like antigen presenting cells in mammals express MHC class II
molecules [173–176], and fish have T cells which express somatically rearranged TCR-α and -β genes
that are expressed and selected in a clonal manner [177,178]. Furthermore, teleost fish have CD4 molecules
with a motif for signaling capacity (CD4-1 and CD4-2; [179–182]), as well as sets of CD3 and signaling
pathway molecules necessary for T cell function [183,184]. Fish CD4 and MHC class II molecules are
expressed at high levels in the thymus in a similar tissue organization as in mammals [174,180,185–189],
suggesting that, like in mammals, the fish thymus generates TH cells that have been negatively selected
against self-antigens. Early thymectomy results in a decreased antibody response against “T-cell dependent
antigens” [190], and anti-hapten B cell responses were found to be supported by carrier-specific aid of
non-B cells in hapten-carrier immunized fish [191–193]. More recently, adaptive transfers of CD4-positive
(CD4-1 positive) lymphocyte fractions of immunized ginbuna crucian carp to syngeneic non-immunized
individuals were found to aid antigen-specific antibody and cell-mediated cytotoxic responses
in vivo [194]. In a zebrafish model support of CD4-1 positive cells to an antigen-specific immune
reaction was suggested by their enhanced cytokine gene expression profiles after zebrafish booster
immunization [195]. Many teleost lymphocytes that express CD4-1 also express CD4-2 [182,194–197],
but within the detection ranges of the applied assays it appears that teleost lymphocytes can also be
single-positive for only CD4-1 or CD4-2 [182,196–199]. It is not sure yet which, if any, of the fish
CD4 molecules can confer mammalian-type CD4 function. Definite evidence for TH functions in fish
may need experiments involving immunizations with different combinations of haptens and carriers
(to reduce the chance of misleading results because of nonspecific immune stimulation by the
antigens), purification of CD4-1+ and/or CD4-2+ lymphocytes, and the ability to manipulate MHC
class II-presentation or -matching by antigen presenting cells; since recently those experiments appear
to be possible, but they haven’t been done yet.
7. TH1-like Responses in Teleost Fish
In this paragraph and the following ones, we try to summarize principle similarities between data
published for i1-i2 axis functions in fish and in mammals, and we will for example not discuss alternative
functions encoded by fish-specific paralogous genes. We also try as much as possible to only reference

Biology 2015, 4 832
those fish studies which allow straightforward conclusions in regard to polarization models, leaving out
for example most studies that only investigated expression of i1 markers, or in which i1 markers were
up-regulated together with markers of other types of immune responses as part of an inflammation reaction.
Important for the discussion of possible type 1 immunity in fish is that they appear to have perforin
and granzyme containing TCRαβ+CD8α+ T cells that can kill virus-infected cells in a specific
manner, as well as “natural killer” cells that display less specificity for their cellular targets (reviewed
by [200,201]. Our definition of fish NK (-like) cells refers to perforin and granzyme containing non-B
non-T lymphocytes with cell-killing ability, although their surface markers may be substantially
different from those of mammalian NK cells [145]. Type 1 immunity pivots around the cytokine
interferon γ (IFNγ), and T-bet is the most important transcription factor. Fish T-bet and IFNG can be
expressed by CD4-1 positive cells [195,202]. The involvement of fish IFNγ in self-amplifying loops, as
known in mammals, was suggested by the observation that in flounder systems recombinantly expressed
IFNγ was able to induce IFNG expression in whole kidney leukocytes and in a permanent cell line [203].
In trout systems, the expression of IFNG by splenic leukocytes and head kidney cells could be
stimulated by recombinant IL-15 [204] and recombinant IL-12 [205], respectively. It was speculated that
the trout IFNG induced by IL-15 was expressed by NK and/or CD8+ T cells [204], because in mammals
these cell types are known to be particularly stimulated by IL-15 and to be able to release considerable
amounts of IFNγ [77,95,206,207]. In its turn, recombinant trout IFNγ was found to enhance the
expression of the i1 cytokine genes IL-15 and IL-12A in trout macrophage and fibroblast cell lines [204]
and in Atlantic salmon head kidney cells [208], respectively. Because under similar conditions some
other trout or Atlantic salmon cytokines were not upregulated [204,205,208], this suggests that also in
fish the IFNγ, IL-15 and IL-12 molecules cooperate in type 1 immunity. Moreover, fish IFNG was found
upregulated by viral dsRNA mimic polyI:C, under conditions in which some other cytokine genes were
not upregulated [202]. In mammals poly(I:C) is known to specifically stimulate type 1 immunity [209]. In
zebrafish spleen and head kidney poly(I:C) was found to enhance transcription of T-bet, while in the same
experiments STAT6 appeared to be downregulated [130]. Direct correlations between fish IFNγ addition
and a transcription factor were reported for STAT1, with Zou et al. [120] reporting upregulation of STAT1
expression in a trout macrophage cell line, and Yabu et al. [210] reporting the induction of human
STAT1 phosphorylation in a human cell line transfected for expression of ginbuna crucian carp IFNγ
receptor chain. STAT4 has hardly been investigated in fish. In summary, possible existence in fish of a
mammalian-like TH1 transcriptional regulation network has not been clarified yet, but the available
fragmentary data do agree with its existence.
A very important feature of type 1 immunity is that it suppresses the other types of immunity, and
vice versa. Indirect indications for such suppression is that, similar as in mammals, expression of fish
IFNG and IL-4/13A can be found up- or down-regulated in opposing manners [166,202]. Furthermore,
under conditions in which recombinant trout IFNγ enhanced the expression of IL-12A in Atlantic salmon
head kidney cells, the expression of some IL-17A/F family genes was slightly downregulated [208].
In conclusion, specific co-regulation of factors important for mammalian type 1 immunity suggests
the existence of a basically similar i1-system in fish. However, because with the establishment of
antibodies recognizing CD4 only recently it became more accessible to isolate (putative) fish helper T cells,
available evidence supporting the existence of fish TH1 cells is still very limited. Definite evidence of TH1

Biology 2015, 4 833
cells will require the establishment of long-term proliferation assays for fish helper T cells and investigations
of to which extent they can be polarized.
8. TH17-Like Responses in Teleost Fish
Important for the possibility of type 3 immunity is that fish do have heterophilic-neutrophilic
granulocytes (neutrophils). In mammals, IL-17A and IL-17F molecules induce the release of
abundant cytokines and chemokines from leukocytes and other cell types, which amongst others attract
neutrophils [211]. Mammalian neutrophils form an important and first line of defense against infiltrating
bacteria [212]. Like their mammalian counterparts, fish neutrophils have granules with the enzyme
peroxidase, they can phagocytose bacteria, they rapidly and extensively migrate to bacterial infection
sites, and have high bactericidal and respiratory burst capacities [213–215]. Furthermore, like mammalian
neutrophils, after their stimulation fish neutrophils can release DNA in the form of neutrophil extracellular
traps (NETs) [216].
Fish have several genes of the IL-17A + IL-17F family, which are called IL-17A/F followed by
a number [217]. Fish IL-17A/F genes can be expressed by CD4-1 positive cells [195,202]. To our
knowledge there is only one study that investigated fish IL-17A/F at the protein level, namely in grass
carp by Du et al. [218]. Du et al. found that recombinant grass carp IL-17A/F stimulates expression of
the genes for the cytokines IL-1β, IL-6 and TNF-α and the chemokine CXCL-8 (alias IL-8) in head kidney
cells. Other studies have shown that in fish CXCL-8, like in humans, can recruit neutrophils [219,220].
Hence, it is likely that fish IL-17A/F can induce neutrophil recruitment, although there is no direct
evidence at the protein level for that in fish yet.
However, a study by Ribeiro et al. [221] provides supportive evidence at the gene expression level
that also in fish IL-17A/F probably is involved in neutrophil recruitment. Ribeiro et al. [221] compared
the infections in common carp of the related protozoan parasites Trypanoplasma borreli and Trypanoplasma
carassii, which cause quite different patterns of disease development. At a time-point of infection in
which T. borreli induced higher levels of IFNγ expression than induced by T. carassii, only T. carassii
induced enhanced IL-17A/F expression, which was accompanied by a marked neutrophil infiltration into
the spleen of only T. carassii-infected fish. Ribeiro et al., furthermore showed that factors derived from
these parasites could ex vivo stimulate expression of IL-23A in head kidney leukocytes from parasite-infected
fish, and that these same factors could efficiently interact with carp toll-like receptor 2 (TLR2) molecules
expressed on a human cell line [221]. Hence, for their T. carassii-infected carp model, the authors
postulated a “TH17-like immune response” model involving the stimulation cascade T. carassii
-TLR2-IL23-TH17-IL17A/F-neutrophil. We are not aware of other studies in fish that suggest a correlation
between IL-23 and IL-17A/F expression.
In Atlantic salmon head kidney cells, under conditions in which recombinant trout IL-1β induced
large increases in expression of the pro-inflammatory cytokine genes IL-1B and TNFA, the expression
of IL-17A/F genes remained unchanged [208]. In the same study recombinant trout IFNγ stimulated the
expression of i1-signature gene IL-12A, while slightly reducing the expression of IL-17A/F genes [208].
In contrast, in this type of experiments, recombinant trout IL-21 was found to enhance expression of
IL-17A/F genes [208], as well as of IL-22 [222], suggesting specific involvement of IL-21 in TH17
polarization as known in mammals.

Biology 2015, 4 834
An antibacterial function of fish IL-22 was indicated by IL-22 upregulation induced in several fish
species through bacterial agents, by the stimulation through recombinant IL-22 of antimicrobial peptide
synthesis in trout and mullet, by the protection of mullet and turbot against bacterial challenge after
injection with recombinant IL-22, and by a decreased resistance against bacterial challenge after IL-22
knockdown in zebrafish embryos [170,223–226]. In zebrafish IL-22 can be expressed by CD4-1 positive
cells [195], and at the tissue level there are a few studies suggesting some correlation between fish IL-22
and IL-17A/F expression ([170]; Table 1).
In our opinion, there are no convincing reports in fish yet linking expression of TH17 signature
cytokine genes to RORC expression.
In summary, from the available evidence it seems likely that also in fish the molecules IL-17A/F, IL-21
and IL-22 (and possibly IL-23) can be orchestrated in an anti-bacterial defense response that involves
recruitment of neutrophils by IL-17A/F-induced chemokine expression. But it is unclear whether this
response also involves RORγ(t) and/or TGF-β molecules, and/or the TH17-like polarization of helper T
cells. We expect that research of fish TH cell polarizations will first concentrate on the possibility of
i1-i2 axis end polarizations, TH1 and TH2, because in mammals these are so pronounced and well-defined,
before possible TH17 polarization will be convincingly addressed.
9. Treg-like Responses in Teleost Fish
Studies have shown that if fish are fed or otherwise treated with antigen preparations that lack PAMPS
or DAMPS, their immune system can develop some level of tolerance against these antigens
(e.g., [227,228]). In mammals immune tolerance is importantly mediated by natural or induced Treg cells,
for which FOXP3 is a master transcription factor and which are immunosuppressive by a variety of
means, including the release of the cytokines IL-10 and TGF-β [229,230].
Fish FOXP3, TGFB1 and IL-10 genes can be expressed by CD4-1 positive cells [202]. Wen et al. [231]
showed that an unknown percentage of the Tetraodon lymphocyte population positive for CD4-2 and
IL-15Rα molecules also expressed TCRα and FOXP3. Fish IL-15Rα is a receptor chain that can bind
IL-2 as well as IL-15 and, although it looks like mammalian IL-15Rα, it corresponds to the evolutionary
precursor form of both mammalian IL-2Rα and IL-15Rα [161,231]. Under non-stimulated conditions,
in mammals, Treg cells are the cell type expressing the highest amount of IL-2Rα and are the most sensitive
to IL-2 [232,233]. The fact that Wen et al. [231] didn’t detect FOXP3 expression in the CD4-2+IL-15Rα−
cells suggests that also in fish IL-2 may have an important function in a negative feedback loop of
immune reactions through activation of Treg (-like) cells [161,234]. Wen et al. [231] reported
immunosuppressive functions of Tetraodon CD4-2+IL-15Rα+ cells observed during in vitro and in vivo
experiments. The in vitro experiments showed that if CD4-2+IL-15Rα+ cells were depleted from Tetraodon
spleen and head kidney leukocytes, the remaining cell population became more effective in executing
non-specific cell-mediated cytotoxicity and in inducing mixed lymphocyte reactions in the respective
assays [231]. Repeated in vivo treatment of Tetraodon with rabbit antibodies binding to Tetraodon IL-15Rα
resulted in bowel inflammation [231], which the authors interpreted as deriving from a depletion of Treg
cells, a model for which they provided some evidence but which may need more investigation.
Quintana et al. [235] found that zebrafish FOXP3 (FOXP3a) was expressed by lymphocytes, and
that in zebrafish embryos overexpression or knockdown of FOXP3 resulted in decreased vs. increased

Biology 2015, 4 835
IL-17A/F expression, respectively [235]; this perfectly agrees with FOXP3 involvement in immunosuppressive
functions as known in mammals. Quintana et al. [235] also showed that zebrafish FOXP3 retained its
capacity to induce Treg-like features upon expression in mammalian cells, because murine T cells
transfected with zebrafish FOXP3 were found to suppress activation of other murine T cells.
In goldfish monocytes recombinant IL-10 suppressed the immune response induced by bacterial
agents as indicated by reduced increase of expression of genes for TNF-α, IL-10, CXCL-8, IFNγ and
several NADPH oxidase subunits, as well as by a reduced increase in production of reactive oxygen
intermediates [236]. Similar results were found for carp, where recombinant IL-10 was shown to reduce
the expression increase induced by LPS in neutrophils and/or macrophages of genes for TNF-α, IL-1β,
IL-6, IL-12A, MHC class I, and MHC class II molecules [237]. Piazzon et al. [237] also found that carp
IL-10 could induce STAT3 phosphorylation, implying similar signaling cascades as in mammals. It is
of note that Piazzon et al. [237] also revealed that carp IL-10 does not only have immunosuppressive
functions, but, like known for multifunctional mammalian IL-10, also has some stimulatory and modifying
effects on the immune system, like the stimulation of proliferation, differentiation, and antibody secretion
by IgM+ B cells.
In regard to TGF-β, it is difficult to distinguish clear regulation patterns from the number of studies
that investigated fish TGFB1 expression. We therefore decided not to try to summarize those studies.
However, it is important that an immunosuppressive function was found for recombinant goldfish TGF-β,
as it was shown to down-regulate the nitric oxide response of TNF- α-activated macrophages [238].
In summary, FOXP3 in fish has been associated with immunosuppressive functions, and, at least in
Tetraodon, FOXP3 is expressed by CD4 positive T cells that constitutively express high levels of IL-2
receptor chain. This suggests the existence of a Treg system in fish similar to that found in mammals. Both
fish IL-10 and TGF-β were found to have immunosuppressive functions. It will need further investigation
whether in fish, as known in mammals, IL-10 and TGF-β are associated with FOXP3 positive cells.
10. TH2-like Responses in Teleost Fish
For the possibility of raising TH2-like responses, it is relevant that fish do have granulocytes other
than neutrophils which have anti-parasite functions. Whether (some of) these cells can be called
eosinophils, basophils and/or mast cells depends on the chosen definition and on the fish species. Our
use of the terms eosinophils and mast cells in the text below is based on the definitions in the indicated
references. Similar to mammalian eosinophils, fish eosinophils express transcription factor GATA-2, and
can migrate to sites of parasite infection and release their peroxidase containing granules upon stimulation
by parasite agents [239,240]. Fish mast cells, which are abundant in the gill and the intestine, can also
accumulate and degranulate at the site of parasitic infection [239–243]. Similar but not identical to
mammals, the granules of fish mast cells contain phosphatases, peroxidase, proteolytic enzymes,
arylsulfatase, 5'-nucleotidase, lysozyme, antimicrobial peptides, and, depending on the species, they can
contain serotonin or histamine [241–244].
Fish GATA-3, IL-4/13-A and -B genes can be expressed by CD4-1 positive cells [195,199,202].
Chettri et al., found that if rainbow trout skin was infected with the parasitic flagellate Ichthyobodo
necator, locally there was substantial increase in GATA-3 but not of FOXP3 or T-bet expression,
concomitant with a substantial decrease in the number of CD8α+ cells and a substantial increase in IgM+

Biology 2015, 4 836
B cells [245]. This might represent a TH2 response, as suggested by the authors, although all investigated
cytokine genes including IFNG were found upregulated. More convincing of a TH2 response are
conditions in which IL-4/13A expression increases while IFNG expression decreases, as could be found
in experiments analyzing (cells of) trout gill [166,246]. Very interestingly, because it suggests that fish
TH2 responses are involved in anti-parasite immunity, the infection of salmon with the parasite
Paramoeba perurans enhanced the expression in infected gill of TH2 signature genes IL-4/13A and
IL-4/13B while the expression of signature genes for TH1, TH17 and Treg like IFNG, IL-17A/F, TGFB1
and IL-10 were downregulated [246]. An opposite regulation of TH1 and TH2 signature genes was also
found by Zhu et al. [247] who showed that injection into zebrafish of recombinant IL-4/13A resulted in
an increase in expression of GATA-3 and STAT6 in the spleen, while concomitantly the expressions of
T-bet and IFNG were decreased; curiously, the authors did not check the effect on IL-4/13-A or -B
expression. Regarding opposite regulations an important observation is also that injection into zebrafish
of recombinant zebrafish “IL-4” (probably IL-4/13A) induced expression of CD209 in blood leukocytes,
while addition of LPS to the IL-4 preparation caused a reduction in the CD209 increase [248]. For carp we
established a clonal (semi-) permanent CD4-1+TCRαβ+ T cell line that expresses readily detectable
amounts of GATA-3 but not of T-bet, thus has a TH2-like profile in regard to its transcription factors [199].
In agreement with TH2-like polarization, this cell line lost the ability to increase its IFNG expression
after suitable stimulation while it retained an ability for upregulation of IL-4/13B; curiously, we were
unable to increase IL-4/13A expression in this cell line [199]. It remains to be determined whether the carp
cell-line phenomenon represents an artefact introduced by prolonged in vitro culture, or that not all fish
TH2-like cells can make significant amounts of IL-4/13A.
An important finding by Zhu et al. [247] analyzing recombinant zebrafish IL-4/13A was that the
cytokine can bind to receptor chain IL-4Rα. The same study also provided evidence that zebrafish IgM+
B cells specifically express IL-4/13Rα and that they can be stimulated by recombinant zebrafish IL-4/13A,
although the extent of the specificity was not investigated [247]. In mammals the stimulation of B cell
activity is not restricted to i2-skewed conditions, but mammalian IL-4 is one of the molecules that can
efficiently stimulate B cell proliferation and the molecule was originally named “B cell growth factor” [249].
Thus although the B cell stimulation by zebrafish IL-4/13A does not provide direct evidence of a TH2
function, it does provide additional evidence that IL-4/13 functions in fish are similar to those of their
mammalian counterparts.
In summary, there is accumulating evidence that in fish the expressions of GATA-3 and IL-4/13A are
correlated, and that their expression suppresses the expression of TH1 signature genes. Although the
regulation mechanisms in fish have not been elucidated yet, it seems likely that fish T cells can polarize
into a TH2 phenotype by mechanisms similar to those in mammals. Fish IL-4/13A has been shown to
stimulate B cells, but it still needs to be investigated whether fish IL-4/13A can stimulate typical i2
functions such as anti-parasite activities of eosinophils and mast cells.
11. M1-like vs. M2-Like Macrophage Polarizations in Fish
Like mammals, fish have macrophages with potent phagocytic and bactericidal abilities that make
use of reactive oxygen and nitrogen intermediates (reviewed by [250]). Also, like in mammals, zebrafish
macrophages are found in healing wounds [251–253] and are important for normal tissue regeneration [254].

Biology 2015, 4 837
The involvement in both M1-like and M2-like functions opens the possibility of differential polarization
towards those functions. As listed below, there is some evidence that fish macrophages can polarize
towards M1- or M2-like phenotypes through similar pathways as known in mammals. The best review
on that has probably been published by Forlenza et al. [255], who importantly realized that also in studies
on fish M2 macrophages it is necessary to conceptually distinguish between M2a (alias “alternatively
activated”) and M2c (alias “deactivated”, or, as Forlenza et al., not unreasonably call it, “regulatory”)
polarizations. For lack of solid polarization data, Forlenza et al. [255] developed a working definition,
based on how fish macrophages were stimulated, to divide them into four “polarization states”, akin to as
how this has been accepted by some researchers studying mammalian macrophages (e.g., [92]) as
a simplification of the classification system by Mantovani et al. [60]. Forlenza et al. [255] defined the
above mentioned M2a and M2c polarizations as those deriving from stimulation with IL-4/13 and from
stimulation with microbial agent + IL-10, respectively. At the far i1-end of the polarization spectrum,
Forlenza et al. [255] conventionally considered macrophages stimulated by both microbial agents plus
IFNγ as “classically activated” (M1). The definition by Forlenza et al., which may not hold in the long
term, but which is practically convenient and seems to define macrophages only somewhat shifted
towards the i1-end (not unlike the mammalian M2b macrophages; [60,92]), concerns “innate activated
macrophages” (“iaM”) which are stimulated with only microbial agents and not with IFNγ. Similar to
mammalian M1 and M2b, fish M1 and iaM express iNOS (reviewed by [255]).
Although not in all cases studied as one among more possible polarizations, there is evidence that M1
phenotypes can be induced by similar agents as in mammals. In synergy with LPS, carp IFNγ was found
to stimulate carp macrophages into expressing higher levels of IL-12A and TNFA [256]. Furthermore,
recombinant rainbow trout IFNγ plus some LPS enhanced respiratory burst activity of rainbow trout
macrophages [120], and recombinant goldfish IFNγ, said to be without LPS contamination, primed
goldfish monocytes/macrophages for enhanced respiratory burst, phagocytic and nitric oxide responses,
while it also stimulated their expression of genes for TNF-α, IL-1β, IL-12α, IL-12β and iNOS [257,258].
Likewise in agreement with M1 differentiation as known in mammals, recombinant goldfish TNF-α
could also prime goldfish monocytes/macrophages for enhanced respiratory burst, phagocytic and nitric
oxide responses [257,259].
There appears to be little direct evidence for the existence of M2c (“Mreg”) polarization of fish
macrophages, but recombinant IL-10 or TGF-β were found able to reduce M1-type macrophage
activations [236,238].
In carp differential polarizations of macrophages using LPS vs. cAMP stimulation have been shown [260]:
while neither LPS nor cAMP stimulated IL-10 expression and both stimulated IL-1B expression, only
LPS stimulated NOS2 (the gene for iNOS) and only cAMP stimulated ARG2 (the gene for arginase 2)
expression. However, although in mammals cAMP is one of the factors contributing to an i2 environment [21],
we are somewhat hesitant to accept the isolated addition of cAMP as an inducer of a natural M2 (-like)
polarization. We would also like more research to be done before concluding that in fish arginase 2
and not arginase 1 is a major marker for M2 differentiation (for a discussion on fish arginase genes
see [260] and [255]).
One of the tasks of macrophages is the removal of cell debris, and in mammals apoptotic bodies are
known to stimulate an M2 phenotype [261,262]. Zymosan is a glucan-rich particle prepared of the
surface of fungi, which in mammals can induce pro-inflammatory responses and in synergy with other

Biology 2015, 4 838
factors can stimulate M1 polarization [263,264]. In vitro analysis of goldfish macrophages showed that
their respiratory burst activity induced by PMA treatment was enhanced by incubation with zymosan
and reduced by incubation with apoptotic bodies [265]. Injection of zymosan or apoptotic bodies in the
goldfish peritoneal cavity, followed by isolation of myeloid cells and analysis of their ability to generate
respiratory burst responses, showed that the in vivo treatment had a similar effect on priming for
respiratory burst activity as the above described in vitro treatments [265].
An interesting study was recently published by Nguyen-Chi et al. [253], who used a double
fluorescent labeling system for zebrafish macrophages (using the MPEG1 promoter) and TNF-α
expression (using the TNFA promoter). They found at the population level that TNFA expression by
zebrafish macrophages positively correlated with their expression of IL-1B and IL-6, and negatively
correlated with their expression of TGFB1 and CXCR4. In mammals both TGFB1 and CXCR4 have been
used as markers for M2 macrophages [266], and Nguyen-Chi designated the TNFA-high macrophages
as “M1-like” and the TNFA-low macrophages as “M2-like”. Nguyen-Chi et al. [253] also found that in
wounded fin of zebrafish larvae the TNFA-high macrophages tended to display a flattened and lobulated
morphology, whereas the TNFA-low macrophages tended to be elongated and dendritic. Other than
creating a wound by fin amputation, Nguyen-Chi et al. [253] also inoculated zebrafish larvae with E.
coli. Based on the abundances of the different macrophages at the relative sites in these experiments,
Nguyen-Chi et al. [253] concluded that zebrafish M1-like macrophages are important in anti-bacterial
combat and initial stages of wound healing and that M2-like macrophages are important in especially
the later stages of wound healing. Nguyen-Chi et al. [253] furthermore showed that in later stages of
wound healing the TNFA-positive (M1-like) macrophages changed towards a phenotype which they call
intermediate to M1 and M2 and which shows high TGFB1 expression besides lowered TNFA expression.
In summary, fish macrophages can be stimulated towards several phenotypes, and at least the M1
phenotype seems to be defined by similar pathways and characteristics as in mammals. The fish
macrophage non-M1 polarizations are not well characterized and appear to be predominantly defined by
reduced M1 features and maybe by the upregulation of ARG2. Hopefully this gap in knowledge on
possible non-M1 polarizations can be closed by future inclusion of recombinant fish IL-4/13 cytokines
in the macrophage polarization assays.
12. I2-Skewed Tissue Milieus in Healthy Mammals and Fish
For fish this paragraph has an overlap with paragraph 5, but we nevertheless like to dedicate a special
paragraph to the comparison between fish and mammals.
Previously we reported that trout and salmon gill and skin appear to have i2-skewed milieus since we
observed rather consistent high ratios of IL-4/13A plus GATA3 vs. IFNG expression [166]. We found
similar high ratios for the thymus of trout, salmon and mouse, but it is discussable whether a primary
immune organ with its unique immune functions can be classified as “i2-skewed” [166]; on the other
hand, it was found in mammals that recent thymic emigrants have a bias towards TH2 polarization [267],
thus at least in some sense the thymus can be seen as “i2-skewed”. High levels of IL-4/13A and/or GATA3
in fish gill were also found by others [121,167,168], and we are glad that in the present paper we could
additionally confirm these findings by SRA dataset analysis for several teleost fish and also for elephant

Biology 2015, 4 839
shark (see paragraph 5). We speculate, as before [166], that i2-skewage of the fish gill helps to protect
it against parasites, but also against possible i1- or i3- type inflammation that might harm this delicate
tissue. There are some data indeed that indicate that it is hard to induce an i1-response in fish gill [166,268],
but other studies suggest that it is possible to induce i1- or i3- responses in this tissue (e.g., [269]); more
research will be needed to clarify the degree of i2-skewage of fish gills.
Our idea that a sensitive fish tissue like gills may be i2-skewed for its protection from other types of
immune reactions actually derives from similar claims made in mammals for the immunity of
pregnancies [270–273] and neonates [33,34,274]. Whether these claims for mammals are actually true,
however, has, at least in a general sense, been disputed [275], and precise locations, conditions and
measured parameters should probably be acknowledged.
We did some preliminary analysis of tissue-specific transcriptomes for mammals available in public
databases to assess the expression levels of i1-i2 axis marker genes, similar to the method followed to
make Table 1, but could not distinguish notable expression patterns (data not shown). However, in
mammals pronounced tissue-specific distinctions were observed by others who investigated the i1-i2
axis positioning of individual cell types. It is intriguing, for example, that IL-13 secreting ILC2 cells can
readily be found in the mouse lung and skin, while these tissues have hardly any ILC3 cells [276]; in
a way this is reminiscent of the i2-skewage observed in fish skin and respiratory tissue (gill). For the
mouse healthy intestine an opposite ratio is found, with abundant ILC3 cells and relatively few ILC2
cells [276]; this agrees well with the facts that also in fish this mucosal tissue is not i2-skewed, and that
in some fish including elephant shark the intestine may be i3-skewed.
Overall, however, it is surprising to us how little work there seems to have been done in mammals to
analyze non-diseased tissues for their immune biases along the i1-i2 axis. It seems to us that this should
be important information when considering how and where to administer vaccines or therapeutic agents.
13. I2-Skewed Tissue Milieus of Tumors in Mammals and Fish
The only mammalian “tissues” for which i2-skewage has been intensively studied and generally
accepted as proven are a variety of tumors. The i2-skewage protects the tumors from eradication by type 1
immunity, and immunotherapy shifting the tumor immune milieu towards type 1 immunity has been
shown helpful in fighting the cancer. The champion results so far are obtained by antibodies that can
block the immunosuppressive functions of the molecules PD-1 and CTLA-4, but many other methods
to induce a shift towards i1-immunity are being tried [116,117,277].
It is important to realize that in tumor studies the term “type 2 immunity” tends to be used for
a combination of immunosuppressive (“Treg-type”) and i2 inflammation (“TH2-type”) conditions, and
that among these the immunosuppressive conditions probably are more pronounced. The difficulty with
changing this immune milieu by therapy is that the cancer cells and the i2-skewed immune cells
reciprocally attract/support each other, so that effects of therapeutic i1-stimuli tend to be undone once
the stimuli fade out after administration.
An example of a situation in which tumor cells and infiltrating immune cells support each other is
found in human and rodent pancreatic ductal adenocarcinoma (PDA). In rodent models, expression of
mutant KRAS oncogene in pancreatic ductal epithelial cells is sufficient to induce their cancerous
proliferation and their expression of factors like STAT3, NF-κB and IL-6 that form part of a (self-)

Biology 2015, 4 840
amplifying loop [278–280]. These tumor cells release abundant amounts of chemokine CCL2 which
attracts monocytes that within the tumor stroma develop into M2 macrophages [281,282]. The tumor is
also invaded by abundant lymphocytes, including many TH2 and Treg cells, while few are TH1 or CD8+
T cells [282–284]. Despite abundant infiltration with immune cells, in a rat PDA model the expressions of
the i1-markers CD8B, IL-15 and granzyme-C were found to be 2-, 5- and 5-fold lower than in healthy rat
pancreas [285]. Meanwhile, in these rat PDA samples, the expressions of i2 markers TGFB1 and IL-33
were found to be 14- and 18-fold higher compared to healthy rat pancreas [285]. Experiments in a mouse
PDA model have shown that the cancer cells and M2 macrophages reciprocally support and stimulate
each other [286].
Unfortunately in fish the immunology of tumor microenvironments has hardly been studied yet, but
there is a nice study by Yan et al. [287] which indicate that also in fish the progress of tumors can be
enhanced by i2 conditions. Yan et al. [287] found that when they induced mutant KRAS expression in
zebrafish hepatocytes, this resulted in rapid recruitment of (fluorescently labeled) neutrophils to the liver
area and in hepatocarcinogenesis. The experimental results of independent knockdowns of GCSFR and
IRF8 let the authors conclude that the infiltrating neutrophils enhanced carcinoma growth. By using
specific stimulators or inhibitors of neutrophils, they concluded that the neutrophils stimulated
proliferation of the mutant KRAS expressing hepatocytes, while reducing their apoptosis. They found
that the nucleus morphology of the infiltrating zebrafish neutrophils resembled that of tumor associated
neutrophils in mammals and that they displayed a modified cytokine gene expression profile, which they
speculated, based on high IL-1B expression, to support angiogenesis. Yan and co-workers [287] also
found that the mutant KRAS expressing hepatocytes expressed increased amounts of TGFB1 (TGFB1a), and
that blocking of TGF-β pathways reduced both the number of neutrophils and carcinoma growth, and
changed the cytokine gene expression pattern of the tumor-associated neutrophils. While some important
questions still remain to be answered in the zebrafish model provided by Yan et al. [287], their study
allows the important conclusion that also in fish tumor development can be supported by TGF-β
expression and by infiltration of immune cells that adapt their phenotypes under influence of TGF-β.
In summary, considering the enormous medical importance of the immune milieus of tumors, it is
surprising how little this matter has been studied in fish. However, the first results appear to confirm that
like in mammals, tumor growth in fish can be stimulated by i2 cytokines and by interaction with
leukocytes. We expect that soon many more studies on the immune milieus of tumors in fish will
be performed.
14. Conclusions and Future Prospects
In the present study we have tried to summarize the i1-i2 axis and its effect on leukocyte polarizations
in mammals in a model (Figure 1) that reflects our interpretation of literature consensus. Although not
unique, the difference from most existing models is the stressing of the continuity between the different
conditions and their associated leukocyte polarizations, in combination with the placement of the i3 and
“Treg-type” conditions/polarizations between the i1 and “TH2-type” conditions/polarizations. We feel we
need this type of model to be able to compare between polarizations of different cell types and beyond
species borders, and that at the very least our model is a good starting point for discussion. The Figure 1
model automatically leads to the question whether it wouldn’t be better if the term “type 2 immunity”

Biology 2015, 4 841
would be split up into Treg-type (“regulatory immunity” alias “i-reg”?) and a narrower definition of type
2 immunity (TH2-type). However, such change of nomenclature would need a thorough discussion on how
entangled Treg-type and TH2-type conditions are, and whether the change of nomenclature would reduce
or increase the confusion.
The strongest evidence that the fish immune system obliges to similar i1-i2 axis principles as known
in mammals comes from the remarkable conservation of many of the most important gene loci. But
beyond that, recent years have also seen an accumulation of functional data that support that fish
leukocytes respond to i1-i2 axis factors in a similar way as they do in mammals. Although these functional
data are still fragmentary, when considered together they are rather convincing. In future research of the
i1-i2 axis regulation of the fish immune system it hopefully will become more commonplace to
simultaneously investigate multiple possible polarizations, so that they can be compared directly.
Furthermore, it should be attempted to maintain long-term T cell cultures under polarizing conditions,
followed by analysis of epigenetic modifications and of the stability of the polarized phenotype. In the
short term we expect the biggest breakthroughs from the research of fish macrophage polarizations,
because a lot of good work has been done already. For research of the tumor microenvironment
(transparent) fish appear to be a great model, and like in mammals, it can be expected that a lot of
i1-i2 research in fish will be dedicated to tumor tissues.
Besides the general discussion on the evolution of the i1-i2 axis, very interesting points of the present
study are the findings that throughout bony as well as cartilaginous fish the gills appear to be i2-skewed,
and that with analysis of the spotted gar genome sequence a canonical type TH2 locus was found in bony
fish that harbors RAD50 as well as genes of both IL-4/13 and IL-3/IL-5/GM-CSF families. Future
functional research should help to clarify the identity of the IL-5fam? sequences in teleost fish.
Acknowledgments
This work was supported by JSPS KAKENHI Grant Number 22580213 for Johannes M. Dijkstra,
and by the EU FP7 grant 311993 (TargetFish) for Takuya Yamaguchi and Uwe Fischer.
Author Contributions
Takuya Yamaguchi and Fumio Takizawa analyzed transcriptome databases. Johannes M. Dijkstra
wrote the manuscript scaffold. Takuya Yamaguchi, Fumio Takizawa, Uwe Fischer and Johannes M. Dijkstra
all investigated literature on the fish immune system and together wrote the current form of
the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Ceredig, R.; Rolink, A.G.; Brown, G. Models of haematopoiesis: Seeing the wood for the trees.
Nat. Rev. Immunol. 2009, 9, 293–300.

Biology 2015, 4 842
2. Akirav, E.M.; Alonso-Gonzalez, N.; Truman, L.A.; Ruddle, N.H. Lymphoid Tissues and Organs.
In Fundamental Immunology; Paul, W.E., Ed.; Lippincott Williams and Wilkins: Philadelphia, PA,
USA, 2012; pp. 47–65.
3. Ikawa, T. Genetic and epigenetic control of early lymphocyte development. Curr. Top. Microbiol.
Immunol. 2014, 381, 1–20.
4. Fields, P.E.; Lee, G.R.; Kim, S.T.; Bartsevich, V.V.; Flavell, R.A. Th2-specific chromatin remodeling
and enhancer activity in the Th2 cytokine locus control region. Immunity 2004, 21, 865–876.
5. Ansel, K.M.; Djuretic, I.; Tanasa, B.; Rao, A. Regulation of Th2 differentiation and Il4 locus
accessibility. Annu. Rev. Immunol. 2006, 24, 607–656.
6. Wilson, C.B.; Rowell, E.; Sekimata, M. Epigenetic control of T-helper-cell differentiation. Nat. Rev.
Immunol. 2009, 9, 91–105.
7. Mosmann, T.R.; Cherwinski, H.; Bond, M.W.; Giedlin, M.A.; Coffman, R.L. Two types of murine
helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted
proteins. J. Immunol. 1986, 136, 2348–2357.
8. Mosmann, T.R.; Coffman, R.L. TH1 and TH2 cells: Different patterns of lymphokine secretion lead
to different functional properties. Annu. Rev. Immunol. 1989, 7,145–173.
9. Heinzel, F.P.; Sadick, M.D.; Holaday, J.; Coffman, R.L.; Locksley, R.M. Reciprocal expression of
interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis.
Evidence for expansion of distinct helper T cell subsets. J. Exp. Med. 1989, 169, 59–72.
10. Murphy, E.; Shibuya, K.; Hosken, N.; Openshaw, P.; Maino, V.; Davis, K.; Murphy, K.; O’Garra, A.
Reversibility of T helper 1 and 2 populations is lost after long-term stimulation. J. Exp. Med. 1996,
183, 901–913.
11. Bird, J.J.; Brown, D.R.; Mullen, A.C.; Moskowitz, N.H.; Mahowald, M.A.; Sider, J.R.; Gajewski, T.F.;
Wang, C.R.; Reiner, S.L. Helper T cell differentiation is controlled by the cell cycle. Immunity
1998, 9, 229–237.
12. McGhee, J.R. The world of TH1/TH2 subsets: First proof. J. Immunol. 2005, 175, 3–4.
13. Lighvani, A.A.; Frucht, D.M.; Jankovic, D.; Yamane, H.; Aliberti, J.; Hissong, B.D.; Nguyen, B.V.;
Gadina, M.; Sher, A.; Paul, W.E.; et al. T-bet is rapidly induced by interferon- in lymphoid and
myeloid cells. Proc. Natl. Acad. Sci. USA 2001, 98, 15137–15142.
14. Afkarian, M.; Sedy, J.R.; Yang, J.; Jacobson, N.G.; Cereb, N.; Yang, S.Y.; Murphy, T.L.;
Murphy, K.M. T-bet is a STAT1-induced regulator of IL-12R expression in naïve CD4+ T cells.
Nat. Immunol. 2002, 3, 549–557.
15. Kaplan, M.H.; Schindler, U.; Smiley, S.T.; Grusby, M.J. Stat6 is required for mediating responses to
IL-4 and for development of Th2 cells. Immunity 1996, 4, 313–319.
16. Ouyang, W.; Ranganath, S.H.; Weindel, K.; Bhattacharya, D.; Murphy, T.L.; Sha, W.C.;
Murphy, K.M. Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent
mechanism. Immunity 1998, 9, 745–755.
17. Ouyang, W.; Löhning, M.; Gao, Z.; Assenmacher, M.; Ranganath, S.; Radbruch, A.; Murphy, K.M.
Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and
commitment. Immunity 2000, 12, 27–37.

Biology 2015, 4 843
18. Takemoto, N.; Kamogawa, Y.; Jun Lee, H.; Kurata, H.; Arai, K.I.; O’Garra, A.; Arai, N.; Miyatake, S.
Cutting edge: Chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific
cytokine gene cluster. J. Immunol. 2000, 165, 6687–6691.
19. Lee, G.R.; Kim, S.T.; Spilianakis, C.G.; Fields, P.E.; Flavell, R.A. T helper cell differentiation:
Regulation by cis elements and epigenetics. Immunity 2006, 24, 369–379.
20. Kidd, P. Th1/Th2 balance: The hypothesis, its limitations, and implications for health and disease.
Altern. Med. Rev. 2003, 8, 223–246.
21. Langston, H.P.; Ke, Y.; Gewirtz, A.T.; Dombrowski, K.E.; Kapp, J.A. Secretion of IL-2 and IFN-,
but not IL-4, by antigen-specific T cells requires extracellular ATP. J. Immunol. 2003, 170, 2962–2970.
22. Pulendran, B.; Artis, D. New paradigms in type 2 immunity. Science 2012, 337, 431–435.
23. Gause, W.C.; Wynn, T.A.; Allen, J.E. Type 2 immunity and wound healing: Evolutionary
refinement of adaptive immunity by helminths. Nat. Rev. Immunol. 2013, 13, 607–614.
24. Koscsó, B.; Csóka, B.; Kókai, E.; Németh, Z.H.; Pacher, P.; Virág, L.; Leibovich, S.J.; Haskó, G.
Adenosine augments IL-10-induced STAT3 signaling in M2c macrophages. J. Leukoc Biol. 2013,
94, 1309–1315.
25. Steinman, L. A rush to judgment on Th17. J. Exp. Med. 2008, 205, 1517–1522.
26. Annunziato, F.; Romagnani, C.; Romagnani, S. The 3 major types of innate and adaptive
cell-mediated effector immunity. J. Allergy Clin. Immunol. 2015, 135, 626–635.
27. Ouyang, W.; Kolls, J.K.; Zheng, Y. The biological functions of T helper 17 cell effector cytokines
in inflammation. Immunity 2008, 28, 454–467.
28. Yang, X.O.; Pappu, B.P.; Nurieva, R.; Akimzhanov, A.; Kang, H.S.; Chung, Y.; Ma, L.; Shah, B.;
Panopoulos, A.D.; Schluns, K.S.; et al. T helper 17 lineage differentiation is programmed by
orphan nuclear receptors ROR α and ROR. Immunity 2008, 28, 29–39.
29. Maloy, K.J.; Kullberg, M.C. IL-23 and Th17 cytokines in intestinal homeostasis. Mucosal Immunol.
2008, 1, 339–349.
30. Denning, T.L.; Norris, B.A.; Medina-Contreras, O.; Manicassamy, S.; Geem, D.; Madan, R.;
Karp, C.L.; Pulendran, B. Functional specializations of intestinal dendritic cell and macrophage
subsets that control Th17 and regulatory T cell responses are dependent on the T cell/APC ratio,
source of mouse strain, and regional localization. J. Immunol. 2011, 187, 733–747.
31. Goto, Y.; Panea, C.; Nakato, G.; Cebula, A.; Lee, C.; Diez, M.G.; Laufer, T.M.; Ignatowicz, L.;
Ivanov, I.I. Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive
mucosal Th17 cell differentiation. Immunity 2014, 40, 594–607.
32. Cohen, C.J.; Crome, S.Q.; MacDonald, K.G.; Dai, E.L.; Mager, D.L.; Levings, M.K. Human Th1
and Th17 cells exhibit epigenetic stability at signature cytokine and transcription factor loci.
J. Immunol. 2011, 187, 5615–5626.
33. Adkins, B.; Bu, Y.; Guevara, P. The generation of Th memory in neonates versus adults: Prolonged
primary Th2 effector function and impaired development of Th1 memory effector function in
murine neonates. J. Immunol. 2001, 166, 918–925.
34. Rose, S.; Lichtenheld, M.; Foote, M.R.; Adkins, B. Murine neonatal CD4+ cells are poised for
rapid Th2 effector-like function. J. Immunol. 2007, 178, 2667–2678.
35. Allen, J.E.; Sutherland, T.E. Host protective roles of type 2 immunity: Parasite killing and tissue
repair, flip sides of the same coin. Semin Immunol. 2014, 26, 329–340.

Biology 2015, 4 844
36. Foster, P.S.; Hogan, S.P.; Ramsay, A.J.; Matthaei, K.I.; Young, I.G. Interleukin 5 deficiency
abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model.
J. Exp. Med. 1996, 183, 195–201.
37. Wynn, T.A. Fibrotic disease and the TH1/TH2 paradigm. Nat. Rev. Immunol. 2004, 4, 583–594.
38. Wynn, T.A. Type 2 cytokines: Mechanisms and therapeutic strategies. Nat. Rev. Immunol. 2015,
15, 271–282.
39. Arora, M.; Chen, L.; Paglia, M.; Gallagher, I.; Allen, J.E.; Vyas, Y.M.; Ray, A.; Ray, P.
Simvastatin promotes Th2-type responses through the induction of the chitinase family member
Ym1 in dendritic cells. Proc. Natl. Acad. Sci. USA 2006, 103, 7777–7782.
40. Loke, P.; Gallagher, I.; Nair, M.G.; Zang, X.; Brombacher, F.; Mohrs, M.; Allison, J.P.; Allen, J.E.
Alternative activation is an innate response to injury that requires CD4+ T cells to be sustained
during chronic infection. J. Immunol. 2007, 179, 3926–3936.
41. Muallem, G.; Hunter, C.A. ParadYm shift: Ym1 and Ym2 as innate immunological regulators of
IL-17. Nat. Immunol. 2014, 15, 1099–1100.
42. Sutherland, T.E.; Logan, N.; Rückerl, D.; Humbles, A.A.; Allan, S.M.; Papayannopoulos, V.;
Stockinger, B.; Maizels, R.M.; Allen, J.E. Chitinase-like proteins promote IL-17-mediated neutrophilia
in a tradeoff between nematode killing and host damage. Nat. Immunol. 2014, 15, 1116–1125.
43. Sica, A.; Saccani, A.; Bottazzi, B.; Polentarutti, N.; Vecchi, A.; van Damme, J.; Mantovani, A.
Autocrine production of IL-10 mediates defective IL-12 production and NF-kappa B activation in
tumor-associated macrophages. J. Immunol. 2000, 164, 762–767.
44. Marie, J.C.; Letterio, J.J.; Gavin, M.; Rudensky, A.Y. TGF-β1 maintains suppressor function and
Foxp3 expression in CD4+CD25+ regulatory T cells. J. Exp. Med. 2005, 201, 1061–1067.
45. Deenick, E.K.; Tangye, S.G. Autoimmunity: IL-21: A new player in Th17-cell differentiation.
Immunol. Cell Biol. 2007, 85, 503–505.
46. Chaudhry, A.; Samstein, R.M.; Treuting, P.; Liang, Y.; Pils, M.C.; Heinrich, J.M.; Jack, R.S.;
Wunderlich, F.T.; Brüning, J.C.; Müller, W.; et al. Interleukin-10 signaling in regulatory T cells is
required for suppression of Th17 cell-mediated inflammation. Immunity 2011, 34, 566–578.
47. Wan, Y.Y.; Flavell, R.A. Regulatory T-cell functions are subverted and converted owing to
attenuated Foxp3 expression. Nature 2007, 445, 766–770.
48. Yang, X.O.; Nurieva, R.; Martinez, G.J.; Kang, H.S.; Chung, Y.; Pappu, B.P.; Shah, B.; Chang, S.H.;
Schluns, K.S.; Watowich, S.S.; et al. Molecular antagonism and plasticity of regulatory and
inflammatory T cell programs. Immunity 2008, 29, 44–56.
49. Lee, Y.K.; Turner, H.; Maynard, C.L.; Oliver, J.R.; Chen, D.; Elson, C.O.; Weaver, C.T. Late
developmental plasticity in the T helper 17 lineage. Immunity 2009, 30, 92–107.
50. Mukasa, R.; Balasubramani, A.; Lee, Y.K.; Whitley, S.K.; Weaver, B.T.; Shibata, Y.; Crawford, G.E.;
Hatton, R.D.; Weaver, C.T. Epigenetic instability of cytokine and transcription factor gene loci
underlies plasticity of the T helper 17 cell lineage. Immunity 2010, 32, 616–627.
51. Wang, Y.; Souabni, A.; Flavell, R.A.; Wan, Y.Y. An intrinsic mechanism predisposes
Foxp3-expressing regulatory T cells to Th2 conversion in vivo. J. Immunol. 2010, 185, 5983–5992.
52. Muranski, P.; Restifo, N.P. Essentials of Th17 cell commitment and plasticity. Blood 2013, 121,
2402–2414.

Biology 2015, 4 845
53. Obermajer, N.; Popp, F.C.; Soeder, Y.; Haarer, J.; Geissler, E.K.; Schlitt, H.J.; Dahlke, M.H.
Conversion of Th17 into IL-17Aneg regulatory T cells: A novel mechanism in prolonged allograft
survival promoted by mesenchymal stem cell-supported minimized immunosuppressive therapy.
J. Immunol. 2014, 193, 4988–4999.
54. Panzer, M.; Sitte, S.; Wirth, S.; Drexler, I.; Sparwasser, T.; Voehringer, D. Rapid in vivo conversion of
effector T cells into Th2 cells during helminth infection. J. Immunol. 2012, 188, 615–623.
55. Sawant, D.V.; Vignali, D.A. Once a Treg, always a Treg? Immunol. Rev. 2014, 259, 173–191.
56. Sakaguchi, S.; Vignali, D.A.; Rudensky, A.Y.; Niec, R.E.; Waldmann, H. The plasticity and stability
of regulatory T cells. Nat. Rev. Immunol. 2013, 13, 461–467.
57. Wang, Y.; Su, M.A.; Wan, Y.Y. An essential role of the transcription factor GATA-3 for the
function of regulatory T cells. Immunity 2011, 35, 337–348.
58. Hatton, R.D.; Weaver, C.T. Duality in the Th17-Treg developmental decision. F1000 Biol. Rep.
2009, doi:10.3410/B1-5.
59. Mathur, A.N.; Chang, H.C.; Zisoulis, D.G.; Stritesky, G.L.; Yu, Q.; O’Malley, J.T.; Kapur, R.;
Levy, D.E.; Kansas, G.S.; Kaplan, M.H. Stat3 and Stat4 direct development of IL-17-secreting Th
cells. J. Immunol. 2007, 178, 4901–4907.
60. Mantovani, A.; Sica, A.; Sozzani, S.; Allavena, P.; Vecchi, A.; Locati, M. The chemokine system
in diverse forms of macrophage activation and polarization. Trends Immunol. 2004, 25, 677–686.
61. Lombardo, E.; Alvarez-Barrientos, A.; Maroto, B.; Boscá, L.; Knaus, U.G. TLR4-mediated survival
of macrophages is MyD88 dependent and requires TNF-α autocrine signalling. J. Immunol. 2007, 178,
3731–3739.
62. Yarilina, A.; Park-Min, K.H.; Antoniv, T.; Hu, X.; Ivashkiv, L.B. TNF activates an IRF1-dependent
autocrine loop leading to sustained expression of chemokines and STAT1-dependent type I
interferon-response genes. Nat. Immunol. 2008, 9, 378–387.
63. Zizzo, G.; Hilliard, B.A.; Monestier, M.; Cohen, P.L. Efficient clearance of early apoptotic cells
by human macrophages requires M2c polarization and MerTK induction. J. Immunol. 2012, 189,
3508–3520.
64. Fadok, V.A.; Bratton, D.L.; Konowal, A.; Freed, P.W.; Westcott, J.Y.; Henson, P.M. Macrophages
that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through
autocrine/paracrine mechanisms involving TGF-β, PGE2, and PAF. J. Clin. Invest. 1998, 101,
890–898.
65. Waddington, C.H. The Strategy of the Genes; A Discussion of Some Aspects of Theoretical
Biology; Allen & Unwin: London, UK, 1957.
66. Rebhahn, J.A.; Deng, N.; Sharma, G.; Livingstone, A.M.; Huang, S.; Mosmann, T.R. An animated
landscape representation of CD4+ T-cell differentiation, variability, and plasticity: Insights into the
behavior of populations versus cells. Eur. J. Immunol. 2014, 44, 2216–2229.
67. Kaplan, M.H.; Hufford, M.M.; Olson, M.R. The development and in vivo function of T helper 9
cells. Nat. Rev. Immunol. 2015, 15, 295–307.
68. Glatman Zaretsky, A.; Taylor, J.J.; King, I.L.; Marshall, F.A.; Mohrs, M.; Pearce, E.J.
T follicular helper cells differentiate from Th2 cells in response to helminth antigens. J. Exp. Med.
2009, 206, 991–999.

Biology 2015, 4 846
69. Ueno, H.; Banchereau, J.; Vinuesa, C.G. Pathophysiology of T follicular helper cells in humans
and mice. Nat. Immunol. 2015, 16, 142–152.
70. Zhou, L.; Lopes, J.E.; Chong, M.M.; Ivanov, I.I.; Min, R.; Victora, G.D.; Shen, Y.; Du, J.;
Rubtsov, Y.P.; Rudensky, A.Y.; et al. TGF-β-induced Foxp3 inhibits TH17 cell differentiation by
antagonizing RORgammat function. Nature 2008, 453, 236–240.
71. Hatton, R.D. TGF-β in Th17 cell development: The truth is out there. Immunity 2011, 34, 288–290.
72. Gorissen, M.; de Vrieze, E.; Flik, G.; Huising, M.O. STAT genes display differential evolutionary rates
that correlate with their roles in the endocrine and immune system. J. Endocrinol. 2011, 209, 175–184.
73. Yu, H.; Pardoll, D.; Jove, R. STATs in cancer inflammation and immunity: A leading role for
STAT3. Nat. Rev. Cancer 2009, 9, 798–809.
74. Stritesky, G.L.; Muthukrishnan, R.; Sehra, S.; Goswami, R.; Pham, D.; Travers, J.; Nguyen, E.T.;
Levy, D.E.; Kaplan, M.H. The transcription factor STAT3 is required for T helper 2 cell development.
Immunity 2011, 34, 39–49.
75. Heltemes-Harris, L.M.; Farrar, M.A. The role of STAT5 in lymphocyte development and
transformation. Curr. Opin. Immunol. 2012, 24, 146–152.
76. Laurence, A.; Tato, C.M.; Davidson, T.S.; Kanno, Y.; Chen, Z.; Yao, Z.; Blank, R.B.; Meylan, F.;
Siegel, R.; Hennighausen, L.; Shevach, E.M.; O’shea, J.J. Interleukin-2 signaling via STAT5
constrains T helper 17 cell generation. Immunity 2007, 26, 371–381.
77. Waldmann, T.A. The biology of interleukin-2 and interleukin-15: Implications for cancer therapy
and vaccine design. Nat. Rev. Immunol. 2006, 6, 595–601.
78. Hazenberg, M.D.; Spits, H. Human innate lymphoid cells. Blood 2014, 124, 700–709.
79. Moro, K.; Koyasu, S. Innate lymphoid cells, possible interaction with microbiota. Semin.
Immunopathol. 2015, 37, 27–37.
80. Bernink, J.H.; Peters, C.P.; Munneke, M.; te Velde, A.A.; Meijer, S.L.; Weijer, K.;
Hreggvidsdottir, H.S.; Heinsbroek, S.E.; Legrand, N.; Buskens, C.J.; et al. Human type 1 innate
lymphoid cells accumulate in inflamed mucosal tissues. Nat. Immunol. 2013, 14, 221–229.
81. Turner, J.E.; Morrison, P.J.; Wilhelm, C.; Wilson, M.; Ahlfors, H.; Renauld, J.C.; Panzer, U.;
Helmby, H.; Stockinger, B. IL-9-mediated survival of type 2 innate lymphoid cells promotes
damage control in helminth-induced lung inflammation. J. Exp. Med. 2013, 210, 2951–2965.
82. Bernink, J.H.; Germar, K.; Spits, H. The role of ILC2 in pathology of type 2 inflammatory diseases.
Curr. Opin. Immunol. 2014, 31, 115–120.
83. Hepworth, M.R.; Fung, T.C.; Masur, S.H.; Kelsen, J.R.; McConnell, F.M.; Dubrot, J.; Withers, D.R.;
Hugues, S.; Farrar, M.A.; Reith, W.; et al. Group 3 innate lymphoid cells mediate intestinal
selection of commensal bacteria-specific CD4+ T cells. Science 2015, 348, 1031–1035.
84. Sad, S.; Marcotte, R.; Mosmann, T.R. Cytokine-induced differentiation of precursor mouse CD8+
T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines. Immunity 1995, 2, 271–279.
85. Harris, D.P.; Haynes, L.; Sayles, P.C.; Duso, D.K.; Eaton, S.M.; Lepak, N.M.; Johnson, L.L.;
Swain, S.L.; Lund, F.E. Reciprocal regulation of polarized cytokine production by effector B and
T cells. Nat. Immunol. 2000, 1, 475–482.
86. Fridlender, Z.G.; Sun, J.; Kim, S.; Kapoor, V.; Cheng, G.; Ling, L.; Worthen, G.S.; Albelda, S.M.
Polarization of tumor-associated neutrophil phenotype by TGF-β: “N1” versus “N2” TAN. Cancer
Cell. 2009, 16, 183–194.

Biology 2015, 4 847
87. Feili-Hariri, M.; Falkner, D.H.; Morel, P.A. Polarization of naive T cells into Th1 or Th2 by distinct
cytokine-driven murine dendritic cell populations: Implications for immunotherapy. J. Leukoc Biol.
2005, 78, 656–664.
88. Qin, H.; Holdbrooks, A.T.; Liu, Y.; Reynolds, S.L.; Yanagisawa, L.L.; Benveniste, E.N. SOCS3
deficiency promotes M1 macrophage polarization and inflammation. J. Immunol. 2012, 189, 3439–3448.
89. Biswas, S.K.; Mantovani, A. Macrophage plasticity and interaction with lymphocyte subsets:
Cancer as a paradigm. Nat. Immunol. 2010, 11, 889–896.
90. Messi, M.; Giacchetto, I.; Nagata, K.; Lanzavecchia, A.; Natoli, G.; Sallusto, F. Memory and
flexibility of cytokine gene expression as separable properties of human TH1 and TH2 lymphocytes.
Nat. Immunol. 2003, 4, 78–86.
91. Wu, C.Y.; Yang, H.Y.; Monie, A.; Ma, B.; Tsai, H.H.; Wu, T.C.; Hung, C.F. Intraperitoneal
administration of poly(I:C) with polyethylenimine leads to significant antitumor immunity against
murine ovarian tumors. Cancer Immunol. Immunother. 2011, 60, 1085–1096.
92. Shime, H.; Matsumoto, M.; Oshiumi, H.; Tanaka, S.; Nakane, A.; Iwakura, Y.; Tahara, H.; Inoue, N.;
Seya, T. Toll-like receptor 3 signaling converts tumor-supporting myeloid cells to tumoricidal
effectors. Proc. Natl. Acad. Sci. USA 2012, 109, 2066–2071.
93. Doherty, T.M.; Seder, R.A.; Sher, A. Induction and regulation of IL-15 expression in murine
macrophages. J. Immunol. 1996, 156, 735–741.
94. Ka, M.B.; Daumas, A.; Textoris, J.; Mege, J.L. Phenotypic diversity and emerging new tools to
study macrophage activation in bacterial infectious diseases. Front Immunol. 2014, doi:10.3389/
fimmu.2014.00500.
95. Kennedy, M.K.; Glaccum, M.; Brown, S.N.; Butz, E.A.; Viney, J.L.; Embers, M.; Matsuki, N.;
Charrier, K.; Sedger, L.; Willis, C.R.; et al. Reversible defects in natural killer and memory CD8
T cell lineages in interleukin 15-deficient mice. J. Exp. Med. 2000, 191, 771–780.
96. Edwards, J.P.; Zhang, X.; Frauwirth, K.A.; Mosser, D.M. Biochemical and functional
characterization of three activated macrophage populations. J. Leukoc. Biol. 2006, 80, 1298–1307.
97. Gerber, J.S.; Mosser, D.M. Reversing lipopolysaccharide toxicity by ligating the macrophage Fc
receptors. J. Immunol. 2001, 166, 6861–6868.
98. Anderson, C.F.; Mosser, D.M. Cutting edge: Biasing immune responses by directing antigen to
macrophage Fc receptors. J. Immunol. 2002, 168, 3697–3701.
99. Modolell, M.; Corraliza, I.M.; Link, F.; Soler, G.; Eichmann, K. Reciprocal regulation of the nitric
oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2
cytokines. Eur. J. Immunol. 1995, 25, 1101–1104.
100. Munder, M. Arginase: An emerging key player in the mammalian immune system. Br. J.
Pharmacol. 2009, 158, 638–651.
101. Whyte, C.S.; Bishop, E.T.; Rückerl, D.; Gaspar-Pereira, S.; Barker, R.N.; Allen, J.E.; Rees, A.J.;
Wilson, H.M. Suppressor of cytokine signaling (SOCS)1 is a key determinant of differential
macrophage activation and function. J. Leukoc Biol. 2011, 90, 845–854.
102. Stempin, C.C.; Dulgerian, L.R.; Garrido, V.V.; Cerban, F.M. Arginase in parasitic infections:
Macrophage activation, immunosuppression, and intracellular signals. J. Biomed. Biotechnol.
2010, doi:10.1155/2010/683485.

Biology 2015, 4 848
103. Miron, V.E.; Boyd, A.; Zhao, J.W.; Yuen, T.J.; Ruckh, J.M.; Shadrach, J.L.; van Wijngaarden, P.;
Wagers, A.J.; Williams, A.; Franklin, R.J.; et al. M2 microglia and macrophages drive
oligodendrocyte differentiation during CNS remyelination. Nat. Neurosci. 2013, 16, 1211–1218.
104. Kurowska-Stolarska, M.; Stolarski, B.; Kewin, P.; Murphy, G.; Corrigan, C.J.; Ying, S.; Pitman, N.;
Mirchandani, A.; Rana, B.; van Rooijen, N.; et al. IL-33 amplifies the polarization of alternatively
activated macrophages that contribute to airway inflammation. J. Immunol. 2009, 183, 6469–6477.
105. Nelson, M.P.; Christmann, B.S.; Werner, J.L.; Metz, A.E.; Trevor, J.L.; Lowell, C.A.; Steele, C.
IL-33 and M2a alveolar macrophages promote lung defense against the atypical fungal pathogen
Pneumocystis murina. J. Immunol. 2011, 186, 2372–2381.
106. Korenaga, H.; Kono, T.; Sakai, M. Isolation of seven IL-17 family genes from the Japanese
pufferfish Takifugu rubripes. Fish Shellfish Immunol. 2010, 28, 809–818.
107. Venkatesh, B.; Lee, A.P.; Ravi, V.; Maurya, A.K.; Lian, M.M.; Swann, J.B.; Ohta, Y.; Flajnik, M.F.;
Sutoh, Y.; Kasahara, M.; et al. Elephant shark genome provides unique insights into gnathostome
evolution. Nature 2014, 505, 174–179.
108. Welch, J.S.; Escoubet-Lozach, L.; Sykes, D.B.; Liddiard, K.; Greaves, D.R.; Glass, C.K. TH2
cytokines and allergic challenge induce Ym1 expression in macrophages by a STAT6-dependent
mechanism. J. Biol. Chem. 2002, 277, 42821–42829.
109. Pepe, G.; Calderazzi, G.; de Maglie, M.; Villa, A.M.; Vegeto, E. Heterogeneous induction of
microglia M2a phenotype by central administration of interleukin-4. J. Neuroinflammation. 2014,
doi:10.1186/s12974-014-0211-6.
110. Ehrchen, J.; Helming, L.; Varga, G.; Pasche, B.; Loser, K.; Gunzer, M.; Sunderkötter, C.; Sorg, C.;
Roth, J.; Lengeling, A. Vitamin D receptor signaling contributes to susceptibility to infection with
Leishmania major. FASEB J. 2007, 21, 3208–3218.
111. Hao, N.B.; Lü, M.H.; Fan, Y.H.; Cao, Y.L.; Zhang, Z.R.; Yang, S.M. Macrophages in tumor
microenvironments and the progression of tumors. Clin. Dev. Immunol. 2012, doi:10.1155/
2012/948098.
112. Lawrence, T.; Natoli, G. Transcriptional regulation of macrophage polarization: Enabling diversity
with identity. Nat. Rev. Immunol. 2011, 11, 750–761.
113. Thomas, A.C.; Mattila, J.T. “Of mice and men”: Arginine metabolism in macrophages. Front
Immunol. 2014, doi:10.3389/fimmu.2014.00479.
114. Sica, A.; Schioppa, T.; Mantovani, A.; Allavena, P. Tumour-associated macrophages are a distinct
M2 polarised population promoting tumour progression: Potential targets of anti-cancer therapy.
Eur. J. Cancer 2006, 42, 717–727.
115. Wynn, T.A.; Chawla, A.; Pollard, J.W. Macrophage biology in development, homeostasis and
disease. Nature 2013, 496, 445–455.
116. Fridlender, Z.G.; Jassar, A.; Mishalian, I.; Wang, L.C.; Kapoor, V.; Cheng, G.; Sun, J.; Singhal, S.;
Levy, L.; Albelda, S.M. Using macrophage activation to augment immunotherapy of established
tumours. Br. J. Cancer 2013, 108, 1288–1297.
117. Zhu, Y.; Knolhoff, B.L.; Meyer, M.A.; Nywening, T.M.; West, B.L.; Luo, J.; Wang-Gillam, A.;
Goedegebuure, S.P.; Linehan, D.C.; DeNardo, D.G. CSF1/CSF1R blockade reprograms
tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in
pancreatic cancer models. Cancer Res. 2014, 74, 5057–5069.

Biology 2015, 4 849
118. Elephant Shark Genome Project. Available online: http://esharkgenome.imcb.a-star.edu.sg/ (accessed
on 10 November 2015).
119. Ensembl Genome Browser. Available online: http://www.ensembl.org/index.html (accessed on 10
November 2015)
120. Zou, J.; Yoshiura, Y.; Dijkstra, J.M.; Sakai, M.; Ototake, M.; Secombes, C. Identification of an
interferon gamma homologue in Fugu, Takifugu rubripes. Fish Shellfish Immunol. 2004, 17, 403–409.
121. Li, J.H.; Shao, J.Z.; Xiang, L.X.; Wen, Y. Cloning, characterization and expression analysis of
pufferfish interleukin-4 cDNA: The first evidence of Th2-type cytokine in fish. Mol. Immunol.
2007, 44, 2078–2086.
122. Ohtani, M.; Hayashi, N.; Hashimoto, K.; Nakanishi, T.; Dijkstra, J.M. Comprehensive clarification
of two paralogous interleukin 4/13 loci in teleost fish. Immunogenetics 2008, 60, 383–397.
123. Igawa, D.; Sakai, M.; Savan, R. An unexpected discovery of two interferon -like genes along with
interleukin (IL)-22 and -26 from teleost: IL-22 and -26 genes have been described for the first time
outside mammals. Mol. Immunol. 2006, 43, 999–1009.
124. Dijkstra, J.M. TH2 and Treg candidate genes in elephant shark. Nature 2014, 511, E7–E9.
125. Wang, T.; Secombes, C.J. The evolution of IL-4 and IL-13 and their receptor subunits. Cytokine
2015, 75, 8–13.
126. Jaillon, O.; Aury, J.M.; Brunet, F.; Petit, J.L.; Stange-Thomann, N.; Mauceli, E.; Bouneau, L.;
Fischer, C.; Ozouf-Costaz, C.; Bernot, A.; et al. Genome duplication in the teleost fish Tetraodon
nigroviridis reveals the early vertebrate proto-karyotype. Nature. 2004, 431, 946–957.
127. Berthelot, C.; Brunet, F.; Chalopin, D.; Juanchich, A.; Bernard, M.; Noël, B.; Bento, P.; da Silva, C.;
Labadie, K.; Alberti, A.; et al. The rainbow trout genome provides novel insights into evolution
after whole-genome duplication in vertebrates. Nat. Commun. 2014, doi:10.1038/ncomms4657.
128. Yoshiura, Y.; Kiryu, I.; Fujiwara, A.; Suetake, H.; Suzuki, Y.; Nakanishi, T.; Ototake, M.
Identification and characterization of Fugu orthologues of mammalian interleukin-12 subunits.
Immunogenetics 2003, 55, 296–306.
129. Bird, S.; Zou, J.; Kono, T.; Sakai, M.; Dijkstra, J.M.; Secombes, C. Characterisation and expression
analysis of interleukin 2 (IL-2) and IL-21 homologues in the Japanese pufferfish, Fugu rubripes,
following their discovery by synteny. Immunogenetics 2005, 56, 909–923.
130. Mitra, S.; Alnabulsi, A.; Secombes, C.J.; Bird, S. Identification and characterization of the
transcription factors involved in T-cell development, t-bet, stat6 and foxp3, within the zebrafish,
Danio rerio. FEBS J. 2010, 277, 128–147.
131. Liongue, C.; Ward, A.C. Evolution of class I cytokine receptors. BMC Evol. Biol. 2007, doi:10.1186/
1471-2148-7-120.
132. Wang, T.; Gorgoglione, B.; Maehr, T.; Holland, J.W.; Vecino, J.L.; Wadsworth, S.; Secombes, C.J.
Fish suppressors of cytokine signaling (SOCS): Gene discovery, modulation of expression and
function. J. Signal Transduct. 2011, doi:10.1155/2011/905813.
133. Skjesol, A.; Liebe, T.; Iliev, D.B.; Thomassen, E.I.; Tollersrud, L.G.; Sobhkhez, M.; Secombes, C.J.;
Joensen, L.L.; Jørgensen, J.B. Functional conservation of suppressors of cytokine signaling
proteins between teleosts and mammals: Atlantic salmon SOCS1 binds to JAK/STAT family
members and suppresses type I and II IFN signaling. Dev. Comp. Immunol. 2014, 45, 177–189.

Biology 2015, 4 850
134. Stolte, E.H.; van Kemenade, B.M.; Savelkoul, H.F.; Flik, G. Evolution of glucocorticoid receptors
with different glucocorticoid sensitivity. J. Endocrinol. 2006, 190, 17–28.
135. Hussain, M.; Wilson, J.B. New paralogues and revised time line in the expansion of the vertebrate
GH18 family. J. Mol. Evol. 2013, 76, 240–260.
136. Huising, M.O.; Stet, R.J.M.; Savelkoul, H.F.J.; Verburg-van Kemenade, B.M.L. The molecular
evolution of the interleukin-1 family of cytokines; IL-18 in teleost fish. Dev. Comp. Immunol.
2004, 28, 395–413.
137. Wang, T.; Bird, S.; Koussounadis, A.; Holland, J.W.; Carrington, A.; Zou, J.; Secombes, C.J.
Identification of a novel IL-1 cytokine family member in teleost fish. J. Immunol. 2009, 183, 962–974.
138. Stansberg, C.; Subramaniam, S.; Olsen, L.; Secombes, C.J.; Cunningham, C. Cloning and
characterisation of a putative ST2L homologue from Atlantic salmon (Salmo salar). Fish Shellfish
Immunol. 2003, 15, 211–224.
139. Köbis, J.M.; Rebl, A.; Kühn, C.; Goldammer, T. Comparison of splenic transcriptome activity of
two rainbow trout strains differing in robustness under regional aquaculture conditions. Mol. Biol.
Rep. 2013, 40, 1955–1966.
140. Gibson, M.S.; Kaiser, P.; Fife, M. The chicken IL-1 family: Evolution in the context of the studied
vertebrate lineage. Immunogenetics 2014, 66, 427–438.
141. Dijkstra, J.M.; Grimholt, U.; Leong, J.; Koop, B.F.; Hashimoto, K. Comprehensive analysis of
MHC class II genes in teleost fish genomes reveals dispensability of the peptide-loading DM
system in a large part of vertebrates. BMC Evol. Biol. 2013, doi:10.1186/1471-2148-13-260.
142. Grimholt, U.; Tsukamoto, K.; Azuma, T.; Leong, J.; Koop, B.F.; Dijkstra, J.M. A comprehensive
analysis of teleost MHC class I sequences. BMC Evol. Biol. 2015, doi:10.1186/s12862-015-0309-1.
143. Nomiyama, H.; Hieshima, K.; Osada, N.; Kato-Unoki, Y.; Otsuka-Ono, K.; Takegawa, S.; Izawa, T.;
Yoshizawa, A.; Kikuchi, Y.; Tanase, S.; et al. Extensive expansion and diversification of the
chemokine gene family in zebrafish: Identification of a novel chemokine subfamily CX. BMC
Genomics 2008, doi:10.1186/1471-2164-9-222.
144. Xu, L.; Yang, L.; Liu, W. Distinct evolution process among type I interferon in mammals.
Protein Cell. 2013, 4, 383–392.
145. Cannon, J.P.; Haire, R.N.; Magis, A.T.; Eason, D.D.; Winfrey, K.N.; Prada, J.A.H.; Bailey, K.M.;
Jakoncic, J.; Litman, G.W.; Ostrov, D.A. A bony fish immunological receptor of the NITR
multigene family mediates allogeneic recognition. Immunity 2008, 29, 228–237.
146. Amemiya, C.T.; Saha, N.R.; Zapata, A. Evolution and development of immunological structures
in the lamprey. Curr. Opin. Immunol. 2007, 19, 535–541.
147. Flajnik, M.F.; du Pasquier, L. Evolution of the immune system. In Fundamental Immunology;
Paul, W.E., Ed.; Lippincott Williams and Wilkins: Philadelphia, PA, USA, 2012 pp. 67–128.
148. Kasahara, M.; Sutoh, Y. Two forms of adaptive immunity in vertebrates: Similarities and
differences. Adv. Immunol. 2014, 122, 59–90.
149. Seumois, G.; Chavez, L.; Gerasimova, A.; Lienhard, M.; Omran, N.; Kalinke, L.; Vedanayagam, M.;
Ganesan, A.P.; Chawla, A.; Djukanović, R.; et al. Epigenomic analysis of primary human T cells
reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility.
Nat. Immunol. 2014, 15, 777–788.

Biology 2015, 4 851
150. Loots, G.G.; Locksley, R.M.; Blankespoor, C.M.; Wang, Z.E.; Miller, W.; Rubin, E.M.; Frazer, K.A.
Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence
comparisons. Science 2000, 288, 136–140.
151. Santangelo, S.; Cousins, D.J.; Winkelmann, N.E.; Staynov, D.Z. DNA methylation changes at
human Th2 cytokine genes coincide with DNase I hypersensitive site formation during CD4+ T
cell differentiation. J. Immunol. 2002, 169, 1893–1903.
152. Sallusto, F.; Reiner, S.L. Sliding doors in the immune response. Nat. Immunol. 2005, 6, 10–12.
153. Zeng, W.P. “All things considered”: Transcriptional regulation of T helper type 2 cell
differentiation from precursor to effector activation. Immunology 2013, 140, 31–38.
154. PHYRE2 Protein Fold Recognition Server. Available online: www.sbg.bio.ic.ac.uk/phyre2/ (accessed
on 10 November 2015).
155. Huising, M.O.; Kruiswijk, C.P.; Flik, G. Phylogeny and evolution of class-I helical cytokines.
J. Endocrinol. 2006, 189, 1–25.
156. Hurley, I.A.; Mueller, R.L.; Dunn, K.A.; Schmidt, E.J.; Friedman, M.; Ho, R.K.; Prince, V.E.;
Yang, Z.; Thomas, M.G.; Coates, M.I. A new time-scale for ray-finned fish evolution. Proc. Biol. Sci.
2007, 274, 489–498.
157. Wells, T.N.; Graber, P.; Proudfoot, A.E.; Arod, C.Y.; Jordan, S.R.; Lambert, M.H.; Hassel, A.M.;
Milburn, M.V. The three-dimensional structure of human interleukin-5 at 2.4-angstroms resolution:
Implication for the structures of other cytokines. Ann. NY Acad. Sci. 1994, 725, 118–127.
158. Rozwarski, D.A.; Diederichs, K.; Hecht, R.; Boone, T.; Karplus, P.A. Refined crystal structure and
mutagenesis of human granulocyte-macrophage colony-stimulating factor. Proteins 1996, 26,
304–313.
159. Feng, Y.; Klein, B.K.; McWherter, C.A. Three-dimensional solution structure and backbone
dynamics of a variant of human interleukin-3. J. Mol. Biol. 1996, 259, 524–541.
160. Rozwarski, D.A.; Gronenborn, A.M.; Clore, G.M.; Bazan, J.F.; Bohm, A.; Wlodawer, A.; Hatada, M.;
Karplus, P.A. Structural comparisons among the short-chain helical cytokines. Structure 1994, 2,
159–173.
161. Dijkstra, J.M.; Takizawa, F.; Fischer, U.; Friedrich, M.; Soto-Lampe, V.; Lefèvre, C.; Lenk, M.;
Karger, A.; Matsui, T.; Hashimoto, K. Identification of a gene for an ancient cytokine, interleukin
15-like, in mammals; interleukins 2 and 15 co-evolved with this third family member, all sharing
binding motifs for IL-15Rα. Immunogenetics 2014, 66, 93–103.
162. Barry, S.C.; Bagley, C.J.; Phillips, J.; Dottore, M.; Cambareri, B.; Moretti, P.; D’Andrea, R.;
Goodall, G.J.; Shannon, M.F.; Vadas, M.A.; et al. Two contiguous residues in human interleukin-3,
Asp21 and Glu22, selectively interact with the alpha- and beta-chains of its receptor and participate
in function. J. Biol. Chem. 1994, 269, 8488–8492.
163. Hercus, T.R.; Bagley, C.J.; Cambareri, B.; Dottore, M.; Woodcock, J.M.; Vadas, M.A.;
Shannon, M.F.; Lopez, A.F. Specific human granulocyte-macrophage colony-stimulating factor
antagonists. Proc. Natl. Acad. Sci. USA 1994, 91, 5838–5842.
164. McKinnon, M.; Page, K.; Uings, I.J.; Banks, M.; Fattah, D.; Proudfoot, A.E.; Graber, P.; Arod, C.;
Fish, R.; Wells, T.N.; et al. An interleukin 5 mutant distinguishes between two functional responses
in human eosinophils. J. Exp. Med. 1997, 186, 121–129.

Biology 2015, 4 852
165. Hansen, G.; Hercus, T.R.; McClure, B.J.; Stomski, F.C.; Dottore, M.; Powell, J.; Ramshaw, H.;
Woodcock, J.M.; Xu, Y.; Guthridge, M.; et al. The structure of the GM-CSF receptor complex
reveals a distinct mode of cytokine receptor activation. Cell 2008, 134, 496–507.
166. Takizawa, F.; Koppang, E.O.; Ohtani, M.; Nakanishi, T.; Hashimoto, K.; Fischer, U.; Dijkstra, J.M.
Constitutive high expression of interleukin-4/13A and GATA-3 in gill and skin of salmonid fishes
suggests that these tissues form Th2-skewed immune environments. Mol. Immunol. 2011, 48,
1360–1368.
167. Kumari, J.; Bogwald, J.; Dalmo, R.A. Transcription factor GATA-3 in Atlantic salmon (Salmo salar):
Molecular characterization, promoter activity and expression analysis. Mol. Immunol. 2009, 46,
3099–3107.
168. Wang, T.; Holland, J.W.; Martin, S.A.; Secombes, C.J. Sequence and expression analysis of two
T helper master transcription factors, T-bet and GATA3, in rainbow trout Oncorhynchus mykiss
and analysis of their expression during bacterial and parasitic infection. Fish Shellfish Immunol.
2010, 29, 705–715.
169. Koppang, E.O.; Fischer, U.; Moore, L.; Tranulis, M.A.; Dijkstra, J.M.; Köllner, B.; Aune, L.;
Jirillo, E.; Hordvik, I. Salmonid T cells assemble in the thymus, spleen and in novel interbranchial
lymphoid tissue. J. Anat. 2010, 217, 728–739.
170. Costa, M.M.; Pereiro, P.; Wang, T.; Secombes, C.J.; Figueras, A.; Novoa, B. Characterization and
gene expression analysis of the two main Th17 cytokines (IL-17A/F and IL-22) in turbot,
Scophthalmus maximus. Dev. Comp. Immunol. 2012, 38, 505–516.
171. Flajnik, M.F. Re-evaluation of the immunological Big Bang. Curr Biol. 2014, 24, R1060–R1065.
172. Star, B.; Nederbragt, A.J.; Jentoft, S.; Grimholt, U.; Malmstrøm, M.; Gregers, T.F.; Rounge, T.B.;
Paulsen, J.; Solbakken, M.H.; Sharma, A.; et al. The genome sequence of Atlantic cod reveals
a unique immune system. Nature 2011, 477, 207–210.
173. Hashimoto, K.; Nakanishi, T.; Kurosawa, Y. Isolation of carp genes encoding major histocompatibility
complex antigens. Proc. Natl. Acad. Sci. USA 1990, 87, 6863–6867.
174. Wittamer, V.; Bertrand, J.Y.; Gutschow, P.W.; Traver, D. Characterization of the mononuclear
phagocyte system in zebrafish. Blood 2011, 117, 7126–7135.
175. Dijkstra, J.M.; Kiryu, I.; Köllner, B.; Yoshiura, Y.; Ototake, M. MHC class II invariant chain
homologues in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 2003, 15, 91–105.
176. Lewis, K.L.; del Cid, N.; Traver, D. Perspectives on antigen presenting cells in zebrafish.
Dev. Comp. Immunol. 2014, 46, 63–73.
177. Rast, J.P.; Litman, G.W. T-cell receptor gene homologs are present in the most primitive jawed
vertebrates. Proc. Natl. Acad. Sci. USA 1994, 91, 9248–9252.
178. Boudinot, P.; Boubekeur, S.; Benmansour, A. Rhabdovirus infection induces public and private T
cell responses in teleost fish. J. Immunol. 2001, 167, 6202–6209.
179. Suetake, H.; Araki, K.; Suzuki, Y. Cloning, expression, and characterization of fugu CD4, the first
ectothermic animal CD4. Immunogenetics 2004, 56, 368–374.
180. Dijkstra, J.M.; Somamoto, T.; Moore, L.; Hordvik, I.; Ototake, M.; Fischer, U. Identification and
characterization of a second CD4-like gene in teleost fish. Mol. Immunol. 2006, 43, 410–419.

Biology 2015, 4 853
181. Laing, K.J.; Zou, J.J.; Purcell, M.K.; Phillips, R.; Secombes, C.J.; Hansen, J.D. Evolution of the
CD4 family: Teleost fish possess two divergent forms of CD4 in addition to lymphocyte activation
gene-3. J. Immunol. 2006, 177, 3939–3951.
182. Taylor, E.B.; Wilson, M.; Bengten, E. The Src tyrosine kinase Lck binds to CD2, CD4–1, and
CD4–2 T cell co-receptors in channel catfish, Ictalurus punctatus. Mol. Immunol. 2015, 66, 126–138.
183. Liu, Y.; Moore, L.; Koppang, E.O.; Hordvik, I. Characterization of the CD3zeta, CD3gammadelta and
CD3epsilon subunits of the T cell receptor complex in Atlantic salmon. Dev. Comp. Immunol.
2008, 32, 26–35.
184. Øvergård, A.C.; Nerland, A.H.; Patel, S. Cloning, characterization, and expression pattern of Atlantic
halibut (Hippoglossus hippoglossus L.) CD4-2, Lck, and ZAP-70. Fish Shellfish Immunol. 2010,
29, 987–997.
185. Fischer, U.; Dijkstra, J.M.; Köllner, B.; Kiryu, I.; Koppang, E.O.; Hordvik, I.; Sawamoto, Y.;
Ototake, M. The ontogeny of MHC class I expression in rainbow trout (Oncorhynchus mykiss).
Fish Shellfish Immunol. 2005, 18, 49–60.
186. Takizawa, F.; Dijkstra, J.M.; Kotterba, P.; Korytář, T.; Kock, H.; Köllner, B.; Jaureguiberry, B.;
Nakanishi, T.; Fischer, U. The expression of CD8α discriminates distinct T cell subsets in teleost fish.
Dev. Comp. Immunol. 2011, 35, 752–763.
187. Toda, H.; Saito, Y.; Koike, T.; Takizawa, F.; Araki, K.; Yabu, T.; Somamoto, T.; Suetake, H.;
Suzuki, Y.; Ototake, M.; et al. Conservation of characteristics and functions of CD4 positive
lymphocytes in a teleost fish. Dev. Comp. Immunol. 2011, 35, 650–660.
188. Picchietti, S.; Guerra, L.; Buonocore, F.; Randelli, E.; Fausto, A.M.; Abelli, L. Lymphocyte
differentiation in sea bass thymus: CD4 and CD8-α gene expression studies. Fish Shellfish Immunol.
2009, 27, 50–56.
189. Picchietti, S.; Abelli, L.; Guerra, L.; Randelli, E.; Serafini, F.P.; Belardinelli, M.C.; Buonocore, F.;
Bernini, C.; Fausto, A.M.; Scapigliati, G. MHC II-β chain gene expression studies define the
regional organization of the thymus in the developing bony fish Dicentrarchus labrax (L.). Fish
Shellfish Immunol. 2015, 42, 483–493.
190. Nakanishi, T. Effects of X-irradiation and thymectomy on the immune response of the marine
teleost, Sebastiscus marmoratus. Dev. Comp. Immunol. 1986, 10, 519–527.
191. Yocum, D.; Cuchens, M.; Clem, L.W. The hapten-carrier effect in teleost fish. J. Immunol. 1975,
114, 925–927.
192. Miller, N.W.; Sizemore, R.C.; Clem, L.W. Phylogeny of lymphocyte heterogeneity: The cellular
requirements for in vitro antibody responses of channel catfish leukocytes. J. Immunol. 1985, 134,
2884–2888.
193. Miller, N.; Wilson, M.; Bengtén, E.; Stuge, T.; Warr, G.; Clem, W. Functional and molecular
characterization of teleost leukocytes. Immunol Rev. 1998, 166, 187–197.
194. Somamoto, T.; Kondo, M.; Nakanishi, T.; Nakao, M. Helper function of CD4+ lymphocytes in
antiviral immunity in ginbuna crucian carp, Carassius auratus langsdorfii. Dev. Comp. Immunol.
2014, 44, 111–115.
195. Yoon, S.; Mitra, S.; Wyse, C.; Alnabulsi, A.; Zou, J.; Weerdenburg, E.M.; van der Sar, A.; Wang, D.;
Secombes, C.J.; Bird, S. First demonstration of antigen induced cytokine expression by CD4-1+
lymphocytes in a poikilotherm: Studies in zebrafish (Danio rerio). PLoS ONE 2015, 10, e0126378.

Biology 2015, 4 854
196. Edholm, E.S.; Stafford, J.L.; Quiniou, S.M.; Waldbieser, G.; Miller, N.W.; Bengtén, E.; Wilson, M.
Channel catfish, Ictalurus punctatus, CD4-like molecules. Dev. Comp. Immunol. 2007, 31, 172–187.
197. Takizawa, F.; Xu, Z.; Gómez, D.; Parra, D.; Sunyer, J.O. Novel T cell subpopulations expressing
CD4-1 and CD4-2 molecules in rainbow trout. Fish Shellfish Immunol. 2013, doi:10.1016/j.fsi.
2013.03.140.
198. Kato, G.; Goto, K.; Akune, I.; Aoka, S.; Kondo, H.; Hirono, I. CD4 and CD8 homologues in
Japanese flounder, Paralichthys olivaceus: Differences in the expressions and localizations of
CD4-1, CD4-2, CD8α and CD8β. Dev. Comp. Immunol. 2013, 39, 293–301.
199. Yamaguchi, T.; Katakura, F.; Someya, K.; Dijkstra, J.M.; Moritomo, T.; Nakanishi, T. Clonal
growth of carp (Cyprinus carpio) T cells in vitro: Long-term proliferation of Th2-like cells.
Fish Shellfish Immunol. 2013, 34, 433–442.
200. Fischer, U.; Koppang, E.O.; Nakanishi, T. Teleost T and NK cell immunity. Fish Shellfish
Immunol. 2013, 35, 197–206.
201. Somamoto, T.; Koppang, E.O.; Fischer, U. Antiviral functions of CD8+ cytotoxic T cells in teleost
fish. Dev. Comp. Immunol. 2014, 43, 197–204.
202. Kono, T.; Korenaga, H. Cytokine gene expression in CD4 positive cells of the Japanese pufferfish,
Takifugu rubripes. PLoS ONE 2013, 8, e66364.
203. Jung, C.Y.; Hikima, J.; Ohtani, M.; Jang, H.B.; del Castillo, C.S.; Nho, S.W.; Cha, I.S.; Park, S.B.;
Aoki, T.; Jung, T.S. Recombinant interferon-γ activates immune responses against Edwardsiella tarda
infection in the olive flounder, Paralichthys olivaceus. Fish Shellfish Immunol. 2012, 33, 197–203.
204. Wang, T.; Holland, J.W.; Carrington, A.; Zou, J.; Secombes, C.J. Molecular and functional
characterization of IL-15 in rainbow trout Oncorhynchus mykiss: A potent inducer of IFN-
expression in spleen leukocytes. J. Immunol. 2007, 179, 1475–1488.
205. Wang, T.; Husain, M.; Hong, S.; Holland, J.W. Differential expression, modulation and bioactivity
of distinct fish IL-12 isoforms: Implication towards the evolution of Th1-like immune responses.
Eur. J. Immunol. 2014, 44, 1541–1551.
206. Berg, R.E.; Cordes, C.J.; Forman, J. Contribution of CD8+ T cells to innate immunity: IFN-
secretion induced by IL-12 and IL-18. Eur. J. Immunol. 2002, 32, 2807–2816.
207. Fauriat, C.; Long, E.O.; Ljunggren, H.G.; Bryceson, Y.T. Regulation of human NK-cell cytokine
and chemokine production by target cell recognition. Blood 2010, 115, 2167–2176.
208. Wang, T.; Jiang, Y.; Wang, A.; Husain, M.; Xu, Q.; Secombes, C.J. Identification of the salmonid
IL-17A/F1a/b, IL-17A/F2b, IL-17A/F3 and IL-17N genes and analysis of their expression following
in vitro stimulation and infection. Immunogenetics 2015, 67, 395–412.
209. Victoratos, P.; Yiangou, M.; Avramidis, N.; Hadjipetrou, L. Regulation of cytokine gene
expression by adjuvants in vivo. Clin. Exp. Immunol. 1997, 109, 569–578.
210. Yabu, T.; Toda, H.; Shibasaki, Y.; Araki, K.; Yamashita, M.; Anzai, H.; Mano, N.; Masuhiro, Y.;
Hanazawa, S.; Shiba, H.; et al. Antiviral protection mechanisms mediated by ginbuna crucian carp
interferon gamma isoforms 1 and 2 through two distinct interferon gamma-receptors. J. Biochem.
2011, 150, 635–648.
211. Korn, T.; Bettelli, E.; Oukka, M.; Kuchroo, V.K. IL-17 and Th17 Cells. Annu. Rev. Immunol. 2009,
27, 485–517.

Biology 2015, 4 855
212. Kumar, V.; Sharma, A. Neutrophils: Cinderella of innate immune system. Int. Immunopharmacol.
2010, 10, 1325–1334.
213. Verburg-van Kemenade, B.M.; Daly, J.G.; Groeneveld, A.; Wiegertjes, G.F. Multiple regulation
of carp (Cyprinus carpio L.) macrophages and neutrophilic granulocytes by serum factors: Influence
of infection with atypical Aeromonas salmonicida. Vet. Immunol. Immunopathol. 1996, 51, 189–200.
214. Afonso, A.; Lousada, S.; Silva, J.; Ellis, A.E.; Silva, M.T. Neutrophil and macrophage responses
to inflammation in the peritoneal cavity of rainbow trout Oncorhynchus mykiss. A light and
electron microscopic cytochemical study. Dis. Aquat. Organ. 1998, 34, 27–37.
215. Lieschke, G.J.; Oates, A.C.; Crowhurst, M.O.; Ward, A.C.; Layton, J.E. Morphologic and functional
characterization of granulocytes and macrophages in embryonic and adult zebrafish. Blood 2001,
98, 3087–3096.
216. Palić, D.; Ostojić, J.; Andreasen, C.B.; Roth, J.A. Fish cast NETs: Neutrophil extracellular traps
are released from fish neutrophils. Dev. Comp. Immunol. 2007, 31, 805–816.
217. Gunimaladevi, I.; Savan, R.; Sakai, M. Identification, cloning and characterization of interleukin-17
and its family from zebrafish. Fish Shellfish Immunol. 2006, 21, 393–403.
218. Du, L.; Feng, S.; Yin, L.; Wang, X.; Zhang, A.; Yang, K.; Zhou, H. Identification and functional
characterization of grass carp IL-17A/F1: An evaluation of the immunoregulatory role of teleost
IL-17A/F1. Dev. Comp. Immunol. 2015, 51, 202–211.
219. De Oliveira, S.; Reyes-Aldasoro, C.C.; Candel, S.; Renshaw, S.A.; Mulero, V.; Calado, A. Cxcl8
(IL-8) mediates neutrophil recruitment and behavior in the zebrafish inflammatory response.
J. Immunol. 2013, 190, 4349–4359.
220. Brugman, S.; Witte, M.; Scholman, R.C.; Klein, M.R.; Boes, M.; Nieuwenhuis, E.E. T lymphocyte-
dependent and -independent regulation of Cxcl8 expression in zebrafish intestines. J. Immunol.
2014, 192, 484–491.
221. Ribeiro, C.M.; Pontes, M.J.; Bird, S.; Chadzinska, M.; Scheer, M.; Verburg-van Kemenade, B.M.;
Savelkoul, H.F.; Wiegertjes, G.F. Trypanosomiasis-induced Th17-like immune responses in carp.
PLoS ONE 2010, 5, e13012.
222. Wang, T.; Diaz-Rosales, P.; Costa, M.M.; Campbell, S.; Snow, M.; Collet, B.; Martin, S.A.;
Secombes, C.J. Functional characterization of a nonmammalian IL-21: Rainbow trout Oncorhynchus
mykiss IL-21 upregulates the expression of the Th cell signature cytokines IFN-, IL-10, and IL-22.
J. Immunol. 2011, 186, 708–721.
223. Corripio-Miyar, Y.; Zou, J.; Richmond, H.; Secombes, C.J. Identification of interleukin-22 in gadoids
and examination of its expression level in vaccinated fish. Mol. Immunol. 2009, 46, 2098–2106.
224. Monte, M.M.; Zou, J.; Wang, T.; Carrington, A.; Secombes, C.J. Cloning, expression analysis and
bioactivity studies of rainbow trout (Oncorhynchus mykiss) interleukin-22. Cytokine 2011, 55, 62–73.
225. Costa, M.M.; Saraceni, P.R.; Forn-Cuní, G.; Dios, S.; Romero, A.; Figueras, A.; Novoa, B. IL-22
is a key player in the regulation of inflammation in fish and involves innate immune cells and PI3K
signaling. Dev. Comp. Immunol. 2013, 41, 746–755.
226. Qi, Z.; Zhang, Q.; Wang, Z.; Zhao, W.; Chen, S.; Gao, Q. Molecular cloning, expression analysis
and functional characterization of interleukin-22 in So-iny mullet, Liza haematocheila. Mol.
Immunol. 2015, 63, 245–252.

Biology 2015, 4 856
227. Mughal, M.S.; Farley-Ewens, E.K.; Manning, M.J. Effects of direct immersion in antigen on
immunological memory in young carp, Cyprinus carpio. Vet. Immunol. Immunopathol. 1986, 12,
181–192.
228. Joosten, P.H.; Engelsma, M.Y.; van der Zee, M.D.; Rombout, J.H. Induction of oral tolerance in carp
(Cyprinus carpio L.) after feeding protein antigens. Vet. Immunol. Immunopathol. 1997, 60, 187–196.
229. Hori, S.; Nomura, T.; Sakaguchi, S. Control of regulatory T cell development by the transcription
factor Foxp3. Science 2003, 299, 1057–1061.
230. Horwitz, D.A.; Zheng, S.G.; Gray, J.D. Natural and TGF-β-induced Foxp3+CD4+CD25+ regulatory
T cells are not mirror images of each other. Trends Immunol. 2008, 29, 429–435.
231. Wen, Y.; Fang, W.; Xiang, L.X.; Pan, R.L.; Shao, J.Z. Identification of Treg-like cells in Tetraodon:
Insight into the origin of regulatory T subsets during early vertebrate evolution. Cell Mol. Life Sci.
2011, 68, 2615–2626.
232. Sadlack, B.; Löhler, J.; Schorle, H.; Klebb, G.; Haber, H.; Sickel, E.; Noelle, R.J.; Horak, I.
Generalized autoimmune disease in interleukin-2-deficient mice is triggered by an uncontrolled
activation and proliferation of CD4+ T cells. Eur. J. Immunol. 1995, 25, 3053–3059.
233. Almeida, A.R.; Legrand, N.; Papiernik, M.; Freitas, A.A. Homeostasis of peripheral CD4+ T cells:
IL-2R α and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers.
J. Immunol. 2002, 169, 4850–4860.
234. Busse, D.; de la Rosa, M.; Hobiger, K.; Thurley, K.; Flossdorf, M.; Scheffold, A.; Höfer, T.
Competing feedback loops shape IL-2 signaling between helper and regulatory T lymphocytes in
cellular microenvironments. Proc. Natl. Acad. Sci. USA 2010, 107, 3058–3063.
235. Quintana, F.J.; Iglesias, A.H.; Farez, M.F.; Caccamo, M.; Burns, E.J.; Kassam, N.; Oukka, M.;
Weiner, H.L. Adaptive autoimmunity and Foxp3-based immunoregulation in zebrafish. PLoS ONE
2010, 5, e9478.
236. Grayfer, L.; Hodgkinson, J.W.; Hitchen, S.J.; Belosevic, M. Characterization and functional analysis
of goldfish (Carassius auratus L.) interleukin-10. Mol. Immunol. 2011, 48, 563–571.
237. Piazzon, M.C.; Savelkoul, H.S.; Pietretti, D.; Wiegertjes, G.F.; Forlenza, M. Carp II10 has
anti-inflammatory activities on phagocytes, promotes proliferation of memory T cells, and regulates B
cell differentiation and antibody secretion. J. Immunol. 2015, 194, 187–199.
238. Haddad, G.; Hanington, P.C.; Wilson, E.C.; Grayfer, L.; Belosevic, M. Molecular and functional
characterization of goldfish (Carassius auratus L.) transforming growth factor beta. Dev. Comp.
Immunol. 2008, 32, 654–663.
239. Balla, K.M.; Lugo-Villarino, G.; Spitsbergen, J.M.; Stachura, D.L.; Hu, Y.; Bañuelos, K.;
Romo-Fewell, O.; Aroian, R.V.; Traver, D. Eosinophils in the zebrafish: Prospective isolation,
characterization, and eosinophilia induction by helminth determinants. Blood 2010, 116, 3944–3954.
240. Fast, M.D. Fish immune responses to parasitic copepod (namely sea lice) infection. Dev. Comp.
Immunol. 2014, 43, 300–312.
241. Mulero, I.; Sepulcre, M.P.; Meseguer, J.; García-Ayala, A.; Mulero, V. Histamine is stored in mast
cells of most evolutionarily advanced fish and regulates the fish inflammatory response. Proc.
Natl. Acad. Sci. USA 2007, 104, 19434–19439.
242. Dezfuli, B.S.; Giari, L. Mast cells in the gills and intestines of naturally infected fish: Evidence of
migration and degranulation. J. Fish Dis. 2008, 31, 845–852.

Biology 2015, 4 857
243. Sfacteria, A.; Brines, M.; Blank, U. The mast cell plays a central role in the immune system of
teleost fish. Mol. Immunol. 2015, 63, 3–8.
244. Prykhozhij, S.V.; Berman, J.N. The progress and promise of zebrafish as a model to study mast
cells. Dev. Comp. Immunol. 2014, 46, 74–83.
245. Chettri, J.K.; Kuhn, J.A.; Jaafar, R.M.; Kania, P.W.; Møller, O.S.; Buchmann, K. Epidermal
response of rainbow trout to Ichthyobodo necator: Immunohistochemical and gene expression
studies indicate a Th1-/Th2-like switch. J. Fish Dis. 2014, 37, 771–783.
246. Benedicenti, O.; Collins, C.; Wang, T.; McCarthy, U.; Secombes, C.J. Which Th pathway is
involved during late stage amoebic gill disease? Fish Shellfish Immunol. 2015, 46, 417–425.
247. Zhu, L.Y.; Pan, P.P.; Fang, W.; Shao, J.Z.; Xiang, L.X. Essential role of IL-4 and IL-4Rα
interaction in adaptive immunity of zebrafish: Insight into the origin of Th2-like regulatory
mechanism in ancient vertebrates. J. Immunol. 2012, 188, 5571–5584.
248. Lin, A.F.; Xiang, L.X.; Wang, Q.L.; Dong, W.R.; Gong, Y.F.; Shao, J.Z. The DC-SIGN of zebrafish:
Insights into the existence of a CD209 homologue in a lower vertebrate and its involvement in
adaptive immunity. J. Immunol. 2009, 183, 7398–7410.
249. Farrar, J.J.; Howard, M.; Fuller-Farrar, J.; Paul, W.E. Biochemical and physicochemical
characterization of mouse B cell growth factor: A lymphokine distinct from interleukin 2.
J. Immunol. 1983, 131, 1838–1842.
250. Grayfer, L.; Hodgkinson, J.W.; Belosevic, M. Antimicrobial responses of teleost phagocytes and
innate immune evasion strategies of intracellular bacteria. Dev. Comp. Immunol. 2014, 43, 223–242.
251. Richardson, R.; Slanchev, K.; Kraus, C.; Knyphausen, P.; Eming, S.; Hammerschmidt, M. Adult
zebrafish as a model system for cutaneous wound-healing research. J. Investig. Dermatol. 2013,
133, 1655–1665.
252. Keightley, M.C.; Wang, C.H.; Pazhakh, V.; Lieschke, G.J. Delineating the roles of neutrophils and
macrophages in zebrafish regeneration models. Int. J. Biochem. Cell Biol. 2014, 56, 92–106.
253. Nguyen-Chi, M.; Laplace-Builhe, B.; Travnickova, J.; Luz-Crawford, P.; Tejedor, G.; Phan, Q.T.;
Duroux-Richard, I.; Levraud, J.P.; Kissa, K.; Lutfalla, G.; et al. Identification of polarized
macrophage subsets in zebrafish. Elife 2015, doi:10.7554/eLife.07288.
254. Petrie, T.A.; Strand, N.S.; Yang, C.T.; Rabinowitz, J.S.; Moon, R.T. Macrophages modulate adult
zebrafish tail fin regeneration. Development 2014, 141, 2581–2591.
255. Forlenza, M.; Fink, I.R.; Raes, G.; Wiegertjes, G.F. Heterogeneity of macrophage activation in
fish. Dev. Comp. Immunol. 2011, 35, 1246–1255.
256. Arts, J.A.; Tijhaar, E.J.; Chadzinska, M.; Savelkoul, H.F.; Verburg-van Kemenade, B.M. Functional
analysis of carp interferon-: Evolutionary conservation of classical phagocyte activation. Fish
Shellfish Immunol. 2010, 29, 793–802.
257. Grayfer, L.; Belosevic, M. Molecular characterization, expression and functional analysis of
goldfish (Carassius aurutus L.) interferon gamma. Dev. Comp Immunol. 2009, 33, 235–246.
258. Grayfer, L.; Garcia, E.G.; Belosevic, M. Comparison of macrophage antimicrobial responses
induced by type II interferons of the goldfish (Carassius auratus L.). J. Biol. Chem. 2010, 285,
23537–23547.
259. Grayfer, L.; Walsh, J.G.; Belosevic, M. Characterization and functional analysis of goldfish
(Carassius auratus L.) tumor necrosis factor-alpha. Dev. Comp. Immunol. 2008, 32, 532–543.