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. 2016 Mar 8;7(10):10917-46.
doi: 10.18632/oncotarget.7295.

First In-Depth Analysis of the Novel Th2-type Cytokines in Salmonid Fish Reveals Distinct Patterns of Expression and Modulation but Overlapping Bioactivities

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Free PMC article

First In-Depth Analysis of the Novel Th2-type Cytokines in Salmonid Fish Reveals Distinct Patterns of Expression and Modulation but Overlapping Bioactivities

Tiehui Wang et al. Oncotarget. .
Free PMC article

Abstract

IL-4 and IL-13 are closely related canonical type-2 cytokines in mammals and have overlapping bioactivities via shared receptors. They are frequently activated together as part of the same immune response and are the signature cytokines produced by T-helper (Th)2 cells and type-2 innate lymphoid cells (ILC2), mediating immunity against extracellular pathogens. Little is known about the origin of type-2 responses, and whether they were an essential component of the early adaptive immune system that gave a fitness advantage by limiting collateral damage caused by metazoan parasites. Two evolutionary related type-2 cytokines, IL-4/13A and IL-4/13B, have been identified recently in several teleost fish that likely arose by duplication of an ancestral IL-4/13 gene as a consequence of a whole genome duplication event that occurred at the base of this lineage. However, studies of their comparative expression levels are largely missing and bioactivity analysis has been limited to IL-4/13A in zebrafish. Through interrogation of the recently released salmonid genomes, species in which an additional whole genome duplication event has occurred, four genomic IL-4/13 loci have been identified leading to the cloning of three active genes, IL-4/13A, IL-4/13B1 and IL-4/13B2, in both rainbow trout and Atlantic salmon. Comparative expression analysis by real-time PCR in rainbow trout revealed that the IL-4/13A expression is broad and high constitutively but less responsive to pathogen-associated molecular patterns (PAMPs) and pathogen challenge. In contrast, the expression of IL-4/13B1 and IL-4/13B2 is low constitutively but is highly induced by viral haemorrhagic septicaemia virus (VHSH) infection and during proliferative kidney disease (PKD) in vivo, and by formalin-killed bacteria, PAMPs, the T cell mitogen PHA, and the T-cell cytokines IL-2 and IL-21 in vitro. Moreover, bioactive recombinant cytokines of both IL-4/13A and B were produced and found to have shared but also distinct bioactivities. Both cytokines rapidly induce the gene expression of antimicrobial peptides and acute phase proteins, providing an effector mechanism of fish type-2 cytokines in immunity. They are anti-inflammatory via up-regulation of IL-10 and down-regulation of IL-1β and IFN-γ. They modulate the expression of cellular markers of T cells, macrophages and B cells, the receptors of IFN-γ, the IL-6 cytokine family and their own potential receptors, suggesting multiple target cells and important roles of fish type-2 cytokines in the piscine cytokine network. Furthermore both cytokines increased the number of IgM secreting B cells but had no effects on the proliferation of IgM+ B cells in vitro. Taken as a whole, fish IL-4/13A may provide a basal level of type-2 immunity whilst IL-4/13B, when activated, provides an enhanced type-2 immunity, which may have an important role in specific cell-mediated immunity. To our knowledge this is the first in-depth analysis of the expression, modulation and bioactivities of type-2 cytokines in the same fish species, and in any early vertebrate. It contributes to a broader understanding of the evolution of type-2 immunity in vertebrates, and establishes a framework for further studies and manipulation of type-2 cytokines in fish.

Keywords: IL-4/13A and 13B; Immune response; Immunity; Immunology and Microbiology Section; bioactivity; expression; rainbow trout Oncorhynchus mykiss; type-2 immunity.

Conflict of interest statement

CONFLICTS OF INTEREST

All authors declare no financial or commercial conflict of interest.

Figures

Figure 1
Figure 1. Gene synteny at the Th2 loci across vertebrates
The KIF3A/IL-4/IL-13/RAD50 loci were analysed using the Genomicus program (genomicus, database release-78.01). The information for frog and elephant shark is extracted from NCBI genomic sequence NW_004668234 (frog Xenopus (Silurana) tropicalis) and NW_006890145 (shark Callorhinchus milii). The spotted gar locus was used as a reference with conserved genes colour-matched. The salmonid IL-4/13 loci were derived from the WGS contigs of Atlantic salmon and rainbow trout (Table 1).
Figure 2
Figure 2. Comparison of the gene organisation of salmonid IL-4/13 genes with IL-4, IL-13 and related genes from other vertebrates
The boxes represent amino acid coding regions and the black bars represent introns. The sizes (bp) of the coding regions are numbered in the boxes. The gene organisation of the salmonid IL-4/13 genes was predicted using the Spidey program based on the sequence information from Tables 1 and Figures S1-S2 and S4-S7. Other genes were extracted from NCBI genomic sequences NW_004668234 (frog IL-4/13) and NW_006890145 (elephant shark IL-4/13-1 and -2) and spotted gar chromosome LG6 (gar IL-4/13). Sequence data representing all other genes was reported by Ohtani et al. [32].
Figure 3
Figure 3. Multiple alignment of mammalian IL-4 and IL-13, and IL-4/13 related molecules from 3R bony fish and other vertebrates A and the patterns of cysteine residues and connectivity prediction in different vertebrate groups
A. The exon-specific multiple alignment was produced using ClustalW. The accession numbers of the amino acid sequences used are as in Figure 2. The four exons are separated and marked above the alignment. The signal peptide, four α-helices and loops known in human IL-4 are marked above human IL-4. The nine cysteine residues conserved in different lineages are denoted under the alignment. B. The lineage specific conserved cysteine residues are schematically shown relative to the four helical structure of human IL-4. The known disulfide-bonds [47-48] in human IL-4 and IL-13 are shown by black lines and the predicted disulfide bonds [49] are indicated by red-lines.
Figure 4
Figure 4. Constitutive expression of trout IL-4/13A, B1 and B2 in vivo in tissues A, in cell lines and purified immune cells B and the ratios of expression levels between each other
A. The transcript expression level of the three trout IL-4/13 paralogues was determined by real time RT-PCR in 17 tissues from six fish. The transcript level was calculated using a serial dilution of references that contained equal molar amounts of the probes for each gene and was normalized against the expression level of EF-1α. The results represent the average +SEM of six fish. B. Total RNA was prepared from cell lines, 4-day old primary HK macrophages, FACS sorted spleen IgM+ B cells, and HK cells after 4 h culture in vitro. The expression analysis was as described in A.. The results represent the average + SEM from four flasks of cells or fish. A paired sample t-test was applied to the tissue samples, macrophages and HK cells, and one-way ANOVA was used for the cell lines. The expression levels between IL-4/13A, B1 and B2 in the same tissues or cell lines are significantly different (p < 0.05) when the letters over the bars are different. C. The ratio of the expression levels between each of the IL-4/13 paralogues. HK = head kidney, AT = adipose tissue, CK = caudal kidney, AF = adipose fin; TF = tail fins, MФ = primary macrophages.
Figure 5
Figure 5. Modulation of the expression of trout IL-4/13A, B1 and B2 in kidney by VHSV infection
Rainbow trout were infected by intraperitoneal (i.p.) injection with 100 μl of VHSV (DK-F1, 1 × 109 TCID50 /ml) while the control fish were mock-infected with the same amount of control media. Fish were sampled at 1 2, 3, 4, 5, 7, 9 and 12 days post challenge, when the kidney was removed for RNA preparation and real-time RT-PCR as described in Figure 4. The relative expression of IL-4/13 paralogues was normalized to the expression of EF-1α, and expressed as arbitrary units where one unit equals the average expression level of the mock-infected fish at day 1 post infection. The results represent the mean + SEM of four fish. The relative significance of a LSD post hoc test after a significant one way-ANOVA between the VHSV and mock-injected groups at the same time point is shown above the bars as: *p < 0.05, **p < 0.01 and ***p < 0.001. The numbers above the bars are the fold changes, calculated as the average expression level of VHSV infected fish divided by that of the time-matched controls. The arrow heads indicate significant increases (*p < 0.05) in the control fish relative to control fish at day 1.
Figure 6
Figure 6. Modulation of the expression of trout IL-4/13A, B1 and B2 in kidney by parasite infection
Kidneys from rainbow trout infected with T. bryosalmonae or from unexposed (control) fish were collected during a natural infection [51-52]. The relative expression of IL-4/13 paralogues was normalized to the expression of EF-1α, and expressed as arbitrary units where one unit equals the average expression level of the control fish. Results are presented as means + SEM. The numbers of fish analysed were 12, 5, 9, 10 and 8 representing control, grade 1, 1-2, 2, and 3, respectively. The expression levels between different groups are statistically significant (p < 0.05) where letters over the bars are different, as tested by one way-ANOVA.
Figure 7
Figure 7. Modulation of the expression of rainbow trout IL-4/13A, B1 and B2 in head kidney A-C and gills (D-F) after vaccination and bacterial challenge
A group of fish was i.p. injected with AquaVacTM ERM (Vac), with naïve fish (Naïve) serving as control. The fish were challenged 60 days later by i.p. injection of Y. ruckeri (0.5 ml/fish, 1×106 cfu/ml; YR) or injected with 0.5 ml of PBS as control (PBS). The fish from each group were killed and the gills and HK collected for total RNA extraction. The gene expression analysis was performed as in Figure 4. The expression level was expressed as arbitrary units where one unit equals the average expression level in the PBS injected control group at 6 h in each tissue. The means + SEM of five fish are shown. The expression levels between different groups of the same tissue and time point are statistically different (p < 0.05) where letters over the bars are different, as determined by one way-ANOVA. The down arrow head indicates a significant down-regulation in the naïve fish after bacterial challenge at the same time point. The black line over the bars indicates a significant difference between the vaccinated and naïve fish after bacterial challenge.
Figure 8
Figure 8. Modulation of the expression of rainbow trout IL-4/13A, B1 and B2 in HK cells
Freshly prepared HK cells were stimulated with formalin killed A. davidanieli (AD, A) and R. salmoninarum (RS, B), a T cell mitogen PHA (C), or the recombinant trout cytokines rIL-2 (D) and rIL-21 (E) for 4 h, 8 h 24 h, 48 h and 96 h. Quantification of gene expression was as described in Figure 4. Modulated expression was expressed as a fold change calculated as the mean expression levels in stimulated cells normalized to that of time-matched controls. The means + SEM of cells from four fish are shown. The relative significance of paired sample T tests between stimulated and time-matched control samples is shown above the bars. * p 0.05, **p 0.01 and ***p 0.001.
Figure 9
Figure 9. Modulation of the expression of rainbow trout IL-4/13A A, B1 B and B2 C in primary HK macrophages
Four day old primary HK macrophages were stimulated with the PAMPs poly IC and peptidoglycan (PGN), or the recombinant cytokines rIL-1β, rIL-6, rIFN-γ and rTNFα for 4 h, 8 h and 24 h. Quantification of gene expression was as described in Figure 4. Modulated expression was expressed as a fold change calculated as the mean expression levels in stimulated cells normalized to that of time-matched controls. The means + SEM of cells from four fish are shown. The relative significance of paired sample T tests between stimulated and time-matched control samples is shown above the bars. *p 0.05.
Figure 10
Figure 10. SDS-PAGE analysis A and dose responses B-G of trout rIL-4/13A and rIL-4/13B2
A. SDS-PAGE analysis of rIL-4/13A and B expressed and purified from E. coli BL21 Star (DE3), and stained with SeeBlue (Invitrogen). 1, a sample from un-induced BL21 cells, 2 and 4, BL21 transformed by IL-4/13A- and IL-4/13B2-expressing plasmid, respectively and induced with 1 mM IPTG for 4 h; 3 and 5, expression product purified from transformed cells expressing IL-4/13A and IL-4/13B2, respectively; and 6, Protein marker, SeeBlue Plus2 (Invitrogen). B.-G. Freshly prepared HK cells were stimulated with rIL-4/13A or rIL-4/13B2 at 0.01, 0.1, 1, 4, 16, 64, 256, and 1000 ng/ml for 4 h. Gene expression was determined as described in Figure 4. The relative gene expression was calculated as the expression levels in stimulated cells normalized to that of un-stimulated controls. The means of cells from four fish are shown. For clarity the SEM bars are not shown here but are provided in Figure S11. The blue arrows and red arrows indicate significant differences (p < 0.05, paired samples T tests) after stimulation with rIL-4/13A and rIL-4/13B2, respectively. A green box indicates significantly different expression levels (p < 0.05, paired samples T tests) between HK cells stimulated with the same doses of rIL-4/13A or rIL-4/13B2.
Figure 11
Figure 11. Modulation of the expression of SAP1 A, Hepcidin B and CATH1 C by rIL-4/13 isoforms
Freshly prepared HK cells were stimulated with rIL-4/13A or rIL-4/13B2 for 2 h to 96 h and gene expression determined as described in Figure 4. Modulated expression was expressed as a fold change, calculated as the expression levels in stimulated cells normalized to that of time-matched controls. The means + SEM of cells from four fish are shown. The relative significance of paired sample T tests between stimulated and time-matched control cell cultures are shown above the bars as: *p 0.05, **p 0.01 and ***p 0.001. The black lines over the bars indicate significantly different expression levels between HK cells stimulated with rIL-4/13A and rIL-4/13B2.
Figure 12
Figure 12. Modulation of the expression of the cytokine receptors for IL-4/13 (IL-4α1 and α2, IL-13Rα1a and α1b, and IL-13Rα2a and α2b), IFNγ (IFNγR1 and 2), IL-6 (IL-6Rα and GP130) and MCSF (MCSFR1 and 2), and cellular markers for T cells (CD4-1, CD8α and CD8β) and dendritic cells (CD83, CLEC4, and CD209L2) by rIL-4/13 isoforms
Freshly prepared HK cells were stimulated with rIL-4/13A or rIL-4/13B2 for 4 h to 96 h and gene expression determined as described in Figure 11. The means + SEM of cells from four fish are shown. The relative significance of paired sample T tests between stimulated and time-matched control cell cultures are shown above the bars as: *p 0.05, **p 0.01 and ***p 0.001. The black lines over the bars indicate significantly different expression levels between HK cells stimulated with recombinant rIL-4/13A and rIL-4/13B2.
Figure 13
Figure 13. Modulation of the expression of the pro-inflammatory cytokines (IFNγ1 and 2, IL-1β1 and LECT2), anti-inflammatory cytokines (IL-10a and b) and negative regulators (SOCS3) by rIL-4/13 isoforms
Freshly prepared HK cells were stimulated with rIL-4/13A or rIL-4/13B2 for 4 h to 96 h and gene expression determined as described in Figure 11. The means + SEM of cells from four fish are shown. The relative significance of paired sample T tests between stimulated and time-matched control cell cultures are shown above the bars as: *p 0.05, **p 0.01 and ***p 0.001. The black lines over the bars indicate significantly different expression levels between HK cells stimulated with recombinant rIL-4/13A and rIL-4/13B2.
Figure 14
Figure 14. Effects of rIL-4/13 isoforms on IgM+ B cells
A. Spleen leucocytes were incubated with/without rIL-4/13A (200 ng/ml), rIL-4/13B2 (200 ng/ml), LPS (100 μg/ml), or LPS in combination of rIL-4/13A or IL-4/13B2 for 3 days at 20°C and IgM secreting cells were determined by ELISPOT. The IgM secreting B cells (means + SEM, n = 5) relative to control (= 1) are shown. B-D. Spleen leucocytes were incubated with/without rIL-4/13A, rIL-4/13B2, or LPS as positive control for 3 days at 20°C. The proliferating cells were then labeled with BrdU and incubated for a further 24 h. The proliferating (BrdU+) cells and IgM+ B cells were then determined by flow cytometry with typical results shown in Figure S12. The relative number (means + SEM, n = 11) of IgM+ B cells (B), proliferating B cells (BrdU and IgM double positive, C) and total proliferating cells (BrdU+, D) are shown. E-H. Spleen leucocytes (n = 6) were incubated with/without rIL-4/13A, rIL-4/13B, or LPS as positive control for 24 h at 20°C. IgM+ cells were sorted into TRIzol. The relative transcript expression (means + SEM, n = 6) of the membrane form of IgM heavy chain (E), secretory form of IgM heavy chain (F), CATH1 (G) and IL-1β (H) in FACS sorted IgM+ B cells are shown. The relative significance of paired sample T tests between stimulated and control samples are shown above the bars as: *p 0.05 **p 0.01 and ***p 0.001.

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