13A expression in zebrafish model

    TET1 and TET3 are essential in induction of Th2-type immunity partly through regulation of IL-4/13 A expression in zebrafish model Chao Yang, Zhuo Li, Wei Kang, Yu Tian, Yuzhu Yan, Wei Chen PII: DOI: Reference:

S0378-1119(16)30551-0 doi: 10.1016/j.gene.2016.07.025 GENE 41458

To appear in:

Gene

Received date: Revised date: Accepted date:

5 May 2016 16 June 2016 9 July 2016

Please cite this article as: Yang, Chao, Li, Zhuo, Kang, Wei, Tian, Yu, Yan, Yuzhu, Chen, Wei, TET1 and TET3 are essential in induction of Th2-type immunity partly through regulation of IL-4/13 A expression in zebrafish model, Gene (2016), doi: 10.1016/j.gene.2016.07.025

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ACCEPTED MANUSCRIPT TET1 and TET3 are essential in induction of Th2-type immunity partly through regulation of IL-4/13A expression in zebrafish model

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Author names and affiliations

Clinical Laboratory, The First Affiliated Hospital of Xi'an Jiaotong University, Xi’an

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a

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Chao Yanga, b, Zhuo Lib, Wei Kangb, Yu Tiana, b, Yuzhu Yana, c, Wei Chena

710061, PR China b

Clinical Laboratory, The First Affiliated Hospital of Xi'an Medical University, Xi’an

c

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710077, PR China

Clinical Laboratory, Xi'an Honghui Hospital, Xi’an 710054, PR China

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Corresponding author Wei Chen

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Clinical Laboratory, The First Affiliated Hospital of Xi'an Jiaotong University, Xi’an

Tel: +8613991119734

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710061, China

Abstract

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E-mail: [email protected]

It has been considered that epigenetic modulation can affect a diverse array of cellular

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activities, in which ten eleven translocation (TET) methylcytosine dioxygenase family members refer to a group of fundamental components involved in catalyzation of 5-hydroxymethylcytosine and modification of gene expression. Even though the function of TET proteins has been gradually revealed, their roles in immune regulation are still largely unknown. Recent studies provided clues that TET2 could regulate several innate immune-related inflammatory mediators in mammals. This study sought to explore the function of TET family members in potential T-helper (Th) cell differentiation involved in adaptive immunity by utilizing a zebrafish model. As shown by results, soluble antigens could induce expression of zebrafish IL-4/13A (i.e. a pivotal Th2-type cytokine essential in Th2 cell differentiation and functions), and further trigger the expression of Th1- and Th2-related genes. It is noteworthy that this response was accompanied by the up-regulation of two TET family members (TET1 1

ACCEPTED MANUSCRIPT and TET3) both in immune organs (spleen and kidney) and cells (peripheral lymphocytes). Knocking-down of TET1 and TET3 will give rise to the decreased responses of IL-4/13A induction against exogenous soluble antigen stimulation, and

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further restrain the expression of Th2-related genes, which indicates a restrained Th2 cell differentiation. Nonetheless, TET2 did not exhibit effect on the modification of

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Th1/Th2 related gene expression. Hence, these data showed that TET1 and TET3 might be two significant epigenetic regulators involved in Th2 differentiation through regulation of IL-4/13A expression. This is the first report to show that TET family

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members play indispensable roles in Th2-type immunity, indicating an epigenetic

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modulation manner involved in adaptive immune regulations and responses.

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Keywords: TET family genes, Th2-type immunity, IL-4/13A, adaptive immunity.

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Highlights:

Soluble antigens can induce IL-4/13A and Th2-type gene expression in zebrafish

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TET1 and TET3 gene expression can respond to soluble antigen stimulation.

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TET1 and TET3 are essential in soluble antigen-induced Th2-type immunity.

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TET2 exerts no effects on the modification of Th1/Th2 related gene expression.

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

Abbreviations:

Th cells, T helper cells; TET, ten-eleven translocation; i.p., intraperitoneally; KLH, keyhole limpet hemocyanin; LPS, lipopolysaccharides.

1. Introduction Th1 and Th2 refer to the two well characterized CD4+ T subsets which are derived from naïve CD4+ Th cells (Gilmour and Lavender, 2008; Liao et al., 2008). These two subsets mediate different immune functions, in which Th1 cells mainly assist cellular immunity, whereas Th2 cells focus on humoral immune responses (Huber and Lohoff, 2014). The mechanisms involved in the differentiation of naïve CD4+ Th cells into effector Th1 or Th2 cells have been intensively studied. It is 2

ACCEPTED MANUSCRIPT generally recognized that the most effective inducer of such differentiation is the cytokine environment (Dong and Flavell, 2000). Additionally, the key Th1 cells differentiation driver is deemed to be IL-12 (Hsieh et al., 1993; Heizmann et al.,

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2008). IL-4, an important class I cytokine family member with conserved structure and function throughout vertebrate evolution, is the main cause of Th2 effector cell

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generation (Swain et al., 1990; Le Gros et al., 2008; Zhu et al., 2012). Nonetheless, explicit genome-level regulatory mechanisms through which Th cell differentiation remain ambiguous.

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Epigenetic modifications play essential roles in cell lineage differentiation and oncogenesis, among which DNA methylation attracts particular attention. In general,

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DNA methylation is regarded as a relatively stable epigenetic modification. As proved by emerging evidences, however, DNA methylation can be removed by passive or

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active pattern, in which active demethylation draws serious attention (Wu and Zhang,

recognized

as

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2010). The recently proposed ten-eleven translocation (TET) family proteins are crucial

enzymes

to

convert

5-methylcytosine

into

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5-hydroxy-methylcytosine to lead to genome-wide DNA demethylation (Wu and Zhang, 2011; Lu et al., 2015). Since their first discovery in mammals in 2009, three members of the TET family (TET1, TET2 and TET3) have been identified (Tahiliani

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et al., 2009). Moreover, they have conserved double-stranded β-helix domain that binds to CpG islands, as well as cysteine-rich catalytic domain that catalyzes hydroxymethylation. Functional studies showed their critical roles in the regulation of zygotic development, maintenance of embryonic stem cells, promotion of somatic cell reprogramming as well as prevention of hematopoietic malignancies (Ito et al., 2010; Gu et al., 2011; Quivoron et al., 2011; Zhang et al., 2013; Hu et al., 2014). Notably, it seems that TET-mediated hydroxymethylation is an ancient genome regulatory mechanism, because their homologous gene members and functions have been implicated in several biological processes in amphibians and bony fish, such as regulation of early eye and neural development in Xenopus as well as early hematopoiesis in zebrafish (Xu et al., 2012; Ge et al., 2014; Gjini et al., 2015). These evidences demonstrated the conserved TET protein family and their related epigenetic 3

ACCEPTED MANUSCRIPT modification mechanisms throughout vertebrate evolution. Even though the roles of TET-mediated hydroxymethylation in organic and cellular development and oncogenesis have been widely concerned, it remains

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unknown concerning whether this epigenetic modification mechanism is involved in immune responses. As demonstrated by a breakthrough study, TET2 selectively

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represses IL-6 transcription in innate myeloid cells so as to resolve inflammation. This finding strongly indicates the essential roles of TET proteins in regulating innate immune responses. Furthermore, it provides clues that TET-mediated epigenetic

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modification might be associated with the process of adaptive immunity, such as Th cell subset differentiation (Zhang et al., 2015). Indeed, Santangelo, S. et al.

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investigated that the selective demethylation of several specific CpG dinucleotides occurred in the IL-4 and IL-13 genes during human Th2 cell differentiation, implying

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the possibility that TET-mediated epigenetic modifications might be involved in Th

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subset fate decision (Santangelo et al., 2009). In this study, we mainly concentrated on the functions and mechanisms of three TET family members in Th2 cell

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differentiation in zebrafish. As expected, both TET1 and TET3 could be induced in response to exogenous antigens. After the knocking-down of TET1 or TET3, the IL-4/13A expression and Th2 cell differentiation were blocked, and oppositely the

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Th1 cell-related genes were up-regulated to a great extent. Nonetheless, TET2 showed no significant effect on Th subset fate decision. To our knowledge, these results were the first observation that TET family members might be indispensable in Th2 cell differentiation.

This

exploration

might

significantly

improve

the

current

understanding on genome-level mechanisms of Th subset fate decision and adaptive immune responses.

2. Materials and Methods 2.1 Animals, strains and plasmids As

previously described,

adult

zebrafish

(Danio

rerio)

maintenance,

developmental staging and in situ analysis were carried out (Shao et al., 2015; Yoon et al., 2015). Briefly speaking, 1-year-old wild-type AB strain zebrafish with both sexes, 4

ACCEPTED MANUSCRIPT ~0.5–1 g in weight and ~3–4 cm in body lengths, was purchased from National Zebrafish Resources of China, and kept in recirculating water at 28 °C and fed with tropical fish flake twice daily as well as frozen brine shrimp and blood worm twice a

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week. All the fishes were held in the laboratory for at least 2 weeks before utilized in experiments to allow for acclimatization. Only healthy fish, as determined by general

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appearance and activity level, was utilized in the study. E.coli BL21(DE3) PLySs competent cells (Takara) and pET28a plasmid (ThermoFisher) was applied for gene

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expression. All primers applied in this study were shown in Table I.

2.2 Recombinant zebrafish IL-4/13A protein preparation

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Zebrafish IL-4/13A gene was amplified by reverse-transcriptional PCR using the cDNA templates reverse transcribed from the spleen total RNA. The forward and

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reverse primers contained 5’-BamHI and 3’-XhoI restriction sites, respectively.

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Moreover, the amplification products were digested and ligated into pET28a. Furthermore, the constructed plasmid was sequenced and then transformed into BL21

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(DE3) PLySs competent cells. Single colony was inoculated into Luria-Bertani medium containing chloramphenicol (100 mg/l) and kanamycin (25 mg/l). Additionally, overnight bacterial cultures were diluted to optical density measured at

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600nm (A600) of 0.2 in shake flasks and grown at 30 ˚C and 160 rpm. At A600 of 0.8, 1 mM Isopropyl β-D-1-thiogalactopyranoside was added to induce gene expression. After ultrasonication, soluble supernatant liquids were collected. Recombinant zebrafish IL-4/13A protein in these supernatant liquids was purified by nickel-nitrilotriacetic acid agarose affinity chromatography in accordance with the QIAexpressionist Manual (Qiagen). The purity of the recombinant protein was more than 95%.

2.3 Tissue and cell preparation According to the methods reported previously, spleen, kidney and peripheral lymphocytes (PBL) were separated (Zhu et al., 2014). Briefly speaking, spleen and kidney were collected in ice-cold D-Hank’s solution with heparin (10 U/mL). Whole 5

ACCEPTED MANUSCRIPT blood cell suspensions were obtained with heparinized capillary tubes, and single cell suspensions of spleens and HKs from 30 fishes were prepared by gently teasing the tissues through an 80 μm nylon mesh filter. Further more, lymphocytes were enriched

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from the cell suspensions by Ficoll-Hypaque (1.080 g/mL) centrifugation at 2500 rpm for 25 min at room temperature, and collected from the interface layer, and then

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washed with ice-cold D-Hank’s solution. Cellular viability was determined by trypan blue (0.4%) exclusion assay.

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2.4 Expression of IL-4/13A and Th1/Th2 related genes upon antigen stimulation For Th1/Th2 related genes and IL-4/13A expression studies upon antigen

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stimulation, fish was injected intraperitoneally (i.p.) with keyhole limpet hemocyanin (KLH, 10 μg) and lipopolysaccharides (LPS, 10 ng). PBS injected group was

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configured as control. After three-day administration, spleen, kidney and PBL were

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collected and meanwhile total RNAs were prepared for cDNA cloning by utilizing RNA extraction solution (TRIzol reagent, Life Technologies BRL) according to the

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manufacturer’s instructions. First-strand cDNA was synthesized from 0.75 μg of total RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). Then, the expression profile of the fish Th1/Th2 related genes and IL-4/13A was analyzed by

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real-time qPCR. The PCR program utilized for amplification was as follows: 1 cycle of 94°C for 10 min, 40 cycles of 94°C for 20 s, annealing at 60°C for 20 s and extension at 72°C for 20 s, followed by 1 cycle of 72°C for 10 min. Additionally, a melting curve for each PCR reaction was established between 72°C and 94°C to ensure that only a single product was amplified. The related gene expression level was calculated using the 2-ΔΔCt methods by normalized to the cycle threshold (Ct) value of endogenous β-actin and PBS injected control groups.

2.5 Expression of Th1/Th2 related genes upon recombinant IL-4/13A stimulation For Th1/Th2 related genes expression studies upon antigen stimulation, fish was injected i.p. with recombinant zebrafish IL-4/13A (0.1 μg) (Zhu et al., 2012). Heat-denatured IL-4/13A protein injected group was configured as control. After 6

ACCEPTED MANUSCRIPT three-day injection, spleen, kidney and PBL were collected. As described in section 2.3, the procedures of tissue RNA isolation, complementary DNA sample synthesis

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and real-time qPCR analysis were carried out.

2.6 Expression of TET1, TET2 and TET3 genes upon antigen stimulation

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For TET1, TET2 and TET3 gene expression studies upon antigen stimulation, fish was injected i.p. with KLH (10 μg) and LPS (10 ng). The PBS injected group was configured as control. As described in section 2.3, the procedures of tissue RNA

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isolation, complementary DNA sample synthesis and real-time qPCR analysis were

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carried out.

2.7 Production and efficiency test of lentiviruses

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The lentiviruses harboring small interfering RNA (siRNA) against TET1, TET2

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or TET3 and scramble siRNA (scr-siRNA) were designed and prepared by the GeneChem Inc. For detecting the effect of these lentiviruses, fish was i.p. injected

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with lentiviral supernatant (2×105 TU/mL) for 3 times with 12-h intervals. After 3 days, total RNA from spleen, kidney and PBL was isolated and transcribed into cDNA. Beyond that, real-time qPCR was performed to detect the TET1, TET2 or TET3

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expression in correspondence with the knock-down genes.

2.8 The role of TETs in the IL-4/13A and Th1/Th2 related gene expression upon antigen stimulation To evaluate the role of TETs in the expression of IL-4/13A and Th1/Th2 related genes upon antigen stimulation, fish was i.p. injected with lentiviral supernatant repeatedly. At the third injection, fish was further stimulated with 10μg KLH and 10ng LPS (the control group received PBS). Three days later after antigen challenge, total RNA from spleen, kidney and PBL was isolated and transcribed into cDNA. Meanwhile, real-time qPCR was conducted to detect the expression of IL-4/13A and Th1/Th2 related genes.

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ACCEPTED MANUSCRIPT 2.9 Statistical analyses Statistical evaluation of differences between means of the experimental groups was performed using ANOVA and multiple student’s t-tests. A p value<0.05 was

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regarded as statistically significant. Additionally, the sample numbers of each group were always 10 fish with almost equal body weight. Data points were taken from at

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least three independent experiments.

3. Results

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3.1 Modification of Th1 and Th2 differentiation by soluble antigen stimulation To validate the polarization of Th0 into Th2 cells upon soluble antigen

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stimulation, the expression of representative Th2-type factors (IL-10, GATA3, c-maf, and STAT6) and Th1-type factors (T-bet, IRF1, STAT1, and IFN-γ) were examined

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after the fish was administrated for 3 days with soluble antigen KLH and

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pathogen-associated molecular pattern LPS. As expected, in comparison with the PBS-injected control group, administration of KLH and LPS can significantly induce

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the expression of Th2-type genes, whereas decrease the expression of Th1-type genes in all the spleen, kidney and PBL (Fig. 1). This result indicated the conserved mechanism of Th subset polarization in evolutionally primitive vertebrates, which

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conforms to the previous reports (Zhu et al., 2012).

3.2 IL-4/13A is involved in Th2 differentiation upon soluble antigen stimulation in zebrafish To ensure whether the antigen-induced Th2 differentiation is dependent on IL-4/13A in zebrafish, we firstly checked the expression of IL-4/13A in response to KLH and LPS stimulation. As shown in Fig.2, the expression of IL-4/13A could be dramatically induced by antigens compared to PBS-injected control group. Subsequently, we administrated the experimental fish with recombinant zebrafish IL-4/13A. As expected, the expressions of Th2-type factors were significantly up-regulated, whereas the expressions of Th1-type factors were significantly down-regulated upon IL-4/13A administration (Fig.3). As suggested by these 8

ACCEPTED MANUSCRIPT observations, the soluble antigen-induced Th2 cell differentiation might be mediated by IL-4/13A in bony fish.

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3.3 KLH and LPS can induce the expression of TET1 and TET3

In vivo administration assay was performed to evaluate whether the expression

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of TET family genes could be modified upon antigen stimulation. After three-day i.p. injection, three TET family genes exhibited a different expression pattern in response to KLH and LPS. Specifically, TET1 and TET3 expression in antigen stimulation

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groups showed ~3–6 fold increase and ~2–4 fold increase respectively than that in PBS injected group (Fig.4). Nonetheless, significant changes of TET2 expression

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were not detectable all in spleen, kidney and PBL. Thus, TET1 and TET3 might be

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involved in the soluble antigen-induced immune responses.

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3.4 TET1 and TET3 play essential roles in the expression of IL-4/13A The in vivo knock-down assay was performed to determine the function of TET

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family members in soluble antigen-induced IL-4/13A expression. First of all, the effectiveness and specificity of the constructed siRNA-harboring lentiviral vectors were assessed in spleen, kidney and PBL of zebrafish following lentivirus

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administration. As shown by the results, compared to scrambled-siRNA control group, the expression level of TET1, TET2 and TET3 reduced to 31.6%±5.4% (spleen), 35.1%±4.9% (kidney) and 29.1%±6.1% (PBL); 26.4%±2.5% (spleen), 45.0%±5.1% (kidney) and 37.5%±2.5% (PBL); 40.1%±5.1% (spleen), 41.3%±3.3% (kidney) and 24.8%±1.5% (PBL) respectively in each siRNA interfering group (Fig. 5A and 5B). Meanwhile, all the TET-siRNA harboring lentivirus introduced to the fish did not elicit cross-inhibitory effects, which suggests the specificity of the method (Fig. 5B). Thus, TET1-siRNA, TET2-siRNA and TET3-siRNA harboring lentiviruses are powerful interfering tools to knock down their target TET family genes in zebrafish. By utilizing these effective tools when fish was treated with KLH and LPS, the induced expression levels of IL-4/13A in response to antigens could be significantly disturbed when the TET1 and TET3 genes were knocked-down in all the spleen, 9

ACCEPTED MANUSCRIPT kidney and PBL. However, knocking down the TET2 gene showed no significant

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3.5 TET1 and TET3 play essential roles in Th2 differentiation

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effect on IL-4/13A expression (Fig. 6).

To investigate whether the TET family members participated in Th2

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differentiation in response to antigens, fish was repeatedly and separately injected with three lentiviruses with siRNA targeting different TET family genes, and then stimulated with KLH and LPS. The KLH and LPS plus TET1-siRNA co-injected

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groups showed significantly declined expression of Th2 related genes as well as increased expression of Th1 related genes in comparison of KLH and LPS plus

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scr-siRNA co-injected groups. In the meantime, KLH and LPS co-injected with TET3-siRNA showed the similar Th1/Th2 related gene expression pattern. Oppositely,

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TET2 knocking-down exhibited less effect on the modification of Th1/Th2 related

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gene expression, which was in consistent with the above results (Fig. 7). On this basis, it could be speculated that TET1 and TET3 might be essential in Th2 differentiation in

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response to antigen infection.

4. Conclusions

dioxygenase

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TET

mediates

conversion

of

5-methylcytosine

to

5-hydroxymethylcytosine has been demonstrated as a key mechanism for DNA demethylation and genome regulation, which provide a significant avenue for the research of active DNA demethylation. It has been demonstrated that TET-mediated epigenetic modification can govern various cellular functions and processes. However, limited studies have explored their roles in adaptive immune process. Bony fish, particularly zebrafish, is believed as an excellent model for the research of immunology and epigenetics (Lieschke and Trede, 2009; Zhu et al., 2013; Zhu et al., 2016). Thus, this study sought to preliminarily investigate the role and mechanism of TET-mediated demethylation modification during the process of Th2 polarization in response to the infection of exogenous soluble antigen by using the zebrafish model. Through in vivo administration assays, we assured the essential roles of IL-4/13A in 10

ACCEPTED MANUSCRIPT KLH and LPS induced Th2 differentiation in zebrafish, which were further corroborated in the previous report (Zhu et al., 2012). Subsequently, the expression of three TET family genes upon antigen stimulation was detected in immune organs and

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cells. The results clearly showed the induced expression pattern of TET1 and TET3 genes. For exploring the roles of these two genes in the process of KLH and LPS

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induced Th2 differentiation, they were knocked-down in vivo by siRNA interference. As expected, the deficiency of TET1 and TET3 considerably decreased the responses of IL-4/13A induction and Th2 polarization against exogenous soluble antigen

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infection.

From these results, we could delineate a supposed pathway, namely, when

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antigens were taken in by antigen-presenting cells (APCs) and recognized by receptors, signals of antigens or pathogen-associated molecular patterns were

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transduced by downstream signal pathways, which could induce the expression of

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TET1 and TET3 genes. Afterwards, TET1 and TET3 proteins removed the methylation inhibition on Th2 immune activation genes in APCs, such as

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co-stimulatory genes. They can participate in the initiation of T cell activation, promote IL-4/13A expression in Th cells, and finally trigger naïve Th0 cells into Th2 cells extensively. To the best of our knowledge, this study firstly indicates that TET1

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and TET3 play essential role in Th2 differentiation partly by orchestrating the expression of Th2-type IL-4 gene. It is expected that these observations might result in a better understanding on the cell-fate decision mechanisms in adaptive immune responses. Also, they will provide novel insight that targeting TET1 and TET3 mediated IL-4 induction may represent a novel therapeutic approach for humoral immune dysfunction or allergy in an epigenetic view.

Acknowledgments This research was supported by Special research projects of Shaanxi Provin cial Education Department (12JK0764)

Additional information 11

ACCEPTED MANUSCRIPT The authors have declared that no competing interests exist.

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Zhang, Q., Zhao, K., Shen, Q., Han, Y., Gu, Y., Li, X., Zhao, D., Liu, Y., Wang, C., Zhang, X., Su, X., Liu, J., Ge, W., Levine, R.L., Li, N. and Cao, X., 2015. Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6. Nature 525, 389-93. Zhang, R.R., Cui, Q.Y., Murai, K., Lim, Y.C., Smith, Z.D., Jin, S., Ye, P., Rosa, L., Lee, Y.K., Wu, H.P., Liu, W., Xu, Z.M., Yang, L., Ding, Y.Q., Tang, F., Meissner, A., Ding, C., Shi, Y. and Xu, G.L., 2013. Tet1 regulates adult hippocampal neurogenesis and cognition. Cell Stem Cell 13, 237-45. Zhu, L.Y., Lin, A.F., Shao, T., Nie, L., Dong, W.R., Xiang, L.X. and Shao, J.Z., 2014. B cells in teleost fish act as pivotal initiating APCs in priming adaptive immunity: an evolutionary perspective on the origin of the B-1 cell subset and 14

ACCEPTED MANUSCRIPT B7 molecules. J Immunol 192, 2699-714. Zhu, L.Y., Nie, L., Zhu, G., Xiang, L.X. and Shao, J.Z., 2013. Advances in research of

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immunity in teleosts. Dev Comp Immunol 39, 39-62.

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fish immune-relevant genes: a comparative overview of innate and adaptive

Zhu, L.Y., Pan, P.P., Fang, W., Shao, J.Z. and Xiang, L.X., 2012. Essential role of IL-4

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and IL-4Ralpha interaction in adaptive immunity of zebrafish: insight into the origin of Th2-like regulatory mechanism in ancient vertebrates. J Immunol 188, 5571-84.

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Zhu, L.Y., Shao, T., Nie, L., Xiang, L.X. and Shao, J.Z., 2016. Evolutionary implication of B-1 lineage cells from innate to adaptive immunity. Mol

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Immunol 69, 123-30.

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ACCEPTED MANUSCRIPT Figure Legends Figure 1. Th2 cell differentiation could be triggered by KLH and LPS stimulation. Transcripts from the spleen, kidney and PBL of zebrafish after i.p. inoculation with

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KLH and LPS for 72 h were analyzed by real-time qPCR. Each gene expression

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pattern was normalized to β-actin. Moreover, each gene expression in each tested

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organs or cells of the PBS-injected control sample was arbitrarily set to 1.0. * represents significant difference (P<0.05). Figure 2.

KLH and LPS stimulation induces the expression of IL-4/13A.

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Transcripts from the spleen, kidney and PBL of zebrafish after i.p. inoculation with KLH and LPS for 72 h were analyzed by real-time qPCR. IL-4/13A expression

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pattern was normalized to β-actin. Furthermore, IL-4/13A expression in each tested organs or cells of the PBS-injected control sample was arbitrarily set

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IL-4/13A promotes Th2 cell differentiation and restrains Th1 cell

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Figure 3.

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represents significantly different (P<0.05).

differentiation. Transcripts from the spleen, kidney and PBL of zebrafish after i.p.

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inoculation with recombinant zebrafish IL-4/13A for 72 h were analyzed by real-time qPCR. Each gene expression pattern was normalized to β-actin. Additionally, each gene expression in each tested organs or cells of the heat-denatured IL-4/13A

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protein-injected control sample was arbitrarily set to 1.0. * represents significant difference (P<0.05). Figure 4.

TET1 and TET3 expression are increased upon KLH and LPS stimulation.

Transcripts from the spleen, kidney and PBL of zebrafish after i.p. inoculation with KLH and LPS for 72 h were analyzed by real-time qPCR. TET family gene expression pattern was normalized to β-actin. Besides, each gene expression in each tested organs or cells of the PBS-injected control sample was arbitrarily set to 1.0. * represents significant difference (P<0.05). Figure 5. Lentiviruses harboring siRNAs refer to effective tools to interfere with their target TET gene expression in vivo. (A) Schematic diagrams of siRNA targeting sites in TET1, TET2 and TET3 mRNAs (Genbank accession numbers: XM_005156709.2, XM_005159903.2 and XM_005168616.2, respectively). The white boxes represent 16

ACCEPTED MANUSCRIPT 5’- and 3’-untranslated regions, while the grey boxes represent open reading frames. The indicated sequence is the siRNA targeting site of each gene. (B) The efficacies of TET1, TET2 and TET3 knockdown by lentiviruses in spleen, kidney and PBL were

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evaluated by real-time qPCR. What is more, scrambled siRNA harboring lentivirus injected group was configured as control. TET family gene expression pattern was

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normalized to β-actin, and each gene expression in each tested organs or cells of the control sample was arbitrarily set to 1.0. * represents significant difference (P<0.05). Figure 6. TET1 and TET3 are involved in antigen-induced IL-4/13A expression

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process. The lentivirus harboring TET1, TET2 or TET3-targeting siRNA was repeatedly delivered into zebrafish 3 times with a 12-h interval of i.p. injection and

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followed by co-inoculation with KLH and LPS on the last injection time. Three days after antigen challenge, relative gene expression of IL-4/13A in spleen, kidney and

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PBL were determined by real-time qPCR. Meanwhile, IL-4/13A gene expression

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pattern was normalized to β-actin. Beyond that, IL-4/13A gene expression in each tested organs or cells of the PBS-injected control sample was set arbitrarily to 1.0. *

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represents significant difference (P<0.05). Figure 7. TET1 and TET3 play essential roles in antigen-induced Th2 cell differentiation. The lentivirus was repeatedly delivered into zebrafish followed by

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co-inoculation with KLH and LPS. Relative gene expression of Th1 and Th2 related genes in spleen, kidney and PBL were determined by real-time qPCR. Each gene expression pattern was normalized to β-actin. Moreover, each gene expression in each tested organs or cells of the PBS-injected control sample was arbitrarily set to 1.0. * represents significant difference (P<0.05).

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ACCEPTED MANUSCRIPT Table 1. Primers used in this study.

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Sequence CGCGGATCCATGCATAAGACTGTATTAAAGG CCGCTCGAGTCAACTTGGTCTTGGGCTTTT CTGAGGAGGAGATGGTG CTTGGTCTTGGGCTTTT GCAACCACTCCCATCCTTCGAC TGTTAGCGACTGACGGGTAGGGAC CCAACAGTGACAGGACGAGG TTGAATAGGGTGAGGGAGCAT CACTCACCCAAGCCGCATATC AGGAGTAAGATTTATGACCCTTATC AGGTCATCACCATCGGCAAT GATGTCCACGTCGCACTTCA AAGATTCTCAGCTACATAATGCACACC ATGCTCATCAGTAGATTCTGCTCAC CCATCAGGAGACGCAAAG GGAGAATGCTGGAAAGTC CTGTCCAGCCAACAAGCG CACCTCCCTCAAACAAACCA TCAAAGGAGGACCTGAACCGC CAACACCTCGGACATCTGACTAATC CTCTGCTCACGCTTCT TAGGGACTGTTTATGTTATG GCTCGCCACTTCTTGAACGAG GTCAGGGCACTCGCCAAACCAG TGATACCTGTCCCGCTTAAAAC GACAGAGTCATTCCACCTTGC CGGTAGTCAGGAAATCAATGC ATCTGTCCAATAGTCTCGTAGG GATCCCCGCGTCAACATAAGCTCTAATTCAAGAG ATTAGAGCTTATGTTGACGCTTTTA AGCTTAAAAAGCGTCAACATAAGCTCTAATCTCT TGAATTAGAGCTTATGTTGACGCGGG GATCCCCGCTACCTCCTACACGGTTTTTCAAGAG AAAACCGTGTAGGAGGTAGCTTTTA AGCTTAAAAAGCTACCTCCTACACGGTTTTCTCT TGAAAAACCGTGTAGGAGGTAGCGGG GATCCCCCCTATGGTCCCATGGGTTATTCAAGAGA TAACCCATGGGACCATAGGTTTTA AGCTTAAAAACCTATGGTCCCATGGGTTATCTCTT GAATAACCCATGGGACCATAGGGGG GATCCCCCCTAACTTCCATCCACAAATTCAAGAG ATTTGTGGATGGAAGTTAGGTTTTA AGCTTAAAAACCTAACTTCCATCCACAAATCTCT TGAATTTGTGGATGGAAGTTAGGGGG

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Primer IL-4/13A-RT-F IL-4/13A-RT-R IL-4/13A-qPCR-F IL-4/13A-qPCR-R TET1-qPCR-F TET1-qPCR-R TET2-qPCR-F TET2-qPCR-R TET3-qPCR-F TET3-qPCR-R β-actin-qPCR-F β-actin-qPCR-R IFN-γ-qPCR-F IFN-γ-qPCR-R IRF1-qPCR-F IRF1-qPCR-R T-bet-qPCR-F T-bet-qPCR-R STAT1-qPCR-F STAT1-qPCR-R IL-10-qPCR-F IL-10-qPCR-R c-maf-qPCR-F c-maf-qPCR-R GATA3-qPCR-F GATA3-qPCR-R STAT6-qPCR-F STAT6-qPCR-R

TET1-siRNA-sense TET1-siRNA-antisense TET2-siRNA-sense TET2-siRNA-antisense TET3-siRNA-sense TET3-siRNA-antisense Scrambled-siRNA-sens e Scrambled-siRNA-antis ense

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