Overexpression of SATB1 Gene Inhibits the Immunosuppressive Function of Regulatory T Cells in Chronic Hepatitis B

Available online at www.annclinlabsci.org Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017 403 Overexpression of SATB1 Gene Inhibits the Immunosuppressive Function of Regulatory T Cells in Chronic Hepatitis B

Yiyin Wang1,2, Xuefen Li1,2, Haishen Kong1,2,3, Qiaoyun Zhu1, Yuejiao Dong1,2, Xia Liu1,2, and Yu Chen1,2,3

1Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province, Hangzhou, 2Department of Labo- ratory Medicine, and 3Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China

Abstract. Special AT-rich sequence-binding protein 1 (SATB1) is a class of nuclear matrix binding protein expressed by T cells and plays an important role in regulatory T cells (Tregs) mediated immune regulation. The immunosuppressive function of Tregs in chronic hepatitis B (CHB) being inhibited by SATB1 has been shown in our previous studies. The objective of this study was to learn the impact of SATB1 on thecellular immune function of CHB. SATB1 isolated from human peripheral blood mononuclear cells (PBMCs) was used as a template of PCR and its product was connected to vector PLV-EF1α-EGFP-N. Reconstructed vector PLV-EF1α-SATB1/EGFP was used to create highly infectious virions and then transduced to Tregs isolated from the CHB patients. Cytokine secreted by Tregs with and without SATB1 overexpression were determined. The results showed that there was a significant increase of Th1 (IFN-γ) and Th2 (IL-4 and IL-5) cytokines following SATB1 overexpression in CHB derived Tregs. It means that overexpression of SATB1 can promote the conversion from Tregs to effector T cells (Teffs) that lose suppressive function and stimulate the secretion of effective cytokines. These dataov pr ide the basis for further research on the mechanism of SATB1 in regulating specific immune response of CHB patients.

Key words: Chronic hepatitis B, regulatory T cell, SATB1, immune response.

Introduction the bottom of the chromosome ring and is involved in biological functions including the regulation of The special AT-rich sequence-binding protein 1 gene expression, development of T cells and apop- (SATB1) is a DNA-binding protein with the ability tosis [4-6]. to bind specific regions of DNA within T-cells that directs lineage differentiation and cell maintenance Regulatory T cells (Tregs) is a type of T-cell subset [1-3]. SATB1 interacts with numerous factors, in- with immunosuppressive functions and can inhibit cluding the Wnt signaling family of proteins and activation and proliferation of CD4+ and CD8+ T GATA-3 to accomplish this process [1]. SATB1, cells by secreting inhibitory cytokines [7,8]. initially discovered by Dickinson et al., is com- Previous data has demonstrated that SATB1 is able prised of 763 amino acids that contain 2 CUT mo- to regulate the function of Tregs as the expression of tifs and is able to combine to matrix attachment the SATB1 gene can convert Tregs into convention- regions (MAR) sequence with high affinity by al T cells (Tconvs) and thereby alter the function of forming a dimer similar to the PDZ structure [3]. Tregs [9]. Studies have shown that the percentage Making use of co-recognition and combination of of Tregs in chronic hepatitis B (CHB) patients is its core sequence and a homologous structure do- relatively higher than that of healthy people [7,10] main to the MAR sequence, SATB1 is anchored to and therefore we speculated the expression of SATB1 can affect the function of Tregs in patients Address correspondence to Xuefen Li, MD, Key Laboratory of Clinical with chronic HBV infection. In Vitro Diagnostic Techniques of Zhejiang Province, Department of Laboratory Medicine, The First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou Previously, we have demonstrated in clinic that 310003, China; phone: 86 571 87236394; fax: 86 571 87236383; e mail: [email protected] SATB1 expressed in Tregs of CHB patients was lower than that in effector T cells (Teffs) and also

Figure 1. Identification of recombinant PLV-EF1α-SATB1/EGFP plasmid. (A) Agarose gel electrophoresis result of PLV-EF1α-SATB1/EGFP after endonuclease digestion by EcoRI and BamHI. Lane 1: λ-EcoT14 I digest DNA ladder marker; Lane 2: EcoRI and BamHI digestion of PLV-EF1α-SATB1/EGFP. (B) Partial comparison result of inserted SATB1 fragment and SATB1 sequence from Genbank.

lower than that in healthy people, indicating that transcription (ReverTra Ace Kit; Toyobo, Osaka, Japan) abnormal expression of SATB1 was very likely to and was used as a template. PCR technology was adopted be closely related to genesis and development of to mutate SATB1 gene from NCBI by eliminating its chronic hepatitis B [11]. However, the mechanism stop codon TGA sequence and inserting EcoRI and that how does SATB1 work in CHB patient re- BamHI restriction sites both before and after the gene sequence. The primer sequences were as follows: forward: mains to be elucidated. In this study, we construct- 5-GCGGAATTCATGGATCATTTGAACGAGG-3’, ed a eukaryotic expression vector carrying the reverse: 5’ SATB1 gene with subsequent transduction of Tregs -GCGGGATCCCGGTCTTTCAAATCAGTATTA-3’. derived from CHB patients to evaluate the changes The reaction conditions included 94°C 5 min, 1 cycle; in immune status. These results provide a basis for 94°C 30 sec→55°C 30 sec→68°C 2 min 30 sec, 35 cy- further research on the mechanism of SATB1 in cles; 68°C 10 min, 1 cycle, using KOD plus master mix regulating specific immune response of CHB (Toyobo). PCR products were visualized by agarose gel patients. electrophoresis. The study protocol, conforming to the guidelines of the Declaration of Helsinki, was approved Materials and Methods by the Ethics Review Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University. Cloning of SATB1. To investigate the immune effects of Plasmid construction and identification. After cloning SATB1 in Tregs, we constructed the eukaryotic plasmid of SATB1, we began to construct the recombinant plas- carrying SATB1 firstly based on cloning of SATB1. mid pLV-EF1α-SATB1-EGFP. PCR products and pLV- Peripheral blood mononuclear cells (PBMCs) were iso- EF1α-EGFP-N plasmid (Invitrogen Tech, Co., Ltd., lated from 10mL human blood samples by Ficoll- Beijing, China) were digested with EcoRI and BamHI re- Hypaque density gradient centrifugation (Amersham striction endonuclease at 37°C for 4h and were isolated Pharmacia, Uppsala, Sweden). Total RNA containing by agarose gel extraction, respectively. The PCR product was isolated from PBMCs by TRIzol extraction (accord- was then subcloned to pLV-EF1α-EGFP-N plasmid at ing to manufacturer’s instructions) followed by reverse SATB1 and chronic hepatitis B 405

Figure 2. Identification of lentivirus vector. (A) HEK293 T-cells transfected with PLV-EF1α-EGFP and PLV-EF1α- SATB1/EGFP under a fluorescence microscope, respectively. (B) Relative mRNA expression of SATB1 in transfected HEK293 T- cells determined by real-time PCR. (C) SATB1 protein expression in HEK293 T-cells following transfection as determined by western blotting.

16°C overnight using the TaKaRa DNA Ligation Kit. utilized through the transfection process. Transfection 100μl of competent E. coli DH5α cells (Tiangen efficiency was assessed by subjective visualization of Biotech, Co., Ltd., Beijing, China) were added to the GFP+ cells utilizing a fluorescence microscope. The lev- ligation product and incubated on ice for 30 min fol- els of SATB1 mRNA and SATB1-EGFP fusion protein lowed by heat-shock treatment at 42°C for 90 sec, and a in 293T cells were evaluated by quantitative PCR and return to ice for 2 min. 400 μl sterile liquid LB medium Western blotting, respectively. was added to the cell/ligation mixture and incubated in a shaker at 37°C for 45 min, and plated on LB agar me- Cells transduction. PBMCs from ten CHB patients dium containing 100 μg/ml ampicillin at 37°C over- were isolated from blood samples (30 mL each) by night. Single colonies were picked and PCR amplifica- Ficoll-Hypaque density gradient centrifugation and were tion was performed to see whether the colonies were used for isolation of CD4+CD25+ Tregs using the Midi contained the SATB1 gene by using the aforementioned Magnetic Cell Sorting (MACS) Separator Unit (Miltenyi primers and PCR reaction conditions. Sequencing Biotec). CD4+CD25+ Tregs were co-cultured with anti- through the Invitrogen Biotechnology Co., Ltd. CD3/CD28 antibodies at the ratio of 1:3 in 5% CO2 at (Shanghai, China) in order to determine which clones 37°C for 24h. The cells were separated into two groups contained the proper SATB1 DNA sequence. Plasmids and centrifuged at 300g for 5 min. Supernatant from the derived from positive colony-based PCR were amplified pLV-EF1α-EGFP transduction was added into one in large liquid cultures and further isolated utilizing the group and the supernatant from the pLV-EF1α-SATB1/ Plasmid Extraction Kit (Tiangen Biotech, Co., Ltd., EGFP transduction was added into another group. Beijing, China). Polybrene was added at the final concentration of 5μg/ ml and was centrifuged at 1170g for 30 to 60 min. 24h Packaging and identification of lentivirus vector. later, IL-2 was added at the concentration of 4ng/ml. When the recombinant plasmid pLV-EF1α-SATB1- The cells were collected on day 3. EGFP was correctly constructed, we packaged a lentivirus vector with this plasmid. HEK 293T cells were plat- RNA isolation, cDNA synthesis, and quantitative real- ed in a 10 cm tissue culture dish until a confluence of time PCR. After transduced cells were collected, we in- 80-90% was reached. After 12~24h in culture, the cells vestigated the changes of immune effects in SATB1- were used for transfection. The V-pL EF1α-SATB1- overexpressed Treg cells from CHB patients by testing EGFP vector 15 μg and lentivirus packaging vectors the secretion of several effective cytokines, including psPAX2 15 μg and PMD2.G 7.5 μg were co-transfected IFN-γ, IL-4, and IL-5 by PCR technology. Total RNA into the HEK 293T cells with 1 ml HBS and 67 μl was isolated from transduced Tregs isolated from blood CaCl2 in 10 ml DMEM and incubated in 5% CO2 at samples (30 mL) of 10 CHB patients by TRIzol extrac- 37°C. The medium was changed at 12h and the superna- tion followed by reverse transcription (ReverTra Ace Kit; tant was collected after filter at 24 and 36h post-infec- Toyobo, Osaka, Japan). RNA levels of cytokines, includ- tion. A control vector, pLV-EF1α-EGFP, was also ing IFN-γ, IL-4, and IL-5, were assessed by quantitative 406 Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017

Figure 3. Gene expression in Tregs from CHB patients after transduction as determined by real-time PCR. (A) Relative mRNA expression of IFN-γ in Tregs after transduction. (B) Relative mRNA expression of IL-4 in Tregs after transduction. (C) Relative mRNA expression of IL-5 in Tregs after transduction. *P<0.05. real-time PCR. The following primers were used: IFN-γ: electrophoresis revealed two linear fragments at sense: 5’-GAGTGTGGAGACCATCAAGGAAG-3’, 9419 bp for the empty plasmid and another at antisense: 2289 bp for SATB1 (Figure 1A). Sequencing re- 5’-TGCTTTGCGTTGGACATTCAAGTC-3’; IL-4: sults revealed that the SATB1 gene was inserted sense: 5’-CCGTAACAGACATCTTTGCTGCC-3’, into the plasmid pLV-EF1α-EGFP in correct read- antisense: 5’-GAGTGTCCTTCTCATGGTGGCT-3’; ing frame. Although mutation occurred in site IL-5:sense:5’-GGAATAGGCACACTGGAGAGTC-3’, antisense:5’-CTCTCCGTCTTTCTTCTCCACAC-3’. 1176 and 1386, the amino acid sequence did not The internal reference GAPDH primers were change in comparison with the gene sequence of 5’-GTCTCCTCTGACTTCAACAGCG-3’ and SATB1 from NCBI by DNAMAN and its biologi- 5’-ACCACCCTGTTGCTGTAGCCAA-3’. The PCR cal effects did not change (Figure 1B). was performed in an ABI Prism 7900 Sequence Detection System, including 95°C 30 sec, 1 cycle; 95°C Cellular SATB1 expression. Following the trans- 5 sec→60°C 34 sec, 40 cycles; 95°C 15 sec, 1 cycle; 60°C fection of the HEK 293T cells with the pLV-EF1α- 1 min, 1 cycle. The relative levels of IFN-γ, IL-4, IL-5, SATB1-EGFP plasmid, green fluorescence with △△Ct and GAPDH were indicated by 2- . strong intensity was subjectively observed in the HEK 293T cells under a fluorescence microscope Statistical analysis. Data were analyzed with GraphPad 5.0 software, and the significance level was set at P< (Figure 2A). Quantitative real-time PCR and west- 0.05. The results of mRNA expression of cytokines in ern blotting were also performed to demonstrate Tregs between SATB1 mRNA overexpression group and the expression of the SATB1 gene (Figure 2B). The control group were compared by a nonparametric Mann- result showed that mRNA level of SATB1 was sig- Whitney rank sum test. nificantly improved than that of control plasmid. Meanwhile, the protein level was also greatly in- Results creased at approximately 112 kD (Figure 2C), which is consistent with the expected molecular Identification ofV- pL EF1α-SATB1-EGFP plas- weight of the SATB1 fusion protein, indicating mid. Correct construction of pLV-EF1α-SATB1- successful expression of the SATB1-EGFP fusion EGFP plasmid was the basis for investigating the protein. immune effects of SATB1 in Tregs of CHB patients. In order to identify SATB1 gene, the pLV- Gene expression and clinical effect of SATB1 on EF1α-SATB1-EGFP plasmid underwent double- regulatory T cells in CHB patients. In order to digestion with EcoRI and BamHI. Agarose gel learn the immune effects of SATB1 in chronic SATB1 and chronic hepatitis B 407 hepatitis B patients, we transduced the packaged lentivirus vector with SATB1 into Tregs from CHB patients to see changes of effective cytokines. Quantitative real-time PCR data from SATB1 transduced CHB T-cells revealed the presence of a change in the cytokine secretion profile. Specifically, SATB1 mRNA overexpression resulted in a significant increase of Th1 cytokine IFN-γ (1.97±0.50, Figure 3A) and Th2 cytokines IL-4 and IL-5 (1.80±0.38 and 1.91±0.32, Figure 3B&C) in comparison with control group without SATB1 overexpression (P<0.05, n=10).

Discussion

SATB1 is a matrix attachment region-binding protein and transcription factor that expresses predom- inantly in T cells and plays pivotal roles in cell development and activation [1]. A study by Beyer et Supplement Figure 1. Model of the mechanism of SATB1 regulation in Tregs. The expression of SATB1 al. indicated that SATB1 acts as a “switch” in im- can be inhibited by FoxP3 directly or through inducing munological regulation mediated by Tregs [9]. combination of microRNA and 3’UTR of SATB1 gene. Implantation the gene of SATB1 may transform Low expression of SATB1 promotes Tregs function. Transduction of PLV-EF1α-SATB1/EGFP vector to over- Tregs to Tconvs, losing its immunological suppres- express SATB1 in CHB derived Tregs may cause Tregs lose sion that contributes to tumor growth inhibition its immunosuppressive function and acquire expression of and chronic inflammation [9,10]. Those findings effector cytokines of Th1 (IFN-γ) and Th2 (IL-4, IL-5). suggested a potential role of SATB1 with Tregs function. In this study, we constructed a lentivirus CHB [15,21]. The results suggested that SATB1 eukaryotic expression vector that constitutively was able to promote the conversion from Tregs to overexpressed SATB1 in order to delineate a possi- Teffs and stimulate the secretion of effective cyto- ble impact of SATB1 interaction with Tregs in pa- kines (Supplement Figure 1). However, compared tients with chronic HBV infection. Results showed with samples from healthy people by the research of that the construction of lentivirus vector PLV- Beyer [9], the secreted cytokines seemed not in- EF1α-SATB1/EGFP provided a useful tool for fur- creased so much, for which the reason might be ther study on the function of the SATB1 protein. that the conversion from Tregs to Teffs was more difficult for CHB patients and Teffs were more dif- Tregs, with the characteristic symbol of FoxP3, can ficult to be activated due to poor immune respons- suppress the function of Teffs responses in CHB pa- es. These indicated that the deletion of SATB1 tients [12,13], such as inhibiting Th1 cells secreting might be an important factor in the pathogenesis of anti-inflammatory cytokines including IFN-γ that CHB. participate in cellular immunity and IL-4 and IL-5 that are involved in humoral immunity [14-16], In conclusion, the clinical effect of SATB1 on Tregs and thereby inhibit virus-specific immune response was preliminarily explored which now provides a in chronic hepatitis B infection and contribute to foundation to further explore the mechanistic role disease pathogenesis [10,17-20]. The expression of of SATB1 in the suppression of HBV-specific im- SATB1 can be inhibited by FoxP3 directly or mune responses. through inducing combination of microRNA and 3’UTR of SATB1 gene [9] (Supplement Figure 1). Acknowledgements In this study, we discovered that the overexpression This work was supported by grants from the General of SATB1 in Tregs may cause Tregs lose its suppres- Program of National Natural Science Foundation of sive function and acquire the expression of Th1 cy- China (No. 81672092), the Zhejiang Provincial Natural Science Foundation of China (No. LY14H030001) and tokines (IFN-γ) and Th2 cytokines (IL-4 and IL-5), the Department of Science and Technology of Zhejiang which are indispensable in the anti-viral response in Province (2016C33133). 408 Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017

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