GARP、Foxp3及TGF-β在桥本甲状腺炎病人PBMC中的表达及意义

程极; 于磊; 裴晓艳; 汪琼; 金国玺

doi:10.13898/j.cnki.issn.1000-2200.2024.01.012

ObjectiveTo investigate the expression level of mRNA and significance of glycoprotein-A repetitions predominant protein (GARP), Forkhead box protein p3 (Foxp3) and transforming growth factor β (TGF-β) in peripheral blood mononuclear cells (PBMC) of Hashimoto's thyroiditis (HT) patients.MethodsThirty-two patients with HT were selected as the observation group and 32 healthy examinees as the control group.The levels of serum thyroid stimulating hormone (TSH), total triiodothyronine (TT3) and total thyroxine (TT4) were measured by chemiluminescence immunoassay, and the levels of thyroid antibodies TmAb and TgAb were detected by radioimmunoassay.Real-time fluorescent quantitative PCR was used to detect the expression levels of GARP, Foxp3 and TGF-β mRNA in PBMC of HT patients.The correlation of GARP, Foxp3 and TGF-β mRNA expression level in PBMC of HT patients with the level of thyroid function index was analyzed.ResultsThere was no significant difference in TT3, TT4 and TSH levels between the two groups (P>0.05).The levels of TgAb and TmAb in the observation group were significantly higher than those in the control group (P < 0.01).The expression levels of GARP, Foxp3 and TGF-β mRNA in the observation group were significantly lower than those in the control group (P < 0.01).Pearson correlation analysis showed that the expression level of GARP mRNA in PBMC of HT patients was negatively correlated with the level of TgAb (P < 0.05) and significantly positively correlated with the level of TT3 (P < 0.01).ConclusionsThe expression of GARP, Foxp3 and TGF-β mRNA in PBMC of HT patients is decreased.GARP mRNA is negatively correlated with the level of TgAb and significantly positively correlated with the level of TT3, which may be related to the occurrence and development of HT.

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桥本甲状腺炎(Hashimoto′s thyroiditis，HT)以自身甲状腺组织为抗原产生特异性抗体，甲状腺内淋巴细胞浸润，从而损伤甲状腺组织结构，在疾病初期表现为甲状腺毒症，随着病情进展最终大多发展为甲状腺功能减退症，多见于30~50岁的女性^[1-2]。HT作为最常见的自身免疫性疾病之一已被证实与调节性T细胞(Treg细胞)亚群失衡有关，且Treg细胞的主要转录因子叉头框蛋白p3(Foxp3)表达减少^[3]。Treg细胞通过抑制机体对自身和外来抗原的免疫应答来调节免疫耐受，维持免疫稳态。

糖蛋白A重复序列为主的蛋白(glycoprotein-A repetitions predominant protein，GARP)表达于活化Treg细胞表面，可与整合素αVβ8结合促进转化生长因子β(transforming growth factor β，TGF-β)的分泌及活化，从而维持Treg细胞的免疫调节功能和稳态^[4]。来自Treg细胞的TGF-β对维持Treg细胞的功能和稳态至关重要。GARP与Foxp3之间的关系仍不明确，研究^[5]表明下调GARP的表达可以部分损害Treg细胞的功能并下调Foxp3的表达，但也有研究^[6]显示沉默GARP并不影响Foxp3的表达。因此，在HT病人中，Treg细胞的GARP表达情况有待深入研究。本研究探讨GARP、Foxp3、TGF-β在HT病人中的表达水平及意义。现作报道。

1. 资料与方法

1.1. 一般资料

选取2020年12月至2021年6月我院内分泌门诊就诊初次诊断、未使用甲状腺相关药物的HT病人32例作为观察组，符合2008年中华医学会内分泌学会《中国甲状腺疾病诊治指南》中HT的诊断标准：(1)超声检查示甲状腺质地不均匀改变，回声不均，可有低回声或甲状腺结节；(2)甲状腺球蛋白抗体(thyroglobulin antibody，TgAb)或甲状腺微粒体抗体(thyroid microsomal antibody，TmAb)阳性。排除标准：(1)合并其他甲状腺疾病, 甲状腺彩超排除其他甲状腺增生性疾病；(2)合并其他自身免疫性疾病或恶性肿瘤；(3)合并有急慢性传染性疾病或肝、肾疾病及低蛋白血症；(4)处于感染、创伤等应激状态；(5)妊娠期及哺乳期妇女。选取同期健康体检者32名作为对照组，入选标准：(1)甲状腺彩超及甲状腺功能正常，TgAb和TmAb均在正常范围内；(2)无甲状腺疾病病史；(3)无自身免疫性疾病病史；(4)无急慢性传染性疾病及肝肾功能、血脂正常。观察组男2例，女30例，年龄24~70岁；对照组男3名，女29名，年龄24~66岁，2组性别、年龄均有可比性。本研究经我院伦理委员会批准，受试者均已签署知情同意书。

1.2. 方法

1.2.1. 样本采集

收集所有受试者的晨起空腹静脉血5 mL于EDTA抗凝试管，1 000 g离心15 min分离血清，用以检测甲状腺功能指标。另采集外周静脉血5 mL，EDTA抗凝，取对应数量的15 mL离心管标号，加入淋巴分离液5 μL，用滴管吸取血样沿管壁缓慢加入到淋巴分离液液面上，然后梯度离心分离外周血单个核细胞(peripheral blood mononuclear cell，PBMC)，放于-80 ℃冰箱保存待检。

1.2.2. 甲状腺彩超

使用PHILIPS HD3彩超诊断系统对HT病人行甲状腺超声检查，HT主要表现为甲状腺体积增大，回声不均，颗粒增粗，可伴低回声区域或甲状腺结节。排除甲状腺恶性肿瘤、甲状腺腺瘤、结节性甲状腺肿。

1.2.3. 甲状腺功能指标检测

采用化学发光免疫法进行测定2组血清促甲状腺激素(TSH)、总三碘甲状腺原氨酸(TT3)、总甲状腺素(TT4)。采用放射免疫法检测甲状腺抗体TmAb、TgAb水平，利用定量的标记抗原测定血清中的抗体滴度，采用二抗分离法，结果采用B/T的比值，正常参考值TmAb＜20%、TgAb＜30%。

1.2.4. PBMC GARP、Foxp3、TGF-β mRNA表达测定

(1) 总RNA提取：采用过柱吸附法，依据EZ-10 Total RNA Mini-Preps Kit试剂盒(上海生工生物工程有限公司)操作说明提取样本总RNA，分光光度计检测提取的RNA浓度。(2)cDNA模板合成：根据NovoScript^® Plus All-in-one 1st Strand cDNA Synthesis SuperMix(gDNA Purge)试剂盒(苏州近岸蛋白质科技股份有限公司)^[7]操作说明将RNA逆转录为cDNA模板。(3)实时荧光定量PCR：采用IQ5实时荧光定量PCR检测系统(美国Bio-rad公司)，根据SYBR Green Fast qPCR Mix试剂盒(武汉爱博泰克生物科技有限公司)^[8]操作说明检测GARP、Foxp3、TGF-βmRNA表达水平，引物序列见表 1，各分子相对表达量采用2^-ΔΔCt法计算。

分子	序列(5′-3′)
GARP F	CCC TGT AAG ATG GTG GAC AAG AA
GARP R	CAG ATA GAT CAA GGG TCT CAG TGT CT
Foxp3 F	GTG GCC CGG ATG TGA GAA G
Foxp3 R	GGA GCC CTT GTC GGA TGA TG
TGF-β F	GGC CAG ATC CTG TCC AAG C
TGF-β R	GTG GGT TTC CAC CAT TAG CAC
β-actin F	CAT GTA CGT TGC TAT CCA GGC
β-actin R	CTC CTT AAT GTC ACG CAC GAT

1.3. 统计学方法

采用t检验、秩和检验、χ²检验和Pearson相关分析。

2. 结果

2.1. 2组甲状腺功能指标水平比较

2组TT3、TT4、TSH水平差异均无统计学意义(P>0.05)，观察组TgAb和TmAb水平均明显高于对照组(P＜0.01)(见表 1)。

分组	n	TT3/(nmol/L)	TT4/(nmol/L)	TSH/(mIU/L)	TgAb/%	TmAb/%
观察组	32	1.51±0.31	93.60±28.70	2.44(1.82, 3.63)	32.59(15.76, 45.98)	24.15(11.44, 32.53)
对照组	32	1.61±0.23	88.10±19.53	2.37(1.40, 3.56)	2.28(0.43, 4.33)	1.69(0.38, 3.85)
t	—	1.41	0.90	0.72^△	5.64^△	5.83^△
P	—	>0.05	>0.05	>0.05	＜0.01	＜0.01
△示u_c值

2.2. 2组PBMC GARP、Foxp3、TGF-β mRNA表达水平比较

观察组GARP、Foxp3、TGF-βmRNA表达水平均明显低于对照组(P＜0.01)(见表 2)。

分组	n	GARP	Foxp3	TGF-β
观察组	32	0.27(0.16, 0.62)	0.21(0.11, 0.32)	0.45(0.29, 0.62)
对照组	32	0.74(0.58, 1.20)	0.66(0.48, 1.16)	1.01(0.88, 1.18)
u_c	—	4.78	4.81	4.96
P	—	＜0.01	＜0.01	＜0.01

2.3. HT病人PBMC中GARP、Foxp3、TGF-βmRNA表达量与甲状腺功能指标水平相关性分析

Pearson相关分析显示，HT病人PBMC中GARP mRNA表达水平与TgAb水平呈负相关关系(P＜0.05), 与TT3水平呈明显正相关关系(P＜0.01)；其他分子间无明显相关关系(P>0.05)(见表 3)。

项目	GARP mRNA	Foxp3 mRNA	TGF-β mRNA
TgAb	-0.421^*	-0.020	-0.100
TmAb	-0.322	0.015	-0.125
TT3	0.453^**	0.283	0.101
TT4	0.243	0.088	0.011
TSH	-0.103	-0.310	0.328
P＜0.05, *P＜0.01

3. 讨论

HT是淋巴细胞介导的自身免疫疾病，甲状腺特异性自身抗体长期处于高水平状态并伴有甲状腺组织损伤和体积增加^[1]。本研究中的病人均具有典型的甲状腺超声表现及显著升高的甲状腺自身抗体(TgAb>30%或TmAb>20%)。

多项研究^[10-11]已证实在HT病人和动物模型中，Treg细胞功能异常发挥重要作用。Treg细胞通过抑制炎症来调控免疫反应，诱导和维持自身免疫耐受^[11]。因此研究Treg细胞的活性在HT发病和进展中的作用已成为热点。

Foxp3作为Treg细胞的特异标志物，对于Treg细胞的发育和功能至关重要^[12]。但单独的Foxp3表达不足以形成稳定的免疫调控表型^[13]，而GARP可以在细胞表面募集TGF-β并增强其活化，从而增强Treg细胞的免疫抑制功能^[14]。已有研究^[5]表明，与CD25⁺GARP^-Treg细胞相比，CD25⁺GARP⁺Treg细胞表现出更强的免疫抑制活性。越来越多的研究^[15-16]证实GARP在多种免疫疾病和移植物抗宿主病中抗炎和免疫调节的治疗潜力，但在HT中尚未有相关的研究。本研究发现HT病人PBMC GARP、Foxp3、TGF-β mRNA表达明显降低，且GARP mRNA水平与TgAb水平呈负相关关系，与TT3水平呈明显正相关关系，提示GARP、Foxp3、TGF-β可能参与了HT疾病的发展过程，其中GARP的结果在以往研究中未见报道，Foxp3、TGF-β的结果与以往HT病人中的研究^[9]结果一致。因此，我们推测GARP的表达减少可引起Th细胞表面TGF-β的募集和通路活性下降，导致Treg细胞分化减少及功能受损，引起自身免疫耐受的紊乱，促进甲状腺炎症发生。由于本研究的病人多数为初诊病人，有的处于炎症的甲状腺毒症期，与长病程HT发展成甲状腺功能减退的病人表现相反，可能是本研究中Foxp3、TGF-β未呈现与甲状腺激素相关性的原因。

综上所述，HT病人PBMC中GARP、Foxp3、TGF-β mRNA表达降低。GARP mRNA与TgAb水平呈负相关关系，与TT3水平呈明显正相关关系，GARP可能通过影响TGF-β通路活性影响Treg细胞的分化和活性。因此，诱导体内Treg细胞高表达GARP或过继传输CD25⁺GARP^-Treg细胞有可能成为今后HT新的治疗思路。本研究虽然初步揭示了GARP、Foxp3、TGF-β与HT发病的相关性，但GARP调节Treg分化及活性的具体分子机制以及是否具有临床干预潜力尚不清楚。

Reference (16)

[1]	陈玉敏, 胡枫湫, 黄慧. Treg与Th17细胞在桥本氏甲状腺炎发病机制中的作用[J]. 西部医学, 2018, 30(10): 1438-.
[2]	周路路, 金国玺, 吕高友. 硒对桥本甲状腺炎的临床疗效及氧化抗氧化系统的作用[J]. 蚌埠医学院学报, 2019, 44(11): 1468-.
[3]	YANG X, LUN Y, JIANG H. SIRT1-regulated abnormal acetylation of FOXP3 induces regulatory T-cell function defect in Hashimoto's thyroiditis[J]. Thyroid, 2018, 28(2): 246-. doi: 10.1089/thy.2017.0286
[4]	LIéNART S, MERCERON R, VANDERAA C. Structural basis of latent TGF-β1 presentation and activation by GARP on human regulatory T cells[J]. Science, 2018, 362(6417): 952-. doi: 10.1126/science.aau2909
[5]	WANG R, KOZHAYA L, MERCER F. Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells[J]. Proc Natl Acad Sci U S A, 2009, 106(32): 13439-. doi: 10.1073/pnas.0901965106
[6]	PROBST-KEPPER M, GEFFERS R, KROGER A. GARP: a key receptor controlling FOXP3 in human regulatory T cells[J]. J Cell Mol Med, 2009, 13(9B): 3343-. doi: 10.1111/j.1582-4934.2009.00782.x
[7]	DONG B, WANG C, ZHANG J. Exosomes from human umbilical cord mesenchymal stem cells attenuate the inflammation of severe steroid-resistant asthma by reshaping macrophage polarization[J]. Stem Cell Res Ther, 2021, 12(1): 204-. doi: 10.1186/s13287-021-02244-6
[8]	LEE M, LI J, LI J. Tet2 inactivation enhances the antitumor activity of tumor-infiltrating lymphocytes[J]. Cancer Res, 2021, 81(8): 1965-. doi: 10.1158/0008-5472.CAN-20-3213
[9]	XUE H, YU X, MA L. The possible role of CD4⁺CD25(high)Foxp3⁺/CD4⁺IL-17A⁺ cell imbalance in the autoimmunity of patients with Hashimoto thyroiditis[J]. Endocrine, 2015, 50(3): 665-. doi: 10.1007/s12020-015-0569-y
[10]	CAO Y, JIN X, SUN Y. Therapeutic effect of mesenchymal stem cell on Hashimoto's thyroiditis in a rat model by modulating Th17/Treg cell balance[J]. Autoimmunity, 2020, 53(1): 35-. doi: 10.1080/08916934.2019.1697689
[11]	ZHANG X, OLSEN N, ZHENG SG. The progress and prospect of regulatory T cells in autoimmune diseases[J]. J Autoimmun, 2020, 111(): 102461-. doi: 10.1016/j.jaut.2020.102461
[12]	LU L, BARBI J, PAN F. The regulation of immune tolerance by FOXP3[J]. Nat Rev Immunol, 2017, 17(11): 703-. doi: 10.1038/nri.2017.75
[13]	ALLAN SE, CROME SQ, CRELLIN NK. Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production[J]. Int Immunol, 2007, 19(4): 345-. doi: 10.1093/intimm/dxm014
[14]	METELLI A, SALEM M, WALLACE CH. Immunoregulatory functions and the therapeutic implications of GARP-TGF-β in inflammation and cancer[J]. J Hematol Oncol, 2018, 11(1): 24-. doi: 10.1186/s13045-018-0570-z
[15]	ESCHBORN M, WEIGMANN B, REISSIG S. Activated glycoprotein A repetitions predominant (GARP)-expressing regulatory T cells inhibit allergen-induced intestinal inflammation in humanized mice[J]. J Allergy Clin Immunol, 2015, 136(1): 159-. doi: 10.1016/j.jaci.2015.04.020
[16]	WANG H, SONG H, PHAM AV. Human LAP⁺GARP⁺FOXP3⁺ regulatory T cells attenuate xenogeneic graft versus host disease[J]. Theranostics, 2019, 9(8): 2315-. doi: 10.7150/thno.30254

Expression and significance of GARP, Foxp3 and TGF-β in PBMC of Hashimoto's thyroiditis patients

Abstract

References

Proportional views

通讯作者: 陈斌, bchen63@163.com

Related

Proportional views

Expression and significance of GARP, Foxp3 and TGF-β in PBMC of Hashimoto's thyroiditis patients

Corresponding author: JIN Guoxi, jyzjyz1999@163.com

HTML

1.1. 一般资料

1.2. 方法

1.2.1. 样本采集

1.2.2. 甲状腺彩超

1.2.3. 甲状腺功能指标检测

1.2.4. PBMC GARP、Foxp3、TGF-β mRNA表达测定

1.3. 统计学方法

2.1. 2组甲状腺功能指标水平比较

2.2. 2组PBMC GARP、Foxp3、TGF-β mRNA表达水平比较

2.3. HT病人PBMC中GARP、Foxp3、TGF-βmRNA表达量与甲状腺功能指标水平相关性分析

Catalog

[1]	陈玉敏, 胡枫湫, 黄慧, 等. Treg与Th17细胞在桥本氏甲状腺炎发病机制中的作用[J]. 西部医学, 2018, 30(10): 1438.
[2]	周路路, 金国玺, 吕高友, 等. 硒对桥本甲状腺炎的临床疗效及氧化抗氧化系统的作用[J]. 蚌埠医学院学报, 2019, 44(11): 1468.
[3]	YANG X, LUN Y, JIANG H, et al. SIRT1-regulated abnormal acetylation of FOXP3 induces regulatory T-cell function defect in Hashimoto's thyroiditis[J]. Thyroid, 2018, 28(2): 246. doi: 10.1089/thy.2017.0286
[4]	LIéNART S, MERCERON R, VANDERAA C, et al. Structural basis of latent TGF-β1 presentation and activation by GARP on human regulatory T cells[J]. Science, 2018, 362(6417): 952. doi: 10.1126/science.aau2909
[5]	WANG R, KOZHAYA L, MERCER F, et al. Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells[J]. Proc Natl Acad Sci U S A, 2009, 106(32): 13439. doi: 10.1073/pnas.0901965106
[6]	PROBST-KEPPER M, GEFFERS R, KROGER A, et al. GARP: a key receptor controlling FOXP3 in human regulatory T cells[J]. J Cell Mol Med, 2009, 13(9B): 3343. doi: 10.1111/j.1582-4934.2009.00782.x
[7]	DONG B, WANG C, ZHANG J, et al. Exosomes from human umbilical cord mesenchymal stem cells attenuate the inflammation of severe steroid-resistant asthma by reshaping macrophage polarization[J]. Stem Cell Res Ther, 2021, 12(1): 204. doi: 10.1186/s13287-021-02244-6
[8]	LEE M, LI J, LI J, et al. Tet2 inactivation enhances the antitumor activity of tumor-infiltrating lymphocytes[J]. Cancer Res, 2021, 81(8): 1965. doi: 10.1158/0008-5472.CAN-20-3213
[9]	XUE H, YU X, MA L, et al. The possible role of CD4⁺CD25(high)Foxp3⁺/CD4⁺IL-17A⁺ cell imbalance in the autoimmunity of patients with Hashimoto thyroiditis[J]. Endocrine, 2015, 50(3): 665. doi: 10.1007/s12020-015-0569-y
[10]	CAO Y, JIN X, SUN Y, et al. Therapeutic effect of mesenchymal stem cell on Hashimoto's thyroiditis in a rat model by modulating Th17/Treg cell balance[J]. Autoimmunity, 2020, 53(1): 35. doi: 10.1080/08916934.2019.1697689
[11]	ZHANG X, OLSEN N, ZHENG SG. The progress and prospect of regulatory T cells in autoimmune diseases[J]. J Autoimmun, 2020, 111: 102461. doi: 10.1016/j.jaut.2020.102461
[12]	LU L, BARBI J, PAN F. The regulation of immune tolerance by FOXP3[J]. Nat Rev Immunol, 2017, 17(11): 703. doi: 10.1038/nri.2017.75
[13]	ALLAN SE, CROME SQ, CRELLIN NK, et al. Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production[J]. Int Immunol, 2007, 19(4): 345. doi: 10.1093/intimm/dxm014
[14]	METELLI A, SALEM M, WALLACE CH, et al. Immunoregulatory functions and the therapeutic implications of GARP-TGF-β in inflammation and cancer[J]. J Hematol Oncol, 2018, 11(1): 24. doi: 10.1186/s13045-018-0570-z
[15]	ESCHBORN M, WEIGMANN B, REISSIG S, et al. Activated glycoprotein A repetitions predominant (GARP)-expressing regulatory T cells inhibit allergen-induced intestinal inflammation in humanized mice[J]. J Allergy Clin Immunol, 2015, 136(1): 159. doi: 10.1016/j.jaci.2015.04.020
[16]	WANG H, SONG H, PHAM AV, et al. Human LAP⁺GARP⁺FOXP3⁺ regulatory T cells attenuate xenogeneic graft versus host disease[J]. Theranostics, 2019, 9(8): 2315. doi: 10.7150/thno.30254

Expression and significance of GARP, Foxp3 and TGF-β in PBMC of Hashimoto's thyroiditis patients

Abstract

References

Proportional views

通讯作者: 陈斌, bchen63@163.com

Related

Proportional views

Expression and significance of GARP, Foxp3 and TGF-β in PBMC of Hashimoto's thyroiditis patients

Corresponding author: JIN Guoxi, jyzjyz1999@163.com

HTML

1.1. 一般资料

1.2. 方法

1.2.1. 样本采集

1.2.2. 甲状腺彩超

1.2.3. 甲状腺功能指标检测

1.2.4. PBMC GARP、Foxp3、TGF-β mRNA表达测定

1.3. 统计学方法

2.1. 2组甲状腺功能指标水平比较

2.2. 2组PBMC GARP、Foxp3、TGF-β mRNA表达水平比较

2.3. HT病人PBMC中GARP、Foxp3、TGF-βmRNA表达量与甲状腺功能指标水平相关性分析

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