Dectin-1过表达对树突状细胞成熟的抑制作用及其对小鼠心脏移植物免疫耐受的诱导作用

doi:10.13481/j.1671-587X.20230421

摘要/Abstract

摘要：

目的探讨过表达Dectin-1基因对树突状细胞（DCs）功能的影响，阐明Dectin-1基因抑制DCs成熟活化发挥免疫学功能的机制及对小鼠心脏移植物的免疫保护作用。方法小鼠骨髓干细胞诱导形成DCs后进行体外培养扩增，采用带有绿色荧光蛋白（GFP）标签的腺病毒载体将Dectin-1基因转染至DCs，经过Dectin-1基因修饰的未成熟DCs（imDCs）为DC-Dectin-1组，同时设未经病毒转染的DCs组和转染GFP（DC-GFP）组，24 h后采用免疫荧光染色法检测腺病毒转染情况，采用Western blotting法检测各组DCs中Dectin-1蛋白表达情况，采用流式细胞术和酶联免疫吸附试验（ELISA）法检测脂多糖（LPS）刺激前后各组DCs表型、功能和细胞培养上清液中白细胞介素10（IL-10）和白细胞介素12（IL-12）水平。建立同种异体小鼠腹部异位心脏移植模型，分为DCs组、DC-GFP组和DC-Dectin-1组，于移植术后第1、3和5天分别输入DCs、DC-GFP和DC-Dectin-1，移植术后第7天HE染色观察各组小鼠心脏移植物病理形态表现，TUNEL染色观察各组小鼠心脏移植物细胞凋亡情况，观察各组小鼠心脏移植物中位存活时间（MST）。结果经基因修饰的Ad5F35载体可提高Dectin-1基因转染效率，转染后GFP可持续在DCs中表达。 LPS刺激前，DCs、DC-GFP和DC-Dectin-1组细胞中CD40、CD80、CD86和人类主要组织相容性复合体Ⅱ（MHC-Ⅱ）表达水平均较低，呈现imDCs表型；与LPS刺激前比较，LPS刺激后DCs组和DC-GFP组细胞中CD40、CD80、CD86和MHC-Ⅱ表达水平升高，呈现成熟DC表型，而DC-Dectin-1组细胞中CD40、CD80、CD86和MHC-Ⅱ表达水平仍较低。LPS刺激前，各组细胞培养上清液中IL-10和IL-12水平比较差异无统计学意义（P>0.05）；LPS刺激后，与DCs组和DC-GFP组比较，DC-Dectin-1组细胞培养上清液中IL-10水平明显升高（P<0.05），IL-12水平明显降低（P<0.05）。移植术后第7天，与DCs组和DC-GFP组比较，DC-Dectin-1组小鼠心脏移植物组织中炎性细胞浸润减少，排斥反应表现明显减轻，TUNEL染色呈阳性的凋亡细胞明显减少。与DCs组和DC-GFP组比较， DC-Dectin-1组小鼠术后心脏移植物MST明显延长（P<0.01）。结论 Dectin-1基因可抑制未成熟DCs在LPS作用下的成熟和活化，减弱其抗原提呈功能，在一定程度上延长小鼠心脏移植物MST，诱导移植物免疫耐受形成。

关键词: Dectin-1, 基因转染, 树突状细胞, 心脏移植物, 免疫耐受

Abstract:

Objective To discuss the effect of over-expression of Dectin-1 gene on the function of the dendritic cells （DCs）， and to clarify the mechanism of immune function of Dectin-1 gene in inhibiting the maturation and activation of DCs and its immune protection on the heart allografts. Methods The bone marrow stem cells of the mice were induced to form DCs and then cultured and expanded in vitro. The Dectin-1 gene was transfected into the DCs by adenovirus vector with green fluorescent protein （GFP）， the immature DCs （imDCs） modified with the Dectin-1 gene were regarded as DC-Dectin-1 group， and DCs group （untransfected with virus）and DC-GFP group（transfected with GFP） were sep up.Immunofluorescence assay used to detect the adenovirus transfection after 24 h； Western blotting method was used to detect the expressions of Dectin-1 protein in DCs in various groups；flow cytometry and enzyme-linked immunosorbent assay （ELISA） were used to detect the phenotype， function， and levels of interleukin-10 （IL-10） and interleukin-12 （IL-12） in cell culture supernatant of DCs in various groups before and after lipopolysaccharide（LPS） stimulation.The model of abdominal heterotopic heart allograft in the allogeneic mice was established， which was divided into DCs group， DC-GFP group，and DC-Dectin-1 group. The DCs， DC-GFP，and DC-Dectin-1 were infused on the first， third and fifth days after transplantation， respectively.On the seventh day after transplantation， HE staining was used to observe the pathomorphology of heart allografts of the mice in various groups； TUNEL staining was used to observe the apoptosis of heart allografts of the mice in various groups；the median survival time （MST） of heart allografts of mice in various groups was observed after transplantation. Results The gene modified Ad5F35 vector could improve the transfection efficiency of Dectin-1 gene， and GFP could continue to be expressed in the DCs after transfection. Before LPS stimulation， the expression levels of CD40， CD80， CD86， and human major histocompatibility complex Ⅱ （MHC-Ⅱ） in the cells in DCs， DC-GFP and DC-Dectin-1 groups were low， which presenting the imDCs phenotype； compared with before LPS stimulation， the expression levels of CD40， CD80， CD86， and MHC-Ⅱ in the cells in DCs and DC-GFP groups were increased after LPS stimulation， which presenting the mature DC phenotype. However， the expression levels of CD40， CD80， CD86， and MHC-Ⅱ in the cells in DC-Dectin-1 group were low. Before LPS stimulation， there were no statistically significant differences in the levels of IL-10 and IL-12 in cell culture supernatant in various groups（P>0.05）； after LPS stimulation， compared with DCs and DC-GFP groups， the IL-10 level in the cell culture supernatant in DC-Dectin-1 group was significantly increased （P<0.05）， while the IL-12 level was significantly decreased （P<0.05）. On the seventh day after transplantation， compared with DCs group and DC-GFP group， the inflammatory cell infiltration in heart allografts tissue of the mice in DC-Dectin-1 group was decreased， the rejection was significantly alleviated， and the number of TUNEL positive apoptotic cells were significantly decreased. Compared with DCs and DC-GFP groups， the MST of heart allografts of the mice in DC-Dectin-1 group was significantly prolonged （P<0.01）. Conclusion Dectin-1 gene can inhibit the maturation and activation of the immature DCs under the action of LPS， weaken their antigen presenting function， prolong the survival time of heart allografts of the mice to a certain extent， and induce the formation of allografts immune tolerance.

Key words: Dectin-1, Gene transfection, Dendritic cells, Heart allografts, Immune tolerance

中图分类号:

R392.4

张轶西,宋飞玉,郭义文,曾志贵. Dectin-1过表达对树突状细胞成熟的抑制作用及其对小鼠心脏移植物免疫耐受的诱导作用[J]. 吉林大学学报(医学版), 2023, 49(4): 994-1000.

Yixi ZHANG,Feiyu SONG,Yiwen GUO,Zhigui ZENG. Inhibitory effect of Dectin-1 over-expression on maturation of dendritic cells and its induction effect on immune tolerance of heart allografts in mice[J]. Journal of Jilin University(Medicine Edition), 2023, 49(4): 994-1000.

图/表 7

图1

图2

图3

表1

图4

图5

图6

参考文献 23

1	REN A， LI Z Q， ZHANG X Z， et al. Inhibition of dectin-1 on dendritic cells prevents maturation and prolongs murine islet allograft survival［J］. J Inflamm Res， 2021， 14： 63-73.
2	DALEY D， MANI V R， MOHAN N， et al. Dectin 1 activation on macrophages by galectin 9 promotes pancreatic carcinoma and peritumoral immune tolerance［J］. Nat Med， 2017， 23（5）： 556-567.
3	LI M， VULTORIUS C， BETHI M， et al. Spatial organization of dectin-1 and TLR2 during synergistic crosstalk revealed by super-resolution imaging［J］. J Phys Chem B， 2022， 126（31）： 5781-5792.
4	WANG M X， LUO W， YE L， et al. Dectin-1 plays a deleterious role in high fat diet-induced NAFLD of mice through enhancing macrophage activation［J］. Acta Pharmacol Sin， 2023， 44（1）： 120-132.
5	LIU R B， YI R W， CHEN X L， et al. Lentivirus-mediated PD-L1 overexpression in bone marrow-derived dendritic cells induces immune tolerance in a rat keratoplasty model［J］. Transpl Immunol， 2022， 74： 101654.
6	HU J， ZHANG W P， XU L J， et al. JAK2 gene knockout inhibits corneal allograft rejection in mice by regulating dendritic cell-induced T cell immune tolerance［J］. Cell Death Discov， 2022， 8（1）： 289.
7	ZHENG Z， YANG Y， LIU H， et al. Inhibition of miR-let-7i induces DC immature cells and improves skin graft tolerance［J］. Dis Markers， 2022， 2022： 8605621.
8	KHAN F U， KHONGORZUL P， RAKI A A， et al. Dendritic cells and their immunotherapeutic potential for treating type 1 diabetes［J］. Int J Mol Sci， 2022， 23（9）： 4885.
9	NAGY N A， CASTENMILLER C， VIGARIO F L， et al. Uptake kinetics of liposomal formulations of differing charge influences development of in vivo dendritic cell immunotherapy［J］. J Pharm Sci， 2022， 111（4）： 1081-1091.
10	SUN L N， ZHAO Y. The biological role of dectin-1 in immune response［J］. Int Rev Immunol， 2007，26（5/6）： 349-364.
11	BODE K， BUJUPI F， LINK C， et al. Dectin-1 binding to annexins on apoptotic cells induces peripheral immune tolerance via NADPH oxidase-2［J］. Cell Rep， 2019， 29（13）： 4435-4446.e9.
12	ZHU G Q， LYU L Y， YANG H， et al. CLEC-1 acts as a negative regulator of dectin-1 induced host inflammatory response signature in aspergillus fumigatus keratitis［J］.Invest Ophthalmol Vis Sci，2021，62（6）： 28.
13	DEERHAKE M E， SHINOHARA M L. Emerging roles of Dectin-1 in noninfectious settings and in the CNS［J］. Trends Immunol， 2021， 42（10）： 891-903.
14	CHENG S C， VAN DE VEERDONK F L， LENARDON M， et al. The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans［J］.J LeukocBiol，2011， 90（2）： 357-366.
15	TONE K， STAPPERS M H T， WILLMENT J A，et al.C-type lectin receptors of the Dectin-1 cluster： physiological roles and involvement in disease［J］. Eur J Immunol， 2019， 49（12）： 2127-2133.
16	VOBOŘIL M， BŘEZINA J， BRABEC T， et al. A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer［J］. Elife， 2022， 11： e71578.
17	SUN L， ZHANG W J， ZHAO L， et al. Self-tolerance of vascular tissues is broken down by vascular dendritic cells in response to systemic inflammation to initiate regional autoinflammation［J］. Front Immunol， 2022， 13： 823853.
18	AGHDAEI H A， GHAVAMI S B， FARMANI M，et al.Overexpression of toll-like receptors and co-stimulatory molecules on immature dendritic cells of Crohn’s disease［J］. Gene Rep， 2022， 27： 101579.
19	YU Y， LIU B， CHEN S Y， et al. Trichostatin A inhibits dendritic cell maturation through down-regulating NF-κB （p65） pathway［J］. Mol Biol Rep， 2022， 49（4）： 2619-2627.
20	BOŠNJAK B， DO K T H， FÖRSTER R， et al. Imaging dendritic cell functions［J］. Immunol Rev， 2021， 306： 137-163.
21	COUTANT F. Shaping of monocyte-derived dendritic cell development and function by environmental factors in rheumatoid arthritis［J］. Int J Mol Sci， 2021， 22（24）： 13670.
22	ROBERTSON H， LI J， KIM H J， et al. Transcriptomic analysis identifies A tolerogenic dendritic cell signature［J］. Front Immunol， 2021， 12： 733231.
23	HAFKAMP F M J， GROOT KORMELINK T， DE JONG E C.Targeting DCs for tolerance induction：don’t lose sight of the neutrophils［J］. Front Immunol， 2021， 12： 732992.

Group	IL-10		IL-12
Group	Before LPS stimulation	After LPS stimulation	Before LPS stimulation	After LPS stimulation
DCs	174.26±18.84	268.54±20.72	123.60±16.58	935.30±48.27
DC-GFP	171.62±19.67	268.54±20.72	121.30±22.42	914.20±62.15
DC-Dectin-1	212.30±25.42	398.50±38.28^*△	113.50±21.39	528.10±59.37^*△

[1]	王丹, 廖丹, 李红, 熊立秋, 武莹, 董营, 盖晓东. 浆细胞样树突状细胞和Foxp3⁺调节性T细胞在结直肠癌组织中的表达及其意义[J]. 吉林大学学报(医学版), 2020, 46(04): 834-838.
[2]	王丹, 宋紫绮, 李怡飞, 历春, 董志恒, 董营, 盖晓东. 人结直肠癌和肿瘤引流淋巴结组织中Foxp3⁺调节性T细胞和髓样树突状细胞的表达及其意义[J]. 吉林大学学报(医学版), 2019, 45(03): 621-626.
[3]	张丽鹏, 荣春书, 谷一鸣, 傅耀文, 高宝山. 肾-骨髓联合移植诱导免疫耐受患者外周血B淋巴细胞重构的动态特点及其意义[J]. 吉林大学学报(医学版), 2018, 44(02): 332-338.
[4]	王睿斌, 赫东芸, 邹积艳, 盛敏佳. DC-CIK免疫治疗对卵巢癌患者凝血功能及D-二聚体水平的影响[J]. 吉林大学学报(医学版), 2017, 43(01): 120-124.
[5]	魏静波, 刘辉, 朱小茼, 刘聪慧, 曲银娥. 药物流产后蜕膜组织中IL-10和IL-12表达水平及其对异常子宫出血的影响[J]. 吉林大学学报(医学版), 2016, 42(01): 134-138.
[6]	王丽英, 杨勇, 李占东, 李东复, 刘力宾, 王敏. siRNA干预树突状细胞CD40表达对大鼠炎症性肠病的治疗作用[J]. 吉林大学学报(医学版), 2015, 41(01): 54-59.
[7]	魏静波，刘辉，谢朝辉，曲银娥. 结直肠癌组织中树突状细胞标志物CD1a和CD83的表达及意义[J]. 吉林大学学报(医学版), 2014, 40(04): 847-850.
[8]	许会静，杜彤华，孙艳，李校堃，肖业臣. FGFR3真核表达载体的构建及其在人白血病细胞系K562中的表达[J]. 吉林大学学报(医学版), 2014, 40(03): 465-470.
[9]	顾延会，欧阳瑶，孙得胜. CCL20抗体对慢性阻塞性肺疾病大鼠外周血和肺泡灌洗液中树突状细胞表面因子表达的影响[J]. 吉林大学学报(医学版), 2013, 39(2): 236-240.
[10]	董肖婷，张斌，刘硕硕，郭婷婷，潘良朋，贾妮. 抑癌基因PTEN真核表达载体的构建及其在人舌鳞癌SCC-4细胞系中的表达[J]. 吉林大学学报(医学版), 2013, 39(2): 282-285.
[11]	乔伟\|冯乐平\|孙情\|熊毅\|黎玉凤. NOD小鼠CD8α⁺抗原基因克隆载体的构建及其相关蛋白在树突状细胞中的表达[J]. J4, 2012, 38(6): 1058-1062.
[12]	王莉,李鹏,杜兆江. 人热敏瞬时受体通道1基因转染对猫角膜内皮细胞P27蛋白表达的影响[J]. J4, 2012, 38(3): 429-432.
[13]	孙际童\|吴静\|贾婷\|刘雪岩\|杨巍. 自身免疫调节因子在小鼠肠系膜和外周淋巴结中的表达及其意义[J]. J4, 2012, 38(1): 1-5.
[14]	武双双, 姜振宇, 武英, 谭业辉, 王畅, 姚程. 卵黄囊内接种诱导免疫耐受大鼠模型的建立及评价[J]. J4, 2011, 37(4): 617-621.
[15]	付京\|陈域\|雷海路\|雷团结\|张鹏. 吉西他滨联合树突状细胞对HepG2 细胞增殖的抑制作用[J]. J4, 2011, 37(3): 460-464.