研究背景

梅毒是由Tp感染所致的严重危害人类健康的性传播疾病，仍在世界范围内广泛传播，被WHO列为严重危害人类健康的全球性公共卫生问题之一。Tp感染过程中可引起血管损伤，累及心血管和中枢神经系统等重要脏器时，如不治疗可导致严重不良结局，甚至死亡。Tp引起血管损伤的机制有待进一步阐明。

近些年来外泌体已成为感染、免疫和肿瘤等领域的研究热点。外泌体可以通过不同机制将生物活性分子从亲本细胞转移到靶细胞，从而介导细胞间通讯。越来越多的研究发现外泌体参与病原微生物感染过程和免疫反应，引起血管并发症。DC是皮肤或粘膜内第一个接触Tp的抗原提呈细胞。目前尚不清楚Tp诱导DC衍生的外泌体（EXOTp）是否会造成血管损伤。

TLR4主要在单核细胞表达，内皮细胞表面也大量表达。TLR4与MyD88结合，激活NF-κB，增加促炎细胞因子的合成。既往研究显示TLR4参与梅毒的感染进程，然而EXOTp是否会引起TLR4介导的血管内皮细胞炎症反应以及调控机制尚不清楚。

细胞焦亡是一种依赖caspase-1/4/5/11的溶解性细胞程序性死亡方式，发生时伴有大量促炎症因子释放，是机体抗菌反应组成部分。焦亡已成为近年感染领域的研究热点。TLR4可激活caspase-1，导致细胞焦亡。尚不清楚EXOTp是否会引起TLR4介导的血管内皮细胞焦亡及其分子机制。

研究目的

本研究的主要目的是探究EXOTp在血管内皮细胞炎症和焦亡中的作用及其分子机制。

研究方法

流式细胞术检测外周血分离和磁珠分选的单核细胞以及诱导后的细胞；TEM检测外泌体的形态，NTA检测外泌体浓度和直径，WB检测外泌体CD63和TSG101的蛋白水平；共聚焦显微镜验证HUVECs内化EXOTp。

DC自然衍生的外泌体（EXO）和EXOTp分别与HUVECs共培养，RT-qPCR和WB分别检测TLR4转录和蛋白水平；RT-qPCR检测HUVECs促炎细胞因子IL-1β、IL-6和TNF-α的转录水平；ELISA检测HUVECs上清和梅毒患者血清IL-1β、IL-6和TNF-α的水平。

TLR4 shRNA慢病毒转染HUVECs, RT-qPCR和WB验证TLR4沉默情况。EXOTp作用于沉默TLR4的HUVECs，RT-qPCR和ELISA分别检测IL-1β、IL-6和TNF-α 转录和分泌水平。 EXOTp与HUVECs、沉默TLR4的HUVECs和NF-κB抑制剂BAY11-7082预处理的HUVECs共培养，WB检测NF-κB p65和MyD88的蛋白水平；RT-qPCR和ELISA分别检测IL-1β、IL-6和TNF-α转录和分泌水平。   

EXO和EXOTp分别与HUVECs共培养，活细胞工作站拍摄HUVECs形态动态变化过程；流式细胞术检测HUVECs焦亡比例；生化实验检测LDH释放水平。

EXOTp与HUVECs、沉默TLR4的HUVECs和BAY11-7082预处理的HUVECs共培养，WB检测NF-κB p65、NLRP3和GSDMD-NT的蛋白水平；流式细胞术检测细胞焦亡比例；生化实验检测LDH水平。

研究结果

EXOTp呈囊泡状，大小约为40-120 nm，表达CD63和TSG101。PKH67标记的外泌体分布在HUVECs内。

EXOTp可显著升高HUVECs TLR4转录和蛋白水平，IL-1β、IL-6和TNF-α转录和分泌水平。

慢病毒转染后HUVECs TLR4转录和蛋白水平降低。沉默TLR4可降低IL-1β、IL-6和TNF-α的表达水平。EXOTp可显著升高 NF-κB p65和MyD88的蛋白水平；沉默TLR4可降低NF-κB p65和MyD88的表达水平；抑制NF-κB 可降低IL-1β、IL-6和TNF-α的表达水平。

EXOTp可使HUVECs出现焦亡特征性的形态，增加细胞焦亡比例和LDH释放水平。

EXOTp可显著增加HUVECs NLRP3和GSDMD-NT的表达水平。沉默TLR4和抑制NF-κB均可降低NF-κB p65、NLRP3和GSDMD-NT的表达水平以及HUVECs焦亡比例和LDH释放水平。

研究结论

HUVECs可内化EXOTp。

EXOTp可诱导HUVECs 炎症反应。

EXOTp通过TLR4/MyD88/NF-κB信号通路介导HUVECs炎症反应。

EXOTp可引起HUVECs焦亡。

阻断TLR4/NF-κB信号通路可降低HUVECs 焦亡相关蛋白NLRP3和GSDMD-NT的表达水平。EXOTp通过TLR4/NF-κB信号通路引起HUVECs GSDMD介导的焦亡。

关键词

梅毒螺旋体；树突状细胞；EXOTp；炎症；焦亡

Background

Syphilis is a sexually transmitted disease caused by Tp infection, which seriously endangers human health. It is still widely spread in the world, and is listed as one of the global public health problems by WHO. Tp infection can cause vascular damage, involving cardiovascular and central nervous system and other important organs, if not treated, it can lead to serious adverse outcomes, even death. The mechanism of vascular injury caused by Tp needs to be further elucidated.

In recent years, exosomes have become a hot topic in the fields of infection, immunity and tumor. Exosomes can mediate intercellular communication by transferring bioactive molecules from parent cells to target cells through different mechanisms. More and more studies have found that exosomes can participate in the process and immune response of pathogenic microorganisms infection, and cause vascular complications. DC are the first immunologically active cells in the skin or mucous membranes that are exposed to Tp. It is currently unclear whether DC-derived exosomes treated by Tp（EXOTp) could cause vascular damage.

TLR4 was mainly expressed in monocytes, but also on the surface of endothelial cells. It binds to MyD88, activates NF-κB, and promotes the synthesis of pro-inflammatory cytokines. TLR4 is involved in the infective process of syphilis. However, whether EXOTp cause TLR4-mediated vascular endothelial cell inflammation and the regulatory mechanisms are unclear.

Pyroptosis is a kind of dissolved programmed cell death, dependent on caspase-1/4/5/11, which occurs with the release of a large number of pro-inflammatory factors and is a component of the body's antibacterial response. Pyroptosis has become a research hotspot in the field of infection in recent years. TLR4 can activate caspase-1 and lead to pyroptosis. It is unclear whether EXOTp cause TLR4-mediated pyroptosis in vascular endothelial cells and its molecular mechanism.

Objectives

To investigate the role and molecular mechanism of EXOTp in inflammation and pyroptosis of vascular endothelial cells.

Materials and methods

Flow cytometry was used to detect monocytes isolated from peripheral blood and sorted by magnetic beads, as well as induced cells, to check whether DC sorting and induction were successful. TEM was used to detect the morphology of exosomes, NTA was used to detect the concentration and diameter, and WB was used to detect the protein levels of CD63 and TSG101. Confocal microscopy was to confirm that HUVECs internalize EXOTp.

DC naturally derived exosomes (EXO) and EXOTp were co-cultured with HUVECs, RT-qPCR and WB were used to detect TLR4 transcription and protein levels, respectively. RT-qPCR was used to detect the transcription levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. ELISA was used to detect the levels of the cytokines in the supernatents and serums of syphilis patients.

TLR4 shRNA lentivirus was transfected into HUVECs, RT-qPCR and WB were used to verify TLR4 silencing. TLR4 shRNA HUVECs were treated with EXOTp, RT-qPCR and ELISA were used to detect the cytokines transcription and secretion levels, respectively. EXOTp were co-cultured with HUVECs, TLR4 shRNA HUVECs and  pretreated HUVECs with NF-κB inhibitor BAY11-7082, and the protein levels of NF-κB p65 and MyD88 were detected by WB. The transcription and secretion levels of the cytokines were detected by RT-qPCR and ELISA, respectively.

EXO and EXOTp were co-cultured with HUVECs, and the dynamic morphological change process was captured by living cell workstation. The death rate of HUVECs was detected by flow cytometry. The release level of LDH was examined by biochemical experiments.

EXOTp were co-cultured with HUVECs, TLR4 shRNA HUVECs and pretreated HUVECs with BAY11-7082, respectively. The protein levels of NF-κB p65, NLRP3 and GSDMD-NT were detected by WB. The cell death rate was detected by flow cytometry. LDH levels were checked by biochemical experiments.

Results

1. EXOTp were vesicular, approximately 40-120 nm in size, and expressed CD63 and TSG101. The PKH67-labeled exosomes are distributed in HUVECs.

2. HUVECs with EXOTp had elevated TLR4 transcription and protein levels, and raised IL-1β, IL-6, and TNF-α transcription and secretion levels. 

3. TLR4 transcription and protein levels of TLR4 shRNA HUVECs were decreased. Silencing TLR4 reduced the expression levels of the cytokines. The levels of NF-κB p65 and MyD88 were increased in HUVECs with EXOTp. Silencing TLR4 decreased the expression of these proteins. Inhibition of NF-κB reduced the expression level of the cytokines.  

4. HUVECs with EXOTp showed characteristic forms of pyroptosis, with increased mortality ratio and LDH release.

5. The protein expression of NLRP3 and GSDMD-NT was increased in HUVECs with EXOTp. Silencing TLR4 and inhibiting NF-κB decreased the expression levels of NF-κB p65, NLRP3 and GSDMD-NT, as well as the death rate and LDH release of HUVECs.

Conclusions

HUVECs could internalize EXOTp.

EXOTp could induce HUVECs inflammatory response. 

EXOTp could cause inflammatory responses in HUVECs via the TLR4/MyD88/NF-κB signaling pathway.

EXOTp could cause pyroptosis of HUVECs.

Blocking TLR4/NF-κB signaling pathway could reduce the expression levels of NLRP3 and GSDMD-NT in HUVECs. EXOTp could induce GSDMD-mediated pyroptosis of HUVECs through the TLR4/NF-κB signaling pathway.

Keywords

Treponema pallidum; Dendritic cells; EXOTp; Inflammation; Pyroptosis