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Abstract：
OBJECTIVE: Abnormal programmed cell death in immune cells is associated with autoimmune diseases, but the patterns of programmed cell death in systemic lupus erythematosus (SLE) and especially lupus nephritis (LN) remain unclear. This study aims to explore the association between SLE, LN, and immune cell death patterns.
METHODS: Bulk RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) data were downloaded from the Gene Expression Omnibus (GEO) database. Bioinformatic analysis was conducted to explore the expression levels of genes related to 3 cell death patterns in peripheral blood mononuclear cells of SLE patients. Key cell subsets involved in the imbalance of cell death patterns were identified through scRNA-seq. Immunofluorescence was used to detect the expression levels of receptor interacting serine/threonine kinase 3 (RIPK3), mixed-lineage kinase domain-like protein (MLKL), phosphorylated MLKL (pMLKL), caspase 1 (CASP1), CD1c molecule (CD1C), C-type lectin domain containing 9A (CLEC9A), and X-C motif chemokine receptor 1 (XCR1) in dendritic cells (DC). scRNA-seq was performed on kidney tissues collected from LN patients and healthy controls (HC) at the Third Xiangya Hospital of Central South University, followed by bioinformatic analysis to identify key cell subsets involved in the imbalance of cell death patterns. Pseudotime analysis and ligand-receptor analysis were used to explore the differentiation direction and cell communication of different DC subsets. Transient transfection was used to transfect RAW264.7 cells with empty plasmid, empty plasmid+dsDNA (HSV-DNA), empty plasmid+200 μmol/L tert-butyl hydroperoxide (TBHP), stimulator of interferon genes (STING) shRNA plasmid, STING shRNA plasmid+dsDNA (HSV-DNA), and STING shRNA plasmid+200 μmol/L TBHP. Annexin V-mCherry and SYTOX Green staining were used to detect cell death in each group. Western blotting was used to detect the activation of CASP1, gasdermin D (GSDMD), RIPK3, and MLKL in each group.
RESULTS: Bioinformatic analysis showed an imbalance in 3 cell death patterns in SLE and LN patients: Pro-inflammatory pyroptosis and necroptosis were activated, while anti-inflammatory apoptosis was inhibited. The key cell subsets involved were DC subsets, particularly focusing on CLEC9A+cDC1. Immunofluorescence results showed that the expression levels of RIPK3, MLKL, and CASP1 in DCs were higher in the SLE group compared to the HC group. pMLKL and CASP1 expression levels in renal cDC1 marked by CLEC9A and XCR1 were higher in the LN group than in the HC group. Pseudotime analysis and ligand-receptor analysis suggested that the CLEC9A+cDC1 subset in LN kidney tissues originated from peripheral circulation. Annexin V-mCherry and SYTOX Green staining results showed that the number of dead cells decreased in the STING shRNA transfection group compared to the empty plasmid group in RAW264.7 cells. Western blotting results showed that the activation of CASP1, GSDMD, RIPK3, and MLKL was decreased in the STING shRNA transfection group compared to the empty plasmid group.
CONCLUSIONS: This study provides novel insights into the role of CLEC9A+cDC1 in the imbalance of cell death patterns in SLE and LN.
目的: 免疫细胞的异常程序性死亡与自身免疫性疾病相关，但系统性红斑狼疮(systemic lupus erythematosus，SLE)，尤其是狼疮肾炎(lupus nephritis，LN)的程序性死亡模式尚不清楚。本研究旨在探究SLE和LN与免疫细胞死亡模式的关联。方法: 在高通量基因表达(Gene Expression Omnibus，GEO)数据库中下载批量RNA测序(bulk RNA sequencing，bulk RNA-seq)和单细胞RNA测序(single-cell RNA sequencing，scRNA-seq)数据，通过生物信息学分析探究SLE患者外周血单个核细胞中3种细胞死亡模式相关基因的表达水平；通过scRNA-seq确定参与细胞死亡模式失衡的关键细胞亚群；采用免疫荧光法检测树突状细胞(dendritic cell，DC)中受体相互作用丝氨酸/苏氨酸激酶3(receptor interacting serine/threonine kinase 3，RIPK3)、混合谱系激酶结构域样蛋白(mixed-lineage kinase domain-like protein、MLKL)、磷酸化MLKL(phosphorylated MLKL，pMLKL)、含半胱氨酸的天冬氨酸蛋白水解酶1(caspase 1、CASP1)、CD1c分子(CD1c molecule、CD1C)、含C型凝集素结构域9A(C-type lectin domain containing 9A、CLEC9A)和X-C基序趋化因子受体1(X-C motif chemokine receptor 1，XCR1)的表达水平；对中南大学湘雅三医院收集的LN患者和健康对照者(healthy control，HC)的肾组织进行scRNA-seq，并通过生物信息学分析参与细胞死亡模式失衡的关键细胞亚群；采用拟时序分析和配受体分析探究不同DC亚群的分化方向和细胞通信。采用瞬时转染技术分别在RAW264.7细胞中转染空质粒、空质粒+dsDNA(HSV-DNA)、空质粒+200 μmol/L叔丁基过氧化氢(tert-butyl-hydroperoxide，TBHP)、STING shRNA质粒、STING shRNA质粒+dsDNA(HSV-DNA)、STING shRNA质粒+200 μmol/L TBHP；采用Annexin V-mCherry和SYTOX Green染色检测各组细胞死亡情况；蛋白质印迹法检测各组CASP1、消皮素D(gasdermin D，GSDMD)、RIPK3和MLKL的活化情况。结果: 生物信息学分析显示SLE和LN患者存在3种细胞死亡模式的失衡：促炎型细胞焦亡和坏死性凋亡被激活，抗炎型细胞凋亡被抑制。其中的关键细胞亚群为DC亚群，并聚焦于CLEC9A+cDC1。免疫荧光法结果显示在外周血中SLE组DC中RIPK3、MLKL和CASP1表达水平较HC组DC升高；通过CLEC9A和XCR1标记肾组织中的cDC1，结果显示LN组cDC1的pMLKL和CASP1表达水平高于HC组。拟时序分析和配受体分析提示LN肾组织中CLEC9A+cDC1亚群存在外周循环起源。Annexin V-mCherry和SYTOX Green染色结果显示，在RAW264.7细胞中，与转染空质粒组相比，转染STING shRNA质粒组的死亡细胞数目减少；蛋白质印迹法结果显示与转染空质粒组相比，转染STING shRNA质粒组CASP1、GSDMD、RIPK3和MLKL的活化减少。结论: 本研究为CLEC9A+cDC1在SLE和LN的细胞死亡模式失衡中的作用提供了新的思路。.
METHODS: Bulk RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) data were downloaded from the Gene Expression Omnibus (GEO) database. Bioinformatic analysis was conducted to explore the expression levels of genes related to 3 cell death patterns in peripheral blood mononuclear cells of SLE patients. Key cell subsets involved in the imbalance of cell death patterns were identified through scRNA-seq. Immunofluorescence was used to detect the expression levels of receptor interacting serine/threonine kinase 3 (RIPK3), mixed-lineage kinase domain-like protein (MLKL), phosphorylated MLKL (pMLKL), caspase 1 (CASP1), CD1c molecule (CD1C), C-type lectin domain containing 9A (CLEC9A), and X-C motif chemokine receptor 1 (XCR1) in dendritic cells (DC). scRNA-seq was performed on kidney tissues collected from LN patients and healthy controls (HC) at the Third Xiangya Hospital of Central South University, followed by bioinformatic analysis to identify key cell subsets involved in the imbalance of cell death patterns. Pseudotime analysis and ligand-receptor analysis were used to explore the differentiation direction and cell communication of different DC subsets. Transient transfection was used to transfect RAW264.7 cells with empty plasmid, empty plasmid+dsDNA (HSV-DNA), empty plasmid+200 μmol/L tert-butyl hydroperoxide (TBHP), stimulator of interferon genes (STING) shRNA plasmid, STING shRNA plasmid+dsDNA (HSV-DNA), and STING shRNA plasmid+200 μmol/L TBHP. Annexin V-mCherry and SYTOX Green staining were used to detect cell death in each group. Western blotting was used to detect the activation of CASP1, gasdermin D (GSDMD), RIPK3, and MLKL in each group.
RESULTS: Bioinformatic analysis showed an imbalance in 3 cell death patterns in SLE and LN patients: Pro-inflammatory pyroptosis and necroptosis were activated, while anti-inflammatory apoptosis was inhibited. The key cell subsets involved were DC subsets, particularly focusing on CLEC9A+cDC1. Immunofluorescence results showed that the expression levels of RIPK3, MLKL, and CASP1 in DCs were higher in the SLE group compared to the HC group. pMLKL and CASP1 expression levels in renal cDC1 marked by CLEC9A and XCR1 were higher in the LN group than in the HC group. Pseudotime analysis and ligand-receptor analysis suggested that the CLEC9A+cDC1 subset in LN kidney tissues originated from peripheral circulation. Annexin V-mCherry and SYTOX Green staining results showed that the number of dead cells decreased in the STING shRNA transfection group compared to the empty plasmid group in RAW264.7 cells. Western blotting results showed that the activation of CASP1, GSDMD, RIPK3, and MLKL was decreased in the STING shRNA transfection group compared to the empty plasmid group.
CONCLUSIONS: This study provides novel insights into the role of CLEC9A+cDC1 in the imbalance of cell death patterns in SLE and LN.
目的: 免疫细胞的异常程序性死亡与自身免疫性疾病相关，但系统性红斑狼疮(systemic lupus erythematosus，SLE)，尤其是狼疮肾炎(lupus nephritis，LN)的程序性死亡模式尚不清楚。本研究旨在探究SLE和LN与免疫细胞死亡模式的关联。方法: 在高通量基因表达(Gene Expression Omnibus，GEO)数据库中下载批量RNA测序(bulk RNA sequencing，bulk RNA-seq)和单细胞RNA测序(single-cell RNA sequencing，scRNA-seq)数据，通过生物信息学分析探究SLE患者外周血单个核细胞中3种细胞死亡模式相关基因的表达水平；通过scRNA-seq确定参与细胞死亡模式失衡的关键细胞亚群；采用免疫荧光法检测树突状细胞(dendritic cell，DC)中受体相互作用丝氨酸/苏氨酸激酶3(receptor interacting serine/threonine kinase 3，RIPK3)、混合谱系激酶结构域样蛋白(mixed-lineage kinase domain-like protein、MLKL)、磷酸化MLKL(phosphorylated MLKL，pMLKL)、含半胱氨酸的天冬氨酸蛋白水解酶1(caspase 1、CASP1)、CD1c分子(CD1c molecule、CD1C)、含C型凝集素结构域9A(C-type lectin domain containing 9A、CLEC9A)和X-C基序趋化因子受体1(X-C motif chemokine receptor 1，XCR1)的表达水平；对中南大学湘雅三医院收集的LN患者和健康对照者(healthy control，HC)的肾组织进行scRNA-seq，并通过生物信息学分析参与细胞死亡模式失衡的关键细胞亚群；采用拟时序分析和配受体分析探究不同DC亚群的分化方向和细胞通信。采用瞬时转染技术分别在RAW264.7细胞中转染空质粒、空质粒+dsDNA(HSV-DNA)、空质粒+200 μmol/L叔丁基过氧化氢(tert-butyl-hydroperoxide，TBHP)、STING shRNA质粒、STING shRNA质粒+dsDNA(HSV-DNA)、STING shRNA质粒+200 μmol/L TBHP；采用Annexin V-mCherry和SYTOX Green染色检测各组细胞死亡情况；蛋白质印迹法检测各组CASP1、消皮素D(gasdermin D，GSDMD)、RIPK3和MLKL的活化情况。结果: 生物信息学分析显示SLE和LN患者存在3种细胞死亡模式的失衡：促炎型细胞焦亡和坏死性凋亡被激活，抗炎型细胞凋亡被抑制。其中的关键细胞亚群为DC亚群，并聚焦于CLEC9A+cDC1。免疫荧光法结果显示在外周血中SLE组DC中RIPK3、MLKL和CASP1表达水平较HC组DC升高；通过CLEC9A和XCR1标记肾组织中的cDC1，结果显示LN组cDC1的pMLKL和CASP1表达水平高于HC组。拟时序分析和配受体分析提示LN肾组织中CLEC9A+cDC1亚群存在外周循环起源。Annexin V-mCherry和SYTOX Green染色结果显示，在RAW264.7细胞中，与转染空质粒组相比，转染STING shRNA质粒组的死亡细胞数目减少；蛋白质印迹法结果显示与转染空质粒组相比，转染STING shRNA质粒组CASP1、GSDMD、RIPK3和MLKL的活化减少。结论: 本研究为CLEC9A+cDC1在SLE和LN的细胞死亡模式失衡中的作用提供了新的思路。.
摘要：
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