Abstract：
BACKGROUND: Mast cell-derived mediators induce vasodilatation and fluid extravasation, leading to cardiovascular failure in severe anaphylaxis. We previously revealed a synergistic interaction between the cytokine IL-4 and the mast cell-derived mediator histamine in modulating vascular endothelial (VE) dysfunction and severe anaphylaxis. The mechanism by which IL-4 exacerbates histamine-induced VE dysfunction and severe anaphylaxis is unknown.
OBJECTIVE: We sought to identify the IL-4-induced molecular processes regulating the amplification of histamine-induced VE barrier dysfunction and the severity of IgE-mediated anaphylactic reactions.
METHODS: RNA sequencing, Western blot, Ca2+ imaging, and barrier functional analyses were performed on the VE cell line (EA.hy926). Pharmacologic degraders (selective proteolysis-targeting chimera) and genetic (lentiviral short hairpin RNA) inhibitors were used to determine the roles of signal transducer and activator of transcription 3 (STAT3) and STAT6 in conjunction with in vivo model systems of histamine-induced hypovolemic shock.
RESULTS: IL-4 enhancement of histamine-induced VE barrier dysfunction was associated with increased VE-cadherin degradation, intracellular calcium flux, and phosphorylated Src levels and required transcription and de novo protein synthesis. RNA sequencing analyses of IL-4-stimulated VE cells identified dysregulation of genes involved in cell proliferation, cell development, and cell growth, and transcription factor motif analyses revealed a significant enrichment of differential expressed genes with putative STAT3 and STAT6 motif. IL-4 stimulation in EA.hy926 cells induced both serine residue 727 and tyrosine residue 705 phosphorylation of STAT3. Genetic and pharmacologic ablation of VE STAT3 activity revealed a role for STAT3 in basal VE barrier function; however, IL-4 enhancement and histamine-induced VE barrier dysfunction was predominantly STAT3 independent. In contrast, IL-4 enhancement and histamine-induced VE barrier dysfunction was STAT6 dependent. Consistent with this finding, pharmacologic knockdown of STAT6 abrogated IL-4-mediated amplification of histamine-induced hypovolemia.
CONCLUSIONS: These studies unveil a novel role of the IL-4/STAT6 signaling axis in the priming of VE cells predisposing to exacerbation of histamine-induced anaphylaxis.
OBJECTIVE: We sought to identify the IL-4-induced molecular processes regulating the amplification of histamine-induced VE barrier dysfunction and the severity of IgE-mediated anaphylactic reactions.
METHODS: RNA sequencing, Western blot, Ca2+ imaging, and barrier functional analyses were performed on the VE cell line (EA.hy926). Pharmacologic degraders (selective proteolysis-targeting chimera) and genetic (lentiviral short hairpin RNA) inhibitors were used to determine the roles of signal transducer and activator of transcription 3 (STAT3) and STAT6 in conjunction with in vivo model systems of histamine-induced hypovolemic shock.
RESULTS: IL-4 enhancement of histamine-induced VE barrier dysfunction was associated with increased VE-cadherin degradation, intracellular calcium flux, and phosphorylated Src levels and required transcription and de novo protein synthesis. RNA sequencing analyses of IL-4-stimulated VE cells identified dysregulation of genes involved in cell proliferation, cell development, and cell growth, and transcription factor motif analyses revealed a significant enrichment of differential expressed genes with putative STAT3 and STAT6 motif. IL-4 stimulation in EA.hy926 cells induced both serine residue 727 and tyrosine residue 705 phosphorylation of STAT3. Genetic and pharmacologic ablation of VE STAT3 activity revealed a role for STAT3 in basal VE barrier function; however, IL-4 enhancement and histamine-induced VE barrier dysfunction was predominantly STAT3 independent. In contrast, IL-4 enhancement and histamine-induced VE barrier dysfunction was STAT6 dependent. Consistent with this finding, pharmacologic knockdown of STAT6 abrogated IL-4-mediated amplification of histamine-induced hypovolemia.
CONCLUSIONS: These studies unveil a novel role of the IL-4/STAT6 signaling axis in the priming of VE cells predisposing to exacerbation of histamine-induced anaphylaxis.
摘要：
背景：肥大细胞（MC）衍生的介体诱导血管舒张和液体外渗，导致严重过敏反应的心血管衰竭。我们先前已经揭示了细胞因子IL-4与MC衍生的介质组胺之间在调节血管内皮（VE）功能障碍和严重过敏反应中的协同相互作用。IL-4加剧组胺诱导的VE功能障碍和严重过敏反应的机制尚不清楚。
目的：确定IL-4诱导的分子过程调节组胺诱导的VE屏障功能障碍的扩增和IgE介导的过敏反应的严重程度。
方法：RNAseq，蛋白质印迹,血管内皮细胞系的Ca2成像和屏障功能分析（EA。hy926）。药理学降解剂（选择性PROTAC（蛋白水解靶向嵌合体）和遗传（慢病毒shRNA）抑制剂用于确定STAT3和STAT6与组胺诱导的低血容量性休克的体内模型系统的联合作用。
结果：IL-4增强组胺诱导的VE屏障功能障碍与VE-Cadherin降解增加有关，细胞内钙通量，磷酸-Src水平和需要转录和从头蛋白合成。对IL-4刺激的VE细胞的RNAseq分析确定了与细胞增殖有关的基因失调，细胞发育,细胞生长和转录因子基序分析表明，差异表达基因（DEG）显着富集，具有推定的STAT3和STAT6基序。EA中的IL-4刺激。hy926细胞诱导STAT3Y705和STAT3S727磷酸化。VESTAT3活性的遗传和药理消融揭示了STAT3在基础VE屏障功能中的作用，然而,IL-4增强和组胺诱导的VE屏障功能障碍主要是不依赖STAT3的.相比之下,IL-4增强和组胺诱导的VE屏障功能障碍是STAT6依赖性的。与这一发现一致，STAT6的药理学敲除消除了IL-4介导的组胺诱导的低血容量的扩增。
结论：这些研究揭示了IL-4/STAT6信号轴在VE细胞引发组胺诱导的过敏反应恶化中的新作用。
目的：确定IL-4诱导的分子过程调节组胺诱导的VE屏障功能障碍的扩增和IgE介导的过敏反应的严重程度。
方法：RNAseq，蛋白质印迹,血管内皮细胞系的Ca2成像和屏障功能分析（EA。hy926）。药理学降解剂（选择性PROTAC（蛋白水解靶向嵌合体）和遗传（慢病毒shRNA）抑制剂用于确定STAT3和STAT6与组胺诱导的低血容量性休克的体内模型系统的联合作用。
结果：IL-4增强组胺诱导的VE屏障功能障碍与VE-Cadherin降解增加有关，细胞内钙通量，磷酸-Src水平和需要转录和从头蛋白合成。对IL-4刺激的VE细胞的RNAseq分析确定了与细胞增殖有关的基因失调，细胞发育,细胞生长和转录因子基序分析表明，差异表达基因（DEG）显着富集，具有推定的STAT3和STAT6基序。EA中的IL-4刺激。hy926细胞诱导STAT3Y705和STAT3S727磷酸化。VESTAT3活性的遗传和药理消融揭示了STAT3在基础VE屏障功能中的作用，然而,IL-4增强和组胺诱导的VE屏障功能障碍主要是不依赖STAT3的.相比之下,IL-4增强和组胺诱导的VE屏障功能障碍是STAT6依赖性的。与这一发现一致，STAT6的药理学敲除消除了IL-4介导的组胺诱导的低血容量的扩增。
结论：这些研究揭示了IL-4/STAT6信号轴在VE细胞引发组胺诱导的过敏反应恶化中的新作用。
