研究背景：
支气管哮喘是多种炎症细胞参与的慢性气道炎症性疾病，发病机制复杂，受宿主易感因素和环境因素的共同影响。T淋巴细胞是人体免疫系统的重要组成部分，在哮喘炎症中发挥着独特作用。CD4+T细胞亚群在正常情况下处于平衡状态，但当失衡时将引发疾病，相关细胞因子可能引起气道结构细胞改变，发展为重症哮喘并伴有气道重塑表型。
研究目的：
本研究旨在探讨Kv1.3在重症哮喘气道炎症和重塑中的作用，研究此条件下Kv1.3参与Th17/Treg细胞平衡及上皮间质转化的分子机制，评价Kv1.3抑制剂对重症哮喘的干预效果。
材料与方法：
本研究分为三个部分。第一部分使用基因表达综合数据库（Gene Expression Omnibus, GEO）中重症哮喘气道组织和外周血样本探讨免疫细胞浸润状况，分析Kv1.3表达、分布定位及CD4+T细胞亚群平衡中Kv1.3表达情况。实验室提取重症哮喘患者外周血CD4+T细胞，评估细胞增殖能力，分析细胞中Kv1.3表达并与CCR7共定位检测。第二部分首先使用野生型小鼠构建卵清蛋白（ovalbumin, OVA）诱导哮喘小鼠模型，评估小鼠行为学变化，无创肺功能仪测定小鼠气道反应，苏木精-伊红、过碘酸-雪夫和马松病理染色观察小鼠气道炎症浸润、黏液分泌和胶原纤维沉积，酶联免疫吸附试验检测小鼠肺泡灌洗液中白细胞介素17A（interleukin 17A, IL-17A）水平，免疫荧光标记肺组织中Kv1.3，免疫组化标记气道上皮间质转化分子标志物，实时定量聚合酶链式反应检测小鼠脾脏CD4+T细胞中RORγt、IL-17A和Foxp3 mRNA的相对表达水平。接着构建Kv1.3基因全敲除小鼠，分组使用OVA诱导，评估气道炎症浸润、黏液分泌和重塑改变，免疫荧光标记肺组织中CD4+CCR7-Kv1.3+T细胞，Fluo-4 AM检测小鼠脾脏CD4+T细胞内钙离子水平，流式细胞术分析小鼠脾脏CD4+T细胞的Th17/Treg亚群，酶联免疫吸附试验测定小鼠肺泡灌洗液中IL-17A水平，小鼠肺组织转录组测序评估哮喘中Kv1.3相关生物过程。最后体外构建辅助性T细胞17（T helper cell 17, Th17）和调节性T细胞（regulatory T cell, Treg）细胞模型及支气管上皮细胞间质转化模型，免疫荧光标记支气管上皮细胞间质转化分子标志物，细胞划痕试验检测上皮细胞迁移能力，免疫印迹检测各细胞模型中Kv1.3与相关信号通路关键分子的水平。第三部分使用5-（4-苯氧基丁氧基）补骨脂素（5-(4-Phenoxybutoxy)psoralen, PAP-1）和克拉霉素分别对OVA诱导哮喘小鼠进行药物干预，评价Kv1.3抑制剂对哮喘气道炎症和重塑的干预效果。
研究结果：
1.GEO数据库中GSE143303显示2型和非2型重症哮喘患者气道组织中CD4+T细胞被活化且可能具有更强的增殖能力，可能涉及辅助性T细胞的分化。GSE147881显示重症哮喘气道组织中Kv1.3表达较高，GSE60679提示Kv1.3在Th17/Treg细胞平衡中具有作用。GSE110551显示，在重症哮喘外周血样本中，CD4+T细胞同样具有活化趋势。Single Cell Portal显示，在外周血免疫细胞中，Kv1.3主要分布在CD4+T细胞上。临床采集的重症哮喘外周血CD4+T细胞的增殖能力较健康对照者更强，Kv1.3 mRNA水平更高且其中CD4+CCR7-Kv1.3+T细胞较多。
2.野生型OVA诱导哮喘小鼠构建成功。与对照组相比，野生型哮喘小鼠气道高反应（P<0.0001），支气管壁增厚，气道上皮肿胀，气道周围、血管周围、肺泡间隙、肺泡腔炎症细胞明显浸润（P<0.001），气道上皮层杯状细胞黏液分泌较多（P<0.05），气道周围胶原沉积更明显（P<0.001）。与对照组相比，蛋白免疫印迹显示野生型哮喘小鼠肺组织中Kv1.3水平较高（P<0.05）。小鼠肺组织CD3、CD4、CCR7和Kv1.3免疫荧光共定位显示，野生型哮喘小鼠肺组织CD4+T细胞浸润，具体表现为CD3+CD4+CCR7-Kv1.3+T细胞增多。小鼠脾脏CD4+T细胞转录因子的mRNA水平检测显示，野生型哮喘小鼠脾脏CD4+T细胞的RORγt（P<0.05）和IL-17A（P<0.05）表达显著升高，Foxp3表达显著降低（P<0.001）。小鼠肺泡灌洗液酶联免疫吸附试验显示，野生型哮喘小鼠支气管肺泡灌洗液中IL-17A增加（P<0.01），IL-10降低（P<0.05）。小鼠肺组织免疫组化标记显示，野生型哮喘小鼠肺组织中N-钙粘蛋白和波形蛋白水平较对照组升高，而E-钙粘蛋白水平降低。使用OVA诱导Kv1.3基因全敲除小鼠后发现，Kv1.3敲除后可以降低OVA诱导所致的气道高反应（P<0.0001），同时可以减少气道周围炎症细胞浸润（P<0.0001）、气道上皮黏液分泌（P<0.05）及气道周围胶原纤维沉积（P<0.0001），并且降低支气管肺泡灌洗液中的IL-17A浓度。在Kv1.3敲除后，即使经OVA诱导也并未见小鼠脾脏CD4+T细胞中钙浓度的明显上升，并且Th17/Treg细胞失衡表现减弱。与此同时，Kv1.3缺失也缓解了OVA诱导的小鼠气道上皮间质转化。小鼠肺组织转录组学测定结果提示，在哮喘中Kv1.3与免疫反应及气道上皮改变均有关。体外构建的Th17和Treg模型免疫印迹结果显示，同样经相关细胞因子诱导，Kv1.3敲除小鼠脾脏CD4+T细胞中Kv1.3、p-JAK2/JAK2和p-STAT3/STAT3水平较野生型小鼠脾脏CD4+T细胞降低。体外构建的气道上皮细胞间质转化模型检测显示，同样经相关细胞因子诱导，Kv1.3被抑制后间充质标志物减少，且PI3K/Akt/mTOR信号通路的关键分子磷酸化减弱。
3.体内药物干预试验结果显示，Kv1.3选择性抑制剂PAP-1可以减轻哮喘气道高反应，缓解气道炎症及重塑表型，降低支气管肺泡灌洗液中的IL-17A浓度。与此同时，对Kv1.3具有抑制效应的克拉霉素在一定程度上可以缓解哮喘气道高反应，减轻气道周围炎症浸润，减弱气道上皮间质转化。
研究结论：
1.重症哮喘中CD4+T细胞活化且Kv1.3水平升高，涉及Th17/Treg细胞失衡。
2.体内和体外试验共同显示，Kv1.3与哮喘炎症中Th17/Treg细胞失衡及气道上皮间质转化密切相关。
3.体内试验显示，Kv1.3抑制剂（如克拉霉素）在一定程度上可以控制哮喘气道炎症及重塑。

BACKGROUND
Bronchial asthma is a chronic inflammatory disease of the airways characterized by the involvement of a diverse array of inflammatory cells. The pathogenesis of this condition is multifaceted, being influenced by both host susceptibility factors and environmental triggers. T lymphocytes constitute a critical component of the immune system and play a pivotal role in asthma-related inflammatory responses. Under normal conditions, the CD4+T cell subpopulation maintains a balanced state; however, any disruption in this equilibrium can lead to associated diseases. Dysregulation of these cells may result in alterations in airway structural cells, potentially progressing to severe asthma with an airway remodeling phenotype.
AIM
The aim of this study was to investigate the role of Kv1.3 in airway inflammation and remodeling in severe asthma, to elucidate the molecular mechanisms by which Kv1.3 influences the Th17/Treg cell balance and epithelial-mesenchymal transition under these conditions, and to assess the therapeutic efficacy of Kv1.3 inhibitors in severe asthma.
MATERIAL & METHODS
Part Ⅰ: Airway tissue and peripheral blood samples from patients with severe asthma were utilized from the Gene Expression Omnibus (GEO) database to investigate immune cell infiltration, Kv1.3 expression, distribution, and localization, as well as Kv1.3 expression in the balance of CD4+T cell subpopulations. Additionally, CD4+T cells were isolated from the peripheral blood of patients with severe asthma in our laboratory to evaluate cell proliferation, analyze Kv1.3 expression, and perform co-localization studies with CCR7.
Part Ⅱ: Wild-type mice were utilized to establish an ovalbumin (OVA)-induced asthma model, and their behavioral changes were assessed. Airway responsiveness was evaluated using a non-invasive pulmonary function apparatus. Hematoxylin-eosin, Periodic Acid-Schiff (PAS), and Masson's trichrome staining were used to assess airway inflammatory infiltration, mucus secretion, and collagen fiber deposition, respectively. The concentration of interleukin-17A (IL-17A) in bronchoalveolar lavage fluid was quantified using an enzyme-linked immunosorbent assay (ELISA). Immunofluorescence labeling was performed to detect Kv1.3 expression in lung tissue, while immunohistochemistry was used to label molecular markers associated with airway epithelial-mesenchymal transition. The relative mRNA expression levels of RORγt, IL-17A, and Foxp3 in CD4+T cells isolated from mouse spleens were quantified by real-time polymerase chain reaction. Subsequently, Kv1.3 gene knockout mice were generated, and OVA induction was employed to assess airway inflammatory infiltration, mucus secretion, and remodeling changes across different groups. Immunofluorescence staining was utilized to label CD4+CCR7-Kv1.3+T cells in lung tissue samples. Intracellular calcium levels in splenic CD4+T cells were measured using Fluo-4 AM. Flow cytometry was conducted to analyze the Th17/Treg subsets of splenic CD4+T cells, while enzyme-linked immunosorbent assay (ELISA) was used to quantify IL-17A levels in bronchoalveolar lavage fluid. Transcriptome sequencing of mouse lung tissue was performed to evaluate Kv1.3-related biological processes in asthma. Finally, in vitro models of T helper cell 17 (Th17) and regulatory T cell (Treg), as well as bronchial epithelial-mesenchymal transition, were constructed. Immunofluorescence was used to label molecular markers of bronchial epithelial-mesenchymal transition. Cell migration was assessed using a scratch assay, and western blot analysis was employed to measure the levels of Kv1.3 and key molecules in relevant signaling pathways in each cell model.
Part Ⅲ: OVA-induced asthmatic mice were treated with 5-(4-phenoxybutoxy)psoralen (PAP-1) and clarithromycin, respectively, to evaluate the therapeutic effects of Kv1.3 inhibitors on airway inflammation and remodeling in asthma.
RESULTS
Part Ⅰ: The GSE143303 dataset in the GEO database revealed that CD4+T cells in the airway tissues of both type 2 and non-type 2 severe asthma patients exhibited activation and potentially enhanced proliferative capacity, which may be associated with the differentiation of helper T cells. The GSE147881 dataset demonstrated elevated expression of Kv1.3 in the airway tissues of severe asthmatic patients, while GSE60679 indicated that Kv1.3 plays a crucial role in maintaining the balance between Th17 and Treg cells. Additionally, GSE110551 showed an activation trend of CD4+T cells in peripheral blood samples from severe asthmatic patients. Data from the Single Cell Portal further elucidated that Kv1.3 is predominantly expressed on CD4+T cells in the immune cells from peripheral blood. Compared to healthy controls, CD4+T cells from the peripheral blood of severe asthmatic patients exhibited significantly stronger proliferative ability, characterized by higher levels of Kv1.3 mRNA and increased numbers of CD4+CCR7- Kv1.3+T cells.
Part Ⅱ: The wild-type asthmatic mouse model was successfully induced by OVA. Compared to the control group, wild-type asthmatic mice exhibited significantly higher airway hyperresponsiveness (P<0.0001), thickened bronchial walls, swollen airway epithelium, and pronounced infiltration of inflammatory cells around the airways, perivascular regions, alveolar spaces, and alveolar cavities (P<0.001). Increased mucus secretion was observed in goblet cells of the upper airway cortex (P<0.05), and collagen deposition was more pronounced around the airways (P<0.001). Western blot analysis revealed elevated levels of Kv1.3 in lung tissues of wild-type asthmatic mice compared to controls (P<0.05). Immunofluorescence co-localization studies demonstrated that CD4+T cells infiltrated the lung tissues of wild-type asthmatic mice, specifically as an increase in CD3+CD4+CCR7-Kv1.3+T cells. The mRNA levels of transcriptional factors  in splenic CD4+T cells showed significant increases in RORγt (P<0.05) and IL-17A (P<0.05), while Foxp3 levels were markedly decreased (P<0.001). ELISA indicated increased IL-17A (P<0.01) and decreased IL-10 (P<0.05) in bronchoalveolar lavage fluid from wild-type asthmatic mice. Immunohistochemical markers in lung tissues revealed higher levels of N-cadherin and vimentin but lower levels of E-cadherin in wild-type asthmatic mice compared to controls. Using OVA to induce Kv1.3 gene knockout mice, it was found that Kv1.3 knockout reduced OVA-induced airway hyperresponsiveness (P<0.0001), decreased inflammatory cell infiltration around the airways (P<0.0001), reduced mucus secretion by airway epithelial cells (P<0.05), and diminished collagen fiber deposition around the airways (P<0.0001). The concentration of IL-17A in bronchoalveolar lavage fluid also decreased. After Kv1.3 knockout, no significant increase in calcium concentration in splenic CD4+T cells was observed even after OVA induction, and the imbalance between Th17 and Treg cells was mitigated. Additionally, Kv1.3 deletion alleviated OVA-induced airway epithelial-mesenchymal transition in mice. Transcriptomic analysis of lung tissues suggested that Kv1.3 is associated with immune responses and airway epithelial changes in asthma. In vitro models of Th17 and Treg cells showed that the levels of Kv1.3, p-JAK2/JAK2, and p-STAT3/STAT3 in splenic CD4+T cells from Kv1.3 knockout mice were lower than those in wild-type mice, after the induction by related cytokines. Inhibition of Kv1.3 in an in vitro airway epithelial-mesenchymal transition model also weakened the phosphorylation of key molecules in the PI3K/Akt/mTOR signaling pathway.
Part Ⅲ: The results of drug intervention trials in vivo demonstrated that PAP-1, a selective inhibitor of Kv1.3, can mitigate airway hyperresponsiveness, alleviate airway inflammation and remodeling, and decrease the concentration of IL-17A in bronchoalveolar lavage fluid in asthma models. Additionally, clarithromycin, which exhibits inhibitory effects on Kv1.3, also significantly reduced airway hyperresponsiveness in asthma, alleviated peribronchial inflammatory infiltration, and attenuated airway epithelial-mesenchymal transition.
CONCLUSIONS
Part Ⅰ: Elevated CD4+T cell activation and increased Kv1.3 expression levels in patients with severe asthma have been related to an imbalance between Th17 and Treg cells.
Part Ⅱ: Both in vivo and in vitro studies have demonstrated a significant association between the Kv1.3 channels and the Th17/Treg cell imbalance, as well as airway epithelial-mesenchymal transition in the context of asthma inflammation.
Part Ⅲ: In vivo studies have demonstrated that Kv1.3 inhibitors, including clarithromycin, can effectively mitigate airway inflammation and remodeling in asthma to a certain extent.