BCL-2保护人和小鼠Th17细胞抵抗糖皮质激素诱导的细胞凋亡
2016/07/07
背景：糖皮质激素抵抗与Th17驱动的炎症相关，但其机制仍不清楚。我们旨在明确人与小鼠Th17细胞是否能抵抗糖皮质激素诱导的细胞凋亡。
方法：新鲜分离人血Th17细胞，体外分化IL-17F红色荧光蛋白标记小鼠的Th17细胞，然后使用强效的糖皮质激素地塞米松来干预Th17细胞。使用annexin V和DAPI染色检测细胞凋亡率。采用PCR阵列筛查细胞凋亡基因。细胞凋亡相关分子水平的测定使用逆转录定量PCR、流式细胞和Western blotting技术。通过逆转录病毒转导敲除小鼠Th17细胞中BCL-2基因。细胞因子使用ELISA检测。应用小鼠Th17驱动的重症哮喘模型检测体内Th17细胞的糖皮质激素敏感性。
结果：人和小鼠Th17细胞及小鼠Th2细胞对激素诱导的细胞凋亡有抵抗性。Th17细胞的糖皮质激素受体水平与其他T效应细胞的受体水平相比具有可比性。Th17细胞有高表达的BCL-2，敲除BCL-2基因后Th17细胞恢复了它对地塞米松诱导细胞凋亡的敏感性。糖皮质激素抑制了IL-22的产生，但对IL-17A和IL-17F无影响。Th17细胞中STAT3磷酸化对糖皮质激素的抑制效应不敏感。糖皮质激素刺激使重症哮喘模型小鼠中肺组织Th17细胞数目增加，而不是降低。
结论：Th17细胞能抵制糖皮质激素诱导的细胞凋亡和细胞因子抑制，这至少有部分原因归于BCL-2的高表达。这些发现支持Th17细胞在诸如哮喘内型的糖皮质激素抵抗的炎症条件中发挥着作用。
BCL-2 protects human and mouse Th17 cells from glucocorticoid-induced apoptosis
J. Banuelos, S. Shin, Y. Cao, B. S. Bochner, L. Morales-Nebreda, G. R. S. Budinger, L. Zhou, S. Li, J. Xin, M. W. Lingen, C. Dong, R. P. Schleimer& N. Z. Lu
Allergy; 2016;71:640-650
Background: Glucocorticoid resistance has been associated with Th17-driven inflammation, the mechanisms of which are not clear. We determined whether human and mouse Th17 cells are resistant to glucocorticoid-induced apoptosis.
Methods: Freshly isolated human blood Th17 cells and in vitro differentiated Th17 cells from IL-17F red fluorescent protein reporter mice were treated with dexamethasone, a potent glucocorticoid. Apoptosis was measured using annexin V and DAPI staining. Screening of apoptosis genes was performed using the apoptosis PCR array. Levels of molecules involved in apoptosis were measured using quantitative RT-PCR, flow cytometry, and Western blotting. Knockdown of BCL-2 in murine Th17 cells was performed via retroviral transduction. Cytokines were measured using ELISA. A murine Th17-driven severe asthma model was examined for Th17 glucocorticoid sensitivity in vivo.
Results: Human and mouse Th17 cells and mouse Th2 cells were resistant to glu- cocorticoid-induced apoptosis. Th17 cells had glucocorticoid receptors levels com- parable to those in other T effectors cells. Th17 cells had high levels of BCL-2, knockdown of which sensitized Th17 cells to dexamethasone-induced apoptosis. Production of IL-22, but not IL-17A and IL-17F, was suppressed by glucocorticoids. STAT3 phosphorylation in Th17 cells was insensitive to glucocorticoid inhibition. Lung Th17 cells in the murine severe asthma model were enhanced, rather than suppressed, by glucocorticoids.
Conclusion: Th17 cells are resistant to glucocorticoid-induced apoptosis and cyto- kine suppression, at least in part due to high levels of BCL-2. These findings sup- port a role of Th17 cells in glucocorticoid-resistant inflammatory conditions such as certain endotypes of asthma.
方法：新鲜分离人血Th17细胞，体外分化IL-17F红色荧光蛋白标记小鼠的Th17细胞，然后使用强效的糖皮质激素地塞米松来干预Th17细胞。使用annexin V和DAPI染色检测细胞凋亡率。采用PCR阵列筛查细胞凋亡基因。细胞凋亡相关分子水平的测定使用逆转录定量PCR、流式细胞和Western blotting技术。通过逆转录病毒转导敲除小鼠Th17细胞中BCL-2基因。细胞因子使用ELISA检测。应用小鼠Th17驱动的重症哮喘模型检测体内Th17细胞的糖皮质激素敏感性。
结果：人和小鼠Th17细胞及小鼠Th2细胞对激素诱导的细胞凋亡有抵抗性。Th17细胞的糖皮质激素受体水平与其他T效应细胞的受体水平相比具有可比性。Th17细胞有高表达的BCL-2，敲除BCL-2基因后Th17细胞恢复了它对地塞米松诱导细胞凋亡的敏感性。糖皮质激素抑制了IL-22的产生，但对IL-17A和IL-17F无影响。Th17细胞中STAT3磷酸化对糖皮质激素的抑制效应不敏感。糖皮质激素刺激使重症哮喘模型小鼠中肺组织Th17细胞数目增加，而不是降低。
结论：Th17细胞能抵制糖皮质激素诱导的细胞凋亡和细胞因子抑制，这至少有部分原因归于BCL-2的高表达。这些发现支持Th17细胞在诸如哮喘内型的糖皮质激素抵抗的炎症条件中发挥着作用。
(郑静 张红萍 王刚 四川大学华西医院中西医结合科呼吸病组 610041 摘译)
（Allergy; 2016;71:640-650）
（Allergy; 2016;71:640-650）
BCL-2 protects human and mouse Th17 cells from glucocorticoid-induced apoptosis
J. Banuelos, S. Shin, Y. Cao, B. S. Bochner, L. Morales-Nebreda, G. R. S. Budinger, L. Zhou, S. Li, J. Xin, M. W. Lingen, C. Dong, R. P. Schleimer& N. Z. Lu
Allergy; 2016;71:640-650
Background: Glucocorticoid resistance has been associated with Th17-driven inflammation, the mechanisms of which are not clear. We determined whether human and mouse Th17 cells are resistant to glucocorticoid-induced apoptosis.
Methods: Freshly isolated human blood Th17 cells and in vitro differentiated Th17 cells from IL-17F red fluorescent protein reporter mice were treated with dexamethasone, a potent glucocorticoid. Apoptosis was measured using annexin V and DAPI staining. Screening of apoptosis genes was performed using the apoptosis PCR array. Levels of molecules involved in apoptosis were measured using quantitative RT-PCR, flow cytometry, and Western blotting. Knockdown of BCL-2 in murine Th17 cells was performed via retroviral transduction. Cytokines were measured using ELISA. A murine Th17-driven severe asthma model was examined for Th17 glucocorticoid sensitivity in vivo.
Results: Human and mouse Th17 cells and mouse Th2 cells were resistant to glu- cocorticoid-induced apoptosis. Th17 cells had glucocorticoid receptors levels com- parable to those in other T effectors cells. Th17 cells had high levels of BCL-2, knockdown of which sensitized Th17 cells to dexamethasone-induced apoptosis. Production of IL-22, but not IL-17A and IL-17F, was suppressed by glucocorticoids. STAT3 phosphorylation in Th17 cells was insensitive to glucocorticoid inhibition. Lung Th17 cells in the murine severe asthma model were enhanced, rather than suppressed, by glucocorticoids.
Conclusion: Th17 cells are resistant to glucocorticoid-induced apoptosis and cyto- kine suppression, at least in part due to high levels of BCL-2. These findings sup- port a role of Th17 cells in glucocorticoid-resistant inflammatory conditions such as certain endotypes of asthma.
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