β2-Adrenergic Biased Agonist Inhibits the Development of Th17 and the Response of Memory Th17 Cells in an NF-κB-Dependent Manner.

Mehri Hajiaghayi, Fatemeh Gholizadeh, Eric Han, Samuel Little, Niloufar Rahbari, Isabella Ardila, Carolina Lopez Naranjo, Kasra Tehranimeh, Steve CC Shih, Peter J. Darlington
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Abstract

Introduction: Adrenergic receptors regulate metabolic, cardiovascular, and immunological functions in response to the sympathetic nervous system. The effect of β2- adrenergic receptor (AR) as a high-expression receptor on different subpopulations of T cells is complex and varies depending on the type of ligand and context. While traditional β2-AR agonists generally suppress T cells, they potentially enhance IL-17A production by Th17 cells. The effects of pharmacological drugs that count as biased agonists of AR like nebivolol are not completely understood. We investigated the impact of nebivolol on human memory CD4+ T (Th1, Th2, Th17) cells and polarized naive Th17 cells highlighting its potential for IL-17A suppression via a non-canonical β2-AR cell-signaling pathway. Methods: The effects of nebivolol were tested on healthy human peripheral blood mononuclear cells, purified memory Th cells, and polarized naive Th17 cells activated with antiCD3/antiCD28/antiCD2 ImmunoCult reagent. IFN‐γ, IL-4, and IL-17A which are primarily derived from Th1, Th2, and Th17 cells respectively, were quantified by ELISA and flow cytometry. IL-10 was measured by ELISA. Gene expression of RORC, ADRB1, ADRB2, and ADRB3 was evaluated by qPCR. The ADRB2 gene was knocked out in memory Th cells using CRISPR/Cas9. Protein expression of phosphorylated-serine133-CREB and phosphorylated-NF-κB p65 was assessed by Western blot. Proliferation was assessed by fluorescent dye loading and flow cytometry. Results: Nebivolol treatment decreased IL-17A and IFN‐γ secretion by activated-memory Th cells and elevated IL-4 levels. Nebivolol reduced the proportion of IL-17A+ Th cells and downregulated RORC expression. Unlike the β2-AR agonist terbutaline, nebivolol inhibited the shift of naive CD4+ T cells towards the Th17 phenotype. IL-10 and proliferation index remained unchanged. Nebivolol-treated β2-knockout memory Th cells showed significant inhibition of β2AR-mediated signaling, evidenced by the absence of IL-17A suppression compared to controls. Phosphorylation of the NF-κB p65 subunit was inhibited by nebivolol, but CREB phosphorylation was not changed, suggesting a selective transcriptional control. Conclusions: The findings demonstrate that nebivolol acts through a β2-AR-mediated signaling pathway, as a distinctive anti-inflammatory agent capable of selectively shifting Th17 cells and suppressing phosphorylation of NF-κB. This highlights nebivolol as a potential for therapeutic interventions in chronic autoimmune conditions with elevated IL-17A levels.
β2-肾上腺素能偏激激动剂以 NF-κB 依赖性方式抑制 Th17 细胞的发育和记忆 Th17 细胞的反应
简介肾上腺素能受体可调节交感神经系统的代谢、心血管和免疫功能。作为一种高表达受体,β2-肾上腺素能受体(AR)对不同亚群T细胞的影响是复杂的,并因配体类型和环境而异。传统的 β2-AR激动剂通常会抑制T细胞,但却有可能增强Th17细胞产生的IL-17A。像奈必洛尔(nebivolol)这样作为AR偏向激动剂的药理药物的作用还不完全清楚。我们研究了奈必洛尔对人类记忆 CD4+ T(Th1、Th2、Th17)细胞和极化的天真 Th17 细胞的影响,强调了其通过非经典 β2-AR细胞信号通路抑制 IL-17A 的潜力:方法:测试了奈必洛尔对健康人外周血单核细胞、纯化的记忆Th细胞和用抗CD3/抗CD28/抗CD2免疫培养基激活的极化幼稚Th17细胞的影响。IFN-γ、IL-4和IL-17A主要分别来自Th1、Th2和Th17细胞,通过酶联免疫吸附和流式细胞术进行量化。IL-10 通过酶联免疫吸附法测定。通过 qPCR 评估了 RORC、ADRB1、ADRB2 和 ADRB3 的基因表达。使用 CRISPR/Cas9 基因敲除记忆 Th 细胞中的 ADRB2 基因。通过 Western 印迹评估磷酸化-丝氨酸-133-CREB 和磷酸化-NF-κB p65 的蛋白表达。通过荧光染料负载和流式细胞术评估增殖情况。结果奈必洛尔治疗降低了活化记忆Th细胞分泌的IL-17A和IFN-γ,并升高了IL-4水平。奈必洛尔降低了IL-17A+ Th细胞的比例,并下调了RORC的表达。与β2-AR激动剂特布他林不同,奈必洛尔抑制了幼稚CD4+ T细胞向Th17表型的转变。IL-10和增殖指数保持不变。与对照组相比,奈必洛尔处理的β2-基因敲除记忆Th细胞显示出对β2AR介导的信号转导的显著抑制,这表现在IL-17A没有受到抑制。奈必洛尔抑制了 NF-κB p65 亚基的磷酸化,但 CREB 磷酸化没有发生变化,这表明转录控制具有选择性。结论:研究结果表明,奈必洛尔通过β2-AR介导的信号通路发挥作用,是一种独特的抗炎药物,能够选择性地转移Th17细胞并抑制NF-κB的磷酸化。这凸显了奈必洛尔作为治疗干预IL-17A水平升高的慢性自身免疫疾病的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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