Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT.
Patrys Ltd., Melbourne, Victoria, Australia.
Immunohorizons. 2022 Jun 13;6(6):356-365. doi: 10.4049/immunohorizons.2100091.
Nuclear-penetrating anti-DNA autoantibodies have therapeutic potential as delivery agents and in targeting DNA and the DNA damage response (DDR). Derivatives of such Abs have advanced to human testing in genetic disease and are in preparation for oncology clinical trials. DNA release associated with neutrophil extracellular traps (NETs) contributes to immunity, inflammation, and the pathophysiology of multiple diseases. The DDR contributes to mechanisms of NETosis, and we hypothesize that anti-DNA autoantibodies that localize into live cell nuclei and inhibit DNA repair will suppress release of NETs by activated neutrophils. In the current study we evaluated the impact of a nuclear-penetrating anti-DNA autoantibody that interferes with the DDR on decondensation and release of DNA and NETs by activated human granulocyte-like differentiated PLB-985 cells and neutrophils isolated from C57BL/6 mice. The response of cells pretreated with control or autoantibody to subsequent stimulators of NETosis, including PMA and the calcium ionophore ionomycin, was evaluated by DAPI and SYTOX Green stains, measurement of DNA release, analysis of histone citrullination by Western blot, or visualization of NETs by immunostaining and confocal fluorescence microscopy. Autoantibody treatment of the cells yielded significant inhibition of NADPH oxidase-dependent and independent NETosis. These findings establish the concept of nuclear-penetrating anti-DNA autoantibodies as modulators of neutrophil biology with potential for use in strategies to suppress NETosis.
核穿透抗 DNA 自身抗体具有作为递药载体和靶向 DNA 及 DNA 损伤反应 (DDR) 的治疗潜力。此类抗体的衍生物已在遗传疾病的人体试验中取得进展，并且正在为肿瘤学临床试验做准备。与中性粒细胞胞外诱捕网 (NETs) 相关的 DNA 释放有助于免疫、炎症和多种疾病的病理生理学。DDR 有助于 NETosis 的机制，我们假设定位于活细胞核内并抑制 DNA 修复的抗 DNA 自身抗体将抑制激活的中性粒细胞释放 NETs。在当前研究中，我们评估了一种核穿透抗 DNA 自身抗体对激活的人粒细胞样分化的 PLB-985 细胞和从小鼠分离的中性粒细胞中 DNA 解凝聚和释放以及 NETs 的影响，该抗体干扰 DDR。通过 DAPI 和 SYTOX Green 染色、DNA 释放测量、Western blot 分析组蛋白瓜氨酸化或免疫染色和共聚焦荧光显微镜观察 NETs，评估了用对照或自身抗体预处理的细胞对随后 NETosis 刺激物（包括 PMA 和钙离子载体离子霉素）的反应。自身抗体处理细胞产生了 NADPH 氧化酶依赖性和非依赖性 NETosis 的显著抑制。这些发现确立了核穿透抗 DNA 自身抗体作为中性粒细胞生物学调节剂的概念，具有用于抑制 NETosis 的策略的潜力。
