Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität Zu Berlin, ECRC Experimental and Clinical Research Center, a Cooperation Between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and Charité-Universitätsmedizin Berlin, Lindenberger Weg 80, 13125, Berlin, Germany.
Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Campus Berlin-Buch GmbH, Robert-Rössle-Straße 10, 13125, Berlin, Germany.
J Neuroinflammation. 2022 Oct 1;19(1):239. doi: 10.1186/s12974-022-02600-0.
In neuromyelitis optica spectrum disorders (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), neutrophils are found in CNS lesions. We previously demonstrated that NMOSD neutrophils show functional deficiencies. Thus, we hypothesized that neutrophil accumulation in the CNS may be facilitated by impairments affecting mechanisms of neutrophil death.
To evaluate cell death in blood neutrophils from aquaporin-4 (AQP4)-IgG-seropositive NMOSD and MOGAD patients as well as matched healthy controls (HC) using in vitro assays.
Twenty-eight AQP4 + NMOSD and 19 MOGAD patients in stable disease phase as well as 45 age- and sex-matched HC were prospectively recruited. To induce cell death, isolated neutrophils were cultured with/without phorbol 12-myristate 13-acetate (PMA). Spontaneous and PMA-induced NETosis and apoptosis were analyzed using 7-AAD and annexin-V by flow cytometry. Caspase-3 was assessed by western blot. Myeloperoxidase-DNA complexes (MPO-DNA), MPO and elastase were evaluated by ELISA, and cell-free DNA (cfDNA) by a fluorescence-based assay. Reactive oxygen species (ROS) were evaluated by a dihydrorhodamine 123-based cytometric assay. Serum GM-CSF, IL-6, IL-8, IL-15, TNF-ɑ and IL-10 were evaluated by multiplex assays, and neurofilament light chain (NfL) by single-molecule array assay.
In response to PMA, neutrophils from AQP4 + NMOSD but not from MOGAD patients showed an increased survival, and subsequent reduced cell death (29.6% annexin V 7-AAD) when compared to HC (44.7%, p = 0.0006). However, AQP4 + NMOSD also showed a mild increase in annexin V 7-AAD early apoptotic neutrophils (24.5%) compared to HC (20.8%, p = 0.048). PMA-induced reduction of caspase-3 activation was more pronounced in HC (p = 0.020) than in AQP4 + NMOSD neutrophils (p = 0.052). No differences were observed in neutrophil-derived MPO-DNA or serum levels of MPO, elastase, IL-6, IL-8 and TNF-ɑ. IL-15 levels were increased in both groups of patients. In AQP4 + NMOSD, an increase in cfDNA, GM-CSF and IL-10 was found in serum. A positive correlation among cfDNA and NfL was found in AQP4 + NMOSD.
AQP4 + NMOSD neutrophils showed an increased survival capacity in response to PMA when compared to matched HC neutrophils. Although the data indicate that the apoptotic but not the NETotic response is altered in these neutrophils, additional evaluations are required to validate this observation.
在视神经脊髓炎谱系疾病（NMOSD）和髓鞘少突胶质细胞糖蛋白抗体相关疾病（MOGAD）中，中性粒细胞存在于中枢神经系统病变中。我们之前已经证明 NMOSD 中性粒细胞存在功能缺陷。因此，我们假设中枢神经系统中中性粒细胞的积累可能是由于影响中性粒细胞死亡机制的损伤所致。
使用体外测定法评估水通道蛋白 4（AQP4）-IgG 阳性 NMOSD 和 MOGAD 患者血液中性粒细胞的细胞死亡。
前瞻性招募了 28 名稳定期 AQP4 阳性 NMOSD 和 19 名 MOGAD 患者以及 45 名年龄和性别匹配的健康对照者（HC）。为了诱导细胞死亡，用/不用佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）培养分离的中性粒细胞。通过流式细胞术用 7-AAD 和 annexin-V 分析自发性和 PMA 诱导的 NETosis 和细胞凋亡。通过 Western blot 评估 caspase-3。通过酶联免疫吸附试验（ELISA）评估髓过氧化物酶-DNA 复合物（MPO-DNA）、髓过氧化物酶和弹性蛋白酶，并通过荧光测定法评估细胞游离 DNA（cfDNA）。通过二氢罗丹明 123 基于细胞的测定法评估活性氧（ROS）。通过多重测定法评估血清 GM-CSF、IL-6、IL-8、IL-15、TNF-ɑ 和 IL-10，通过单分子阵列测定法评估神经丝轻链（NfL）。
与 HC（44.7%，p=0.0006）相比，AQP4 阳性 NMOSD 但不是 MOGAD 患者的中性粒细胞在 PMA 作用下显示出更高的存活率，随后细胞死亡减少（29.6% annexin V 7-AAD）。然而，与 HC（20.8%，p=0.048）相比，AQP4 阳性 NMOSD 患者早期凋亡的 annexin V 7-AAD 中性粒细胞也略有增加（24.5%）。HC 中 PMA 诱导的 caspase-3 激活减少更为明显（p=0.020），而 AQP4 阳性 NMOSD 中性粒细胞中减少较少（p=0.052）。中性粒细胞来源的 MPO-DNA 或血清 MPO、弹性蛋白酶、IL-6、IL-8 和 TNF-ɑ 水平无差异。两组患者的 IL-15 水平均升高。在 AQP4 阳性 NMOSD 中，血清中发现 cfDNA、GM-CSF 和 IL-10 增加。在 AQP4 阳性 NMOSD 中，cfDNA 和 NfL 之间存在正相关。
与匹配的 HC 中性粒细胞相比，AQP4 阳性 NMOSD 中性粒细胞在 PMA 作用下显示出更高的存活能力。尽管数据表明这些中性粒细胞的凋亡而非 NETotic 反应发生改变，但需要进一步评估来验证这一观察结果。
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