Impaired response of blood neutrophils to cell-death stimulus differentiates AQP4-IgG-seropositive NMOSD from MOGAD.
Impaired response of blood neutrophils to cell-death stimulus differentiates AQP4-IgG-seropositive NMOSD from MOGAD.
复制标题
DOI：
10.1186/s12974-022-02600-0
复制
发表时间：
2022-10-01
影响因子：
9.3
通讯作者：
中科院分区：
文献类型：
--
作者：
在视神经肌萎缩症谱系疾病（NMOSD）和髓鞘少突胶质细胞糖蛋白抗体相关疾病（MOGAD）中，在CNS病变中发现中性粒细胞。我们以前证明，NMOSD中性粒细胞显示功能缺陷。因此，我们假设中枢神经系统中的中性粒细胞蓄积可能是由影响中性粒细胞死亡机制的损伤促进的。使用体外试验评价水通道蛋白4（AQP 4）-IgG血清阳性NMOSD和MOGAD患者以及匹配的健康对照（HC）血液中性粒细胞中的细胞死亡。前瞻性招募了28例AQP 4 + NMOSD和19例MOGAD稳定期患者以及45例年龄和性别匹配的HC。为了诱导细胞死亡，分离的中性粒细胞与/不与佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）一起培养。使用7-AAD和膜联蛋白-V通过流式细胞术分析自发和PMA诱导的NETosis和细胞凋亡。通过蛋白质印迹法评估Caspase-3。通过ELISA评价髓过氧化物酶-DNA复合物（MPO-DNA）、MPO和弹性蛋白酶，并通过基于荧光的测定评价游离DNA（cfDNA）。通过基于二氢罗丹明123的细胞计数法评估活性氧（ROS）。血清GM-CSF、IL-6、IL-8、IL-15、TNF-α和IL-10采用多重检测法，神经丝轻链（NfL）采用单分子阵列检测法。与HC（44.7%，p = 0.0006）相比，来自AQP 4 + NMOSD而非MOGAD患者的中性粒细胞对PMA的反应显示出存活率增加，随后细胞死亡减少（29.6%膜联蛋白V+ 7-AAD+）。然而，与HC（20.8%，p = 0.048）相比，AQP 4 + NMOSD还显示膜联蛋白V+ 7-AAD−早期凋亡中性粒细胞（24.5%）轻度增加。PMA诱导的caspase-3活化减少在HC中（p = 0.020）比在AQP 4 + NMOSD中性粒细胞中（p = 0.052）更明显。在嗜中性粒细胞衍生的MPO-DNA或MPO、弹性蛋白酶、IL-6、IL-8和TNF-α的血清水平中未观察到差异。两组患者的IL-15水平均升高。在AQP 4 + NMOSD中，在血清中发现cfDNA、GM-CSF和IL-10的增加。在AQP 4 + NMOSD中发现cfDNA和NfL之间呈正相关。与匹配的HC中性粒细胞相比，AQP 4 + NMOSD中性粒细胞对PMA的存活能力增强。虽然数据表明，在这些中性粒细胞中，凋亡反应而不是NETotic反应发生了改变，但需要进行额外的评价来验证这一观察结果。在线版本包含补充材料，可通过10.1186/s12974-022-02600-0获得。
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. The online version contains supplementary material available at 10.1186/s12974-022-02600-0.
登录
查看更多内容
影响因子：
3.4
作者：
Borisow N;Mori M;Kuwabara S;Scheel M;Paul F
通讯作者：
Paul F
影响因子：
6.4
作者：
Jarius, Sven;Wildemann, Brigitte
通讯作者：
Wildemann, Brigitte
影响因子：
4.1
作者：
Girard, D;Paquin, R;Beaulieu, AD
通讯作者：
Beaulieu, AD
影响因子：
7.3
作者：
Fresneda Alarcon M;McLaren Z;Wright HL
通讯作者：
Wright HL
影响因子：
4.4
作者：
Jarius, S.;Probst, C.;Wandinger, K. P.
通讯作者：
Wandinger, K. P.