Ma Jianjuan, Dong Cong, Wei Shushan, Qiu Minzhi, Wu Penghui, Ou Changxing, Zhang Bomeng, Zhang Xueyan, Yan Jie, Zhang Qingling, Zhong Nanshan
Department of Pathophysiology, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, China.
Pulmonary and Critical Care Medicine, Guangzhou Institute of Respiratory Health, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, State Key Laboratory of Respiratory Diseases, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Front Mol Biosci. 2021 Apr 27;8:653461. doi: 10.3389/fmolb.2021.653461. eCollection 2021.
Eosinophilic granulomatosis with polyangiitis (EGPA) prognosis is generally favorable and is treated with combined corticosteroids/immunosuppressor(s) therapy. However, disease flares increase the number of clinical visits. Therefore, discovering new serum biomarkers for early identification of active EGPA is crucial. To identify reliable serum biomarkers to measure EGPA activity. The expression of 160 proteins was compared in sera from 15 inactive and 13 active EGPA patients by antibody-based microarray. Network-based analysis identified patterns in the different groups. Differentially expressed proteins (DEPs) in active disease were identified, and the correlation between their serum levels and clinical parameters was assessed. DEPs were further analyzed for GO enrichment and KEGG pathways. Finally, DEP marker candidates were validated by ELISA and Bio-plex as well as against a second cohort of 22 inactive and 18 active EGPA patients. The active group presented higher peripheral and sputum eosinophil counts, FeNO, and FEV1 (% predicted) ( < 0.05). Network-based analysis showed scattered expression patterns in active subjects, but no significant bias in inactive subjects. Significant differences were observed in serum levels of 19 candidate markers, all of which were higher in active EGPA ( < 0.05). KEGG analysis indicated that DEPs were mainly involved in the MAPK, PI3K-Akt, RAS and Rap1 related pathways. Nine out of 19 candidate markers were positively correlated with peripheral eosinophil counts including FGF-7, SCF, GDNF, β-NGF, IGFBP-4, Axl, PIGF, Insulin, NT-4, ErbB3, OPN and BMP-4 ( = 0.693, = 0.692, = 0.687, = 0.683, = 0.671, = 0.606, = 0.571, = 0.570, = 0.516, respectively; < 0.05), while two, CD14 and MCP-3, were negatively correlated ( = -0.644 and = -0.515; < 0.05). The higher expression of Axl, OPN, HCC-4, GDNF, and MCP-3 in active EGPA subjects was confirmed by ELISA and Custom Multiplex Bio-plex analyses. The serum protein profiles were significantly different between active and inactive EGPA. The expression of the candidate proteins correlated with peripheral blood eosinophil count. Serum Axl, OPN, HCC-4, GDNF, and MCP-3 levels were consistently higher in active EGPA, independent of the assessment methods. Finally, Axl had the largest AUC, indicating that this cytokine may serve as novel biomarker for the diagnosis of active EGPA.
嗜酸性肉芽肿性多血管炎（EGPA）的预后通常良好，采用糖皮质激素/免疫抑制剂联合治疗。然而，疾病复发会增加临床就诊次数。因此，发现用于早期识别活动性EGPA的新血清生物标志物至关重要。为了鉴定可靠的血清生物标志物以衡量EGPA的活动情况。通过基于抗体的微阵列比较了15例非活动性EGPA患者和13例活动性EGPA患者血清中160种蛋白质的表达。基于网络的分析确定了不同组中的模式。鉴定出活动性疾病中差异表达的蛋白质（DEP），并评估了它们的血清水平与临床参数之间的相关性。对DEP进行了进一步的GO富集和KEGG通路分析。最后，通过ELISA和生物芯片以及针对另一组22例非活动性EGPA患者和18例活动性EGPA患者对DEP标记候选物进行了验证。活动组的外周血和痰液嗜酸性粒细胞计数、呼出气一氧化氮（FeNO）和第一秒用力呼气容积（FEV1，预测值%）更高（P<0.05）。基于网络的分析显示活动组受试者的表达模式分散，但非活动组受试者无明显偏差。在19种候选标志物的血清水平上观察到显著差异，所有这些标志物在活动性EGPA中均较高（P<0.05）。KEGG分析表明，DEP主要参与丝裂原活化蛋白激酶（MAPK）、磷脂酰肌醇-3激酶-蛋白激酶B（PI3K-Akt）、RAS和Rap1相关通路。19种候选标志物中有9种与外周血嗜酸性粒细胞计数呈正相关，包括成纤维细胞生长因子7（FGF-7）、干细胞因子（SCF）、胶质细胞源性神经营养因子（GDNF）、β-神经生长因子（β-NGF）、胰岛素样生长因子结合蛋白4（IGFBP-4）、酪氨酸蛋白激酶受体Axl（Axl）、胎盘生长因子（PIGF）、胰岛素、神经营养因子4（NT-4）、表皮生长因子受体3（ErbB3）、骨桥蛋白（OPN）和骨形态发生蛋白4（BMP-4）（r分别为0.693、0.692、0.687、0.683、0.671、0.606、0.571、0.570、0.516；P<0.05），而两种，即CD14和单核细胞趋化蛋白3（MCP-3），呈负相关（r分别为-0.644和-0.5-15；P<0.05）。ELISA和定制多重生物芯片分析证实了活动性EGPA受试者中Axl、OPN、HCC-4、GDNF和MCP-3的高表达。活动性和非活动性EGPA之间的血清蛋白质谱有显著差异。候选蛋白的表达与外周血嗜酸性粒细胞计数相关。无论评估方法如何，活动性EGPA患者血清中Axl、OPN、HCC-4、GDNF和MCP-3水平始终较高。最后，Axl的曲线下面积（AUC）最大，表明这种细胞因子可能作为诊断活动性EGPA的新型生物标志物。
