嗜酸性粒细胞相关核糖核酸酶A家族成员2参与狼疮小鼠肾脏损害的机制研究

Pathogenic role of eosinophil-associated ribonuclease A family member 2 in renal damage in lupus mice

目的通过嗜酸性粒细胞相关核糖核酸酶A家族成员2(Ear2)髓系细胞条件性敲除小鼠探讨其在狼疮发病中的作用及参与肾脏损害的可能机制。方法利用规律间隔成簇短回文重复序列关联基因9(CRISP/Cas9)技术构建Ear2髓系细胞条件性敲除小鼠模型,应用PCR鉴定小鼠基因型。实验分为3组:CKO+R848组,对照+R848组,对照组。R848处理敲除鼠和同型对照鼠,评估小鼠狼疮样病变发生情况。荧光实时定量PCR法检测小鼠肾脏中Toll样受体(TLR)7/8和炎症因子的表达。流式细胞术检测狼疮小鼠肾脏中巡逻单核细胞(PMOs)的比例,免疫荧光法分析Ear2和PMOs在肾组织中的空间分布。R848体外刺激条件性敲除(CKO)和对照小鼠的骨髓细胞,流式细胞术检测PMOs比例变化。采用单因素方差分析对3组进行比较。结果髓系细胞条件敲除Ear2小鼠构建后PCR显示基因型为Lyz2^(ki/wt)Ear2^(fl/fl),敲除鼠骨髓细胞中Ear2 mRNA水平显著下调[(1.03±0.26)与(0.22±0.15),t=6.65,P<0.001]。与对照+R848组相比,CKO+R848组小鼠狼疮表型得到改善,生存率有上升趋势(6/10与7/8,χ^(2)=1.51,P=0.220),病理结果提示CKO+R848组小鼠肾脏病变减轻。CKO+R848小鼠肾脏组织中TLR7表达水平降低[(1.02±0.09)与(0.53±0.04),t=5.13,P=0.003],同时PMOs浸润减少[(62.00±3.38)%与(52.36±0.68)%,t=2.80,P=0.023],免疫荧光结果显示Ear2和PMOs在肾组织共定位。体外R848刺激引起对照组PMOs比例的增多[(3.99±0.59)%与(33.48±1.38)%,t=-33.84,P<0.001],敲除Ear2可以抑制TLR7引起的PMOs的增多[(14.33±1.72)%与(16.10±1.44)%,t=-1.37,P=0.220]。结论Ear2条件性敲除可减轻狼疮小鼠发病,尤其是肾脏损害。其机制与抑制TLR7通路激活、减少PMOs局部浸润有关。

ObjectiveTo explore the role of eosinophil associated ribonuclease A family member 2(Ear2)in the pathogenesis of lupus and its possible mechanisms involved in renal damage by conditional knockout of myeloid cells in mice.MethodsAn Ear2 myeloid conditional knockout mouse model was constructed using CRISP/Cas9 technology,and PCR was applied to identify mice genotype.The experiment was divided into 3 groups:CKO+R848 group,control+R848 group,and control group.R848(Resiquimod)was used to treat the knockout mice and homozygous control mice to evaluate the occurrence of lupus-like features.Quantitative real-time PCR was performed to detect the expression of Toll-like receptor 7/8(TLR7/8)and its related inflammatory factors in the kidneys of mice.Flow cytometry(FCM)was used to detect the proportion of patrolling monocytes in the kidneys,and immunofluorescence was used to analyze the spatial distribution of Ear2 and PMOs in renal tissues.In addition,R848 was used to stimulate myeloid cells of conditional knockout(CKO)and control mice in vitro,with changes in the proportion of PMOs detected by flow cytometry.Variance(ANOVA)was used to compare the differences between groups,t-test was used for two-by-two comparisons,and one-way analysis of ANOVA was used for comparisons between multiple groupscant.ResultsPCR of myeloid conditioned knockout Ear2 mice showed a genotype of Lyz2^(ki/wt)Ear2^(fl/fl)and significant down-regulation of Ear2 mRNA levels in bone marrow cells of the knockout mice[(1.03±0.26)vs.(0.22±0.15),t=6.65,P<0.001].Compared with the control+R848 group,lupus related phenotype presentations of mice was improved and the survival rate tended to increase in the CKO+R848 group(6/10 vs.7/8,χ^(2)=1.51,P=0.220).The pathological results examination suggested that renal lesions of mice in the CKO+R848 group were also attenuated.The expression level of TLR7 was reduced in the renal tissues of CKO+R848 mice[(1.02±0.09)vs.(0.53±0.04),t=5.13,P=0.003],accompanied by a decrease in PMOs infiltration[(62.00±3.37)%vs.(52.36±0.68)%,t=2.80,P=0.023],and immunofluorescence results showed that Ear2 and PMOs were co-localized in renal tissues.In vitro,R848 stimulation caused an increase in the proportion of PMOs in the control group[(3.99±0.59)%vs.(33.48±1.38)%,t=-33.84,P<0.0001],yet had no effect on CKO mice[(14.33±1.72)%vs.(16.10±1.44)%,t=-1.37,P=0.220].ConclusionConditional knockdown of Ear2 attenuates the development of lupus in mice,especially renal impairments,which is related to the inhibition of TLR7 pathway and reduction of local infiltration of PMOs.