Objective To 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.Methods An 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.Results PCR of myeloid conditioned knockout Ear2 mice showed a genotype of Lyz2ki/wtEar2fl/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,x2=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].Conclusion Conditional 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.
Lupus erythematosus,systemicToll-like receptor 7MonocytesEosinophil-associated ribonuclease A family member 2
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