The Expressing Of CXCL9/10/11 In Human Mesangial Cells And The Role Of CXCL9/10/11 In The Diagnosis Of Patients With Lupus Nephritis

Objective:To study the effect and mechanism of interferon-gamma (IFN-y) on the expression of CXCL9、CXCL10 and CXCL11 in human mesangial cells (HMC), and to investigate the role of CXCL9、CXCL10、CXCL11 in the diagnosis of patients with lupus nephritis, and lay a theoretical foundation for looking for a new noninvasive and high sensitivity kidney damage detection index.Methods:1.HMC were cultured and divided into three groups:the control group, IFN-y treatment group and AG490 treatment group. Different concentrations of IFN-y were applied to stimulus HMC 6 h, then 1000U/ml IFN-γ was used to stimulus HMC for different time, and the specificity antagonists of JAK2—AG490 was apply to intervene the inducing effect of IFN-y. The expression of CXCL9, CXCL10 and CXCL11 mRNA were measured by real-time quantitative PCR and the levels of CXCL9, CXCL10 and CXCL11 in culture supernatant were tested by enzyme-linked immunosorbent assay (ELISA). The expression of JAK1、 JAK2、STAT1 and their phosphorylated protein p-JAK1、p-JAK2、p-STAT1 were measured by western blot.2.This study included patients with SLE (fulfilling American College of Rheumatology (ACR) 1997 criteria) recruited from the outpatient and inpatient services of People’s hospital affiliated to fujian university of traditional Chinese medicine from June 2015 to December 2015. Age-and gender-matched healthy controls were also recruited. CXCL9. CXCL10 and CXCL11 levels in stored serum and urine were determined by ELISA. and the correlation between this three chemokines with systemic lupus erythematosus disease activity index(SLEDAI),anti-dsDNA antibody. C3. C4. microalbuminuria(mAlb) analysis by linear correlation analysis.Results:1. Basic research:(1) With the increase of IFN-y concentration, the expression of CXCL9. CXCL10, CXCL11 mRNA and the levels of CXCL9.CXCL10.CXCL11 proteins in HMC were up-regulated, and gradually increased with progress of time, and peaked at 1000U/ml(P<0.01 or P<0.05). Application of 1000 U/ml IFN-y stimulate HMC, as the extension of time, the expression of CXCL9, CXCL10, CXCL11 mRNA reached the peak at 24h(P<0.01 or P<0.05); The levels of CXCL9 and CXCL10 proteins peaked at 48h(P<0.01 and P<0.05), the levels of CXCL11 peaked at 72h(P<0.01).(2) The CXCL9, CXCL10, CXCL11 were down-regulated by 98.43%、99.27% and 99.29% (P<0.01 for both) at mRNA level and 80.67%、49.89%、81.50%(P<0.01 for both) respectively, at protein level when pretreated with AG490 for 40 min and then incubated with IFN-y.(3) The phosphorylation levels of JAK1、JAK2 and STAT1 were significantly up-regulated in IFN-y treatment group (P<0.01 for both). The phosphorylation levels of JAK1、JAK2 and STAT1 were down-regulated when HMC were pretreated with AG490 and then incubated with IFN-y (P<0.01 or P<0.05). 2. Clinical study:(1) Both serum levers of CXCL9、CXCL10 and CXCL11 were higher in SLE group compared to control group(P<0.01 for both), and they were correlated significantly with the anti-dsDNA antibody and SLEDAI. respectively(P<0.01 or P<0.05). Besides, in lupus nephritis group compared to control group, there was a rise in serum levels(P<0.01 for both), but there were no difference between lupus nephritis group and non-nephritis lupus group(P>0.05 for both).(2) Both urinary levers of CXCL10 and CXCL11 were higher in SLE group compared to control group (P<0.01 and P<0.05), but there was no difference in urinary levers of CXCL9(P>0.05); and urinary CXCL10 was higher in lupus nephritis group and non-nephritis lupus group compared to control group(P<0.01).(3) In SLE group, there was no correlation between urinary levers of CXCL10 and mAlb (r =0.077, P>0.05). On ROC analysis, urinary CXCL10 was similar to mAlb. When mAlb≥ 62.3mg/mmol-Cre, (AUC (95% confidence interval (CI))= 0.758(0.589-0.928) P<0.05), CXCL10≥5.82 ng/mmol-Cre, AUC= 0.717(0.528～0.905, P<0.05).Conclusion:1. IFN-γ could up-regulate the expression of CXCL9、CXCL10 and CXCL11 in HMC through JAK/STAT1 signal pathway.2. Both serum levers of CXCL9、CXCL10、CXCL11 and urinary levers of CXCL10、 CXCL11 were higher in SLE group compared to control group. Given that CXCL9、CXCL10 and CXCL11 play an import role in SLE. And urinary CXCL10 were higher in lupus nephritis group, suggesting that CXCL10 may involved in the development process of SLE renal damage. Besides, our study provides evidence that the chemokine CXCL10 is potentially useful biomarker in SLE.