The Role And Mechanism Of LCN2 In Lupus Nephritis And Diffuse Alveolar Hemorrhage

Background:Systemic lupus erythematosus(SLE)is a chronic systemic autoimmune disease.Lupus nephritis(LN)and diffuse alveolar hemorrhage(DAH)are severe SLE complications,which are the leading causes of death.The pathogenesis of SLE is yet to be understood.SLE is considered to be a multifactorial disease,in which genetic factors,immune dysregulation,and environmental factors,are involved.Conventional therapies including glucocorticoids and immunosuppressants have been used with limited efficacy and significant side effects in SLE.It is urgent to investigate the pathogenesis of SLE to propose effective therapy.Lipocalin-2(LCN2)is a biomarker of LN,which is important for the early diagnosis,disease monitoring and prognosis evaluation of LN.In addition,LCN2 is also a lipid delivery protein closely related to inflammation,which plays an important role in immune response.Objective:The purpose of this study was to investigate the role of LCN2 in SLE.By studying the role of LCN2 in LN and DAH,the potential contribution of LCN2 to the pathogenesis of SLE was explored.Methods:1.To determine the differentially expressed genes in SLE peripheral blood mononuclear cells(PBMCs),RNAs purified from fresh PBMCs of SLE patients and healthy control(HC)subjects were hybridized to a GeneChip Human Gene 1.0 ST Array.Five upregulated genes were verified by RT-PCR.LCN2 gene expression in naive CD4+T cells was detected by RT-PCR.Correlation analysis was conducted between the expression level of LCN2 in naive CD4+T cells and SLEDAI,proteinuria and serum creatinine levels in SLE patients.2.Immunohistochemistry and immunofluorescence were used to detect the expression levels of LCN2 in renal tissues and different renal cells(T cells,macrophages,neutrophils and tubular epithelial cells)in LN patients.Besides,the correlation between LCN2 expression level in renal tissue of LN patients and acute activity index,chronic activity index and tubulointerstitial inflammation index was also analyzed.3.MRL/lpr mice were intraperitoneally injected with recombinant LCN2 or antiLCN2 antibody.LCN2-/-mice and wild type(WT)C57BL/6 mice were injected with 0.5ml pristane intraperitoneally.The weight of mice,spleen and urine were measured before and after treatment.Microalbumin and serum anti-doublestranded DNA(ds-DNA)antibody were detected by ELISA,and creatinine level was detected by microplate.HE and PAS staining were used to assess the renal pathological changes.The deposits of IgG and C3 were detected by immunofluorescence.The inflammatory genes in the kidney were detected by RTPCR.Immunohi stochemi stry was used to detect the infiltration of macrophages and neutrophils in the kidney.The percentage of T cell subsets in the spleen,kidney and lymph nodes was detected by flow cytometry.4.To detect whether LCN2 was involved in the differentiation of Th1 cells,the WT and LCN2-/-na?ve CD4+T cells(with or without LCN2 pretreatment)were differentiated into Th1 cells under specific skewing conditions.Flow cytometry was used to detect the percentage of Th1 cells,RT-PCR was used to detect the expression of T-bet and IFN-γ,and ELISA was used to detect IFN-γ levels in the culture supernatant.Western blotting(WB)was used to detect the expression of transcription activator STAT4 and phosphorylated(P)-STAT4.5.The frozen lung tissues of WT C57BL/6 mice and DAH mice were selected for whole-genome sequencing.The gene and protein levels of LCN2 in lung of DAH mice were detected by RT-PCR and WB,respectively.ELISA was used to detect LCN2 levels in serum and bronchoalveolar lavage fluid(BALF)of DAH mice.6.In LCN2-/-mice and WT C57BL/6 mice,0.5ml pristane was injected intraperitoneally.Two weeks later,the mice were killed.The mortality and the severity of alveolar hemorrhage were assessed.HE staining was used to evaluate the pathological changes of the lungs.The protein levels of inflammation-related factors in BALF were measured by ELISA.The percentage of T cell subsets,macrophages,neutrophils and apoptotic rate of macrophages in the lungs and BALF were measured by flow cytometry.Results:1.The expression level of LCN2 in naive CD4+T cells of LN patients was significantly higher than that in healthy control group and SLE patients without LN,and LCN2 expression level of in naive CD4+T cells in SLE patients was positively correlated with SLEDAI score,proteinuria and serum creatinine level.2.The expression level of LCN2 in the renal tissues of LN patients was significantly higher than that of the control group.The correlation analysis showed that the expression of LCN2 in the kidney was positively correlated with the acute activity index,chronic activity index and tubulointerstitial inflammation index.The expression of LCN2 in renal T cells,macrophages,neutrophils and tubular epithelial cells of LN patients was significantly increased compared with the control group.3.After LCN2 treatment,the albuminuria level of lupus mice increased significantly,proliferative glomerulonephritis and tubulointerstitial inflammation aggravated,the expression of C3 in glomerulus increased,and the expression of proinflammatory factors and the infiltration of macrophages and neutrophil in the kidney increased.In lupus mice treated with anti-LCN2 antibody,the albuminuria level decreased significantly,histological analysis showed less cellular proliferation and mesangial matrix deposition in glomeruli and improved interstitial lesions,and the expression of inflammatory factors and infiltration of macrophages and neutrophils decreased.Compared with WT C57BL/6 mice,the glomerulonephritis of LCN2-/-mice was alleviated in pristane-induced lupus.4.The differentiation of LCN2-/-T cells into Th1 cells was inhibited,and the phosphorylation level of STAT4 was down-regulated.The inhibition was reversed after LCN2 supplementation.In vivo experiments also confirmed that Th1 cells increased in lupus mice treated with LCN2,but decreased after neutralization of LCN2 in lupus mice.5.In pristane-induced DAH,LCN2 levels in the lung increased significantly.Apoptotic rate of alveolar macrophages(AM)in BALF increased with progression of DAH.6.In LCN2-/-mice,diffuse hemorrhage,exudation and inflammatory cell infiltration in the lung tissues decreased.AM increased while neutrophils and inflammatory cytokines decreased in BALF of LCN2-/-mice.In addition,apoptotic rate of AM in BALF decreased in LCN2-/-mice.Conclusion:The expression of LCN2 increased in patients with LN.LCN2 promoted Th1 cell differentiation through IL-12/STAT4 pathway and was involved in the pathogenesis and progression of LN.LCN2,which is highly expressed in the lungs of DAH mice,can promote apoptosis of AM,induce neutrophil recruitment to the lungs and further aggravate DAH.These findings suggest that antagonizing LCN2 may be effective in the treatment of LN and DAH.