Study On Interferon-Inducible Protein 16 As A Biomarker For Disease Activity And Prognosis In Lupus Nephritis

Background and ObjectiveLupus nephritis(LN)is the most common and severe complication of systemic lupus erythematosus(SLE),and is also one of the main causes of death in SLE patients.Current conventional laboratory tests for LN diagnosis,such as proteinuria and anti-double stranded DNA(anti-ds DNA)antibodies,etc,are very limited in sensitivity and specificity.Invasive renal biopsy still remains the "gold standard" for the diagnosis of LN.However,LN pathological indexes defined by the National Institutes of Health(NIH)in predicting long-term prognosis for LN patients was limited,and was not confirmed by the Lupus Nephritis Collaborative Study Group.Due to the lack of reliable and precise LN markers,current management of LN remains unsatisfactory.In this study,the rapidly developing bioinformatics combined with machine learning algorithms was first utilized to screen the potential LN diagnostic biomarkers.The results showed that interferon-inducible protein 16(IFI16)was identified as a biomarker for LN.IFI16 belongs to the interferon induced p200 protein family and may regulate a variety of biological processes,including inhibiting cell transcription,inhibiting cell proliferation,and inducing cell apoptosis,etc.IFI16 and systemic autoimmune disease have been connected by evidence reports.IFI16 protein and anti-IFI16 antibodies were detected in the serum of patients with SLE,Sjogren’s syndrome,systemic sclerosis,and rheumatoid arthritis.Significant high expression of IFI16 protein was detected in lesioned skin from both patients with SLE and those with systemic sclerosis.Although studies have shown abnormal IFI16 expression was related to SLE,it is still unclear whether IFI16 can serve as a biomarker for diagnosis and/or monitoring of LN.Moreover,there is no information on IFI16 expression in renal biopsies from LN patients.Based on the screening of IFI16 as a diagnostic marker for LN in bioinformatics,this study focused on IFI16 detection in patients with LN,explored whether IFI16 could be a biomarker for LN,and explored the possible mechanism of IFI16 expression in LN kidneys.Materials and Methods1.Datasets GSE32591 and GSE99339 from LN and controls were selected and downloaded from the GEO database.These 2 sets used as training sets to determine LN differentially expressed genes(DEGs).LN diagnostic models were constructed by using three machine learning algorithms: Least Absolute Shrinkage and Selection Operator(LASSO),Random Forest(RF),and Support Vector Machine Recursive Feature Elimination(SVM RFE).Genes identified by all three diagnostic models were selected as potential biomarkers for LN diagnosis.Diagnostic efficacy of the selected biomarkers was verified by using the GSE104948 dataset as the validation set.2.104 renal biopsy specimens of LN patients were selected.12 cases with diabetes nephropathy(DKD),12 with minimal change(MCD)and 12 Ig A nephropathy(Ig AN)were obtained as the disease control.Normal control(NC)samples were taken from the kidney tissues next to renal cancerous tissue from 14 patients with renal cell carcinoma.Immunohistochemistry staining method was used to detect the expression of IFI16 in renal tissue.The correlation between renal IFI16 expression and severity of renal pathology,as well as LN patients’ clinical parameters were analyzed.Followed up 26 months,the relationship between renal IFI16 expression and LN patient’s prognosis was also analyzed.3.Multiple immunofluorescence staining examined the expression atlas of IFI16 in various renal cell populations,as well as relationship between localization of IFI16 expression and proliferating cells.4.34 serum samples were collected from LN patients diagnosed by renal biopsy,and20 normal serum samples were used as healthy controls.The serum IFI16 levels were measured using enzyme-linked immunosorbent assay(ELISA).The correlation between serum IFI16 levels and renal pathology,as well as LN patients clinical parameters were analyzed.5.Gene Set Variation Analysis(GSVA)and Gene Set Enrichment Analysis(GSEA)on IFI16 were performed in LN,and the correlation between IFI16 expression in LN kidney and different immune cell infiltration was analyzed using the CIBERSORT algorithm.Results1.The LN diagnostic model constructed by combining bioinformatics with machine learning.LASSO algorithm includes a total of 14 genes;the SVM-RFE algorithm only includes two genes;RF includes 30 genes.The IFI16 gene is a gene that was included in the diagnostic model by all three algorithms.The validation set showed significant high expression of IFI16 in the LN group,indicating that IFI16 can be a new biomarker for diagnosing LN.2.Immunohistochemical staining showed that IFI16 was clear expressed in the nuclei of cells in the glomerular and tubulointerstitial regions of the kidney in LN patients.In comparison to MCD,DKD,Ig AN and NC samples,IFI16 expression was considerably greater in the glomerular and tubulointerstitial regions of LN patients.The glomerular IFI16 expression was significantly higher in class Ⅳ LN patients compared to class Ⅱ,Ⅲ andⅤ.However,no significant difference of IFI16 expression was found in the tubulointerstitium between different LN pathological class.3.Multiple immunofluorescence staining results showed that IFI16 protein co-localized well with podocytes(WT1),mesangial cells(ITGA8),and endothelial cells(CD31)of glomerular capillaries in the glomerulus.In addition,IFI16 co-localized well with glomerular infiltrating inflammatory cells,including monocytes/macrophages(CD68),T-lymphocytes(CD34),B-lymphocytes(CD20),and neutrophils(MPO).In renal tubulointerstitial area of LN,IFI16 also co-localized well with infiltrating inflammatory cells(monocytes/macrophages,T,B lymphocytes,and neutrophils).Multiple immunofluorescence staining of IFI16 and Ki-67(labeled proliferating cells),as well as IFI16 and p53 proteins were also performed.The results demnostrasted that Ki-67 and IFI16 co-localized cells were only accounted for 2.7% of the number of IFI16 positive cells.This indicate that renal cells with IFI16 expression were most of non-proliferating cells in LN patients.Meanwhile,IFI16 and p53 co-localized well in the renal cells,suggesting the interaction between IFI16 and p53 protein in the cells.4.Clinical pathological significance of renal IFI16 expression were analyzed.Glomerular IFI16 expression was positively correlated with the total pathological activity index(AI),as well as several activity indices,including endocapillary hypercellularity,neutrophils/karyorrhexis,and cellular-fibrocellular crescents.However,no significant correlation between glomerular IFI16 expression and total pathological chronic index(CI)was found.Although there was no significant correlation between IFI16 expression in the tubulointerstitial region and total AI,while the higher tubulointerstitial IFI16 expression has been found in the LN with interstitial inflammation than those without interstitial inflammation.Additionally,IFI16 expression in the tubulointerstitium was higher in the LN patients with renal pathological chronicity than those without chronicity.The tubulointerstitial IFI16 expression was correlated with pathological CI.In the clinical parameters evaluations,glomerular IFI16 expression was positively correlated with SLE disease activity index(SLEDAI),serum creatinine,and amount of hematuria(red blood cell count per high-power field),while negatively related to serum complement C3,C4 and the estimated glomerular filtration rate(eGFR)values.The expression of IFI16 in tubulointerstitium was positive correlated with SLEDAI and serum creatinine,negative correlated with eGFR.In summary,renal IFI16 expression was associated with disease activity and severity in LN patients.5.Among the 104 LN cases,a total of 52 patient were analyzed for prognosis.After 26 months follow-up,conversion of anti-ds DNA antibodies from positive to negative and a~3 50% reduction from baseline eGFR were included as renal outcome endpoints.After adjusting for age,gender,proteinuria,basal creatinine,and hypertension,multivariate Cox risk analysis showed that high glomerular IFI16 expression was an independent risk factor for predicting eGFR decline in LN patients.Higher IFI16 expression in glomerulus or in the glomerulus combined with tubulointerstitium were independent risk factors for predicting a decrease conversion rate of anti-dsDNA antibodies from positive to negative.It suggests that renal IFI16 can be a new marker in predicting the prognosis of LN patients.6.The serum IFI16 protein by ELISA showed that the serum IFI16 levels in LN patients were significantly higher than those in the healthy control.However,no significant correlation was found between the serum IFI16 levels with LN patients clinical parameters,including SLEDAI,serum creatinine,hematuria,serum complement C3 and/or C4 levels,and eGFR.Meanwhile,no significant correlation was found between serum IFI16 with LN renal pathological indexes,including AI and CI.7.GSEA analysis showed that high IFI16 expression in LN was enriched with active immune response,adaptive immune response,and activation of α / β T-cells.The CIBERSORT algorithm showed a significant positive correlation between IFI16 and infiltration of monocytes and activated dendritic cells in the kidney.These suggested that renal IFI16 expression in LN patients maybe involved in local adaptive immune responses.ConclusionRenal IFI16 expression was positive correlated with the degree of renal involvement,disease activity and severity of LN patients.The higher IFI16 expression was closely related to poorer prognosis of LN patients.Renal IFI16 expression can serve as a new biomarker for disease activity and clinical prognosis in LN patients.