Background
Plasmacytoid dendritic cells (pDCs) play a key role in the activation of the autoimmune response in LN “G. De Palma – 2011 [1], ” N. Fiore – 2008 “ [2]. However, the effects of pDCs, the major producer of IFN-alpha, on the renal tubular epithelial cells (RPTEC) is unknown. The aim of the study was to investigate the effects of IFN-alpha in RPTEC, in vitro and ex vivo.
Patients and Methods
- Microarray analyses (Illumina) were conducted to analyze differential gene expression profiles of RPTEC cells, stimulated with IFN-alpha 100U/ml for 48h, versus cells without stimuli. The genes that were significantly modulated were studied using the Gene Ontology database, Ingenuity™ software and Gene Enrichment Analysis (GSEA).
- Validation of some significantly regulated genes was made using quantitative real-time (RT)-PCR (DTXL,FBOX6, PSMB8) and flow cytometry ( LMP7, HLA-I).
- The study enrolled 10 patients with lupus nephritis, 6 with a histological diagnosis of lupus nephritis class IV and 4 with lupus nephritis class I.
- Immunohistochemical (IH) analysis was effectuated on renal biopsies of class I and IV LN patients. Confocal analysis was effectuated on renal biopsies of class I and IV LN patients.
Results
Microarray analysis showed 108 significantly up-regulated genes and only 7 down-regulated genes with a fold-change >2 (false discovery rate, FDR<0.05). Gene set enrichment analysis identified 123 processes differentially regulated in IFN-α treated cells. The most represented processes resulted interferon response pathway, antigen presentation and inflammatory pathways. Among over-expressed genes, contributing to these processes, we identified the HLA-I, the ubiquitin (in particular FBXO6 and DTX3L) and the immunoproteasome subunits LMP7(PSMB8 gene)(Figura 1). After validation by RT-PCR, flow cytometry experiments on RPTEC cells confirmed a significant increase of the antigen presentation pathway (HLA I : 75%±2/MnI 7.8±3 basal vs 90%±3/MnI 11±2 48h 100 U/ml IFN-alpha) and the inflammatory signaling (LMP7 49.75%±2 basal vs 72.4%±2 48h 100 U/ml IFN-alpha) (Figura 2). IH analysis on renal biopsies of patients with LN showed a significant increase of LMP7 expression, at tubular interstitial level, (LMP7 5% ± 2 NL class I vs 16%± 5 class IV, p <0.0001) proportional to MXA protein, a specific marker of IFN-alpha (MXA 0% ± 1.0 class I vs 4.5% ± 1.2 class IV, P <0.0001)(Figura 3). Confocal microscopy confirmed the colocalization of LMP7-MXA proteins expression at tubular epithelial cells (Figura 4) and the activation of inflammatory signaling via NF-kB(p65) in the MXA+ tubules (Figura 5).
Conclusions
Our data demonstrate a critical role of pDC-derived IFN-alpha in the activation of RPTEC. Therefore we hypothesize that inhibition of IFN-alpha pathway may be a novel therapeutic strategy to reduce renal tubular damage in patients with LN.