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Local synthesis of interferon-alpha in lupus nephritis is associated with type I interferons signature and LMP7 induction in renal tubular epithelial cells.

Castellano G, Cafiero C, Divella C, Sallustio F, Gigante M, Pontrelli P, De Palma G, Rossini M, Grandaliano G, Gesualdo L - Arthritis Res. Ther. (2015)

Bottom Line: Type I interferons signature was characterized by MXA-specific staining in renal tubular epithelial cells; in addition, in situ hybridization showed that renal tubular epithelial cells were the major producers of interferon-alpha, indicating a potential autocrine effect.Whole-genome expression profile showed interferon-alpha induced up-regulation of genes involved in innate immunity, protein ubiquitination and switching to immunoproteasome.Our data indicate that type I interferons might have a pathogenic role in lupus nephritis characterized by an autocrine effect of interferon-alpha on renal tubular epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Renal, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari, Piazza Giulio Cesare 11, 70124, Bari, Italy. giuseppe.castellano@uniba.it.

ABSTRACT

Introduction: Type I interferons are pivotal in the activation of autoimmune response in systemic lupus erythematous. However, the pathogenic role of interferon-alpha in patients affected by lupus nephritis remains uncertain. The aim of our study was to investigate the presence of a specific interferon signature in lupus nephritis and the effects of interferon-alpha at renal level.

Methods: We performed immunohistochemical analysis for MXA-protein and in situ hybridization to detect interferon-alpha signature and production in human lupus nephritis. Through microarray studies, we analyzed the gene expression profile of renal tubular epithelial cells, stimulated with interferon-alpha. We validated microarray results through real-time polymerase chain reaction, flow cytometry on renal tubular epithelial cells, and through immunohistochemical analysis and confocal microscopy on renal biopsies.

Results: Type I interferons signature was characterized by MXA-specific staining in renal tubular epithelial cells; in addition, in situ hybridization showed that renal tubular epithelial cells were the major producers of interferon-alpha, indicating a potential autocrine effect. Whole-genome expression profile showed interferon-alpha induced up-regulation of genes involved in innate immunity, protein ubiquitination and switching to immunoproteasome. In accordance with the in vitro data, class IV lupus nephritis showed up-regulation of the immunoproteasome subunit LMP7 in tubular epithelial cells associated with type I interferon signature.

Conclusions: Our data indicate that type I interferons might have a pathogenic role in lupus nephritis characterized by an autocrine effect of interferon-alpha on renal tubular epithelial cells. Therefore we hypothesize that inhibition of type I interferons might represent a therapeutic target to prevent tubulo-interstitial damage in patients with lupus nephritis.

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Quantitative analysis of IFN-alpha up regulated genes and proteins involved in antigen presentation pathways. Validation of differential expression of PSMB8 (A), DTX3L (B) and FBOX6 (C) in RPTEC stimulated with IFN-alpha. Expression levels were quantified using RT-PCR. The genes’ relative expressions were normalized to the expression of ß actin. The histograms represent the mean ± SEM. The intracellular expression of LMP7 was evaluated by flow cytometry analysis (D) (LMP7 48.09% ± 2 basal versus 76.18% ± 2, 48 hours, 100 U/ml INF-alpha) and western blot analysis (LMP7 basal versus 48 hours, 100 U/ml INF-alpha P <0.02) (E). The surface expression of HLA I (F) in RPTEC stimulated with IFN-alpha showed a significant increase of mean fluorescence intensity (MnI) (19.5 ± 3 basal versus 43.7 ± 2, 48 hours, 100 U/ml IFN-alpha stimulation). Data shown were gated on RPTEC cells and histograms were based on RPTEC staining with isotype control mAbs. The data presented for both HLA-I and LMP7 are representative of three independent experiments performed using RPTEC cells (P <0.02 and P <0.05, respectively). mAbs, monoclonal antibodies; RPTEC, renal proximal tubular cells; SEM, standard error of the mean.
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Fig3: Quantitative analysis of IFN-alpha up regulated genes and proteins involved in antigen presentation pathways. Validation of differential expression of PSMB8 (A), DTX3L (B) and FBOX6 (C) in RPTEC stimulated with IFN-alpha. Expression levels were quantified using RT-PCR. The genes’ relative expressions were normalized to the expression of ß actin. The histograms represent the mean ± SEM. The intracellular expression of LMP7 was evaluated by flow cytometry analysis (D) (LMP7 48.09% ± 2 basal versus 76.18% ± 2, 48 hours, 100 U/ml INF-alpha) and western blot analysis (LMP7 basal versus 48 hours, 100 U/ml INF-alpha P <0.02) (E). The surface expression of HLA I (F) in RPTEC stimulated with IFN-alpha showed a significant increase of mean fluorescence intensity (MnI) (19.5 ± 3 basal versus 43.7 ± 2, 48 hours, 100 U/ml IFN-alpha stimulation). Data shown were gated on RPTEC cells and histograms were based on RPTEC staining with isotype control mAbs. The data presented for both HLA-I and LMP7 are representative of three independent experiments performed using RPTEC cells (P <0.02 and P <0.05, respectively). mAbs, monoclonal antibodies; RPTEC, renal proximal tubular cells; SEM, standard error of the mean.

Mentions: We next performed, in a separate set of experiments, RT-PCR to confirm and validate the increased expression of candidate genes identified by microarray analysis. PSMB8, DTX3L and FBOX6 genes showed a significantly increased expression following IFN-alpha stimulation (P <0.05; Figure 3A-C). We also quantified intracellular tubular LMP7 by flow cytometer analysis and western blotting. After 48 hours of activation, the synthesis of β5i proteasome subunit LMP7 significantly increased when compared to basal conditions as shown by flow cytometry (Figure 3D) and by western blotting analysis (Figure 3E). In addition, we performed phenotypic analysis of HLA I expression on IFN-activated-RPTEC, confirming the increased surface expression of HLA class I molecules compared to basal conditions (Figure 3F; P <0.02).Figure 3


Local synthesis of interferon-alpha in lupus nephritis is associated with type I interferons signature and LMP7 induction in renal tubular epithelial cells.

Castellano G, Cafiero C, Divella C, Sallustio F, Gigante M, Pontrelli P, De Palma G, Rossini M, Grandaliano G, Gesualdo L - Arthritis Res. Ther. (2015)

Quantitative analysis of IFN-alpha up regulated genes and proteins involved in antigen presentation pathways. Validation of differential expression of PSMB8 (A), DTX3L (B) and FBOX6 (C) in RPTEC stimulated with IFN-alpha. Expression levels were quantified using RT-PCR. The genes’ relative expressions were normalized to the expression of ß actin. The histograms represent the mean ± SEM. The intracellular expression of LMP7 was evaluated by flow cytometry analysis (D) (LMP7 48.09% ± 2 basal versus 76.18% ± 2, 48 hours, 100 U/ml INF-alpha) and western blot analysis (LMP7 basal versus 48 hours, 100 U/ml INF-alpha P <0.02) (E). The surface expression of HLA I (F) in RPTEC stimulated with IFN-alpha showed a significant increase of mean fluorescence intensity (MnI) (19.5 ± 3 basal versus 43.7 ± 2, 48 hours, 100 U/ml IFN-alpha stimulation). Data shown were gated on RPTEC cells and histograms were based on RPTEC staining with isotype control mAbs. The data presented for both HLA-I and LMP7 are representative of three independent experiments performed using RPTEC cells (P <0.02 and P <0.05, respectively). mAbs, monoclonal antibodies; RPTEC, renal proximal tubular cells; SEM, standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Fig3: Quantitative analysis of IFN-alpha up regulated genes and proteins involved in antigen presentation pathways. Validation of differential expression of PSMB8 (A), DTX3L (B) and FBOX6 (C) in RPTEC stimulated with IFN-alpha. Expression levels were quantified using RT-PCR. The genes’ relative expressions were normalized to the expression of ß actin. The histograms represent the mean ± SEM. The intracellular expression of LMP7 was evaluated by flow cytometry analysis (D) (LMP7 48.09% ± 2 basal versus 76.18% ± 2, 48 hours, 100 U/ml INF-alpha) and western blot analysis (LMP7 basal versus 48 hours, 100 U/ml INF-alpha P <0.02) (E). The surface expression of HLA I (F) in RPTEC stimulated with IFN-alpha showed a significant increase of mean fluorescence intensity (MnI) (19.5 ± 3 basal versus 43.7 ± 2, 48 hours, 100 U/ml IFN-alpha stimulation). Data shown were gated on RPTEC cells and histograms were based on RPTEC staining with isotype control mAbs. The data presented for both HLA-I and LMP7 are representative of three independent experiments performed using RPTEC cells (P <0.02 and P <0.05, respectively). mAbs, monoclonal antibodies; RPTEC, renal proximal tubular cells; SEM, standard error of the mean.
Mentions: We next performed, in a separate set of experiments, RT-PCR to confirm and validate the increased expression of candidate genes identified by microarray analysis. PSMB8, DTX3L and FBOX6 genes showed a significantly increased expression following IFN-alpha stimulation (P <0.05; Figure 3A-C). We also quantified intracellular tubular LMP7 by flow cytometer analysis and western blotting. After 48 hours of activation, the synthesis of β5i proteasome subunit LMP7 significantly increased when compared to basal conditions as shown by flow cytometry (Figure 3D) and by western blotting analysis (Figure 3E). In addition, we performed phenotypic analysis of HLA I expression on IFN-activated-RPTEC, confirming the increased surface expression of HLA class I molecules compared to basal conditions (Figure 3F; P <0.02).Figure 3

Bottom Line: Type I interferons signature was characterized by MXA-specific staining in renal tubular epithelial cells; in addition, in situ hybridization showed that renal tubular epithelial cells were the major producers of interferon-alpha, indicating a potential autocrine effect.Whole-genome expression profile showed interferon-alpha induced up-regulation of genes involved in innate immunity, protein ubiquitination and switching to immunoproteasome.Our data indicate that type I interferons might have a pathogenic role in lupus nephritis characterized by an autocrine effect of interferon-alpha on renal tubular epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Renal, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari, Piazza Giulio Cesare 11, 70124, Bari, Italy. giuseppe.castellano@uniba.it.

ABSTRACT

Introduction: Type I interferons are pivotal in the activation of autoimmune response in systemic lupus erythematous. However, the pathogenic role of interferon-alpha in patients affected by lupus nephritis remains uncertain. The aim of our study was to investigate the presence of a specific interferon signature in lupus nephritis and the effects of interferon-alpha at renal level.

Methods: We performed immunohistochemical analysis for MXA-protein and in situ hybridization to detect interferon-alpha signature and production in human lupus nephritis. Through microarray studies, we analyzed the gene expression profile of renal tubular epithelial cells, stimulated with interferon-alpha. We validated microarray results through real-time polymerase chain reaction, flow cytometry on renal tubular epithelial cells, and through immunohistochemical analysis and confocal microscopy on renal biopsies.

Results: Type I interferons signature was characterized by MXA-specific staining in renal tubular epithelial cells; in addition, in situ hybridization showed that renal tubular epithelial cells were the major producers of interferon-alpha, indicating a potential autocrine effect. Whole-genome expression profile showed interferon-alpha induced up-regulation of genes involved in innate immunity, protein ubiquitination and switching to immunoproteasome. In accordance with the in vitro data, class IV lupus nephritis showed up-regulation of the immunoproteasome subunit LMP7 in tubular epithelial cells associated with type I interferon signature.

Conclusions: Our data indicate that type I interferons might have a pathogenic role in lupus nephritis characterized by an autocrine effect of interferon-alpha on renal tubular epithelial cells. Therefore we hypothesize that inhibition of type I interferons might represent a therapeutic target to prevent tubulo-interstitial damage in patients with lupus nephritis.

Show MeSH
Related in: MedlinePlus