Limits...
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.

Show MeSH

Related in: MedlinePlus

Activation of NF-kB pathways in tubular epithelial cells with type I IFN signature. (A) RPTEC were stimulated at different time points with IFN-alpha 100 U/ml and stained for p65 (green) and pNIK (red). At basal conditions both proteins were detectable only in the cytoplasm; after five minutes of stimulation p65 and pNIK began to move from the cytoplasm to the nucleus with significant translocation after 15 and 30 minutes. (B) Specific p65 and pNIK fluorescence intensity inside the nuclei was quantified as described in Methods section. Data are averages ± SD for n = 10 cells from one field on one slide; *P <0.05 and °P <0.05 for comparison pNIK and p65 versus basal, respectively. The images and results are representative of at least three independent experiments. (C) Double staining for MXA (green), p65 (red) and merge analysis (yellow) on class IV lupus nephritis showing translocation of p65 from cytoplasm to nuclei. NF-kB, nuclear factor-kB; RPTEC, renal proximal tubular cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4389585&req=5

Fig5: Activation of NF-kB pathways in tubular epithelial cells with type I IFN signature. (A) RPTEC were stimulated at different time points with IFN-alpha 100 U/ml and stained for p65 (green) and pNIK (red). At basal conditions both proteins were detectable only in the cytoplasm; after five minutes of stimulation p65 and pNIK began to move from the cytoplasm to the nucleus with significant translocation after 15 and 30 minutes. (B) Specific p65 and pNIK fluorescence intensity inside the nuclei was quantified as described in Methods section. Data are averages ± SD for n = 10 cells from one field on one slide; *P <0.05 and °P <0.05 for comparison pNIK and p65 versus basal, respectively. The images and results are representative of at least three independent experiments. (C) Double staining for MXA (green), p65 (red) and merge analysis (yellow) on class IV lupus nephritis showing translocation of p65 from cytoplasm to nuclei. NF-kB, nuclear factor-kB; RPTEC, renal proximal tubular cells.

Mentions: As indicated in Additional file 3, the pathway analysis suggested a possible role of IFN-alpha in the activation of the NF-kB pathway in tubular epithelial cells. Therefore, we tested the activation of canonical and non-canonical NF-kB pathways in vitro by p65 and pNIK analysis [20]. At basal conditions, p65 was detectable only in the cytoplasm with low pNIK activation (Figure 5A). After five minutes of stimulation with IFN-alpha, p65 and pNIK began to move from the cytoplasm to the nucleus and completely transmigrated in to the nucleus at 15 and 30 minutes (Figure 5A). Interestingly, pNIK presented an intensive migration to the nucleus upon IFN-alpha stimulation [21,22] as indicated by confocal microscopy (Figure 5A). Quantification of specific nuclear fluorescence indicated that nuclear translocation of p65 and pNIK was statistically significant (Figure 5B).Figure 5


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)

Activation of NF-kB pathways in tubular epithelial cells with type I IFN signature. (A) RPTEC were stimulated at different time points with IFN-alpha 100 U/ml and stained for p65 (green) and pNIK (red). At basal conditions both proteins were detectable only in the cytoplasm; after five minutes of stimulation p65 and pNIK began to move from the cytoplasm to the nucleus with significant translocation after 15 and 30 minutes. (B) Specific p65 and pNIK fluorescence intensity inside the nuclei was quantified as described in Methods section. Data are averages ± SD for n = 10 cells from one field on one slide; *P <0.05 and °P <0.05 for comparison pNIK and p65 versus basal, respectively. The images and results are representative of at least three independent experiments. (C) Double staining for MXA (green), p65 (red) and merge analysis (yellow) on class IV lupus nephritis showing translocation of p65 from cytoplasm to nuclei. NF-kB, nuclear factor-kB; RPTEC, renal proximal tubular cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389585&req=5

Fig5: Activation of NF-kB pathways in tubular epithelial cells with type I IFN signature. (A) RPTEC were stimulated at different time points with IFN-alpha 100 U/ml and stained for p65 (green) and pNIK (red). At basal conditions both proteins were detectable only in the cytoplasm; after five minutes of stimulation p65 and pNIK began to move from the cytoplasm to the nucleus with significant translocation after 15 and 30 minutes. (B) Specific p65 and pNIK fluorescence intensity inside the nuclei was quantified as described in Methods section. Data are averages ± SD for n = 10 cells from one field on one slide; *P <0.05 and °P <0.05 for comparison pNIK and p65 versus basal, respectively. The images and results are representative of at least three independent experiments. (C) Double staining for MXA (green), p65 (red) and merge analysis (yellow) on class IV lupus nephritis showing translocation of p65 from cytoplasm to nuclei. NF-kB, nuclear factor-kB; RPTEC, renal proximal tubular cells.
Mentions: As indicated in Additional file 3, the pathway analysis suggested a possible role of IFN-alpha in the activation of the NF-kB pathway in tubular epithelial cells. Therefore, we tested the activation of canonical and non-canonical NF-kB pathways in vitro by p65 and pNIK analysis [20]. At basal conditions, p65 was detectable only in the cytoplasm with low pNIK activation (Figure 5A). After five minutes of stimulation with IFN-alpha, p65 and pNIK began to move from the cytoplasm to the nucleus and completely transmigrated in to the nucleus at 15 and 30 minutes (Figure 5A). Interestingly, pNIK presented an intensive migration to the nucleus upon IFN-alpha stimulation [21,22] as indicated by confocal microscopy (Figure 5A). Quantification of specific nuclear fluorescence indicated that nuclear translocation of p65 and pNIK was statistically significant (Figure 5B).Figure 5

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