Methods Lupus Patient and healthy subjects: Lupus patients were enrolled from outpatient department (OPD) of rheumatology clinic, PGIMER, Chandigarh.
Flowcytometric analysis PBMC isolated were incubated with antibodies conjugated to APC, PE, PerCP/cy5.5 for surface staining of antigens CD3,CD4,CD8 and CD25(Biolegend,USA). Primary antibodies for Keap1/Nrf2 (from Santacruz biotech,USA) were utilized for intracellular staining followed by FITC labelled Goat anti-mouse IgG(santacruz biotech,USA). DCFDA dye method used for analysis of oxidative stress.
Magnet -associated cell sorting for isolation of CD4+ and CD8+ T-cells: CD4+ and CD8+ T-cells were sorted using isolation Kits manufactured by StemcellTM, USA,
Quantitative real-time polymerase chain reaction cDNA was synthesized from RNA separated from these sorted subtypes and were utilized for evaluating expression of genes Keap1/Nrf2/HMOX-1. Quantitative PCR was done on the StepOnePlus RealTime PCR Systems and was analyzed with StepOne Software V2.1 (Applied Biosystems New York, USA).
Statistical analysis Quantitative data were presented as mean ± standard error mean (SEM), were analyzed using unpaired Student’s T-tests for parametric quantitative data and Mann-Whitney U test for non-parametric data. Likewise, the correlational study of parametric data was done with Pearson’s correlation and for non-parametric data Spearman’s rank correlation. The software Graphpad prism version5.1 was used for analysis
Intracellular oxidative stress was higher in T-cell subtypes: CD3+CD4+, CD3+CD8+ and CD4+ CD25hi cells of lupus patients.
Keap1levels were significantly higher in CD3+CD8+ and CD4+ CD25Hi in SLE patients.
Intracellular concentration of Nrf2 were significantly higher in CD3+CD8+ of SLE patients.
Relative mRNA expression of Nrf2 in CD8+ cells were higher in SLE patients as compared to healthy controls
Relative mRNA expression of HMOX-1 was higher in CD4+ and CD8+ of SLE patients
Proportion of CD3+CD4+ , CD3+CD8+ and CD4+ CD25hi were significantly reduced in SLE patients.
Median Fluorescence Intensity (MFI)of Dcfda(or ROS levels) were directly correlated with disease activity score in CD3+CD4+ & CD3+CD8+ of SLE patients.
Intracellular levels of Nrf2 directly correlates with proportion of CD3+CD4+ and CD3+CD8+ cells.
Concentration of Keap1 in CD3+CD4+ and CD3+CD8+ and relative mRNA of Keap1 expression in CD4+ & CD8+ cells and relative mRNA expression of Nrf2 positively correlated with SLEDAI score.
Conclusion Our study clearly elucidates that intracellular oxidative stress was elevated in the subtypes of T-cells and their was alteration in Keap1/Nrf2/HMOX-1 and proportions, found to be associated with disease activity score(SLEDAI).
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