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Thursday 06 October 2022 from 13:00 to 14:10
PO.2 E- poster session 2: adaptive immunity including autoantibodies, APS, diagnostic and classification criteria
PO.2.28 Intracellular oxidative stress in t-cells is associated with the disease activity of lupus
  1. A Tandon1,
  2. A Sharma1 and
  3. A Bhatnagar2
  1. 1PGIMER ~ Chandigarh ~ India
  2. 2Department of Biochemistry, Panjab University ~ Chandigarh ~ India there is significant implication of oxidative stress in pathology of SLE. The abnormality in cell mediated immunity largely contributes to many autoimmune diseases like SLE. To understand the association between intracellular oxidative stress and antioxidant status, alterations in proportions (TH, Tc and Treg) and severity of Lupus, the cell-specific study was carried out in T-cell subtypes of SLE patients

Abstract

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

Results

  • 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.

Abstract PO.2.28 Figure 1
Abstract PO.2.28 Figure 1

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).

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

http://dx.doi.org/10.1136/lupus-2022-elm2022.58

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