Background/Purpose: Studies suggested that defective or inefficient DNA double strand break (DSB) repair results in failure to preserve genomic integrity leading to apoptotic cell death, a hallmark of lupus. Compelling evidence linked environmental factors that increase oxidative stress with lupus risk and the formation of DSBs. Histone H2AX is robustly phosphorylated in the chromatin micro-environment at DSB loci resulting in the accumulation of essential repair proteins. The aim of this study is to measure DSB levels as a marker linking the effect of environmental stressors and the chromatin micro-environment in lupus.

Methods: Peripheral-blood mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation from lupus patients and age-, sex-, and ethnicity-matched healthy controls. Flow cytometry using intracellular staining was used to measure H2AX phosphorylation levels in monocytes, CD4+ T cells, and CD8+ T cells. Median fluorescence intensity of anti-phospho-H2AX represents relative accumulation of DSBs within cells. Propidium iodide DNA staining was simultaneously used to assess cell cycle phase. A dose and time-effect relationship of hydrogen peroxide (H₂O₂) exposure, as an extrinsic environmental stressor, on the formation of DSBs was studied. Statistically significant differences (P < 0.05) between patients and controls were determined using a two-tailed t-test, and correlation analysis with lupus disease activity as measured by SLEDAI was assessed by Pearson’s correlation coefficient.

Results: We observed increased DSBs in monocytes, CD4+ T cells, and CD8+ T cells in lupus patients compared to healthy controls (P=0.0008, 0.001, and 0.001, respectively). This difference in DSBs between patients and controls was independent of the cell cycle phase. A significant positive correlation between DSB accumulation and SLEDAI scores was observed among lupus patients (CD4+ T cells: r²= 0.64, P= 0.03; CD8+ T cells: r²= 0.64, P= 0.009; Monocytes: r²= 0.57, P= 0.01). SLEDAI also correlated positively with DSB in lupus patients during all cell cycle phases in T cells and monocytes. A time-dependent effect (0, 2, 5, 10, 30 and 60 min, n=4) of 0.5uM H₂O₂ revealed a significant increase of DSBs with time in both patients and controls in all cell types, and a significantly greater accumulation of DSBs in lupus CD4+ T cells compared to healthy controls. A similar trend was also observed in lupus CD8+T cells and monocytes.

Conclusion: Our data indicate that DSBs occur more frequently and independent of the cell cycle in lupus monocytes and T cells compared to controls, and that accumulation of DSBs in lupus correlates with disease activity. Further, lupus T cells are more sensitive and accumulate more DSBs upon exposure to extrinsic oxidative stress compared to healthy T cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/histone-h2ax-phosphorylation-as-a-measure-of-dna-double-strand-breaks-and-a-marker-of-environmental-stress-and-disease-activity-in-lupus/