Discussion
SLE is characterised by increased levels of cellular apoptosis, inefficient clearance of apoptotic cells, and the production of autoantibodies, which may arise from increased exposure to nuclear self-antigens derived from apoptotic debris.17–20 This SLE-associated increase in apoptosis is thought to be contributed by defective DNA repair mechanisms. Indeed, rare variants associated with SLE were shown to impair protein function of RNase H2, an essential enzyme that removes misincorporated ribonucleotides during DNA replication. The ineffective DNA damage repair that ensued resulted in increased expression of interferon (IFN)-regulated genes and an enhanced type-I IFN response in fibroblasts from patients.21 Furthermore, defective repair of DSBs has been associated with EBV-transformed B-cell lines in paediatric SLE and PBMCs in lupus nephritis.7 ,8 More recently, a polymorphism in the DNA repair gene RAD51B has been associated with increased risk of SLE.2
In this study, we explored DSB accumulation for the first time in SLE CD4+ T cells, CD8+ T cells and monocytes, compared with healthy controls and patients with RA. In addition, we measured intracellular DSB accumulation with detailed analyses of phospho-H2AX by flow cytometry, which can quantify DSBs with high specificity and sensitivity to genotoxicity (91% and 89%, respectively), while offering statistical superiority to other phospho-H2AX assays by analysing greater numbers of cells.22 ,23 The anti-phospho-H2AX antibody used in this study specifically detects phosphorylation of H2AX at serine 139, and has been previously validated and repeatedly used to accurately assess H2AX phosphorylation.21 ,24 ,25
For each of the analysed cell types, we revealed significantly increased DSB accumulation in patients with SLE compared with healthy controls. Further, DSB accumulation was significantly higher in SLE compared with RA, an inflammatory disease control. It remains to be seen, however, how this increase in DSBs in SLE compares with other conditions known to be associated with increased oxidative stress, such as sepsis and radiation therapy. As phospho-H2AX levels are known to be decreased in G1 compared with both S and G2 phases,26 ,27 we also assessed DSB accumulation at G0/G1, S and G2 phases to show significant DSB accumulation at each cell-cycle phase in SLE T cells and at G0/G1 and G2 phases in SLE monocytes. In combination, these findings suggest that increased DSB accumulation in SLE immune cells is independent of lineage and cell cycle.
Our analysis of the relationship between disease activity and DSB levels in patients with SLE showed a significant positive correlation in CD4+ T cells and CD8+ T cells, independent of the cell-cycle phases. When the SLEDAI scores were calculated without criteria for low complement binding and increased anti-dsDNA antibodies, the correlation was also significant in CD4+ T cells, CD8+ T cells and monocytes, independent of cell cycle. These data might support a proof of concept for the potential use of phospho-H2AX as a biomarker of disease activity in SLE. Although the utility of this biomarker will require replication efforts with a larger cohort for validation, the benefits of using phospho-H2AX as a biomarker for early detection, prognosis and treatment efficacy have been already described in cancer.28 ,29
Previous studies have shown that SLE lymphocytes and neutrophils are sensitive to oxidative stress.30 ,31 In this study, we assessed the DNA damage response to oxidative stress in specific immune cell types using a time-course treatment of H2O2. In CD4+ and CD8+ T cells from healthy individuals, our data showed that phospho-H2AX levels did not increase further with H2O2 exposures longer than 10 min. In addition, phospho-H2AX levels of healthy monocytes did not increase appreciably at any of the evaluated exposure times, indicating that monocytes may have an alternate or enhanced ability to handle oxidative stress. However, SLE phospho-H2AX levels continued to increase with H2O2 exposure times for each of the three immune cell subsets. These data suggest that SLE is associated with defective DSB repair or an intrinsically higher susceptibility for DNA damage in CD4+ T cells, CD8+ T cells and monocytes.
Taken together, these data provide evidence for defective repair of endogenous and oxidative stress-induced DSBs in SLE CD4+ T cells, CD8+ T cells and monocytes. In addition, DSB levels correlate with disease activity, and suggest further investigation of phospho-H2AX as a disease biomarker linking environmental exposures to the chromatin microenvironment in SLE. Importantly, our findings add to our current understanding of SLE, and offer further evidence for the role of aberrant DSB repair in disease pathogenesis.