Oxidative stress and Treg depletion in lupus patients with anti-phospholipid syndrome

Abstract

Anti-phospholipid antibodies (APLA) represent a diagnostic criterion of systemic lupus erythematosus (SLE) and cause morbidity, termed anti-phospholipid syndrome (APS). Activation of the mechanistic target of rapamycin (mTOR) has been recently associated with APS. mTOR is a sensor of oxidative stress. Therefore, we examined mitochondrial mass, superoxide production, mTOR activity and FoxP3 expression in 72 SLE patients, twelve of whom also had APS, and 54 healthy controls by flow cytometry. Mitochondrial mass was increased in CD4(-)CD8(-) double-negative (DN) T cells of SLE patients with APS (2.7-fold) in comparison to those without APS (1.7-fold; p = 0.014). Superoxide production was increased in all lymphocyte subsets of APS patients. FoxP3(+) cells were depleted within CD4(+)CD25(+) Tregs in patients with APS (28.4%) relative to those without APS (46.3%, p = 0.008). mTOR activity was similar between SLE patients with and without APS. Thus, oxidative stress and Treg depletion rather than mTOR activation underlie APS in patients with SLE.

Keywords: Anti-phospholipid syndrome; Mechanistic target of rapamycin; Oxidative stress; Systemic lupus erythematosus; Treg; mTOR.

Figure 1

Increased oxidative stress in SLE patients with APS. Peripheral blood lymphocytes (PBL) of 72 lupus patients were freshly isolated and studied in parallel with 54 healthy control subjects matched for age within 10 years, gender, and ethnicity for each blood donation, as earlier described (4;5). A, Mitochondrial mass was measured by MitoTracker Green (MTG) fluorescence in CD3⁺, CD4⁺, CD8⁺, and CD3⁺CD4⁻CD8⁻ double-negative (DN) T cells and CD19⁺ B cells. Left panels show histogram overlays of healthy (black) and SLE subjects with (red) and without APS (blue). Bar chart represents mean ± SEM of fold changes relative to paired controls normalized at 1.0. B, Oxidative stress, that is, the production of reactive oxygen intermediates (ROI) was measured by hydroethidine (HE) fluorescence. Left panels show representative histogram overlays, while bar chart depicts the mean ± SEM of fold changes relative to paired controls normalized at 1.0. p values reflect comparison between SLE patients with and without APS using unpaired t-test.

Figure 2

Comparable mTOR pathway activity in lupus T cells of patients with or without APS. mTORC1 activity was measured by the frequency of cells with elevated intracellular phosphorylated S6 ribosomal protein levels (%pS6RP^hi). Left panels show representative histogram overlays, while bar chart depicts the mean ± SEM of fold changes relative to paired controls normalized at 1.0. p values reflect comparison between SLE patients with and without APS using unpaired t-test.

Figure 3

Treg depletion in SLE patients with APS. CD25⁺FoxP3⁺ Tregs were enumerated within CD4⁺ T cells and FoxP3 expression was also assessed by intracellular staining of permeabilized CD4⁺CD25⁺ T cells (4;5). * reflect p values < 0.05 comparing groups of subjects marked by brackets. Exact p values of 0.043 and 0.008 reflect comparisons between SLE patients with and without APS using unpaired t-test.