Dissertation committee:
Πέτρος Σφηκάκης, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Βασίλειος Γοργούλης, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Βασίλειος Σουλιώτης, Ερευνητής Α', Ινστιτούτο Χημικής Βιολογίας, Εθνικό Ίδρυμα Ερευνών
Μαρία Τεκτονίδου, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Ευάγγελος Λυμπερόπουλος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Κλειώ Μαυραγάνη, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Γεώργιος Φραγκούλης, Επίκουρος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Systemic autoimmune diseases comprise an heterogenous group and are characterized by aberrant chronic aberrant immune activation against self-antigens, production of autoantibodies and tissue injury. Although their pathophysiology remains unclear, several lines of evidence suggest that the imbalance between the oxidant and antioxidant cellular systems after exposure to deleterious stimuli, termed oxidative stress, is shown to be involved. Reactive oxygen and nitrogen species are key mediators of oxidative stress and may damage cellular DNA. To protect the genome, cells have developed several DNA repair mechanisms, composing the DNA damage response and repair network (DDR/R). On the other hand, chronological age, associated with a decline of the immune system functionality, leading to increased susceptibility to cancer and autoimmunity. Herein, we aimed to examine whether systemic autoimmunity is associated with aberrations in oxidative stress formation and DNA damage accumulation, as well as whether these aberrations may be attributed to the person’s chronological age.
We studied 78 consecutive patients with systemic autoimmune diseases, including Rheumatoid Arthritis (RA) (n=9), Systemic Lupus Erythematosus (SLE) (n=14), Systemic Sclerosis (SSc) (n=9), Adamantiades – Behcet’s disease (ABD) (n=6) and Antiphospholipid Syndrome (APS) (n=40). A total of 212 apparently healthy individuals (HC) were studied in parallel. Peripheral blood mononuclear cells (PBMCs) were isolated using standard methods. Oxidative stress formation was assessed by quantifying intracellular glutathione oxidation with chemiluminescent methods , DNA damage levels were measured by alkaline single-cell gel electrophoresis (single- and double- strand DNA breaks) and abasic (apurinic/apyrimidinic) sites using a chemiluminescent assays, while DNA damage repair efficacy was assessed by immunofluorescence antigen staining and confocal laser microscopy (double-strand break repair / DSB-R). Results from each patient subgroup were compared with corresponding results derived from an HC subgroup, matched 1:3 for age and sex.
Firstly, increased oxidative stress levels were observed in RA, SLE, SSc, ABD and APS patients compared to HC (glutathione oxidation: RA: 46.2 ± 8,56 / HC: 70±10.7 [p<0.001], SLE: 49.86 ± 12.6 / HC: 70 ±8.6 [p<0.001], SSc: 52 ± 15.68 / HC: 67.29 ± 10.1, [p<0.01], ABD: 30.83 ± 8.33/ HC: 74.2 ± 8.54 [p<0.001] , APS: 44.86 ± 11.54 / HC: 69±11.33 [p<0.001]) Secondly, increased abasic site formation was observed in RA, SLE, SSc, ABD and APS patients compared to HC (abasic site formation: RA: 16.3 ± 3.2/ HC: 7.8±2.4 [p<0.001], SLE: 14.8±4 / HC: 7±2.4 [p<0.001], SSc: 12.56 ± 3.2 / HC: 7 ±2.4 [p<0.01], ABD: 23 ± 8.12/ HC: 5.7 ± 1.4 [p<0.001], APS: 16.6 ± 6.7/ HC: 7.3 ± 3.04 [p<0.001]). Abasic site formation strongly associated with intracellular oxidative stress levels in HCs and patients with systemic autoimmune diseases (HC: r=-0.555, p<0.001, Patients: r=-0.418, p<0.001). Thirdly, endogenous double-strand and single-strand DNA breaks were highly elevated in the PBMCs of patients with systemic autoimmune diseases, compared to HC (Olive Tail Moment: RA: 15.7±8.5 / HC: 6.2±1.7 [p<0.001], SLE: 9.7± 5.4 / HC: 4.7 ± 1.5 [p<0.001], SSc: 11.8 ± 8 / HC: 4.8± 1.8 [p<0.001], ABD: 9.4± 5.7 / HC: 4± 1.3 [p<0.01], APS: 14.5 ± 7.4 / HC: 5.3 ± 2 [p<0.001]). Collectively within patients, these elevated DNA damage levels strongly correlated with both the oxidative stress (r=-0.388 , p<0.001) and abasic site levels (r=0.455, p<0.001) underlining a possible mechanistic link. Notably, a lack of correlation was observed between DNA damage repair capacity and oxidative stress levels in patients. DNA damage repair capacity correlated strongly with oxidative stress levels (r=-0.500, p<0.001) in healthy individuals, indirectly confirming the deficient DDR capacity in patients with systemic autoimmunity. Finally, chronological age was found to strongly associate with oxidative stress (r=0.636, p<0.001), DNA damage levels (r=0.641, p<0.001) and DDR capacity (r=0.781 , p<0.001) in HCs, but not in patients with systemic autoimmune diseases.
In conclusion, we show that patients with systemic autoimmune diseases display increased oxidative stress, leading to increased DNA damage formation, and decreased DNA damage repair capacity. Such aberrations are not systemic autoimmune disease specific and are not influenced by the chronological age of the patient, as happens in the absence of autoimmunity.
