THU0233 In vitro induced regulatory t-cells can ameliorate severity of pristane induced lupus (PIL)

Abstract

Background: Pristane induced lupus (PIL) is a well-established murine model for environmentally induced systemic lupus erythematosus (SLE). Mice develop specific autoantibodies and show symptoms of SLE including arthritis, diffuse proliferative immune complex glomerulonephritis and haemorrhagic pulmonary capillaritis. Objectives: To investigate the therapeutic effects of in vitro -induced regulatory T cells (iTreg) in the murine model of PIL. Methods: BALB/c mice were injected i.p. with either 0.5ml of pristane (PIL) or PBS (controls). Naive CD4+ thymocytes were sorted and cultured and cell suspensions with >80% of CD4+FoxP3+ cells (iTreg) were injected intravenously (i) once when PIL was induced (5x106 iTreg (iTreg-single)) or (ii) every 4 weeks (1x106 iTreg, iTreg-rep). Animals were monitored for paw swelling and grip strength. After 8 months histological analysis evaluated for cartilage degradation, number of osteoclasts and the extent of inflammation and bone erosion. Glomerulonephritis and pneumonitis were quantified using the kidney biopsy score and a newly adapted histomorphometric image analysis system; inflammatory tissue was further analyzed by tissue cytometry. Serum levels of anti-dsDNA, anti-histone and anti-chromatin antibodies were measured by ELISA. Results: Monthly injections of 1x106 iTreg reduced the clinical as well as the histological severity of PIL-arthritis, seen by a higher mean grip strength (2.96±0.02 vs. 2.73±0.06, p<0.01), less mean paw swelling (0.04±0.02 vs. 0.36±0.07, p<0.01) and retardation of the symptom onset (Figure A). 62% of PIL-mice and 33% of iTreg-rep mice had erosive arthritis. There was a significant reduction of arthritis severity in all histological parameters (inflammatory area in mm2 0.19±0.06 vs. 0.69±0.11; erosive area in mm2 0.01±0.01 vs. 0.07±0.02; number of osteoclasts 2±1.13 vs. 9.14±2; cartilage degradation in mm2 0.06±0.01 vs. 0.19±0.03, all p<0.01, Figure B). The single boost of 5x106 iTreg could not prevent joint manifestations. However, a slight retardation in “loss of grip strength” and a significantly less erosive area was observed. In regards to the cellular composition of the inflammatory tissue in paws, a significantly increased relative amount of CD4+Foxp3+ cells was seen in the iTreg-rep group compared to the PIL group (in % 5.2±2.3 vs. 0.6±0.2, p<0.01). Repeatedly injected mice (iTreg-rep) had significant less pulmonary involvement (perivascular inflammatory area in mm2/mm2 lung area 0,003±0,001 vs. 0,007±0,001, p<0.01) and renal disease (glomerular activity score 1,17±0,31 vs. 2,43±0,39, p<0.05) compared to PIL (Figure C). Corresponding, iTreg-rep mice had significantly lower serum levels of disease-associated auto-antibodies (Figure D). Conclusions: Repeated injections of iTreg ameliorate the clinical and histological severity of PIL- manifestations. A single boost of iTreg at the time of disease induction does not prevent manifestations, but retards the onset of symptoms. Thus iTreg have significant positive effects on PIL, which may have consequences for future approaches in treating SLE (Figure Presented).