603-P: Circulating Inflammatory Cells and Inflammasome Activation in Diabetics with Diabetic Retinopathy (DR)

Increasing evidence points to inflammation as a key component in diabetes-associated retinal perturbations, however, the underlying molecular mechanisms are not yet fully understood. The aims of this study were to characterize peripheral blood mononuclear cells (PBMC) in diabetics with and without DR and to elucidate the biologic role of NLRP3 inflammasome in these cells. PBMC from 33 diabetics (T1D n=9 and T2D n=24) and 18 controls were assessed by flow cytometry. To examine the inflammasome activation, monocytes and neutrophils were treated with the TLR-2 agonist, peptidoglycan (PGN) (10µg/ml) for 3 hours followed by stimulation with a potassium ionophore, nigericin. NLRP3, IL-1β, Caspase-1, IL-18 levels were assessed. Neutrophil counts were significantly higher (1.6 folds, p<0.0002) in diabetics with proliferative diabetic retinopathy (PDR)(45.88%) compared with nondiabetics (28.21%). Increased classical monocytes (88.66%, p<0.0580) were observed in subjects with DR compared to controls (77.66%). Diabetics demonstrated decreased lymphocytes (43.36%, p<0.0007) compared with nondiabetics (58.77%). Lymphocytes were most reduced in T2D with PDR (38.88%) compared to non-DR subjects (43.21%). Expression analysis of inflammasome genes in response to PGN treatment showed a significant increase in monocytes and neutrophil IL-β mRNA expression in diabetic and controls, but no difference whether DR was present or not. Neutrophil IL-β was increased in diabetics at baseline compared to controls. This study demonstrates that increased neutrophils and classical monocytes and reduced lymphocytes are associated with DR. In vitro inflammasome activation studies could not distinguish differences in the level of activation between neutrophils and monocytes obtained from complication-free diabetics and diabetic with DR suggesting the need for repeated measurements of their activation status if these parameters are being used as biomarkers of DR progression.