Receptor Interacting Protein-2 (RIP2) Mediated Caspase-1 Activation in Müller Cells Under Hyperglycemic Conditions

Abstract

Purpose: Purpose: Activation of the pro-inflammatory caspase-1/Interleukin-1β (IL-1β) pathway plays an important role in the development of diabetic retinopathy. In order to develop therapies targeting this pathway, a better understanding of the mechanisms leading to caspase-1 activation under diabetic conditions is necessary. Receptor Interacting Protein-2 (RIP2) is a known activator of caspase-1 in other systems, and this study tests the hypothesis that caspase-1 is activated by (RIP2) under hyperglycemic conditions and by IL-1β signaling.

Methods: Methods: Primary human Müller cells (hMC), a known source of active caspase-1 and IL-1β in diabetic retinopathy, were isolated and cultured from retinas of non-diabetic healthy donors. hMCs (1x10⁶ cells) were treated with normal (5 mM) or high (25 mM) glucose for 48 hours or treated with recombinant IL-1β (2ng/ml) for 24 hours in the presence or absence of siRNA against RIP2. Scramble siRNA samples served as controls. Caspase-1 activity was measured using a caspase-1 specific fluorescent substrate (YVAD-AFC; 2.5μM) and expressed as the mean ± SDEV (pmol AFC/mg protein/min). IL-1β release was measured by ELISA. ANOVA followed by Tukey test was used for statistical analysis of data.

Results: Results: Caspase-1 activity was significantly increased in Müller cells treated with high glucose to 16.8±0.3 pmol AFC/mg/min, compared to 11.0±0.6 pmol AFC/mg/min for those treated with normal glucose, an increase of 53±3.0% (p<0.05). siRNA against RIP2 decreased high glucose-induced caspase-1 activity from 16.8±0.3 pmol AFC/mg/min to 11.9±0.7 pmol AFC/mg/min (p<0.05). Accordingly, high glucose-induced IL-1β release was significantly attenuated from 6.2±0.3 pg/ml/mg to 1.4±0.9 pg/ml/mg (normal: 2.0±0.4 pg/ml/mg) with siRNA against RIP2. Treatment with recombinant IL-1β itself led to increased caspase-1 activity (18.1±0.6 pmol AFC/mg/min) compared to control (10.8±1 pmol AFC/mg/min) (p<0.05). siRNA against RIP2 also abrogated IL-1β-induced caspase-1 activity by 62±1.5% (p<0.05).

Conclusions: Conclusion: Our results demonstrate that RIP2 indeed mediates hyperglycemia-induced caspase-1 activation in Müller and thus, might be responsible for the onset of retinal inflammation seen in diabetic retinopathy. Therefore, RIP2 represents a valid therapeutic target to treat diabetic retinopathy.