Thrombospondin-1 expression controls macrophage survival in the subretinal space

Abstract

Purpose: Thrombospondin-1 (TSP1) plays a crucial role in the maintenance of the immune privilege of the eye as TSP1-/- mice show increased microglial activation and accumulation in retinal injury. We here study the influence of macrophage-expressed TSP1 on their survival in the subretinal space.

Methods: C57B6 and TSP1-/- mice were exposed to light-induced retinal injury during 4 days. Choroidal and retinal flatmounts were immunostained for the presence of IBA1 positive macrophages. Macrophage clearance from the subretinal space (SR) was studied in wildtype recipient eyes 24h after subretinal injection of thioglycolate elicited CFSE stained C57B6 or TSP1-/- peritoneal macrophages (PM). In vitro, the potential of TSP1 to induce macrophage apoptosis was assessed by TUNEL quantification of C57B6 and TSP1-/- PM incubated for 6h with 1 µg/ml recombinant human TSP1 (rhTSP1).

Results: A significant increase of subretinal macrophages was quantified in light-injured TSP1-/- as compared to C57B6 eyes. In accordance, clearance of TSP1-/- macrophages in the subretinal space was significantly inhibited 24h after injection, resulting in a 3-fold increase of TSP1-/- macrophages compared to controls. Co-injection of macrophages with 10 µg/ml rhTSP1 strongly decreased the number of subretinal TSP1-null macrophages. In vitro, apoptosis in TSP1-/- macrophages was inhibited compared to C57B6 macrophages. Normal apoptosis was restored in TSP1-/- macrophages using 1 µg/ml rhTSP1.

Conclusions: Our results show that macrophage secretion of TSP1 inhibits their survival in the subretinal space, by promoting their apoptotic clearance. This might explain the observed subretinal macrophages accumulation in light-injured TSP1-/- mice. Altogether, these data suggest that macrophages control subretinal inflammation via TSP1.

Keywords: 557 inflammation • 449 cell survival • 561 injection