HTRA1 inactivates Thrombospondin-1 mediated subretinal immune-suppression

Abstract

Purpose : The molecular mechanism responsible for the linkage of the major Age-related Macular Degeneration (AMD)-risk haplotype of the 10q26 locus is poorly understood. We previously showed that the breakdown of the physiological immune-suppression and an accumulation of pathogenic macrophages (Mphis) in AMD is due in part by the lack of CD47 activation by the C-terminal domain of Thrombospondin 1 (TSP-1). We here investigated the influence of the 10q26 locus on PLEKHA1, ARMS2 and HTRA1 expression in human monocytes and evaluated the effect of HTRA1 on TSP-1 CD47 signaling.

Methods : PLEKHA1, ARMS2 and HTRA1 expression was analyzed in human monocytes (Mo) from 18 homozygous 10q26 AMD-risk carriers and 18 homozygous non-carriers by RT-PCR and Elisa. The effect of HTRA1 on recombinant TSP1 and its C-terminal domaine was analyzed on electrophoreses gels, WB, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The biological effect of HTRA1-induced TSP1 hydrolysis was evaluated in Mo/retinal pigment epithelial (RPE) co-cultures, subretinal monocyte injections and light-induced subretinal Mphi-accumulation in vivo.

Results : We here show that circulating Mo from homozygous carriers of the major AMD-risk haplotype of the 10q26 locus significantly overexpress HTRA1, but not ARMS2 or PLEKHA1. Mphis in AMD donor eyes strongly express the High-Temperature Requirement A Serine Peptidase 1 (HTRA1) independently of the risk-haplotype. Mechanistically, we demonstrate that HTRA1 hydrolyses TSP-1 separating its two CD47-binding VVM sites necessary for efficient CD47 activation. We show that HTRA1-induced exaggerated Mo survival in RPE co-cultures in vitroand subretinal MPs accumulation in vivo, is dependent on TSP-1 deactivation. This pathogenic effect of HTRA1 was reversible by synthetic CD47 agonists.

Conclusions : Our study reveals that HTRA1 is strongly expressed in Mphis in AMD donor and over-expressed in 10q26 AMD-risk carriers. We show that HTRA1-mediated hydrolyses the C-terminal domain of TSP-1 inhibits its immune-suppressive activity, thereby leading to the exaggerated survival and accumulation of pathogenic Mphi. Our work uncovers a comprehensive mechanism how HTRA1 participates in the pathogenesis of AMD and opens new therapeutic avenues to restore subretinal immunosuppressivity and inhibit the pathogenic subretinal inflammation.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.