Abstract
Purpose :
Cells from the retinal pigment epithelium (RPE) sequentially activate various types of cell surface receptors to initiate and complete the daily rhythmic phagocytosis of photoreceptor outer segments (POS). We recently showed that among the myriad of receptors expressed at the RPE apical surface, class A and class B scavenger receptors (SR) appear to be implicated in the daily elimination of POS by RPE cells. We thus investigated how and when these receptors where recruted in vitro and in vivo.
Methods :
We investigated class A –SR-A and MARCO– and B –CD36, SR-BI and SR-BII– SRs. We specifically inhibited each candidate using siRNAs or blocking antibodies and assessed the effect on phagocytosis after 1.5 and 3 hours of POS challenge. We used co-labeling immunofluorescent and biochemistry assays to characterize the association of SRs with POS and specific membrane domains at 1, 3 and 5 hours of phagocytosis. We studied their circadian expression in vivo by qPCR and immunoblotting.
Results :
Functional inhibition assays confirm that CD36 is regulating POS internalization speed at 1.5 hours. SR-BI and SR-BII inhibition slightly decreased binding and internalization at all time-points, while MARCO and SR-A inhibition diminished POS internalization at 3 hours. Times of implication were confirmed by variation of the co-localization of SR-A, MARCO, CD36 and SR-BII receptors with POS during phagocytosis, but no connection was ever observed for SR-BI. All 4 receptors associated with lipid raft domains, as assessed by co-localization with caveolin and flotillin, and the dynamic of this association appears to match the timings identified in our other experiments. CD36 expression follows a marked bimodal expression pattern with peaks 2 hours before light onset and 2 hours after the phagocytic peak. In contrast, variation of other receptors expression was more limited in amplitude: MARCO expression increased slightly before light onset and offset, SR-BII before and after light offset while SR-BII expression did not change.
Conclusions :
Our results indicate that SRs appear to participate in POS clearance globally as a family, possibly in separate steps and with different tasks. Current studies aim at analyzing their potential role as co-receptors with other known members of the phagocytic machinery such as MerTK or other receptor candidates in vitro and in vivo.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.