Abstract
Purpose :
In the retina, the circadian elimination of aged photoreceptor outer segment (POS) by cells from the retinal pigment epithelium (RPE) is based on the recognition of phosphatidylserines, apoptotic eat-me signals displayed by their most aged extremities. Cluster of Differentiation 14 (CD14) and the InterCellular Adhesion Molecule 3 (ICAM3) are involved in the clearance of apoptotic cells by macrophages and are also expressed by RPE cells. We thus set out to characterize their potential participation in the regulation of the daily phagocytosis of photoreceptor outer segment (POS), alone or as co-receptors.
Methods :
In vitro, inhibition assays (siRNA, blocking antibodies and pharmacological molecules) and immunofluorescence co-labelings of candidate receptors with POS were performed on the rat RPE-J cell line. In vivo, circadian mRNA expression patterns of candidates were studied using RT-qPCR on mouse RPE/choroid tissues from control mice sacrificed at different times of the light:dark cycle.
Results :
CD14 siRNA transfection decreased POS phagocytosis, an effect that was counteracted by the co-transfection of ICAM3 or TLR4 siRNAs. Similarly, the use of the baicalin and IAXO-102 antagonists –that affect CD14-TLR4 signaling by decreasing the expression of CD14 and inhibiting the CD14-TLR4 signaling pathways, respectively– modified POS binding and internalization by RPE cells. The ICAM inhibitor BMS 688521 only affected POS tethering and to a limited extent. Upon POS challenge, CD14 expression levels at the cell surface increase, the receptors form clusters and directly co-localize with POS at 3 and 5 hours of phagocytosis. In contrast, ICAM3 receptors do not seem to directly interact with POS. In vivo, CD14 qPCR analysis show 2 peaks of expression at ZT3, just after the time of peak phagocytosis, and at ZT8.
Conclusions :
Taken together, our current data suggest that CD14 may be involved in the regulation of POS phagocytosis by RPE cells, potentially in association with other receptors such as TLR4 and/or ICAM3. Further investigations such as in vitro receptor co-localization during phagocytosis and in vivo expression analysis by immunoblots and immunolabelings on RPE flatmounts at different times of the day are being performed in order to fully understand the exact participation of both receptors to this crucial function.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.