Single-cell RNA sequencing study of retinal immune regulators identified CD47 and CD59a expression in photoreceptors-Implications in subretinal immune regulation.

The neuroretina is protected by its own defense system, that is microglia and the complement system. Under normal physiological conditions, microglial activation is tightly regulated by the neurons although the underlying mechanism remains elusive. Using published single-cell RNA sequencing data sets, we found that immune regulatory molecules including CD200, CD47, CX3CL1, TGFβ, and complement inhibitor CD59a are expressed by various retinal neurons. Importantly, we found that photoreceptors express higher levels of CD47 and CD59a, which was further confirmed in cultured 661W cells, WERI-Rb1 cells, and microdissected photoreceptors from human eyes. The expression of CD59a mRNA in 661W cells was upregulated by TNFα and hypoxia, whereas LPS, hypoxia, and IL-4 upregulated CD47 mRNA expression in 661W cells. Immunofluorescence staining detected strong CD59a immunoreactivity in the outer nuclear layer, inner/outer segments, and discrete staining in ganglion cell layer (GCL), inner plexiform layer (IPL), and outer plexiform layer. The expression of CD59a in photoreceptors was increased in the detached retina, but decreased in retinas from experimental autoimmune uveoretinitis (EAU) mice. In EAU retina, CD59a was highly expressed by active immune cells. CD47 was detected in GCL, IPL, and inner nuclear layer and some photoreceptors. The expression of CD47 in photoreceptors was also increased in the detached retina but decreased in EAU retina. In a coculture system, 661W enhanced arginase-1 and reduced IL-6 mRNA expression in BV2 microglial cells. Our results suggest that photoreceptors express immune regulatory molecules and may have the potential to regulate immune activation in the outer retina/subretinal space under pathophysiological conditions.