T cell immunoglobulin and mucin domain family and galectin proteins: novel targets for RPE-mediated immune regulation

Abstract

Purpose : Pro-inflammatory immune cells are common in the subretinal space in AMD although functional intraocular lymphatic vessels and B and T cells are absent in the normal human eye. The presence of these cells in AMD retina may reflect altered immune regulatory functions in retinal pigment epithelium (RPE). However, the mechanisms by which RPE exerts its immune regulatory effects are not completely understood. Our prior work implicates G-protein coupled receptor HCAR2/GPR109A, a receptor expressed highly in RPE and macrophages, as a key regulator of immunity/inflammation in RPE under normal conditions. Gene microarray conducted in wildtype (WT) and Hcar2/Gpr109a^-/- (knockout, KO) RPE revealed T cell immunoglobulin mucin domain 3 (TIM3) and galectin 9 as key differentially regulated genes. TIM/galectin pathways have been shown to enhance the clinical/pathological severity of autoimmune diseases by altering immune cell activation however, little is known about TIM and galectin expression in retina/RPE. Here, we evaluated the expression of these genes in WT and KO retina/RPE.

Methods : qPCR, in situ hybridization, Western blotting and immunolocalization studies were performed to evaluate TIM1-4 and galectin 1-9 expression in the retina/RPE of WT and KO C57BL/6J mice. Flow cytometry was performed in conjunction with anti-CD45, anti-CD11b, and anti-F4/80 antibodies on homogenized retinal samples obtained from WT and KO mice treated with LPS +/- HCAR2/GPR109A ligand treatment to identify specific populations of immune cells.

Results : TIM and galectin expression increased and robust increases in inflammatory markers and pro-inflammatory immune cell numbers in KO control and in LPS-treated WT and KO eyes. Treatment with HCAR2/GPR109A ligands reduced the LPS-induced inflammatory effects in WT but not KO retinas. TUNEL-positive cells and associated caspase 3 protein levels were similarly modulated in LPS-treated WT eyes, with ligand treatment having no effect in KOs.

Conclusions : Our findings support the potential of GPR109A as a therapeutic target for curtailing pathologic inflammation in the retina, thereby impacting the development and progression of retinal neurodegenerative diseases.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.