Abstract
Purpose :
CIITA is a founding member of the NLR protein family and a master regulator of MHC Class II immune responses. We previously demonstrated increased expression of MHC class II genes and CIITA in a mouse model of RPE degeneration due to histone deacetylase-1 and -2 deficiency (HDAC1/2). Moreover, HDAC1/2 expression is downregulated in the RPE of post-mortem eyes with advanced dry AMD. Here, we studied differential gene expression levels of CIITA in eyes with dry AMD vs age-matched controls and identified epigenetic mechanisms that regulate CIITA expression in human RPE (hRPE).
Methods :
We analyzed RNA-sequencing data from RPE/choroid samples acquired from postmortem eyes with dry AMD and age-matched controls (n=6-8). Meta-analyses were also performed on existing RNA-sequencing datasets. Differential gene expression of MHC class II genes and CIITA was quantified and compared between groups (Deseq2). To investigate epigenetic mechanisms of CIITA gene regulation in hRPE, CIITA mRNA expression was measured by qPCR in early passage primary hRPE cells after broad and class-specific small molecule HDAC inhibition (dose ranges including trichostatin-A (TSA, broad-class), romidepsin (class I), droxinostat (class II), and Ex527 (class III) n=3 per group). In ARPE19 cells, CRISPR-mediated HDAC 1/2 knockout lines were generated, and CIITA mRNA expression was measured by qPCR (n=3).
Results :
Transcriptomic analyses identified several upregulated MHC II transcripts along with CIITA (1.86-fold, P=0.05) in RPE/choroid from dry AMD eyes compared to age-matched controls. In hRPE cells, broad-class HDAC inhibition with TSA significantly increased CIITA expression (P <0.0001). Class I HDAC inhibition with romidepsin also induced CIITA transcription (P <0.0001), whereas Class II and III HDAC inhibition did not. CRISPR-mediated knockout of HDAC1/2 in ARPE19 cells also expressed significantly more CIITA compared to control (pCas9 vs pNull, P <0.001).
Conclusions :
Our findings reveal that CIITA gene expression is increased in the RPE/choroid in post-mortem human eyes with AMD compared to age-matched controls. In vitro models of class-specific HDAC inhibition strongly suggest that CIITA transcription is heavily regulated by HDAC1/2. These novel findings identify CIITA as a potential therapeutic target and biomarker in the pathogenesis of dry AMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.