High Throughput RNA Sequencing of Germ- Free Mouse Retina Reveals Metabolic Pathways Involved in the Gut-Retina Axis

Abstract

Background and aimsConnections between the gut microbiome and retinal diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP), and primary open-angle glaucoma (POAG) are recently being established. Communication between the gut microbiome and retina, referred to as the gut-retina axis, has been proposed; however, the biologic pathways and mediators involved in the interactions have not yet been elucidated. Using high-throughput RNA sequencing (RNA-seq) of whole retinas, we compare the retinal transcriptome from germ-free (GF) and specific pathogen-free (SPF) mice to investigate the effects of the gut-microbiome on both retinal gene expression and biologic pathways. MethodsRNA was extracted from whole retinas of GF and SPF mice (four animals per group) and cDNA libraries were created. RNA-seq was performed on NovaSEQ6000 using the paired-end method. After preprocessing the RNA-seq data, gene expression value was calculated by count per million (CPM). The differentially expressed genes (DEGs) were identified with the limma package from Bioconductor on the expression data. Functional enrichment and protein-protein interaction STRING protein-protein association network analyses were created for the differentially expressed genes (DEGs). ResultsRNA-sequencing reveals a cohort of 396 DEGs, of which, 173 are upregulated and 223 are downregulated in GF mouse retina. Enrichment analysis reveals that the DEGs are involved in glucocorticoid effects, transcription factor binding, cytoskeletal stability, lipid metabolism, and mitogen-activated protein kinase (MAPK). Multiple biologic pathways, including obesity/metabolic syndrome, longevity, insulin-like growth factor (IGF) signaling pathway, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1 transcription pathway, and 5 AMP-activated protein kinase (AMPK) signaling pathway are affected in the GF retinas. PPARG1a (PGC1a) gene is involved in 13 of the 35 significantly modulated pathways. Proteins with the greatest number of interactions in the PPI are E1A binding protein P300(EP300), forkhead box O3(FOXO3), and PGC1a. ConclusionsTo our knowledge, this is the first study demonstrating the involvement of the gut microbiome in driving the retinal transcriptome, providing evidence for the presence of a gut-retina axis. Future studies are needed to define the precise role of the gut-retina axis in the pathogenesis of retinal diseases.