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 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 edgeR Bioconductor analysis of expression data. Functional enrichment and protein-protein interaction (PPI) 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, and 5’ AMP-activated protein kinase (AMPK) signaling pathway are affected in the GF retinas. PPARG1a (PGC1a) gene is involved in 12 of the 16 significantly modulated pathways. Proteins with the most 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.

Recent Insights into the Role of Gut Microbiota in Diabetic Retinopathy

“… 15 The relationship between gut microbiota and ocular abnormalities including uveitis, glaucoma, and age-related macular degeneration (AMD) has been increasingly revealed, and the concept of a “microbiota–gut–retina axis” has been put forward. 16 , 17 However, little has been understood regarding the molecular mechanisms of intestinal dysbiosis and how they induce retinopathy in patients with DM. Recently, data indicate that changes in the gut microbiome act as a key internal environmental modulator which favorably affects the pathogenetic processes underlying the micro-complications of DM such as DR. 18 …”

High-Fat Diet Alters the Retinal Transcriptome in the Absence of Gut Microbiota

“…To further elucidate the biological connections between the diet-gut-retina axis, we aimed to investigate how diet affects the retinal transcriptome independently of the gut microbiome [30]. Germ-free mice, raised without exposure to any microbes, provide an ideal model to investigate this hypothesis [46].…”

Gut microbiome and retinal diseases: an updated review

“…Although some associative studies have been performed over the last decade, we have only recently begun to explore the retinal transcriptional signatures linking the microbial organ and retinal homeostasis and physiology relating to AMD. Using high-throughput RNA sequencing, our team detailed the transcriptional profile of retinas from germ-free mice revealing significant differentially expressed genes (DEGs) and altered retinal pathways, including VEGF, mitochondrial biogenesis via peroxisome proliferatoractivated receptor gamma coactivator 1-alpha (PGC-1a), oxidative stress and AMP-activated protein kinase (AMPK) signalling critical to AMD pathogenesis [45]. These studies not only reinforce the complexity of the gut-retina axis but also highlight its potential integral role in AMD pathogenesis at even the transcriptomic level.…”