Aim: To investigate genes and pathways involved in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using RNA sequencing. Methods: Using paired human donor eyes, human organ-cultured anterior segment (HOCAS) was established in one eye to characterize GC responsiveness based on intra ocular pressure (IOP) change and, in the other eye, primary HTM cell culture was established. For RNA sequencing, total RNA extracted from GC-responder (GC-R) and non-responder (GC-NR) cells after dexamethasone (DEX) or ethanol (ETH) treatment for 7d was used. Differentially expressed genes (DEGs) were compared among five groups and validated. Results: In total, 616 and 216 genes were identified as significantly dysregulated in Group #1 and #2 (#1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR), respectively. Around 80 genes were commonly dysregulated in Group #3 (overlapping DEGs between #1 and #2), whereas 536 and 136 genes were uniquely expressed in GC-R (#4) and GC-NR HTM (#5) cells, respectively. Pathway analysis revealed that WNT signaling, drug metabolism cytochrome p450, cell adhesion, TGF-β signaling, and MAPK signaling were associated with GC responsiveness. Conclusion: This is the first study reporting distinct gene signatures and their associated pathways for GC-R and GC-NR HTM cells. WNT and MAPK signaling are potential therapeutic targets for the management of GC-induced glaucoma.

Glucocorticoids (GCs) are known to regulate several physiological processes and are the mainstay in the management of inflammatory eye diseases. The long-term use of GC causes raised intraocular pressure (IOP) or ocular hypertension (OHT) in about 30–50% of the susceptible individuals depending on the route of administration, and can lead to steroid-induced secondary glaucoma. The present study aims to understand the role of microRNAs (miRNAs) in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. The human organ-cultured anterior segment (HOCAS) model was used to identify whether donor eyes were from GC-responders (GC-R; n = 4) or GC-non-responders (GC-NR; n = 4) following treatment with either 100 nM dexamethasone (DEX) or ethanol (ETH) for 7 days. The total RNA was extracted from cultured HTM cells with known GC responsiveness, and the differentially expressed miRNAs (DEMIRs) were compared among the following five groups: Group #1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR; #3: overlapping DEGs between Group #1 and #2; #4: Unique DEMIRs of GC-R; #5: Unique DEMIRs of GC-NR; and validated by RT-qPCR. There were 13 and 21 DEMIRs identified in Group #1 and Group #2, respectively. Seven miRNAs were common miRNAs dysregulated in both GC-R and GC-NR (Group #3). This analysis allowed the identification of DEMIRs that were unique to GC-R (6 miRNAs) and GC-NR (14 miRNAs) HTM cells, respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. This is the first study to reveal a unique miRNA signature between GC-R and GC-NR HTM cells, which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma.

Bacterial keratitis caused by Pseudomonas aeruginosa is a destructive disease of cornea. Pseudomonas keratitis progresses rapidly and leads to vision loss if untreated. Even with adequate treatment, many patients show poor visual outcomes. The virulence factors or multiple drug-resistant (MDR) mechanisms of the ocular strains responsible for poor clinical outcomes remain largely unknown. Here, we performed whole-genome sequencing of five P. aeruginosa strains cultured from corneal scrapings of the healed and corneal buttons of the poor outcome keratitis patients. We investigated the distribution of virulence factors, resistance genes and resistance-associated mutations, the efflux-pump system in all five genomes, and as groups between poor and good clinical outcome as well as MDR vs. non-MDR. We detected several resistance genes and mutations associated with drug resistance in MDR groups; however, a large number of virulence genes were detected in all our genomes. Among the virulence genes, exoU and exoS exotoxin of the Type III secretion system detected in MDR and non-MDR strains, respectively, considered as main virulence contributors of keratitis pathogenesis. Despite this fact, this study did not show an association between MDR with exoU and poor clinical outcomes. However, strain-specific resistance and virulence genes were observed in this study, suggesting their role in the clinical outcome. Mainly, the flagellar genes fliC and fliD, reported to altering the host immune response, might impact the clinical outcome. This comparative study may provide new insights into the genome of ocular strains and requires further functional studies.