Abstract
Purpose :
Air pollution is associated with increased incidence of dry eye disease and meibomian gland dysfunction (MGD), but the mechanisms are not well understood. Our earlier study showed that PM2.5 from diesel-exhaust (DEP) are toxic to human meibomian gland cells in vitro, but that intracellular lipid droplets are induced at low DEP concentrations. Here, we utilise transcriptomic analyses to investigate molecular mechanisms underlying the impact of DEP on primary human meibomian gland cells (pHMGC).
Methods :
pHMGC (3 biological replicates) were grown to 80% confluence in proliferating medium, then switched to serum-free differentiating medium (DMEM/F12 + 30μM Rosiglitazone) with or without 25μg/ml DEP. After 3 days, RNA was extracted from cells, followed by sequencing. Identification of differentially expressed genes (DEG), functional annotation and pathways analysis (Gene Ontology, GO and Gene Set Enrichment analysis, GSEA) were performed in R, using appropriate packages and models.
Results :
DEP exposure significantly induced the expression of 808 genes and downregulated 421 genes (fold change > 1.5, adjusted p-value < 0.05), including many previously reported markers of MGD, such as S100 calcium binding protein A7-9, small proline rich protein 2 and 3, cornulin, keratinisation-associated proteins, keratin 4, and Interleukin 1. GO pathway analyses revealed significant changes in cellular processes including cell differentiation, keratinization, migration and regulation of lipid metabolic and biosynthetic process. GSEA using The Molecular Signatures Database, highlighted significant decreases in DNA repair, cell cycle and cell growth via mTORC1 pathway, adipogenesis and cholesterol homeostasis. Meanwhile, upregulation pathways involved immune and inflammatory responses, especially via STAT proteins signalling and genes up regulated by reactive oxygen species.
Conclusions :
Increased intracellular lipid production in DEP-exposed cells may be caused by metabolic regulation and inflammatory response. This study provides insights into the transcriptional regulation of pHMGC, and identifies novel mechanisms which may be involved in MG inflammation. The molecular and cellular pathways identified provide future potential targets for MGD therapeutic intervention.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.