MicroRNA-762 Is Upregulated in Human Corneal Epithelial Cells in Response to Tear Fluid and Pseudomonas aeruginosa Antigens and Negatively Regulates the Expression of Host Defense Genes Encoding RNase7 and ST2

James Mun,David Evans,Jong Hun Kim,Connie Tam,Gary Chan,Suzanne Fleiszig,David M Ojcius

doi:10.1371/journal.pone.0057850

Abstract

Mucosal surfaces regulate defenses against infection and excessive inflammation. We previously showed that human tears upregulated epithelial expression of genes encoding RNase7 and ST2, which inhibited Pseudomonas aeruginosa invasion of human corneal epithelial cells. Here, microRNA microarrays were used to show that a combination of tear fluid exposure (16 h) then P. aeruginosa antigens (3 h) upregulated miR-762 and miR-1207, and down-regulated miR-92 and let-7b (all > 2-fold) in human corneal epithelial cells compared to P. aeruginosa antigens alone. RT-PCR confirmed miR-762 upregulation ∼ 3-fold in tear-antigen exposed cells. Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization. However, P. aeruginosa invasion was increased > 3-fold by a miR-762 mimic which reduced RNase7 and ST2 gene expression. Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir. Combination of tear fluid and miR-762 antagomir increased RNase7 and ST2 gene expression. These data show that mucosal fluids, such as tears, can modulate epithelial microRNA expression to regulate innate defense genes, and that miR-762 negatively regulates RNase7, ST2 and Rab5a genes. Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria. These data also suggest a complex relationship between tear induction of miR-762, its modulation of innate defense genes, and P. aeruginosa internalization.

Highlights

Many mucosal epithelia in humans and animals have evolved to co-exist with a rich and complex microbiota, but have retained the ability to respond to potential pathogens, and regulate inflammatory responses to avoid tissue damage and disease [1,2,3,4]
We have shown that tear fluid protects ocular surface epithelial cells against bacterial virulence mechanisms in vitro [5,6] and in vivo [6], and that in vitro protection can be independent of direct antimicrobial activity [5,7,8]
The results of this study show that mucosal fluid can influence epithelial microRNA expression to regulate expression of innate defense genes

Summary

Introduction

Many mucosal epithelia in humans and animals have evolved to co-exist with a rich and complex microbiota, but have retained the ability to respond to potential pathogens, and regulate inflammatory responses to avoid tissue damage and disease [1,2,3,4] All of these mucosal epithelial surfaces are bathed in fluids, and yet little is known about how these fluids influence epithelial regulation of innate defense and inflammation. Tear fluid enhanced activation of NFkB and AP-1 transcription factors in response to bacterial antigens, and upregulated epithelial-derived innate defense genes The latter included genes encoding RNase and ST2, both of which reduced bacterial internalization by corneal epithelial cells [9]. ST2 is constitutively expressed in the corneal epithelium, and its immunomodulatory role is important for resolution of P. aeruginosa corneal infections in murine models by promoting Th2-mediated immune responses [16,17]

Objectives

Methods

Results

Conclusion