The miR-183/96/182 cluster regulates corneal resident macrophages and early response to Pseudomonas aeruginosa infection

Abstract

Purpose : Corneal resident macrophages (ResMΦ) are critical in corneal homeostasis, wound healing, immune/inflammatory responses to injury and microbial infection, and overall ocular immune privilege. However, the roles of microRNAs (miRNAs) in regulating corneal ResMΦ are utterly unknown. The purpose of this project is to determine the role of the miR-183/96/182 cluster (miR-183/96/182) in the regulation of ResMΦ and the early response of the cornea to Pseudomonas aeruginosa (PA) infection.

Methods : Young adult (8-18 weeks old) miR-183/96/182 knockout (ko) and wild-type control (wt) mice were employed. The central corneas of anesthetized mice were scarred and 5 ml of 1X10⁶ CFU/ml of PA (strain 19660; ATCC) was topically applied. Corneal RNA was harvested at 3 and 6 hours post-infection (hpi) for quantitative (q)RT-PCR analysis. For ResMΦ study, corneas and spleens of naïve ko and wt mice were dissociated for fluorescence activated cell sorting (FACS). To easily trace and isolate ResMΦ, we also produced and used miR-183/96/182 ko and wt mice in the background Csf1r-EGFP (Jackson Laboratory), which express enhanced green fluorescent protein (EGFP) in MΦ and trophoblast cell lineages.

Results : 1) Inactivation of miR-183/96/182 resulted in an increased production of pro-inflammatory cytokines, including members of the IL-17 family, in the cornea at 3 and 6 hpi. Intriguingly, anti-inflammatory cytokine, IL-10, was also increased in the cornea of ko mice in these early stages; 2) Csf1r-EGFP is a reliable marker for corneal ResMΦ. Csf1r-EGFP⁺ cells can be detected in naïve developing cornea as early as embryonic day (E) 12.5. 3) Inactivation of miR-183/96/182 resulted in an increased number of corneal ResMΦ and changes in inflammatory cytokine production by splenic ResMΦ. 4) Preliminary results suggested an increase in the CD45⁺CD11b⁺Ly6G⁺ population, a signature of myeloid derived suppressor cells (MDSCs), in ResMΦ of miR-183/96/182 ko mice.

Conclusions : miR-183/96/182 modulates the early innate immune response of the cornea to PA infection, including innate IL-17 and IL-10 production. miR-183/96/182 regulates the number and characteristics of corneal ResMΦ which may contribute to its overall function in the innate immune response to PA infection.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.