Abstract
Purpose :
Myeloid-derived corneal resident innate immune cells (MDCRIICs) play significant roles in the homeostasis of the cornea, including ocular immune privilege, transplant graft survival, wound healing, corneal nerve regeneration and inflammatory/immune responses following tissue damage and/or microbial infection and orchestrate adaptive immune responses. However, the roles of microRNAs (miRNAs) in MDCRIICs 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 MDCRIICs.
Methods :
Young adult (8-18 weeks old) miR-183/96/182 knockout (ko) and wild-type control (wt) mice were used. To trace MDCRIICs, we also produced miR-183/96/182 ko and wt mice on the background Csf1r-EGFP (Jackson Laboratory), which express enhanced green fluorescent protein (EGFP) in MDCRIICs. To detail the dynamics of these cells, central corneas of anesthetized mice were scarred and 5 ml of 1X106 CFU/ml of Pseudomonas aeruginosa (PA. Strain 19660; ATCC) was topically applied. Naïve and PA-infected corneas were harvested 3 and 6 hours post-infection (hpi) for flat-mount confocal microscopic studies. In addition, corneas of naïve mice were dissociated for fluorescence activated cell sorting (FACS). Total RNA was isolated from FACS-purified resident macrophages (ResMΦ) for quantitative (q)RT-PCR analysis.
Results :
1) Inactivation of miR-183/96/182 resulted in an increased number (~54%) of steady-state corneal resident innate immune cells, including MDCRIICs and ResMΦ; 2) MDCRIICs are highly dynamic in PA-infected and non-infected contralateral eyes. At 3 hpi, the number of MDCRIICs was significantly decreased (~82%) in PA-infected eyes of both ko and wt mice; however, at 6 hpi, ko vs. wt mice showed significantly enhanced “come-back” in the infected eyes (~217% vs. 124%); 3) Corneal ResMΦ simultaneously express IL-10 and IL-17F; miR-183/96/182 regulates IL-10 and IL-17F production by targeting the genes encoding key transcriptional regulators.
Conclusions :
Corneal ResMΦ are innate IL-10- and IL-17F-producing cells. miR-183/96/182 plays important roles in corneal innate immunity by regulating the number, dynamics and functions of MDCRIICs.
This is a 2020 ARVO Annual Meeting abstract.