Abstract
Purpose :
Wounds of the corneal stroma produce vision-disrupting scar tissue. Topical treatment of experimental corneal wounds with mesenchymal stem cells from the limbal stroma (CSSC) resolves scars inducing regeneration of transparent native stromal tissue. We found previously that CSSC therapy prevents infiltration of neutrophils into cornea 24 hr after wounding and that neutrophil infiltration is essential for scar formation. This study examined the hypothesis that CSSC therapy alters additional immunological events in the wound healing process.
Methods :
Mouse corneal wounds were generated by debridement with Algerbrush II as previously described. Corneas were collected at 1,3,5,10 and 15 days after wounding from CSSC-treated and untreated eyes (n=12) and total RNA prepared. Mouse RNA expression was screened using Nanostring analysis for 1500 genes involved in cancer and immune response. Differential expression at days 1, 3 and 5 was determined and confirmed using qPCR. Presence of neutrophils, monocytes, and platelets was determined in collagenase digests of wounded corneas with or without CSSC treatment. qPCR and cytometry samples were carried out in triplicate and significance determined using ANOVA, p<0.05.
Results :
Nanostring array analysis revealed a number of genes differentially regulated in CSSC-treated corneas 1-3 days after wounding. Genes associated with monocytes (CD14, Plaur, Cxcr2, S100a8) were 4-5 fold more abundant in wounds than in treated wounds. Surprisingly, genes characteristic of platelets (Ppbp, Cxcl1, Ccl3, Cxcl3, Il1b, Ccl2, Ccl7) were 5-20 fold more abundant in the wounded tissue than in native cornea. Upregulation was suppressed by CSSC treatment. Flow cytometry of corneal cells 3 days after wounding showed a 100-fold increase in CD45+ cells compared to naïve corneas. 90% of the CD45+ cells were neutrophils, 2.5% were monocytes/macrophages, and 2.5% were T-cells. CD41+ platelets were also strongly upregulated and as abundant as neutrophils. All four of these cell populations were reduced to the level of naïve cornea in CSSC treated corneas.
Conclusions :
Platelets have not been previously linked with corneal scarring but are strongly induced in healing wounds and express genes involved in recruitment of immune cells into the tissue. Suppression of this robust platelet response by CSSC may be a key mechanism by which these cells mediate scar formation in healing wounds.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.