Vitamin D is an important regulator of immune function and largely acts to dampen chronic inflammatory events in a variety of tissues. There is also accumulating evidence that vitamin D acts to enhance initial inflammation, beneficial during both infection and wound healing, and then promotes resolution and prevention of chronic, damaging inflammation. The current study examines the effect of topical vitamin D in a mouse of model of corneal epithelial wound healing, where acute inflammation is necessary for efficient wound closure. At 12 and 18 hours post-wounding, vitamin D treatment significantly delayed wound closure by ~17% and increased infiltration of neutrophils into the central cornea. Basal epithelial cell division, corneal nerve density, and levels of VEGF, TGFβ, IL-1β, and TNFα were unchanged. However, vitamin D increased the production of the anti-microbial peptide CRAMP 12 hours after wounding. These data suggest a possible role for vitamin D in modulating corneal wound healing and have important implications for therapeutic use of vitamin D at the ocular surface.

The purpose of this study was to use a mouse model of diet-induced obesity to determine if corneal dysfunction begins prior to the onset of sustained hyperglycemia and if the dysfunction is ameliorated by diet reversal. Methods Six-week-old male C57BL/6 mice were fed a high fat diet (HFD) or a normal diet (ND) for 5-15 weeks. Diet reversal (DiR) mice were fed a HFD for 5 weeks, followed by a ND for 5 or 10 weeks. Corneal sensitivity was determined using aesthesiometry. Corneal cytokine expression was analyzed using a 32-plex Luminex assay. Excised corneas were prepared for immunofluorescence microscopy to evaluate diet-induced changes and wound healing. For wounding studies, mice were fed a HFD or a ND for 10 days prior to receiving a central 2mm corneal abrasion. Results After 10 days of HFD consumption, corneal sensitivity declined. By 10 weeks, expression of corneal inflammatory mediators increased and nerve density declined. While diet reversal restored nerve density and sensitivity, the corneas remained in a heightened inflammatory state. After 10 days on the HFD, corneal circadian rhythms (limbal neutrophil accumulation, epithelial cell division and Rev-erbα expression) were blunted. Similarly, leukocyte recruitment after wounding was dysregulated and accompanied by delays in wound closure and nerve recovery.

After corneal epithelial injury, the ensuing inflammatory response is necessary for efficient wound healing. While beneficial healing effects are attributed to recruited neutrophils and platelets, dysregulated inflammation (too little or too much) is associated with impaired wound healing. The purpose of this study was to use an established C57BL/6J mouse model of corneal injury to evaluate the potential modulatory role of interleukin-20 (IL-20) on the inflammatory and healing responses to epithelial wounding. In the uninjured cornea, immunofluorescence staining for IL-20 and its receptor, IL-20RA, was observed on basal epithelial cells at the limbus. After a 2mm central epithelial abrasion, IL-20 staining was also observed in stromal keratocytes and ELISA studies showed a significant increase (nearly 3-fold) in IL-20 expression. Injured corneas healed more slowly when treated with a topical application of a neutralizing anti-IL-20 antibody. While corneal epithelial cell division and epithelial nerve recovery measured at 24 hours post-injury were reduced compared to controls, neutrophil influx into the cornea was increased. In contrast, topical application of recombinant IL-20 (rIL-20) decreased corneal inflammation as evidenced by reductions in limbal vessel dilatation, platelet extravasation, neutrophil recruitment and CXCL1 expression. In wild type mice, topical rIL-20 had a limited effect on corneal wound healing and resulted in only a slight increase in epithelial cell division and epithelial nerve recovery; the rate of wound closure was unaffected. To clarify the effect of IL-20 on corneal wound healing, rIL-20 was topically applied to neutropenic wild type (WT) mice and mutant mice (γδ T cell deficient mice and CD11a deficient mice), all of which have well characterized reductions in neutrophil recruitment and delayed wound healing after corneal injury. In each case, rIL-20 restored corneal wound healing to baseline levels while neutrophil recruitment remained low. Thus, it appears that IL-20 plays a beneficial and direct role in corneal wound healing while negatively regulating neutrophil and platelet infiltration.

Corneal abrasion not only damages the epithelium but also induces stromal keratocyte death at the site of injury. While a coordinated cascade of inflammatory cell recruitment facilitates epithelial restoration, it is unclear if this cascade is necessary for keratocyte recovery. Since platelet and neutrophil (PMN) recruitment after corneal abrasion is beneficial to epithelial wound healing, we wanted to determine if these cells play a role in regulating keratocyte repopulation after epithelial abrasion. A 2 mm diameter central epithelial region was removed from the corneas of C57BL/6 wildtype (WT), P-selectin deficient (P-sel-/-), and CD18 hypomorphic (CD18hypo) mice using the Algerbrush II. Corneas were studied at 6h intervals out to 48h post-injury to evaluate platelet and PMN cell numbers; additional corneas were studied at 1, 4, 14, and 28 days post injury to evaluate keratocyte numbers. In WT mice, epithelial abrasion induced a loss of anterior central keratocytes and keratocyte recovery was rapid and incomplete, reaching ~70% of uninjured baseline values by 4 days post-injury but no further improvement at 28 days post-injury. Consistent with a beneficial role for platelets and PMNs in wound healing, keratocyte recovery was significantly depressed at 4 days post-injury (~30% of uninjured baseline) in P-sel-/- mice, which are known to have impaired platelet and PMN recruitment after corneal abrasion. Passive transfer of platelets from WT, but not P-sel-/-, into P-sel-/- mice prior to injury restored anterior central keratocyte numbers at 4 days post-injury to P-sel-/- uninjured baseline levels. While PMN infiltration in injured CD18hypo mice was similar to injured WT mice, platelet recruitment was markedly decreased and anterior central keratocyte recovery was significantly reduced (~50% of baseline) at 4–28 days post-injury. Collectively, the data suggest platelets and platelet P-selectin are critical for efficient keratocyte recovery after corneal epithelial abrasion.

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the β2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.

Platelets and neutrophils are often recruited at sites of microvascular inflammation. While neutrophil extravasation in inflammation is well characterized, platelet extravasation in inflammation is poorly understood and has only been documented in a small number of studies. We showed previously that mice expressing low levels of the leukocyte integrin CD18 (CD18hypo) had reduced total recruitment of platelets (intravascular and extravascular) in the corneal limbus following corneal epithelial wound injury. In this study we sought to determine the role of CD18 in platelet extravasation. Following corneal wound injury, wild‐type (WT) mice had evidence of platelet extravasation across limbal venules; however, CD18hypo mice had markedly reduced platelet extravasation. In view of isolated reports that platelets express CD18, we compared blood platelet counts and ex vivo platelet function under shear stress between CD18hypo and WT mice. Blood platelet counts were measured with a Scil Vet ABC hematology analyzer (Scil animal care company). Flow chamber assays were conducted using a microfluidic BioFlux System (Fluxion Biosciences) with plates coated with type I collagen (25 μg/ml; Helena) and blocked with 5% Bovine serum albumin. The labeled blood was perfused over the collagen‐coated surfaces at two values of fixed shear stress (16 dyne/cm2 and 30 dyne/cm2). Platelet counts and platelet adhesion to collagen at both levels of shear stress were comparable between CD18hypo and WT mice. Since a variety of leukocytes express CD18, including mast cells and neutrophils, we performed experiments to assess the role of these leukocytes in platelet extravasation. We found that CD18hypo mice had a small but statistically significant decrease in mast cell number and a marked decrease in degranulated mast cells as compared to WT mice following wound injury. Further, corneal epithelial wound injury in mice deficient in mast cells (KitW‐sh/W‐sh) resulted in a marked decrease in platelet extravasation as compared to WT mice. Finally, antibody‐induced depletion of circulating neutrophils resulted in a marked decrease in platelet extravasation relative to control mice. Overall, these studies demonstrate that platelet extravasation in a corneal wound injury model of inflammation depends on CD18, mast cells, and neutrophils. Support or Funding Information Supported by: NIH EY018239, EY17120, HL079368 and VA BLR&D Merit Review Award I01 BX002551. M.A.B. was supported by NHLBI T32 HL139425 Extravascular platelets (green) are evident surrounding venules 24 hours postwounding in wild type mice. Platelets from CD18 hypomorphic mice had similar adhesion under shear as those from Wild‐type mice; blood platelet counts were similar in both strains