Heme oxygenase-1 induction attenuates corneal inflammation and accelerates wound healing after epithelial injury

Abstract

Purpose: Heme oxygenase (HO) is considered a fundamental endogenous immunomodulatory, cytoprotective, and anti-inflammatory system. This protective function is primarily ascribed to the inducible HO-1. The authors examined the effect of HO-1 induction on corneal inflammation and wound healing in mice undergoing epithelial injury.

Methods: C57BL6 mice were treated with SnCl(2) the day before epithelial injury and once daily thereafter. The corneal epithelium was removed with the use of a corneal rust ring remover in anesthetized mice. Reepithelialization was measured by fluorescein staining. The inflammatory response was examined by histology and was quantified by the myeloperoxidase assay. Inflammatory lipid mediators were detected and quantified by LC/MS/MS-based lipidomic analysis. HO-1 expression was assessed by real-time PCR, and HO activity was determined by measuring HO-dependent carbon monoxide production.

Results: Epithelial injury caused a time-dependent transient increase in HO-1 expression and HO activity that was significantly amplified by treatment with SnCl(2), resulting in a twofold to threefold increase in mRNA levels and a similar increase in corneal HO activity. Induction of HO-1 was associated with a significant acceleration of wound healing when compared with a vehicle-treated group and with attenuation of the inflammatory response, evidenced by a significant decrease in the number of infiltrating cells and by a significant reduction in the expression and production of proinflammatory lipid mediators and cytokines.

Conclusions: Increased expression of HO-1 provides a mechanism that modulates inflammation and promotes wound closure; pharmacologic amplification of this system may constitute a novel strategy to treat corneal inflammation while accelerating wound repair after injury.

Figure 1

Effect of SnCl₂ treatment on HO-1 expression and activity. (A) Corneal HO-1 mRNA levels in vehicle- and SnCl₂-treated mice 1 day before (−d1) and 0 to 7 days (d0–d7) after injury. Results are the mean ± SE; n = 3 to 5. *P < 0.05 from corresponding vehicle-treated mice. (B) Corneal HO activity in vehicle- and SnCl₂-treated mice at −d1 and at d0 to d7 after injury. Results are expressed as the amount of CO produced per milligram protein per hour and are the mean ± SE; n = 3 to 5. *P < 0.05 from corresponding vehicle-treated mice.

Figure 2

Effect of SnCl₂ treatment on wound healing. (A) Fluorescein-stained corneas after injury in vehicle- and SnCl₂-treated mice (16×). (B) Wound closure as percentage change from d0. The area of the injury (d0) in vehicle- and SnCl₂-treated mice was 6.60 ± 0.05 and 6.70 ± 0.04 mm², respectively. Results are mean ± SE; n = 14 to 50. *P < 0.005 from vehicle-treated mice. †P < 0.05 from vehicle-treated mice.

Figure 3

Effect of SnCl₂ treatment on the inflammatory response. (A) Corneal MPO activity in vehicle- and SnCl₂-treated mice at −d1 and at d0 to d7 after injury. Results are expressed as the number of PMNs per cornea and are the mean ± SE; n = 3 to 5. *P < 0.05 versus vehicle-treated mice. (B) H&E staining of corneas at d2 (a, b), d4 (c, d), and d7 (e, f) after injury.

Figure 4

Effect of SnCl₂ treatment on inflammatory lipid mediator pathways. (A) Representative LC/MS/MS profiles of lipid mediators in injured corneas (d3) from vehicle- and SnCl₂-treated mice. Upper: elution profile of authentic standards. Middle: elution profile of lipids in vehicle-treated corneas 3 days after injury. Lower: elution profile of lipids in SnCl₂-treated corneas 3 days after injury. (B) Quantitative lipidomic analysis. Results are mean ± SE; n = 4. *P < 0.05 from vehicle-treated mice. (C) Corneal CYP4B1 mRNA levels in vehicle- and SnCl₂-treated mice at −d1 and at d0 to d7 after injury. Results are the mean ± SE; n = 3 to 5. *P < 0.05 from corresponding vehicle-treated mice.

Figure 5

Effect of SnCl₂ treatment on levels of IL-1 (A) and MIP-2 (B) after epithelial injury. Corneas were harvested at the indicated time, homogenized, and assayed for levels of IL-1 and MIP-2 by ELISA. Results are the mean ± SE; n = 3. *P < 0.05 from corresponding vehicle-treated eyes.