Effect of topical azithromycin on corneal innate immune responses

Abstract

Purpose: To determine the effect of azithromycin (AZM) in a murine model of corneal inflammation.

Methods: The effect of topical AZM was studied in murine corneal inflammation. Corneal inflammation was induced by thermal cautery in BALB/c mice. Leukocyte infiltration at different time points was analyzed by flow cytometry. At set time points, real-time polymerase chain reaction was performed to quantify the expression of different inflammatory cytokine transcript in the cornea. Corneal samples were analyzed immunohistochemically for the expression of intercellular adhesion molecule-1 (ICAM-1). Corneal neovascularization (CNV) was induced by micropellet (VEGF-A) placement. Mice were then treated topically with either AZM or vehicle. CNV was evaluated morphometrically.

Results: Eyes receiving AZM showed a significant decrease in corneal infiltration compared with the vehicle-treated group. AZM also significantly decreased messenger RNA expression levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and ICAM-1 in the cornea. There was no significant difference in CNV between the AZM- and vehicle-treated groups.

Conclusions: After an inflammatory insult, topical AZM significantly reduced leukocyte infiltration into the cornea. This was further supported by an associated decrease in expression of IL-1β, TNF-α, and ICAM-1 in the cornea, indicating AZM may have a potential anti-inflammatory effect on corneal inflammation.

Figure 1.

Amelioration of corneal inflammation by topical AZM. Corneas were harvested, and single-cell suspensions were prepared and triple stained with (A) anti–CD45 PE-Cy5, (B) anti–Gr-1 FITC, (C) anti–CD11c PE, and (D) anti–CD11b FITC. (E) Representative flow cytometric data plots show CD11c⁺/CD45⁺ and Gr-1⁺/CD45⁺ cell (in all corneal cells) infiltration to the cornea at day 7 after cautery. Topical AZM treatment led to a significant decrease in total cell infiltration, specifically in CD11c⁺ cells. Data shown are representative of three independent experiments. Results depicted are the mean number of total cell infiltrations ± SEM.

Figure 2.

Time course of corneal cytokine expression. RT-PCR analysis showing transcript levels of IL-1β, IL-10, IL-6, and TNF-α in the cornea. Treatment with topical AZM significantly decreased relative expression of (A) IL-1β and (B) TNF-α transcripts. Corneas treated with AZM increased the expression level of (C) IL-10 at all time points. The relative expression of (D) IL-6 was variable at different time points. Horizontal line: results normalized to messenger RNA (mRNA) expression levels in the corneas of naive mice. Bars represent SEM.

Figure 3.

Suppression of vascular endothelial cell ICAM-1 expression by AZM. Eyes of BALB/c mice were treated with topical AZM or vehicle alone after the induction of corneal inflammation. Corneal sections were immunostained with anti–ICAM-1 antibody at different time points. Seven days after corneal cauterization, limbal vessels of eyes treated with (A) vehicle alone showed significant ICAM-1 staining, in contrast to (B) AZM-treated eyes in which the vascular endothelium remained ICAM-1 negative. (C) RT-PCR analysis showed transcript levels of ICAM-1 in the cornea. Relative expression of ICAM-1 in the cornea was significantly decreased in the AZM-treated eyes at day 7. Horizontal line: normalized to mRNA expression levels in the corneas of naive mice. Bars represent SEM.

Figure 4.

Analysis of corneal angiogenesis after treatment with AZM. After the induction of new blood vessels, the eyes of BALB/c mice were treated with either AZM or vehicle. Corneal whole mounts were immunostained with anti–CD31 antibody (A). The area covered by CD31^hi BV was evaluated by measuring the percentage of the total corneal area covered by BV (B). Data are presented as mean ± SD.