Lipoxin A4 (LXA4) Reduces Alkali-Induced Corneal Inflammation and Neovascularization and Upregulates a Repair Transcriptome

Abstract

Purpose: To investigate the anti-inflammatory and anti-angiogenic effects of the bioactive lipid mediator LXA4 on a rat model of severe corneal alkali injury.

Methods: To induce a corneal alkali injury in the right eyes of anesthetized Sprague Dawley rats. They were injured with a Φ 4 mm filter paper disc soaked in 1 N NaOH placed on the center of the cornea. After injury, the rats were treated topically with LXA4 (65 ng/20 μL) or vehicle three times a day for 14 days. Corneal opacity, neovascularization (NV), and hyphema were recorded and evaluated in a blind manner. Pro-inflammatory cytokine expression and genes involved in cornel repair were assayed by RNA sequencing and capillary Western blot. Cornea cell infiltration and monocytes isolated from the blood were analyzed by immunofluorescence and by flow cytometry.

Results: Topical treatment with LXA4 for two weeks significantly reduced corneal opacity, NV, and hyphema compared to the vehicle treatment. RNA-seq and Western blot results showed that LXA4 decreased the gene and protein expression of pro-inflammatory cytokines interleukin (IL)-1β and IL-6 and pro-angiogenic mediators matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGFA). It also induces genes involved in keratinization and ErbB signaling and downregulates immune pathways to stimulate wound healing. Flow cytometry and immunohistochemistry showed significantly less infiltration of neutrophils in the corneas treated with LXA4 compared to vehicle treatment. It also revealed that LXA4 treatment increases the proportion of type 2 macrophages (M2) compared to M1 in blood-isolated monocytes.

Conclusions: LXA4 decreases corneal inflammation and NV induced by a strong alkali burn. Its mechanism of action includes inhibition of inflammatory leukocyte infiltration, reduction in cytokine release, suppression of angiogenic factors, and promotion of corneal repair gene expression and macrophage polarization in blood from alkali burn corneas. LXA4 has potential as a therapeutic candidate for severe corneal chemical injuries.

Keywords: RNA-seq; angiogenesis; corneal chemical injury; lipoxin A4; macrophage polarization; proinflammatory cytokines.

Figure 1

Clinical evaluation of alkali-burned rat corneas treated topically with LXA4 (black circles) or vehicle (white circles) for 2 weeks. (A–C) Clinical scores, LXA4-treated eyes had significantly less corneal opacity, NV, and hyphema than vehicle-treated eyes. (A,B) Values are expressed as Mean ± SEM of 12 eyes and subjected to paired t-test, **** p < 0.0001; for hyphema, data are presented as Mean ± SEM and subjected to chi-square test. * p < 0.05. (D) Representative photographs. For comparison, images of normal rat eyes are shown.

Figure 2

(A) RNA-seq analysis of the effect of LXA4 on the gene expression of inflammatory cytokines and angiogenic mediators. Data are expressed as Mean ± SD (n = 5–6 corneas/each group; control means normal cornea removed from rats without injury). * p < 0.01 (ANOVA). (B) Jess Western blot analysis of the effect of LXA4 on the protein expression of inflammatory and angiogenic mediators. Data represented as Mean ± SD; each sample consists of 2 corneas with 3 repetitions. * p < 0.01. C: Control, normal corneas from rats without injury; V: vehicle; L: LXA4. The numbers on the sides of the gel represent molecular weights (kDa).

Figure 3

The corneal transcriptome analysis unravels the mechanism of LXA4 to induce cornea restoration after alkali burn damage. (A) Venn analysis of up- and downregulated genes detected in three groups: (i) vehicle vs. control (normal cornea), (ii) vehicle vs. LXA4 treatment, and (iii) LXA4 treatment vs. control. The shared genes between groups (i) and (ii) refer to the genes modulated by the injury and rescue by the LXA4 treatment. They were named as “LXA4-rescued functions” genes. The shared genes from all three groups regarding genes changed by the injury that could not be rescued by the LXA4 treatment were named as “Injury-dysfunctions” genes. (B) Gene sets enrichment analysis of two lists: “LXA4-rescued functions” and “Injury-dysfunctions”. The normalized enrichment was plotted in the bar chart with a negative score for the downregulation pathways (orange) and a positive score for the upregulation pathways (blue). Only significant pathways (adjusted p-value < 0.05) were shown. The “Injury-dysfunctions” list just gives one significant pathway (Metapathway biotransformation), as highlighted by a blue box. (C) Heatmap of RNA-seq data for all genes belonging to significant pathways such as the keratinization mechanism (R-RNO-6805567) and ErbB pathway (WP1299), Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell (R-RNO-198933), and antimicrobial peptides (R-RNO-6803157).

Figure 4

Effects of LXA4 treatment on inflammatory cell infiltration in alkali-burned corneas. (A,B) show that treatment for 14 days with LXA4 significantly reduced the number of CD45+ cells, especially neutrophils (CD11b+HIS-48+), in the alkali-burned corneas (Mean ± SEM, * p < 0.05). There were no significant differences in the number of macrophages (CD68+/CD11b+) in corneas treated with LXA4 (n = 8 corneas per group). (C) Immunofluorescence showed fewer inflammatory cells in LXA4-treated corneas than in vehicle-treated corneas. (D) Effects of LXA4 treatment on the phenotype of macrophages from the blood of alkali-burned corneas. Isolated monocytes from the blood of rat corneas treated with LXA4 or vehicle were stained with CD68, CD86, and CD163 and analyzed by flow cytometry. There was a higher percentage of macrophages compared to vehicle and non-injured (control) rats. Percentage of M2 cells (CD68+/CD163+) was higher than M1 (CD68+/CD86+) in LXA4-treated corneas. Data were expressed as Mean ± SEM; * p < 0.05; *** p < 0.001, ANOVA with Tukey’s multiple comparison.

Figure 5

Schematic diagram of the action of LXA4 after a cornea alkali burn.