Alpha-1 antitrypsin promotes re-epithelialization by regulating inflammation and migration

Abstract

Purpose: Regulation of inflammation and re-epithelialization are critical for efficient wound healing. This study explores the role of human α1-antitrypsin (hAAT), an immunomodulatory protein, in modulating inflammation and promoting re-epithelialization across various epithelial cell types.

Methods: In-vitro, epithelial gap closure and migration assays were performed using two human epithelial cell lines-HaCaT and A549 cells with and without mitomycin C treatment. These cell lines were also used in an in-vitro gel-directed epithelial migration assay. Cells were treated with hAAT, and the gap area was measured using image analysis. Gene expression of inflammatory markers (IL-1β, IL-6, and TNFα) and adhesion molecules (desmoglein-1, plectin, and integrin α6β4) were analyzed using qPCR. In-vivo, corneal abrasions were induced in C57BL/6 mice using an Ophthalmic Burr. Mice received topical hAAT treatment immediately after injury and every 6 hours thereafter. Wound closure was assessed by applying the standard ophthalmic staining technique, fluorescein, and image analysis. Inflammatory markers and adhesion molecule expression were evaluated using qPCR and immunohistochemistry.

Results: In-vitro, hAAT accelerated epithelial gap closure and increased migration distance, independent of cell proliferation. hAAT-treated cells also exhibited earlier peak expressions of IL-1β and IL-6. In-vivo, hAAT treatment accelerated corneal wound closure and resulted in a preference for IL-1Ra over IL-1β expression. hAAT also enhanced the expression of desmoglein-1, plectin, and integrin α6β4, both in-vitro and in-vivo, and increased desmoglein-1 expression in the epithelial migration zone of mouse cornea.

Conclusions: hAAT enhances re-epithelialization by modulating inflammation, promoting epithelial cell migration, and regulating expression of adhesion molecules.

Keywords: adhesion molecules; corneal abrasion; desmosomes; gene expression; hemidesmosomes; inflammation; wound Healing.

Figure 1

In-vitro epithelial cell gap closure and migration . (A–C) Epithelial cell gap repair assay using (A) HaCaT cells (human keratinocyte cell line) in 10% serum 2×105 cells/well in 12-well plates in triplicates) and (B) HaCaT cell in 0% serum ( top , representative photomicrographs; bottom , phosphate-buffered saline (PBS) treatment set at 100% gap area, data pooled from 2 independent experiments), and (C) A549 cells (human lung epithelial cell) in 5% serum (1×10⁵ cells/well in 24-well plates in triplicates) ( left , serial measurements, indicated time points),data pooled from 2 independent experiments. EGF , epidermal growth factor; MitC , mitomycin C. (D) Migration assay using A549 cells, set 1 mm apart from lysed A549 cells with or without added adenosine triphosphate (ATP), illustrated inset. Left , representative photomicrographs; right , percent area from initial gap area, representative data out of 3 independent experiments). Arrow , distance marking in image analysis. Mean±SEM. * p<0.05, **** p<0.0001.

Figure 2

Gene expression profile in epithelial gap closure assay. A549 cells treated with phosphate-buffered saline (PBS) or hAAT 0.5 mg/ml in triplicates, gap inflicted at 0 hr. Cells were harvested for gene expression analysis at indicated time points. Data presented as (A) fold from sham and (B) percent from peak expression. Representative data out of 2 independent experiments. Mean± SEM; ns, not significant, *p<0.05, **p<0.01 and ****p<0.001.

Figure 3

In-vivo wound area in mouse corneal abrasion model. Corneal abrasion performed at time 0, topical treatments provided at the time of wounding and then every 6 hours. CT, control (saline 7 µl, n=15); hAAT (4 μg/eye, n=12); DEX (dexamethasone, 4 μg/eye, n=10). Wound area was quantified at 6,10 and 16 hours, represented as percent of initial wound area. Data pooled from 4 independent experiments. Mean ± SEM, deviation represented by solid fill. Box and whiskers represent mean (min to max); * p<0.05, **p<0.01, ***p<0.001, ****p< 0.0001.

Figure 4

In-vivo re-epithelialization: corneal abrasion model. (A) Untreated abrasion, Hematoxylin and Eosin (H&E), 10 hrs. Representative microscopic images out of 8 samples. Radial plane. Black arrow, epithelial leading cell. (B) Desmoglein-1 immunofluorescent staining. Representative images. Migration zone, 50 µm from wound edge; corneal epithelium, adjacent corneal layer. Right, mean fluorescent intensity per 10 µm² tiles, pooled data from 3 slides per group. Mean, ****p<0.0001.

Figure 5

Gene expression profile in mouse corneal abrasion model. Corneal abrasion was performed at time 0, and topical treatments were applied at the time of injury and at 6, 10 and 16 hours post-injury. Complete eyes were collected at indicated time points for gene expression analysis. (A) Expression of inflammation-related genes; interleukin-1 beta (IL-1β), interleukin-1 receptor antagonist (IL-1Ra), and IL-1Ra/IL-1β ratio. (B) Expression of genes related to adhesion molecules; Desmoglein-1, Plectin, and Integrin α6β4. Data are presented as fold change from sham or as ratio per sample. Representative results from three independent experiments. Mean ± SEM; ns, not significant; *p < 0.05, ****p < 0.0001.