(T1530-05-32) Topical Dexpanthenol Modulates Microbially Induced Inflammation in a 3D Corneal Model: A Proteomic Analysis

Purpose: Dexpanthenol is a common active pharmaceutical ingredient (API) in topical and ophthalmic formulations [1]. Dexpanthenol eye drops and ointments facilitate epithelium hydration and accelerate wound healing in treating dry eyes and corneal erosions [2-4]. Dexpanthenol might also exert anti-inflammatory effects in inflamed tissues. Its anti-inflammatory effect on lipopolysaccharide (LPS)-mediated lung injuries, sepsis-induced liver and kidney injuries, and experimental ultraviolet-induced erythema has been already established [5-7]. However, the potential anti-inflammatory properties of dexpanthenol against ocular inflammation remains hitherto under-investigated. Accordingly, the prime objective of the present study was to investigate the impact of dexpanthenol upon the expression of microbially induced inflammation markers in EpiCorneal™ cell culture model.

Methods: Experiments were carried out on in vitro human three-dimensional (3D) corneal epithelial tissue model “EpiCorneal™” (MatTek Life Sciences) [8]. Inserts were removed from the embedding agarose gel and pre-equilibrated in culture medium at 37°C for 24 h. Subsequently, the integrity of the cellular barrier was examined by measuring the transepithelial electric resistance (TEER). The inserts were then moved on hangtops in 12-well plates with 5 ml medium in the receptor compartment. Inflammation was induced by incubating the apical side with LPS solution (10 µg/ml) for 24 h. Following the removal of the medium in donor and acceptor compartments, the cells were washed twice with Krebs-Ringer Buffer (KRB) from both sides. The inserts were then transferred back to the hangtops and medium was added to the receptor compartment. The apical side was incubated with either medium (control) or dexpanthenol solution (treatment). The medium in the receptor compartment was collected after 24 h and 48 h and subjected to proteome profiling using Proteome Profiler Human XL Cytokine Array Kit (R&D systems) according to the manufacturer’s instructions. Briefly, membranes were blocked with blocking buffer, and incubated with the samples and the reconstituted human cytokine array detection antibody cocktail. Following 3 washing steps, the membranes were treated with streptavidin-HRP solution, washed three times, placed in the ChemiDoc™XRS + molecular imager® (Biorad), where they were treated with a Chemi Reagent Mix. After incubation in the dark, the membranes were imaged, and the mean pixel density was measured using Image Lab® (Bio-Rad). Data were normalized to the protein content of the cell models associated with each sample. While proteome profiling allowed the identification of a total of 96 markers, only those with mean pixel densities surpassing 600 pixels in at least one condition (control or treated), were selected for further analysis. Fold change (FC) and enrichment analyses were carried out using the statistical programming language R [9] with the packages UniprotR [10] and gProfiler2 [11]. Enrichment analyses were performed for up- (log₂ FC > 1), down- (log₂ FC < -1) and de-regulated proteins (abs(log₂ FC) > 1) separately (p < 0.05).

Results: TEER measurements proved the integrity of the models. LPS treatment did not exert a significant effect on the TEER of the models (TEER recovery = 100.4±11.0 % and 92.4±17.4 for medium and LPS treated models, respectively). In comparison with the untreated control, dexpanthenol treatment led to an increased secretion (FC > 2) of platelet-derived growth factor (PDGF)-AA and PDGF-AB/BB after 24 h, while several proteins showed FCs of 0.5 and smaller (Figure 1). After 48 h, increased secretion was found for Serpin E1 only. In contrast to 24 h, PDGF-AB/BB secretion was less than that of the samples without treatment. FC of 0.5 and smaller was observed also for interleukin (IL)-22, IL-1a and interferon gamma-induced protein-10 (IP-10). Proteins exhibiting reduced secretion after 24 h in the treated samples are associated with the categories “production of molecular mediator of immune response” and “regulation of cytokine production” (p < 0.05). Upregulated proteins after 48 h are associated with “positive regulation of leukotriene production involved in inflammatory response”, while those downregulated are involved in “acute-phase response”. For all de-regulated proteins after 48 h, one of the biological processes modified by the treatment is “cellular response to lipopolysaccharide” (p < 0.01).

Conclusion: Our results indicate a positive effect of dexpanthenol treatment on LPS-induced inflammation for the corneal epithelial tissue model. PDGF-BB increases the speed of re-epithelialization in diabetic and varicose ulcers [12], IL-1a has a "repulsive chemotactic effect on corneal fibroblasts” [13]. Treatment also increased the level of the inflammatory mediator Serpin E1, which can reverse the LPS-induced migration of neutrophils [14].

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Acknowledgements: The authors report no conflict of interest.

Figure 1: log2 Fold Change (FC) of LPS-induced proteins after 24 h of treatment with 5% dexpanthenol in 3D EpiCornealTM cell culture model