Investigation of Skin Epithelial Innate Immune Barrier Functions using a Xenopus laevis Cell Line

Abstract

Frog skin functions as both a physical and immunological barrier to the external environment. In addition to protection by antimicrobial peptides (AMPs) present at the skin surface, epithelial cells recognize pathogen-associated molecular patterns and coordinate local immune responses. While amphibian AMPs are well known for antimicrobial activity, their roles in regulating epithelial immunity and barrier function remain unclear, partly due to limited in vitro models. The objectives of this thesis were to use the Xenopus laevis epithelial-like cell line Xela DS2 to (1) explore whether two X. laevis AMPs, magainin II and PGLa, exert immunomodulatory effects on epithelial cells, and (2) develop an in vitro epithelial barrier model for studying anuran skin immunity. To support the first objective, Xela DS2 were examined for the presence of magainin II and PGLa transcripts and treated with AMPs or a synthetic analogue of viral double stranded RNA [poly(I:C)] to determine non-cytotoxic concentrations. Xela DS2 demonstrated little to no transcription of target AMPs. Magainin II or PGLa at concentrations ≥32 µM, or poly(I:C) at concentrations above 250 ng/mL, were cytotoxic to Xela DS2, and established non-cytotoxic concentrations for use in experiments. Initial attempts to study potential immunomodulatory activity of AMPs were unsuccessful due to an unanticipated cellular response to the vehicle control. In parallel, an air-liquid interface model was established, where cells formed highly restrictive barriers with TEER values >10,000 Ω·cm² prior to airlift and maintained above functional thresholds for 6-8 days post airlift. Barrier formation depended on seeding density and passage number, with lower-passage cells performing better. Collagen coating was not essential, and use of a mammalian ALI supplement appeared detrimental. Barrier integrity was supported by organized ZO-1 localization and reduced paracellular permeability. Together, these findings establish Xela DS2 as a platform to study epithelial barrier function, AMP activity, and host-pathogen interactions in frog skin.