IL-4/-13 are central to AD pathophysiology and dispose of three receptors. Previously undetected in keratinocytes, the IL2Rγ receptor chain, participates in the type 1 receptor together with IL4Rα, and is induced in AD skin and in RHE treated with IL-4/-13. Furthermore, the role and function of the IL13Rα2 receptor also inducible is largely unexplored and poorly understood. This master's thesis aimed to investigate the induction mechanisms of IL2Rγ and IL13Rα2 in keratinocytes upon in vitro IL-4/-13 treatment, with a specific focus on the type 2 receptor (IL4Rα/IL13Rα1) signaling. The study utilized immortalized N/TERT keratinocytes, which offer advantages in scalability and genetic-editing while closely resembling primary cells. Protein levels were measured through WB and gene expression levels were measured through RT-qPCR.
Initial investigations focused on monolayer cultures under different conditions of differentiation and stratification to investigate if the induction could happen in a non-epidermis setting. We showed that the IL-4/-13 treatments induced STAT6 phosphorylation and led to the overexpression of AD markers (CA2 and NELL2), irrespective of the differentiation stage. Yet, alterations in barrier markers (FLG, IVL, and LOR) showed less pronounced changes, and STAT3, ERK, and AKT were phosphorylated at baseline. However, the induction of IL2Rγ and IL13Rα2 receptors was not observed, suggesting that neither differentiation nor stratification are the driving factors for their upregulation.
The study then transitioned to a 3D culture model of RHE, using genetically edited N/TERT keratinocytes deficient for the IL13Rα1 gene to eliminate the type 2 receptor. In contrast to monolayer cultures, treatment induced the expression of IL2Rγ and IL13Rα2 along with the overexpression of AD markers. Using the IL13Rα1 -/- cells, we show that these changes were dependent on the type 2 receptor. Reflecting on monolayer cultures, STAT6 phosphorylation was found to be mediated by the type 2 receptor, while STAT3, ERK, and AKT were phosphorylated at baseline. Furthermore, we showed that IL-4/-13 treatment led to impaired barrier integrity depending on the type 2 receptor, evidenced by increased barrier permeability and downregulation of barrier markers.
Challenges were encountered with experiments, requiring optimization for more robust data. Further studies are warranted to explore the role of the type 1 and IL13Rα2 receptors. In conclusion, we show that IL2Rγ and IL13Rα2 are induced through the type 2 receptor in keratinocytes exclusively in an epidermis setting, and that regulation of AD markers, STAT6 phosphorylation, and barrier impairment also rely on the type 2 receptor.
Characterization of IL-4 and IL-13 dependent signaling in N/TERT keratinocytes
VAN DOOREN, N. (Auteur). 25 août 2023
Student thesis: Master types › Master en sciences biomédicales à finalité spécialisée en recherche préclinique