P21 Defining the mechanism of skin barrier restoration by <i>Lactobacillus rhamnosus</i>

Abstract Introduction and aims The beneficial effects of probiotics on skin health has stimulated recent exploration. The bacterium, Lactobacillus rhamnosus GG (LGG), an established probiotic in gut health, is a promising therapeutic candidate for treating skin barrier diseases. Previous work suggests that lysates of LGG may enhance the skin barrier, in part by increasing expression of tight junction (TJ) and keratinocyte differentiation proteins. We aim to identify the underlying molecular mechanisms behind this barrier enhancement and to test if the LGG lysate can restore the skin barrier function in an in vitro model of atopic dermatitis. Methods A phosphoproteomic study was performed using normal human epithelial keratinocytes (nHEK), three LGG lysate-treated samples vs. three control samples. Mass spectrometry and bioinformatic analysis was used to identify differentially phosphorylated proteins. We also developed an in vitro ‘barrier impaired’ model using short hairpin RNA filaggrin knockdown nHEK. The expression and localization of TJ and desmosomal proteins was studied through Western blot analysis and immunostaining expression. Results Our results showed reduced molecular target of rapamycin (mTORC1) activity in the LGG lysate-treated cells. Through mTOR being the most enriched upstream kinase in the phosphoproteomic data and validated through Western blot analysis of phosphorylation of both mTOR and a downstream effector protein S6 kinase. Additionally, we showed that filaggrin knockdown significantly decreased the expression of TJ and desmosomal proteins (≥ n = 3). Furthermore, LGG lysate increased TJ expression in nHEK (≥ n = 3). Our results highlight the essential role of filaggrin in skin barrier function and suggests the mechanism by which LGG lysate restores skin barrier function may be in part through its effect of increasing TJ expression. Conclusions Together, these findings suggest likely molecular mechanisms behind the effects of the LGG lysate and suggests a possible mechanism by which it could help to restore barrier function in an impaired model.