O12 Inhibition of mechanistic target of rapamycin signalling by Lacticaseibacillus rhamnosus GG in human keratinocytes

Introduction and aims The epidermal barrier is essential for protection against environmental insults and water loss, yet it is compromised in disorders such as eczema. Probiotics have emerged as candidates for treatment of various skin conditions, but their molecular mechanisms in epidermis remain poorly defined. The aim of this study is to understand how an established probiotic, Lacticaseibacillus rhamnosus GG (LGG) is able to enhance and possibly restore the skin barrier. Methods Normal human epidermal keratinocytes (nHEKs) were treated with a lysate of LGG (n = 3) and phosphoproteomic analysis using mass spectrometry was performed to assess cell signalling pathways affected by the lysate. Findings were validated by Western blot analysis in nHEK and nTERT cell lines. In addition, human 3D skin organotypic models were grown and immunofluorescence analysis was performed to further validate the changes. To model barrier dysfunction, short hairpin (sh)RNA filaggrin-knockdown keratinocytes were generated and treated with the LGG lysate. Heat- and trypsin-treated LGG lysates were applied to nTERTS in order to understand the properties of the active in the lysate. Results Kinase enrichment analysis of the phosphoproteomic data revealed the kinase mechanistic target of rapamycin (mTOR) to be differently regulated in keratinocytes treated with the LGG lysate. A reduction in mTOR phosphorylation and its downstream effector, ribosomal protein S6 was seen in Western blot analysis of LGG-treated nHEKs and nTERTS (n = 3), suggesting an inhibition of mTORC1. This reduction was also seen in the three-dimensional skin models (n = 3). shRNA filaggrin knockdown keratinocytes had a hyperphosphorylation of S6, which was rescued with the addition of the LGG lysate (n = 3). Both heat- and trypsin-treated lysates induced a similar effect to the mTORC1 pathway (n = 3) suggesting an active, nonprotein component within the lysate. Conclusions These findings provide mechanistic insight into probiotic-derived enhancement of epidermal barrier repair and support further development of microbiome-inspired topical therapies.