Non-specific lipid-transfer proteins trigger TLR2 and NOD2 signaling and undergo ligand-dependent endocytosis in epithelial cells.

BACKGROUND: Allergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. OBJECTIVE: We show the molecular details and the effects of three non-specific lipid transfer proteins (nsLTPs; Mal d 3, Cor a 8 and Pru p 3) upon epithelial cell uptake and transport. METHODS: We used fluorescent imaging, flow cytometry, proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. RESULTS: NsLTPs are transported across the epithelium without affecting cell membrane stability or viability and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis (CME). Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent upon these ligands in the protein complex.nsLTPs were found to initiate cellular signaling via TLR2 but not the CD1d receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the three allergens induced intracellular stress signaling through activation of the NOD2 pathway. CONCLUSIONS: Our work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. Additionally, we have developed a versatile toolbox to extend these investigations to other allergens and cell types.

Surfactants for stabilization of dermal emulsions and their skin compatibility under UVA irradiation: Diacyl phospholipids and polysorbate 80 result in high viability rates of primary human skin cells.

Phospholipids are versatile formulation compounds with high biocompatibility. However, no data on their effect on skin in combination with UVA radiation exist. Thus, it was the aim of this work to (i) develop o/w nanoemulsions (NEs) differing in surfactant type and to investigate their physicochemical stability at different storage temperatures, (ii) establish a standardized protocol for in vitro phototoxicity testing using primary human skin cells and (iii) investigate the phototoxicity of amphoteric phospholipids (S45, S75, E80, S100, LPC80), sodium lauryl ether sulfate (SLES) and polysorbate 80 (PS80). Satisfying systems were developed with all surfactants except S100 due to low zeta potential (-21.4 mV ± 4.69). SLES and PS80-type NEs showed the highest stability after eight weeks; temperature-dependent variations in storage stability were most noticeable for phospholipid surfactants. For phospholipid-based NEs, higher phosphatidylcholine content led to unstable formulations. Phototoxicity assays with primary skin fibroblasts confirmed the lack of UVA-related phototoxicity but revealed cytotoxic effects of LPC80 and SLES, resulting in cell viability as low as 2.7 % ±0.78 and 1.9 % ±1.57 compared to the control. Our findings suggest that surfactants S45, S75 and PS80 are the most promising candidates for skin-friendly emulsifiers in sensitive applications involving exposure to UV light.