Plant lipid transfer proteins' ligand enhances allergic sensitization to non-related proteins in murine models

Allergic sensitization is a complex process, characterized by the development of a heightened immune response to harmless substances, known as allergens. Identifying contributors to this response is crucial to understand its mechanisms and to develop effective prevention and treatment strategies. In this context, protein ligands have gained attention since the ligand carried by some plant pathogenesis-related proteins (PRs) are implicated in allergic sensitization.1, 2 Plant lipid transfer proteins (Plant-LTPs), main food allergens, transport a specific lipid ligand (LTPs' ligand) consisting of a polar head derived from camptothecin, linked through an amide bond to a phytosphingosine tail (PHS).3 Previous studies have shown its relevant role in allergic sensitization to peach in complex with its natural allergen, Pru p 3.3, 4 To confirm whether this adjuvant capacity of the LTPs' ligand is a non-dependent protein feature, two different unrelated proteins, bovine serum albumin (BSA) and ovalbumin (OVA), were chosen to study their immunological properties in the presence of the ligand, comparing to the previously described for Pru p 3.4 In silico studies predicted that BSA binds five molecules of LTPs' ligand, while OVA, only binds two (Figure 1A; Appendix S1). This was confirmed in vitro by thin-layer chromatography (TLC, Figure 1B). In addition, both complexes showed a significant immune capacity when cultured with THP1-XBlue™ cells (Figure 1C). Next, to analyze the adjuvant capacity of the ligand, two mouse models were developed, following previous studies (Appendix S1).4 Two different mouse strains (C3H and BALB/c) were used, and both complexes were employed as skin sensitizers, and compared with Pru p 3-Ligand complex (Figure 1D). For all groups, only mice sensitized with LTPs' ligand in complex suffered a drop in body temperature after challenge (Figure 1E). In a more detailed characterization of the systemic response the expression of several genes associated with food allergies and atopic dermatitis5 was evaluated (Appendix S1). In the case of the BSA model (Figure 2B,C), Il1b, Ccl5, Nos2, Il10, Il1r1l, Cd36, Dusp1 and S100a9 were overexpressed in the skin (Figure 2D). This local inflammatory state led to CD45+ cell infiltration, observed only in the complex sensitized group (Figure 2E–G), being the majority of those cells positive for the expression of Sphingosine-1-Phosphate-Receptor 1 (S1PR1, Figure 2F). Finally, the systemic immune response induced the production of specific humoral antibodies, with a higher ratio IgG1/IgG2a (Figure 2H). Previous reports, described that the PHS tail of LTPs' ligand can be phosphorylated by human sphingosine-1-kinase in vitro, inducing the cell migration by its product, PHS-1P.3 So, to study the immunological mechanism of the LTPs' ligand as an adjuvant, a non-phosphorylable analogue of PHS (non-PLigand) was employed in the BSA model (Appendix S1; Figure 2A). The results showed that no immunological response was observed in mice treated with non-PLigand in complexed with BSA (Figure 2B–H). In detail, any mice treated with non-PLigand complex had a drop in body temperature (Figure 2C); nor humoral response (Figure 2H) and lower CD45+ skin infiltration compared to mice treated with BSA-Ligand (Figure 2E,F). While the protein itself is capable of inducing a small skin CD45+ infiltration, the results showed that the presence of the ligand leads to a significantly greater cell infiltration. Thus, the adjuvant capacity of the LTPs' ligand seems to be based on its ability to induce immune cell migration. To confirm this, media from THP1 cells, preincubated with BSA-Ligand or BSA-non-PLigand, were utilized in a migration assay, in the presence of Fingolimod, a well-characterized S1PR inhibitor6 (Figure 2I; Appendix S1). As shown in Figure 2J, monocyte migration was observed only in the case of BSA-Ligand supernatant, while non-PLigand showed no effect. Furthermore, Fingolimod treatment blocked migration using BSA-Ligand media. All these data confirm the relevance of the LTPs' ligand in the allergic sensitization process. Mice exposed to LTPs' ligand with non-related proteins were systemically sensitized, independent of the protein or mouse strain used. Additionally, in the short term, the LTPs' ligand seems to promote local inflammation, increasing immune cell infiltration (CD45+ S1PR1+); favouring systemic allergic sensitization in the long term. These findings shed light on the mechanisms underlying ligand–receptor interactions, cell migration and the development of allergic reactions. Although further studies are needed, the results suggest that the presence of the PHS tail and its phosphorylation, are key to the induction of allergic sensitization. ADP, JTA and MGA designed the study. ZGK, DPC, JLRG, GVP, HBH, SFB, LPG and JTA performed experimental assays. Data were analyzed by ZGK, JTA, VE, ADP and MGA. Manuscript was written and revised by JTA, ADP and MGA. All authors reviewed and approved the manuscript. This research was supported by the Spanish Ministry of Science and Innovation through the project LISENTRA, granted by the Spanish Research State Agency (PID2020-113629RB00/AEI/10.13039/501100011033) and FIS grant PI21/00158 (ISCIII). DP-C was granted by Universidad Politécnica de Madrid and Banco Santander for a predoctoral Programa Propio grant. SFB was granted from the ISCIII (FI22/00046). GVP was granted by PRE2021-100446 funded by MCIN/AEI/10.13039/501100011033 and by ESF+. ZG-K and JT-A were granted by funding from the Community of Madrid in the framework of the FOODAL project (S2018/BAA-4574). The CBGP was granted ‘Severo Ochoa’ Distinctions of Excellence by the Spanish Ministry of Science and Innovation (SEV-2016-0672 and CEX2020-000999-S). The authors declare there is no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.