P12-33 Unveiling the immunosuppressive potential of the emerging Alternaria mycotoxin alterperylenol: From cellular impact to molecular mechanisms

Alternaria molds can produce a diverse array of mycotoxins, some of which may pose a significant threat to human health due to the wide range of toxic effects reported in vivo and in vitro , as well as their high occurrence in food. Despite this, Alternaria mycotoxins are still not regulated due to the lack of toxicological data. In this context, critical information on the immunomodulatory properties of the emerging mycotoxin alterperylenol (ALTP) remains scarce. This underscores the urgent need to acquire data to assess the health risks associated with consuming foods contaminated by this mycotoxin. Building upon this knowledge, the present study aimed to assess the immunomodulatory properties and underlying mechanisms of action of the mycotoxin ALTP using a range of toxicological and molecular techniques. In particular, the immunosuppressive properties of ALTP were evaluated by applying the NF-κB gene reporter assay to THP-1 Lucia monocytes within a co-culture model. In this model, THP-1 cells were cultured alongside intestinal Caco-2 and HT29-MTX-E12 cells, with a semipermeable membrane separating them to better simulate the intestinal environment. To acquire mechanistic information, changes in the expression of key components of the NF-κB signaling cascade (e.g., IκB, p-IκB, TLR-4, p-IKK, NF-κB, and p-NF-κB) were examined at the mRNA level (qRT-PCR) and/or protein level (Western blot analysis and confocal microscopy). Finally, the expression levels of selected cytokines (IL-6, IL-8, IL-10, and TNF-α) were assessed in THP-1 Lucia monocytes at the mRNA level (qRT-PCR). Results from the NF-κB assay revealed the ability of ALTP to effectively suppress the LPS-induced NF-κB activation in THP-1 Lucia monocytes starting at 5 µM. Notably, this suppression was not due to cytotoxic effects, as confirmed by the CellTiter Blue® viability assay. Furthermore, TEER measurements and the Lucifer Yellow permeability assay demonstrated the preservation of intact Caco-2/HT29-MTX-E12 monolayers following exposure to the mycotoxin, highlighting ALTP's potential for absorption and systemic immunosuppressive effects. The results of qRT-PCR, confocal microscopy and Western blot analysis further revealed the ability of ALTP to modulate key NF-κB pathway proteins, particularly targeting the inhibitory protein IκB, its phosphorylated form (p-IκB), and the IKK enzyme complex. Finally, cytokine quantification confirmed the downregulation of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α, supporting the immunosuppressive properties of ALTP [1] . In conclusion, the findings of this study highlight the immunosuppressive potential of the emerging Alternaria mycotoxin ALTP in vitro . Given that the results suggest possible absorption at the intestinal level, and thus, a possible systemic effect, studies on the mechanisms of action and effects of the mycotoxin in vivo are essential to accurately assess the actual risk of human exposure to this mycotoxin through food.