Modulation of NF-κB signaling by Alternaria mycotoxins: in vitro and in silico insights into molecular mechanisms of immunosuppression in THP-1 monocytes.

Alternaria fungi produce various structurally diverse mycotoxins, several of which exhibit immunomodulatory properties. Among these, alternariol monomethyl ether (AME), alternariol (AOH), alterperylenol (ALTP), altertoxin I (ATX-I), and altersetin (AST) have been reported to suppress lipopolysaccharide (LPS)-induced inflammatory responses. However, the precise molecular mechanisms underlying these effects remain unclear. The present study aimed to elucidate how these selected Alternaria mycotoxins (0.1-50 μM) target the NF-κB signaling pathway in THP-1 monocytes. Key components of the NF-κB cascade were analyzed by immunofluorescence microscopy, Western blotting and qRT-PCR. Nuclear translocation of NF-κB p65 and its phosphorylated form (p- NF-κB p65) was assessed by Western blot, while cytokine responses were determined at transcript (qRT-PCR) and protein (ELISA) levels. Moreover, in silico docking analyses were performed to investigate potential interactions of the toxins with IKKβ, and receptor-mediated crosstalk was studied using the glucocorticoid receptor (GR) antagonist RU486. Co-treatment with RU486 attenuated the immunosuppressive effects of 1 and 5 μM AOH, indicating partial involvement of GR-dependent mechanisms. AME, AOH, ALTP, ATX-I, and AST increased total IκBα levels while reducing its phosphorylated form. Additionally, AST and ALTP decreased the protein levels of Toll-like receptor 4 (TLR4), the IκB kinase (IKK) complex, NF-κB p65, and p- NF-κB p65. While AOH (5 μM) and AST (25 μM) reduced nuclear translocation of p65 and p-p65, ALTP (2 μM) enhanced nuclear localization despite decreasing cytokine expression. Together, these findings suggest toxin-specific interference at multiple regulatory levels of NF-κB signaling and provide novel mechanistic insight into the immunomodulatory effects of Alternaria mycotoxins.