| dc.description.abstract | Introduction: Mycotoxins are toxic secondary metabolites produced by fungi (Aspergillus, Penicillium, Fusarium, and Alternaria) and pose a significant concern in the realm of food safety, occupational and public health. Exposure (i.e. inhalation, dermal, ingestion) to mycotoxins in agriculture and industrial settings such as compound feed mills, farming, and animal husbandry is prevalent and an occupational health concern. Certain mycotoxins have been described to be immunosuppressive by inhibiting the triggering effect of lipopolysaccharide (LPS) on the NF-κB-signaling pathway, that is necessary to engage the immune system.
Objective: This study investigates the immunotoxic effects and inflammasome activation of Alternaria toxins—specifically Alternariol (AOH) and Alternariol monomethyl ether (AME)—in an in vitro model using engineered HEK-293 Toll-Like Receptor (TLR2 & TLR4) and IL-1β reporter cells, as well as THP-1 cells.
Methods: Experimental parameters involve exposing THP-1 cells and HEK-293 reporter cells encompassing HEK null, TLR2, and TLR4, and HEK IL-1β, HEK Null 1v cells to varying concentrations of mycotoxins (i.e., AOH- 30, 6, 1.2, 0.24 µM; AME- 10, 2.5, 0.625, 0.156 µM), with dimethyl sulfoxide (DMSO 0.1%) as vehicle control. A TLR2 ligand LTA and LPS for TLR4, and THP-1, were used to activate the response/signaling pathway. Assessment methods include alamar-Blue for cell viability and QuantiBlue assays for detecting and quantifying SEAP activity, that serves as a reporter for NF-κB activation. Levels of proinflammatory cytokine IL-1β secretion are measured by ELISA. Bioactive IL-1β is measured by the HEK IL-1β inflammasome assay, and qPCR is used to quantify the gene expression of IL-1β.
Result: LPS and LTA activation of TLR receptors led to an increase in the NF-κB signal. However, combination exposure of LPS and LTA with mycotoxins reduced the increased NF-κB signal in both receptors (TLR2 and TLR4) for both mycotoxins (AOH and AME). AOH and AME also reduced the secretion and gene expression of the key pro-inflammatory cytokine IL- 1β in the inflammasome THP-1/IL-1β assay, when co-exposed to LPS. Some effect of exposure to LPS/LTA and mycotoxins on the cell viability was also seen though no dose-dependent significant reduction was apparent.
Conclusion: The results indicate an immunomodulating effect of AOH and AME mycotoxins on LPS- or LTA-induced activation of TLR via the downstream NF-κB signaling pathway. This study highlighted the need for improved monitoring and control of these mycotoxins to ensure the safety of food supply and protect the health of people related to agriculture and food industry. | |
