Fungicides enhance inflammatory response and hamper gastrointestinal barrier integrity in in vitro human models

Fungal infections represent a major challenge for agricultural production, driving the extensive use of fungicides to prevent crop losses. Although these compounds effectively control fungal pathogens, increasing evidence suggests that they may also exert unintended effects on human health. Indeed, their impact on human immune cells and intestinal epithelial function remains poorly characterized. In this study, we investigated the effects of four widely used agricultural fungicides (azoxystrobin, fluxapyroxad, cyazofamid and cyflufenamid) on inflammatory responses, macrophage metabolism and epithelial barrier integrity using human in vitro models. PBMC-derived macrophages were exposed to fungicides at concentrations ranging from 1 to 50 μM to assess inflammatory and metabolic responses, while Caco-2 cells were used to evaluate epithelial barrier function. None of the tested fungicides induced significant toxicity. However, all four compounds enhanced the expression of the pro-inflammatory markers CXCL10, TNF and CD80, and increased total NF-κB p65 protein levels. Moreover, cyazofamid promoted a shift from an anti-inflammatory M2-like state toward a pro-inflammatory profile and induced the M1-like polarization of GM-CSF-treated non-polarized macrophages. NMR-based metabolomic analyses revealed profound metabolic reprogramming, characterized by increased glycolytic metabolism, enhanced succinate release favoured by succinate dehydrogenase inhibition and reduced extracellular amino acid levels. Furthermore, all fungicides impaired epithelial barrier function by reducing transepithelial electrical resistance, increasing paracellular permeability and altering occludin localization. Overall, these findings demonstrate that agricultural fungicides with distinct chemical structures and mechanisms of action converge on common pro-inflammatory and metabolic pathways, promoting macrophage activation and intestinal barrier dysfunction. These results provide new mechanistic insight into the pro-inflammatory effects of fungicide exposure and highlight the need for further studies to evaluate their potential impact on human health.