Advanced integrated human in vitro approach to explore gut-brain barrier interactions with gut microbiota metabolites and pesticides

The gut microbiota (GM) plays a central role in host barrier homeostasis. Alterations in GM due to chronic dietary pesticide residues can have far-reaching consequences on human health. Although in vitro models are increasingly used to study organophosphate effects, especially Chlorpyrifos (CPF), accurately modeling complex GM-host interactions remains challenging. Here we investigated how CPF-altered GM metabolites influence epithelial and brain barrier integrity. An integrated human-relevant in vitro approach combining three complementary models: the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) under control and CPF-exposed conditions to evaluate both short-term and long-term effects (15 and 30 days), a Caco-2-based intestinal barrier (IB) and a blood-brain barrier (BBB) co-culture. Two parallel experimental setups (SHIME-IB and SHIME-BBB) were developed: SHIME®-derived microbial metabolites and pesticide residues supernatants were directly applied to each barrier model for 24 h. Confocal imaging revealed discontinuous localization of tight junction proteins (occludin, claudin-5 and ZO-1) without concomitant increase in FITC-dextran apparent permeability or overt cytotoxicity. This structural disorganization occurred despite unchanged transcriptional expression of tight junctions, except for a significant reduction of CLDN5 mRNA in the BBB at CPF15, suggesting early molecular signals. In contrast, we showed a selective decrease in P-GP (P-glycoprotein) expression in both barriers, confirmed at the protein level. Concurrently, IL-8 (Interleukin-8) secretion increased markedly in IB, particularly at CPF15, highlighting its potential as an early biomarker chemokine-driven inflammatory activation. This integrated approach provides an ethical model to study environmental contaminants' effects on human barriers, revealing microbiota-mediated disruption by CPF.