INTESTINAL AND BLOOD–BRAIN BARRIER PERMEABILITY IN AUTISM SPECTRUM DISORDER

Autism Spectrum Disorder (ASD) is a multifactorial neurodevelopmental condition characterized by impairments in social communication and behavior, frequently associated with gastrointestinal dysfunctions. Increasing evidence highlights the central role of the microbiota–gut–brain axis (MGBA), where alterations in biological barrier integrity may contribute to systemic inflammation and neurodevelopmental alterations. In particular, increased intestinal permeability (“leaky gut”) has been consistently reported in ASD and linked to microbial dysbiosis and immune dysregulation. The intestinal epithelial barrier (IEB) plays a key role in maintaining homeostasis by regulating the selective passage of luminal components into the systemic circulation. Based on our previous promising results from a genetic ASD murine model, here we explored intestinal permeability in three environmental ASD murine models: Maternal Immune Activation (MIA), Post-natal Immune Activation (PIA) and Early Immune Activation (EIA). Both male and female were included in the study, allowing for the evaluation of potential sex-related differences in barrier dysfunction and tight junction gene expression. The expression of key tight junction (TJ) components (claudin-1, claudin-3, occludin and zonula occludens-1), was analyzed by RT-qPCR, revealing significant molecular alterations across three models. Microbiota modulation through administration of food-derived Lactiplantibacillus plantarum has been shown to partially restore TJs gene expression and improve barrier integrity in all in vivo models, reducing intestinal permeability and supporting a protective role of microbiota-targeted interventions in preserving epithelial function. In parallel, dysbiosis-associated alterations promote the systemic dissemination of pro-inflammatory mediators and microbial metabolites through MGBA, which may subsequently affect the integrity of the blood–brain barrier (BBB). To further investigate these interactions, advanced microfluidic in vitro platforms will be developed using bioreactors developed by IVTech (IVTech srl, Pisa, Italy) to recreate physiologically relevant dynamic conditions. These systems enable real-time monitoring of structural and functional changes in barrier permeability under controlled conditions. The IEB will be modeled using NCM460 (Normal Colon Mucosa 460) and HT29-MTX (Human Colorectal Adenocarcinoma Methotrexate) cell lines, chosen for their ability to recapitulate intestinal epithelial and mucus-secreting properties, respectively, while the BBB will be reconstructed using Human Pericytes (Human Brain Vascular Pericytes), Human Astrocytes (Human Brain Astrocytes) and hCMEC/D3 (Human Cerebral Microvascular Endothelial Cell line/D3) cells, providing a multicellular representation of the neurovascular unit. By integrating microbiota-derived signals and barrier-forming cells, these models could help to dissect the mechanistic links between gut and brain interfaces and to identify key biomarkers involved in barriers dysfunction.