Breaking barriers: exploring gut-brain axis dysfunction in autism with advanced in vitro models - Segnali che attraversano la vita: dal neurosviluppo alla neurodegenerazione

Autism Spectrum Disorder (ASD) is a multifactorial neurodevelopmental condition characterized by impairments in social communication and behavior, frequently associated with gastrointestinal dysfunctions. Increasing evidence supports a central role of the gut-brain axis (GBA) in ASD pathophysiology, whereby simultaneous dysfunction of the intestinal epithelial barrier (IEB) and the blood-brain barrier (BBB) may promote systemic dissemination of pro-inflammatory mediators, microbial metabolites and neurotoxic molecules, ultimately contributing to neuroinflammation and neurodevelopmental alterations. In this framework, the microbiota-gut-brain axis (MGBA) represents a critical bidirectional communication network linking the intestinal microenvironment to the central nervous system, and its dysregulation has been increasingly implicated in ASD pathogenesis. Food-derived microorganisms, such as Lactiplantibacillus plantarum, have shown promising capacity to modulate gut microbiota composition, restore barrier integrity, intestinal inflammation positioning it as potential candidate for microbiota-targeted interventions in ASD. Despite growing interest, the mechanistic interplay between IEB and BBB dysfunction and the role of the MGBA in ASD remain poorly characterized, and effective targeted therapeutic strategies are still lacking. To address this, advanced microfluidic in vitro platforms are currently being established using IVTech (IVTech srl., Pisa, Italy), bioreactors to recreate dynamic physiological conditions and model the bidirectional communication between gut and brain compartments under controlled perfusion flow. The IEB is being reconstructed through a co-culture system of human cells, while the BBB is being modeled using a tri-culture approach providing a multicellular representation of the neurovascular unit. A monocytic component will be also integrated to simulate immune cell trafficking and its impact on barrier integrity.Within this dynamic system, the MGBA is being modeled by exposing the IEB compartment to selected food-derived Lactiplantibacillus plantarum strains, previously characterized for their probiotic and anti-inflammatory properties, in order to evaluate their capacity to modulate barrier permeability, TJ protein expression and the inflammatory cytokine profile under conditions mimicking ASD-related symptoms such as the higher intestinal permeability, also known as “leaky gut”, intestinal and neuroinflammation. So far the barrier function in the IEB model has been assessing by establishing a co-culture of human intestinal cell lines, specifically NCM460 cells, derived from normal human colonic mucosa, and HT29-MTX cells, a mucin-secreting goblet cell variant capable of producing a physiologically relevant mucus layer. To evaluate intestinal permeability and confirm tight barrier establishment, transepithelial electrical resistance (TEER) measurements and Lucifer Yellow transwell permeability assays were performed. TEER values progressively increased over time, reflecting the formation of a confluent and functional monolayer, while Lucifer Yellow assay showed a passage rate below 5%, confirming the establishment of a functional and tight cellular barrier suitable for downstream experimental challenges. Expression of key tight junction (TJ) proteins, mucin components and pro- and anti-inflammatory cytokines will be further quantified using nCounter® Analysis System (NanoString Technologies) and ELISA.These models aim to dissect the molecular mechanisms underlying gut-brain barrier dysfunction in ASD, to evaluate the therapeutic potential of microbiota-targeted strategies through the MGBA, and to identify novel peripheral biomarkers with translational relevance for early diagnostic and intervention strategies. Framed within the One Health paradigm, this research recognizes the interconnection between intestinal health, neurological integrity, immune regulation and microbiota composition, embracing a systemic vision of human health that goes beyond the individual.