Synergistic Modulation of the Gut–Brain–Immune Axis by a Dual <i>Lactobacillus</i> Combination in a Murine IBS Model

The gut microbiota is a key regulator of immune and neuroendocrine pathways along the gut-brain axis. Disruption of this bidirectional communication contributes to irritable bowel syndrome (IBS), a multifactorial disorder associated with gastrointestinal dysfunction and psychiatric comorbidities. Although microbiota-targeted therapies are promising, most current studies rely on single-strain interventions with limited efficacy, and the bioactive components as well as their host-mediated mechanisms remain insufficiently characterized. Here, we demonstrate that oral co-administration of <i>Lactobacillus paracasei</i> KBL382 and <i>Lactobacillus plantarum</i> KBL396 synergistically ameliorates IBS-like symptoms in a zymosan-induced mouse model. The combination therapy outperformed individual strains in reducing colonic shortening, abnormal cecal morphology, mucosal inflammation, and anxiety-like behaviors. These effects were accompanied by bidirectional neurobiological changes, including downregulation of colonic brain derived neurotrophic factor (BDNF) and serotonin 3A (5-HT3A), and restoration of hippocampal serotonergic signaling. Immunologically, the treatment decreased pro-inflammatory M1 macrophages and inflammatory dendritic cells (DCs), while increasing tolerogenic DCs and regulatory T cells in mesenteric lymph nodes. Furthermore, >100 kDa macromolecular fractions isolated from both strains enhanced the IL-10/IL-6 ratio and serotonin transporter (SERT) expression in vitro. These effects were abolished by protease or mutanolysin treatment, implicating structurally integrated peptidoglycan-protein complexes as key immunoregulatory and neuroactive components. The complexes engaged MyD88-dependent signaling pathways, promoting regulatory immune phenotypes. Importantly, therapeutic effects were preserved in microbiota-depleted mice, demonstrating a microbiota-independent, host-targeted mechanism. These findings demonstrate that defined <i>Lactobacillus</i> strains synergistically modulate neuroimmune pathways via bioactive macromolecules, offering a host-directed strategy for managing the multifactorial symptoms of IBS.