Objective: Neonatal enteric infection disrupts development of the microbiota–gut–brain (MGB) axis, yet the immunological mechanisms driving persistent gut dysfunction remain unclear. While prior work implicates intestinal epithelial cell (IEC) nucleotide-binding oligomerization domain-1 (NOD1) signaling in long-term MGB outcomes, its role in neonatal mucosal immunity, epithelial repair, and enteric nervous system (ENS) integrity is unknown. Here, we define IEC-immune-neuronal responses to neonatal enteropathogenic Escherichia coli (EPEC) infection and test whether probiotic-derived muropeptides (MP) improve these outcomes. Hypothesis: IEC NOD1 signaling coordinates neonatal mucosal immunity, epithelial remodeling, and ENS protection after neonatal enteric infection, and probiotic-derived MP enhance these host responses. Methods: IEC Nod1 conditional knockout (Nod1ΔIEC; Cre+) and WT (Cre–) pups were infected at postnatal day (P)7 with EPEC by oral gavage. Colonization was assessed at P14 and P28. Intestinal inflammation (qPCR), immune infiltration (flow cytometry), and enteric neuronal density (HuC/HuD ANNA-1 whole-mount immunofluorescent IF confocal imaging) were assessed at P14. Goblet cells were quantified by periodic acid–Schiff (PAS) staining and IEC proliferation by Ki67 + IEC counts along the crypt–villus axis. MP were isolated from Lactobacillus spp. and administered from P7-14 to test their immunomodulatory potential, with qPCR, PAS, and IF used to assess epithelial remodeling. Results: Neonatal EPEC infection induced a robust pro-inflammatory response in WT pups, with significant increases in cytokines (Il1β, Il6, Il12, and Il22) and chemokines (Ccl2, Ccr2, Cxcl1, and Cxcr1) vs WT sham pups (P < 0.001), in contrast to Nod1ΔIEC infected pups. Flow cytometry demonstrated increased recruitment of monocytes and macrophages in WT, but not Nod1ΔIEC, EPEC-infected pups (P < 0.001). WT EPEC pups displayed significant myenteric neuronal loss following infection (HuC/HuD + neurons vs WT sham, P < 0.0001), whereas Nod1ΔIEC mice were fully protected. WT EPEC pups also showed decreased IEC proliferation (Ki67 + cells per crypt, P < 0.01) and increased Muc2 and Slc26a3 expression (P < 0.01). WT EPEC pups had an increased density of PAS + goblet cells in the distal ileum vs WT sham pups (P < 0.05), an effect absent in Nod1ΔIEC EPEC pups. MP supplementation did not alter EPEC burden, but significantly reduced infection-induced pro-inflammatory cytokines (P < 0.01) and chemokines (P < 0.05). In contrast, MP administration did not restore goblet cells (PAS + cells and Muc2 expression) in EPEC+MP groups (P < 0.001), suggesting preserved or enhanced mucus barrier function. Importantly, MP-treated mice showed normalization of crypt–villus length and Ki67 + IEC counts vs EPEC+vehicle (P < 0.05), indicating improved epithelial repair. Lastly, MP supplementation partially normalized stress- and serotonergic-related transcripts (Crfr2 and 5htr2c) in the ileum (P < 0.05 vs EPEC+vehicle), aligning with preservation of enteric neurons and suggesting a host-directed mechanism that may mitigate downstream MGB and behavioral alterations. Conclusions: IEC NOD1 serves as a key regulator of neonatal mucosal immunity, coordinating inflammatory signaling, immune-cell recruitment, epithelial repair, and preservation of the ENS following neonatal enteric infection. Infection-induced enteric neuronal loss emerges as a previously unrecognized consequence of neonatal EPEC exposure. Probiotic-derived MP mitigated intestinal inflammation, highlighting a promising new strategy to promote gut resilience during early development. Funding: NIH R01AT009365, NIH R21MH108154 This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Park et al. (Fri,) studied this question.