Long‐term Microbiota‐Gut‐Brain Axis Deficits Following Neonatal EPEC Infection

Abstract

BackgroundDiarrheal diseases remain a leading cause of death in children under age five worldwide. In addition, repeated early life exposures to diarrheal pathogens can result in co‐morbidities such as impaired growth and cognitive deficits. Given that neural development, differentiation of the gut, and colonization of the microbiota are interconnected and develop concurrently, it is critical to understand the mechanisms that connect them and how early life dysbiosis can lead to pathology of the microbiota‐gut‐brain (MGB) axis.HypothesisNeonatal enteropathogenic Escherichia coli (EPEC) infection disrupts the developing MGB axis leading to long‐term behavioral deficits, impaired intestinal physiology, and dysbiosis.MethodsNeonatal C57BL/6 mice were infected with EPEC (strain e2348/69), ΔescV (T3SS mutant), or vehicle (LB broth) via orogastric gavage (105CFU) at post‐natal day (P7). Behavior, intestinal physiology, and the microbiota were assessed in adulthood (6–8 weeks). A battery of three behavioral tests (novel object recognition [NOR] task, light/dark [L/D] box, and open field test [OFT]), 16S Illumina sequencing of fecal samples, Ussing chambers, immunofluorescence (IF), and qPCR were utilized to determine the long‐term effects of neonatal bacterial infection on the MGB axis.ResultsEPEC‐infected mice had impaired memory (NOR task) without evidence of anxiety‐like behavior (L/D box) or impaired locomotor activity (OFT) compared to ΔescV or sham‐infected controls. This was accompanied by increased expression of pro‐inflammatory cytokines (TNF‐α, IL‐12, IL‐1β, IL‐22, IL‐6), pattern recognition receptors (PRR: Nod1 and Nod2), and the neuroprotective brain‐derived neurotropic factor (BDNF) in the hippocampus of EPEC‐infected mice. Hippocampal IF revealed increased neurogenesis in the dentate gyrus (DG; cell proliferation [Ki67] and number of immature neurons [DCX]) and neuroinflammation (increased microglia activation [Iba1]) in the DG and CA1 regions following neonatal EPEC infection. Intestinal pathophysiology in EPEC‐infected mice was characterized by increased secretory state (short circuit current; Isc), permeability (conductance; G), and FITC‐dextran flux in the ileum and colon. In addition, EPEC‐infected mice had increased expression of pro‐inflammatory cytokines (TNF‐α, IL‐22, IL‐12, and IL‐6) and increased PRR expression (Nod1, Nod2) in the ileum, but no change in expression in the colon. Using 16S rRNA sequencing, we determined that neonatal infection significantly remodeled the composition of microbiota and decreased alpha diversity.Conclusion and SignificanceNeonatal EPEC infection disrupts the development of the MGB axis leading to long‐lasting physiological and behavioral deficits. These findings may have important clinical implications for pediatric patients exposed to bacterial enteric pathogens during early development.Support or Funding InformationThis work was funded by NIH 5R21MH108154‐01 (MGG), U54 HD079125 (MGG); T32 Animal models of infectious diseases NIH AI060555 (CH).