The Notorious M.G.B.: The effects of neonatal enteric infection on the microbiota-gut-brain axis with insights into prevention and signaling mechanisms

Enteric infections remain a global health problem, with diarrheal diseases still a leading cause of death in children under 5 worldwide. Unfortunately, children exposed to multiple enteric infections suffer from additional comorbidities that lead to long-term malnutrition and cognitive delays. These findings suggest that establishment of the microbiota-gut-brain (MGB) axis in children is impaired by enteric infection. Therefore, characterizing the effects on the MGB axis and identifying mechanisms to target for therapies is essential to improving recovery from enteric infection. Current therapies focus on acute symptoms due to dehydration, relying on the self-limiting nature of the infection to clear in most children. The overall goal of the research outlined in this dissertation is to determine the impacts of neonatal enteric infection on development of the MGB axis in mice, identify mechanisms of signaling leading to the long-term impairments, and finally outline the potential for administration of probiotics to ameliorate those symptoms. In order to accomplish these goals, neonatal C57BL/6 mice were infected with enteropathogenic Escherichia coli (EPEC), an attaching and effacing enteric pathogen, in Chapter 2. The effects of the infection on the MGB axis were characterized during the infection (post-natal day 7 [P7] – P21) and once the mice reached adulthood (6-8wks old). Behavior tests, intestinal physiology, and composition of the gut microbiota were used to characterize the long-term effects of neonatal enteric infection on the MGB axis. Gene expression and confocal imaging were used to assess the changes in inflammation and neurogenesis in the axis. Following neonatal EPEC infection, adult mice had recognition memory deficits with increased neurogenesis and neuroinflammation in the hippocampus. These brain deficits were accompanied by intestinal pathophysiology and dysbiosis of the gut microbiota. Once the effects were characterized, a probiotic cocktail of Lactobacillus rhamnosus and Lactobacillus helveticus was administered during the infection (P7-P21) to ameliorate the MGB axis deficits found in adulthood following neonatal EPEC infection in Chapter 3. Again, behavior and intestinal physiology were measured to discover if this cocktail of probiotics could prevent the long-term effects of neonatal EPEC infection. Unfortunately, lack of robust memory in control mice made it impossible to determine if probiotics improved recognition memory deficits in EPEC infected mice. Similarly, intestinal physiology experiments were also inconclusive. Together, this data suggests that this cocktail of probiotics in the timeframe chosen do not alleviate the long-term effects of neonatal EPEC infection on the MGB axis. In an effort to identify signaling mechanisms along the MGB axis, nucleotide oligomerization domain (Nod)-like receptors, important in maintaining innate immunity following enteric pathogen infection, were studied in Chapter 4. Nod1 conditional knock-out (cKO) mice were bred in house using the Cre-Lox system, by crossing Nod1f/f mice with intestinal epithelial cell (IEC)-specific (villin: Vil.Cre) or hippocampal neuron specific (CaMIIK: Cam.Cre) Cre-expressing mice. Behavior and gene expression were assessed in adult cKO mice following neonatal EPEC infection. Like the probiotic supplementation studies, behavior tests were inconclusive due to lack of recognition memory in the control animals. In contrast, gene expression of neuroinflammation- and neurogenesis-related genes in the hippocampus of Vil.Cre cKO mice suggest a role for IEC Nod1 in the MGB axis. Overall, the findings in this dissertation demonstrate that neonatal enteric infection can significantly impair the MGB axis in adulthood in mice. While probiotics supplementation did not effectively alleviate the EPEC-induced deficits in the MGB axis, future studies with other probiotic species and alternative time frames of administration, including pre-treatment, may prove more effective. Finally, Nod1 expression in IECs was identified as a potential mechanism of communication in the MGB axis during neonatal EPEC infection. Taken together, these studies highlight the crucial role of the MGB axis in the downstream response to infection with enteric bacterial pathogens and may provide insight into novel treatment strategies for children to prevent these long-lasting impairments in the future