Influence of Microbial Products on the Developmental Programming of the Enteric Nervous System

Abstract

Bacterial colonization of the gastrointestinal (GI) tract takes place during the perinatal period, thus coinciding with a critical window of enteric nervous system (ENS) development. Previous work has found that the myenteric plexus of germ free (GF) mice exhibits structural and functional aberrancies in the early postnatal period as compared to specific pathogen free (SPF) and altered Schaedler flora (ASF) mice. These early life disruptions in ENS development in GF mice compared to SPF mice, and more specifically ASF mice, support the notion that a simple intestinal flora is sufficient for directing perinatal ENS development. It has previously been believed that the intrauterine environment during fetal development is sterile. Recent evidence showing successful isolation of microbial communities from embryonic cord blood and newborn meconium that are not of maternal origin suggests that the intrauterine environment is not sterile and is unique to the fetus. Coinciding with this timeline of fetal microbial colonization is the development of the ENS through a population of precursors known as enteric neural crest derived cells (ENCDCs). The prenatal period is characterized by rapid expansion and differentiation of ENCDCs into the many enteric neuron subtypes that comprise the ENS. Terminal differentiation of ENCDCs continues into the early postnatal period. In the current study, we tested the hypothesis that ENCDCs interact directly with microbial products during ENS development. Further, these ENCDC-bacterial product interactions influence the proliferation, apoptosis, and chemical coding of enteric neuron precursors. These objectives were carried out in an in vitro model of ENCDCs isolated from the prenatal period that was established for the first time in our lab using immunoselection. Further, this model was characterized at key timepoints for proliferation, apoptosis, and differentiation. Our results are suggestive of direct ENCDC interactions with lipopolysaccharide (LPS), a TLR4 ligand, and flagellin, a TLR5 ligand, in stimulating ENCDC proliferation and differentiation into early born neurons of nitrergic and serotonergic subtypes. Peptidoglycan derivatives, muramyl dipeptide (MDP) and ƴ-D-Glu-mDAP (iE-DAP), ligands for NOD2 and NOD1 respectively, appear to mainly stimulate differentiation into nitrergic neurons, and possibly serotonergic neurons. The lack of apoptosis in all conditions is consistent with the notion that apoptosis is not an important characteristic of ENCDC maturation and ENS development. Finally, the lack of significance for differentiation into dopaminergic neurons could be further evidence of their late born nature, which has previously been reported to be stimulated by serotonin after the emergence of serotonergic neurons.