Influence of bacterial components on the developmental programming of enteric neurons

Abstract

Background: Intestinal bacteria have been increasingly shown to be involved in early postnatal development. Previous work has shown that intestinal bacteria are necessary for the structural development and intrinsic function of the enteric nervous system in early postnatal life. Furthermore, colonization with a limited number of bacteria appears to be sufficient for the formation of a normal enteric nervous system. We tested the hypothesis that common bacterial components could influence the programming of developing enteric neurons.

Methods: The developmental programming of enteric neurons was studied by isolating enteric neural crest-derived cells from the fetal gut of C57Bl/6 mice at embryonic day 15.5. After the establishment of the cell line, cultured enteric neuronal precursors were exposed to increasing concentrations of a panel of bacterial components including lipopolysaccharide, flagellin, and components of peptidoglycan.

Key result: Exposure to bacterial components consistently affected proportions of enteric neuronal precursors that developed into nitrergic neurons. Furthermore, flagellin and D-gamma-Glu-mDAP were found to promote the development of serotonergic neurons. Proportions of dopaminergic neurons remained unchanged. Proliferation of neuronal precursor cells was significantly increased upon exposure to lipopolysaccharide and flagellin, while no significant changes were observed in the proportion of apoptotic neuronal precursors compared to baseline with exposure to any bacterial component.

Conclusions and interfaces: These findings suggest that bacterial components may influence the development of enteric neurons.

Keywords: bacterial components; development; enteric nervous system; enteric neural crest-derived cells.

Figure 1

Characterization of ENCDC cultures. Flow cytometry of ENCDC cultures stained with antibodies against the immature neuronal marker, p75^NTR+‐FITC demonstrated 95.1% p75^NTR+‐positive, 97.3% viability in subculture 3, and 96.1% p75^NTR+‐positive, 98.2% viability in subculture 4 (a). Proportion of ENCDC cultures characterized for expression of p75NTR+, pH3, 5‐HT, nNOS, and TH. Scale bar = 20 µm (b). There was a significant increase in the proportion of serotonergic neurons and nitrergic neurons in subculture 5 (gray) compared to subculture 4 (black), but no significant difference in proliferating cells or dopaminergic neurons across the cultures. *p ≤ .05. Values are presented as mean ± SEM (c)

Figure 2

Role of LPS, flagellin, MDP, and iE‐DAP on ENCDC programming. LPS had a significant effect on increasing enteric neuronal precursor proliferation and development into nitrergic neurons, but no significant effect on apoptosis or development into dopaminergic neurons. LPS exhibited a significant effect on increasing serotonergic neurons although this was not significant with the control (a). Flagellin had a significant effect on increasing enteric neuronal precursor proliferation and development into serotonergic and nitrergic neurons. Proliferation appeared to be concentration‐dependent. Flagellin had no significant effect on apoptosis or development into dopaminergic neurons (b). MDP had a significant effect on promoting the development of nitrergic neurons. MDP had no significant effect on enteric neuronal precursor proliferation, apoptosis, or development into dopaminergic neurons. Development of serotonergic neurons was only significant between 0.01 µg/ml and 1 µg/ml, but not compared to the vehicle control (c). iE‐DAP had a significant effect on promoting the development of serotonergic and nitrergic neurons. iE‐DAP did not have a significant effect on enteric neuronal precursor proliferation, apoptosis, or development into dopaminergic neurons (d). *p ≤ .05. Values are presented as mean ± SEM