Deciphering how CD71+ erythroid cells influence the microbiome and enteric nervous system in newborns

Introduction: Mounting preclinical and clinical evidence strongly supports a crucial role for the gut microbiota in health. Dysbiosis of the microbiome is associated with a spectrum of disorders, including inflammatory bowel disease (IBD) and necrotizing enterocolitis. We have found that CD71+ erythroid cells (CECs) are abundant in the gut of newborns. The removal of CECs from the intestinal tissues by the anti-CD71 antibody disrupts immune homeostasis and results in inflammation. This suggests an essential role for CECs in the adaptation of newborns to colonization with microbial communities. Additionally, we have observed that CECs secrete artemin, a neurotrophic factor. Artemin acts on neurons by binding to the tyrosine kinase membrane receptor (RET) and GDNF receptor-α 3 (GFRα3) complex. Notably, we have observed a lower frequency of CECs in the cord blood of C-Section compared to vaginally delivered twins. Similarly, preterm deliveries have a lower frequency of CECs in their cord blood. These observations may, in part, explain the underlying mechanisms associated with dysbiosis and other GI tract complications in these infants. Our objective is to explore the short and long-term effects of CECs on establishing the microbiome, whether the microbiome determines the generation of CECs, and the role of CEC-derived artemin on the development of the enteric nervous system (ENS). Methods: CECs were depleted using the anti-CD71 antibody in Balb/c mice (3-6 days old); control mice were treated with the isotype control antibody. To examine the short-term effects of CECs on the microbiome, mice were sacrificed two days post-treatment; for long-term effects, the mice were sacrificed two weeks post weening. Bacterial genomic DNA was isolated for 16S rRNA gene amplicon sequencing. The small intestinal and colonic tissue were collected separately and subjected to RNA isolation. RNA samples were submitted for RNAseq analysis and qRT-PCR to evaluate the pattern of gene expression profile in the absence/presence of CECs in the gut. The 2-∆∆Ct method was used to calculate the relative fold gene expression of samples. Results: At day 3, germ-free mice had significantly reduced levels of CECs in the small and large intestines when compared to the wild-type counterpart (p < 0.005). Moreover, there was a higher frequency of CECs in the gut of neonatal mice compared to adults (p < 0.05). Depletion of CECs resulted in a significant downregulation of SOX10, FOXP3, and PHOX2b, which are involved in the ENS development at day 4 and day 22 (p < 0.05). Interestingly, depletion of CECs at day 4 did not cause downregulation of RET, however, at day 22 RET expression was significantly downregulated (p < 0.005). Conclusion: The frequency of CECs are elevated in the gut of neonatal mice. Furthermore, preliminary data suggests a role for CECs in relation to the proper development of the ENS through differential expression of transcriptional factors. We anticipate that these proposed studies provide a comprehensive understanding of the role of CECs in the health of the GI tract in the newborn. Despite the complexity of the cellular biology of the gut, our data will provide a novel role for CECs in the dialogue between the microbiota, immune system, and the ENS.