Changes in the gut intra-epithelial lymphocyte landscape induced by erythropoietin treatment in a non-diseased mouse model

Erythropoietin (EPO) is a glycoprotein hormone primarily known for its central role in red blood cell production, but it is now also recognized for its immunomodulatory effects on a range of immune effectors. Several studies indicate that EPO helps regulate intestinal barrier integrity, although its effect on intraepithelial lymphocytes (IELs) is still unknown. Using multiparameter flow cytometry and both conventional and unsupervised analyses, we conducted an in-depth phenotypic assessment of IELs from the murine duodenum, jejunum, ileum and colon. Our results revealed that in EPO-treated C57BL/6 mice, the absolute IEL count in the colonic epithelium was more than twice that observed in control mice without changes in the frequencies of the various subpopulations, indicating a global increase. This was accompanied by integrin α4β7 overexpression. Furthermore, EPO treatment alters the intestinal microbiota, increasing bacterial load while reducing diversity, with a notable decrease in Muribaculaceae, a taxon involved in microbial cross-feeding. This alteration may contribute to epithelial imbalance and indirectly to the observed increase in IEL density. Importantly, EPO treatment did not trigger colonic mucosal inflammation, although the affected IELs show a microbiota-dependent decrease in CD8αα expression (known to downregulate IEL activity). The present study is the first to provide a phenotypic mapping of IEL populations in the digestive tract of C57BL/6 mice and highlighted a novel regulatory role of EPO on colonic IELs. These results raise important questions about the impact of exogenous EPO supplementation and endogenous EPO fluctuations on intestinal immune responses - particularly in the context of inflammatory diseases.