Treg cells retain stable lineage commitment during pregnancy in mice after late gestation inflammatory challenge

Inflammation is a major driver of preterm birth, a common pregnancy disorder and the leading cause of childhood death. T regulatory (Treg) cells are essential mediators of maternal fetal tolerance and are critical for constraining uterine inflammation. In some tissue settings, loss of Foxp3 expression can cause instability in Treg cell lineage commitment, elevated production of proinflammatory cytokines, and compromised suppressive function. Whether preterm birth susceptibility is associated with loss of lineage fidelity and adoption of pro-inflammatory phenotypes in Treg cells is unknown. In this study, we investigated the lineage stability of Treg cells in vivo in mice using a Foxp3 fate-mapping system and models of preterm birth induced by late-gestation inflammatory challenge with lipopolysaccharide (LPS) or interleukin-1β (IL-1β). Ex-Foxp3-expressing (exFoxp3) cells were observed in the uterus-draining lymph nodes (udLNs) in non-pregnant mice and in similar abundance across normal gestation, and a proportion expressed pro-inflammatory cytokines IFNγ and/or IL-17A. Bulk RNA-sequencing of sorted Treg and exFoxp3 cells from late-gestation udLNs revealed substantial loss of the Treg cell lineage program in exFoxp3 cells, characterised by reversal in expression of canonical Treg cell genes and pathways. Late gestation LPS or IL-1β administration to induce preterm birth did not expand the exFoxp3 cell population. We conclude that uterine Treg cells exhibit a high level of lineage stability in pregnancy regardless of proinflammatory challenge. Whether there is any biological or pathophysiological significance of exFoxp3 cells in gestational tissues remains to be defined.