Proenkephalin produced by neonatal T-bet+ Treg cells promotes periportal hepatocyte maturation.

The mechanisms that govern postnatal hepatocyte differentiation and maturation in the periportal region, where Notch signaling is high, remain incompletely understood. Here we demonstrate that 2-week-old mice deficient in lymphocytes, CD4+ T cells or T-bet+ regulatory T (Treg) cells exhibit comparable increases in ductal plate cell/cholangiocyte differentiation and impaired periportal hepatocyte maturation. We found that the transient enrichment of periportal region-localized T-bet+ Treg cells and their expression of proenkephalin (PENK) are crucial for coordinating hepatocyte and cholangiocyte differentiation in this region. T-bet deletion in Treg cells impacts their acquirement of a tissue residence signature and their PENK expression. Depletion of Penk in Tregs or the administration of naltrexone, an antagonist of the opioid growth factor receptor (OGFr), results in similar liver defects. The supplementation of PENK-derived methionine enkephalin (Met-ENK) reduces ductal plate cholangiocytes and enhances hepatocyte maturation in mice lacking T-bet+ Tregs and in 3-dimensional hepatocyte culture. Molecularly, the Met-ENK/OGFr axis counteracts periportal Notch signaling by downregulating Adam10 expression and promotes adult splicing program by upregulating Esrp2 expression. Our findings uncover a previously unrecognized mechanism through which periportal T-bet+ Treg cells foster liver maturation independently of their immune regulatory functions. It also underscores the critical role of Treg-associated immune zonation in facilitating periportal hepatocyte maturation in neonatal mice.