Single-cell profiling of mouse spleen myeloid cells

Neonates are at higher risk of inflammatory disorders due to their immature immune system. The timely appearance of regulatory immune cells in early life contributes to the control of inflammation in neonates via induction of immunotolerance, the mechanisms underlying which remain poorly understood. In this study, we identified a subset of neonatal monocytes with high expression of transmembrane protein Neuropilin-1 (Nrp1), termed Nrp1high monocytes. Nrp1high monocytes displayed potent immunosuppressive activity and their frequencies declined in age-dependent manner. Deletion of Nrp1 in myeloid cells aggravated the severity of neonatal inflammatory disease necrotizing enterocolitis (NEC). Adoptive transfer of Nrp1high monocytes led to remission of NEC. Mechanistic studies showed that Nrp1, via binding with its ligand semaphorin-4a (Sema4a), induced intracellular p38-MAPK/mTOR signaling and activated transcription factor KLF4. KLF4 activation led to transcription of Nos2 and enhanced the production of nitric oxide, a key mediator of immunosuppression in monocytes. These observations uncover an important immunosuppressive axis in neonatal monocytes, and provides potential therapeutic strategy for inflammatory disorders in neonates. Overall design: Spleen was harvested from C57BL/6J mice (7 days) and treated with ACK lysis buffer. CD3-CD19-CD11b+ cells were sorted with a MoFlo Astrios EQ Cell Sorter and performed single cell RNA-seq using a Chromium Controller instrument (10× Genomics).