Introduction Sepsis and the ensuing immune dysfunction, continues to be a major contributor to neonatal morbidity and mortality. Neonatal sepsis also is associated with profound immune dysfunction. We have recently identified a role for a family of co-inhibitory molecules that are altered in murine sepsis and in critically ill adult patients, which may be a target for development of novel therapies. There is, however, a paucity of data pertaining to the role of co-inhibitory check point proteins in the control and modulation of neonatal sepsis. Methods The cecal slurry (CS) model consists of harvesting the cecal contents of an adult wild type (Wt) male mouse and combining it with 5% dextrose to create a cecal slurry (CS) with a concentration of 80mg/ml (LD70 at 7 days). Neonatal mice (5–7 days of age) underwent intraperitoneal injection (IP) of the CS or IP of 0.9% saline for sham procedure (Sh). Wild Type (C57BL/6) or PD-1−/− mice were used. 7 day survival study was undertaken. Cytometric bead array was used for cytokine expression. Blood and peritoneal fluid was cultured for bacterial burden. Flow cytometry was used to assess the peritoneal cavity cell populations. Results There was no mortality following Sh in either WT or PD-1−/− pups. PD-1 markedly affected sepsis survival with significantly improved survival in the PD-1−/− pups (40% versus 80%; p<0.01). This survival improvement was not associated with any difference in bacterial clearance. The bacterial burden was equivalent between WT and PD-1−/− pups at 24 hours following CS. However, PD-1−/− pups did display an increased circulating cytokine response to the CS compared with WT, with increased expression of IL-6, IL-10 and TNF-α levels. Within the peritoneal cavity, sepsis induced an influx of neutrophils, a finding that was increased in PD-1−/− pups. Although the T-cell response was unaffected by PD-1, it was noted that CS induced a loss of peritoneal B-cells in WT, while the peritoneal B-cell population was preserved in PD-1−/− pups. Conclusion Our data suggests that the checkpoint protein, PD-1, plays an important role in controlling the immune response to sepsis in the neonate, ultimately affecting sepsis related mortality in this neonatal murine model of sepsis. Akin to adult studies, this data further emphasizes the potential therapeutic target for PD-1 across a spectrum of septic patients.

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Invariant natural killer T-cells (iNKT) are a subset of T-cells that play a regulatory role in sepsis. Following cecal ligation and puncture (CLP), iNKT cells emigrate from the liver and into the circulation and peritoneum in a manner dependent upon co-inhibitory molecule Programmed Cell Death Receptor 1 (PD-1). We hypothesized that the effect of PD-1 on iNKT cell emigration was dependent upon the direct PD-1:PD-L1 interaction and that PD-1 and PD-L1 would play a role in chemotaxis and chemokine receptor expression. Adoptive transfer of Vybrant-labelled wild type (WT) cells showed the donor iNKT cells migrated from the liver to the peritoneum following CLP, but PD-L1 deficient donor iNKT cells did not. In a chemotaxis assay, WT iNKT cells chemotaxed to CXCL12, but PD-1 and PD-L1 deficient iNKT cells did not. Using flow cytometry to evaluate chemokine receptor expression, peritoneal iNKT expression of CXCR4 increased following CLP in the WT, PD-1, and PD-L1 deficient animals, and CXCR6 increased in the WT and PD-1 deficient animals. In conclusion here we document that the hepatic emigration of iNKT cells following CLP to the peritoneum appears dependent upon the direct PD-1:PD-L1 interaction, however, while PD-1 and PD-L1 appear to play a role in chemotaxis, this is unlikely a reflection of iNKT cell chemokine receptor expression changes.