A role for Programmed cell death receptor-1 (PD-1) in regulating iNKT-cell modulation of neonatal sepsis

Author(s):

Eleanor Fallon, Brown University / Rhode Island Hospital; Daithi Heffernan; Tristen Chun, Rhode Island Hospital – Brown University; Christopher Muratore, Brown University; William Cioffi, Rhode Island Hospital; Alfred Ayala, RI Hospital

Background:

Neonatal sepsis induces immune dysfunction and organ failure. We previously demonstrated a role for PD-1 in mediating mortality in murine adult and neonatal sepsis as well as in surgical patients. In adult sepsis models, iNKT cells mediate end-organ changes and immune responses, including macrophage bacterial phagocytosis, mechanistically regulated by PD-1.

Hypothesis:

Given our previous findings, that PD-1 modulates neonatal sepsis survival, we hypothesize that this effect is due to iNKT mediated immune and end organ dysfunction.

Methods:

We utilized a polymicrobial sepsis model involving an intra-peritoneal injection of a cecal slurry(CS) or saline sham into 7 day old wild type (WT), iNKT^-/- or PD-1^-/- neonatal pups. Tissues were harvested at 24 hrs. Liver was assessed for histologic changes. Western blots were performed for both PECAM-1, assessing for hepatic endothelial gap junction integrity and Vinculin, a marker of hepatic focal adhesion. Peritoneal macrophage influx and phenotype was quantified via flow cytometry.

Results:

Histologically, CS in WT induced liver architectural distortion including marked vacuolization. These effects were intermediately present in PD-1^-/- pups, and were markedly attenuated in the iNKT^-/- pups. On Western blot, PECAM-1 expression was unchanged in the WT pups following CS; however PECAM-1 expression was increased in iNKT^-/- pups. Vinculin was decreased in WT. This decline was more marked in iNKT^-/- pups. PD-1^-/- pups were then assessed to ascertain a potential mechanism modulating these findings. Similarly, CS in PD-1^-/- pups was associated with increased PECAM-1 and decreased vinculin expression. To further delineate our prior observations of PD-1 attenuating peritoneal microbial clearance, we characterized the peritoneal macrophage phenotypes. CS vs Sham induced greater proportional influx of macrophages into the peritoneal cavity in the WT (49% vs 3%;p<0.01), with decreased levels of activation and the emergence of Ly6C^low subpopulation. This macrophage influx was markedly decreased in the iNKT^-/- mice (11% vs 4%;p=0.03). However compared to WT, iNKT^-/- macrophages displayed a higher level of activation and greater percentage of Ly6C^highmacrophages.

Conclusions:

We demonstrate a key role for iNKT cells in affecting end organ damage as well as alterations in phagocytes phenotypes in neonatal sepsis. This iNKT cell mediated effect is driven by the central checkpoint protein PD-1, a ligand for which therapeutic agents are currently clinically available.