Society: AGA

Background: Postoperative ileus (POI) is a common consequence of abdominal surgery and a neuroinflammation-based disorder of the gastrointestinal tract, which results in motility disturbances compromising patients' recovery. The underlying neuroinflammation involves the activation of resident macrophages and monocyte-derived macrophages (MDM) infiltration into the intestine's muscularis externa (ME). Immune checkpoint proteins, including PD-1 and its ligands PD-L1 and PD-L2, play a prominent role in cancer immunology, but their role in non-oncological inflammation-driven diseases is still elusive. Herein, we studied the role of PD-1/PD-L1/PD-L2 signaling in POI.
Materials and methods: POI was induced by a surgical intestinal manipulation in wildtype, PD-1^-/-, PD-L1^-/-and PD-L2^-/-mice. We performed flow cytometry to identify the cellular sources of PD-1 and PD-L1 and used a blood cell transfer model to determine the role of a leukocyte-restricted PD-1 deficiency. qPCR and SMART-Seq2 analyses were used to analyze transcriptomes of sorted CX3CR1^GFP MDM from wildtype and PD-1^-/- macrophage populations. Immunofluorescence microscopy visualized neuronal cell loss, PD-1 and PD-L2 expression and in vivo macrophage phagocytosis assay and GI transit measurements were used as functional readouts of PD-1 deficiency.
Results: PD-1, PD-L1 and PD-L2 mRNA levels were induced in the postoperative ME upon intestinal manipulation. PD-1^-/- and PD-L1^-/- but not PD-L2^-/- mice were protected from POI- PD-1^-/- mice showed a 50% reduction of MDM infiltration and reduced inflammation-mediated neuronal loss. Ly6C^-Cx3CR1⁺ resident macrophages and Ly6C⁺Cx3CR1⁺ MDM were identified as the primary cellular source of PD-1. Both cell populations showed reduced in vivo phagocytosis under PD-1 deficiency, and a cell transfer experiment revealed a more than 60% reduced postoperative ME infiltration of PD-1-deficient compared to wildtype controls. The primary PD-L1 source are infiltrating MDM, monocytes and neutrophils. Gene ontology (GO) analysis within the ME of PD-1^-/- mice revealed lower enrichment levels of genes regulating metabolic respiratory chain processes, oxidative stress, and immune functions. Moreover, sorted CX3CR1⁺Ly6C⁺ MDMs of PD-1^-/- mice showed stronger enrichment of genes associated with various immune functions, host defense and negative regulation of immune and neuronal cell death.
Conclusion: Our data provide new evidence on the role of PD-1 signaling in MDM during postoperative intestinal neuroinflammation following abdominal surgery. PD-1 deficiency prevents POI, results in a metabolic switch, and reduces infiltration capacity and phagocytosis of MDM. We conclude that interaction in PD-1 signaling might be a potential target in prevetion of POI or other immune-driven intestinal disorders.

Mona Breßer

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