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Updated: May 5, 2026
Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
Sepsis-induced potentiation of peritoneal macrophage migration is mitigated by programmed cell death receptor-1 gene
Alfred Ayala1, Gwendolyn F Elphick, Ye Sul Kim
1Department of Surgery, Division of Surgical Research, the Alpert School of Medicine at Brown University/Rhode Island Hospital, Providence, R.I., USA.
Abstract:
The effect of programmed cell death receptor-1 (PD-1) on phagocyte function has not been extensively described. Here we report that experimental mouse sepsis, cecal ligation and puncture (CLP), induced a marked increase in peritoneal macrophage random migration, motility and cell spread, but these changes were lost in the absence of PD-1. Alternatively, phagocytic activity was inversely affected. In vitro cell culture imaging studies, with the macrophage cell line J774, documented that blocking PD-1 with antibody led to aggregation of the cytoskeletal proteins α-actinin and F-actin. Further experiments looking at ex vivo peritoneal macrophages from mice illustrated that a similar pattern of α-actinin and F-actin was evident on cells from wild-type CLP mice but not PD-1-/- CLP mouse cells. We also observed that fMLP-induced migration by J774 cells was markedly attenuated using PD-1 blocking antibodies, a nonselective phosphatase inhibitor and a selective Ras-related protein 1 inhibitor. Finally, peritoneal macrophages derived from CLP as opposed to Sham mice demonstrated aspects of both cell surface co-localization with CD11b and internalization of PD-1 within vacuoles independent of CD11b staining. Together, we believe the data support a role for PD-1 in mediating aspects of innate macrophage immune dysfunction during sepsis, heretofore unappreciated.
Insights
Programmed cell death receptor-1 (PD-1) influences macrophage function during sepsis. Blocking PD-1 impairs cytoskeletal organization and migration, suggesting a role in innate immune dysfunction.
Area of Science:
- Immunology
- Cell Biology
- Sepsis Research
Background:
- The role of programmed cell death receptor-1 (PD-1) in phagocyte function, particularly macrophages, during sepsis remains poorly understood.
- Sepsis is a life-threatening condition characterized by dysregulated host response to infection, often involving immune cell dysfunction.
Purpose of the Study:
- To investigate the impact of PD-1 on macrophage behavior and function during experimental sepsis.
- To elucidate the molecular mechanisms by which PD-1 influences macrophage migration, cytoskeletal dynamics, and phagocytosis.
Main Methods:
- Induction of experimental sepsis using cecal ligation and puncture (CLP) in wild-type and PD-1 knockout mice.
- In vitro studies using the J774 macrophage cell line with PD-1 blocking antibodies.
- Confocal microscopy to analyze cytoskeletal protein aggregation (α-actinin, F-actin).
- Assessment of cell migration, motility, cell spread, and phagocytic activity.
Main Results:
- Sepsis induced increased macrophage migration and motility, which was lost in PD-1 deficient mice.
- PD-1 blockade in vitro led to aggregation of α-actinin and F-actin in macrophages.
- fMLP-induced migration was attenuated by PD-1 blockade and inhibitors of phosphatases and Ras-related protein 1.
- PD-1 was found on the surface and within vacuoles of macrophages from septic mice.
Conclusions:
- PD-1 plays a significant role in regulating macrophage cytoskeletal organization and migration during sepsis.
- PD-1 signaling appears to contribute to innate macrophage immune dysfunction in the context of sepsis.
- These findings reveal a novel, previously unappreciated role for PD-1 in sepsis-induced immune impairment.
