Neutrophil CEACAM1 determines susceptibility to NETosis by regulating the S1PR2/S1PR3 axis in liver transplantation
Hirofumi Hirao, Hidenobu Kojima, Kenneth J. Dery, Kojiro Nakamura, Kentaro Kadono, Yuan Zhai, Douglas G. Farmer, Fady M. Kaldas, Jerzy W. Kupiec-Weglinski
Hirofumi Hirao, Hidenobu Kojima, Kenneth J. Dery, Kojiro Nakamura, Kentaro Kadono, Yuan Zhai, Douglas G. Farmer, Fady M. Kaldas, Jerzy W. Kupiec-Weglinski
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Research Article

Neutrophil CEACAM1 determines susceptibility to NETosis by regulating the S1PR2/S1PR3 axis in liver transplantation

Abstract

Neutrophils, the largest innate immune cell population in humans, are the primary proinflammatory sentinel in the ischemia-reperfusion injury (IRI) mechanism in orthotopic liver transplantation (OLT). Carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1, CC1, or CD66a) is essential in neutrophil activation and serves as a checkpoint regulator of innate immune-driven IRI cascade in OLT. Although CC1 alternative splicing generates two functionally distinct short and long cytoplasmic isoforms, their role in neutrophil activation remains unknown. Here, we undertook molecular and functional studies to interrogate the significance of neutrophil CC1 signaling in mouse and human OLT recipients. In the experimental arm, we employed a mouse OLT model to document that ablation of recipient-derived neutrophil CC1-long (CC1-L) isotype aggravated hepatic IRI by promoting neutrophil extracellular traps (NETs). Notably, by regulating the S1P–S1PR2/S1PR3 axis, neutrophil CC1-L determined susceptibility to NET formation via autophagy signaling. In the clinical arm, liver grafts from 55 transplant patients selectively enriched for neutrophil CC1-L showed relative resistance to ischemia-reperfusion (IR) stress/tissue damage, improved hepatocellular function, and clinical outcomes. In conclusion, despite neutrophils being considered a principal villain in peritransplant tissue injury, their CC1-L isoform may serve as a regulator of IR stress resistance/NETosis in human and mouse OLT recipients.

Authors

Hirofumi Hirao, Hidenobu Kojima, Kenneth J. Dery, Kojiro Nakamura, Kentaro Kadono, Yuan Zhai, Douglas G. Farmer, Fady M. Kaldas, Jerzy W. Kupiec-Weglinski

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Figure 4

CC1 regulates NETosis via the S1PR2/S1PR3 signaling axis.

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CC1 regulates NETosis via the S1PR2/S1PR3 signaling axis. (A) WB of CC1,...
(A) WB of CC1, H3Cit (lysate), and VCL in WT or CC1-KO neutrophil cultures after S1P stimulation (100 nM or 1 μM, 4 hours). VCL was used as an internal control. (B) WB of H3Cit in the culture media of WT or CC1-KO neutrophils stimulated with S1P (1 μM, 4 hours). (C) Representative (n = 3) IF images of CC1, MPO, and DAPI in WT versus CC1-null neutrophils stimulated with S1P (1 μM, 4 hours) and quantitated for NET+ cells. Arrowheads indicate nucleus extrusion. Data are represented as mean ± SEM. Original magnification, ×200. *P < 0.05, Student’s t test, n = 3/group. (D) WB-assisted detection and relative intensity ratio of CC1, S1PR2, and S1PR3 expression in LPS-treated WT and CC1-KO neutrophils (500 ng/ml, 3 hours). VCL was used as an internal control for protein analysis. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA followed by Tukey’s HSD test. n = 3/group. (E) Representative (n = 3) IF images of MPO (green), H3Cit (red), and Hoechst 33342 (blue) in WT versus CC1-null neutrophils stimulated with S1P (1 μM, 4 hours) in the presence of JTE-013 (10 μM, 0.5 hours) or TY52156 (10 μM, 0.5 hours) and quantification of NET+ cells. Original magnification, ×200. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA followed by Tukey’s HSD test. n = 3/group.