OX40 expression in neutrophils promotes hepatic ischemia/reperfusion injury
Hua Jin, Chunpan Zhang, Chengyang Sun, Xinyan Zhao, Dan Tian, Wen Shi, Yue Tian, Kai Liu, Guangyong Sun, Hufeng Xu, Dong Zhang
Hua Jin, Chunpan Zhang, Chengyang Sun, Xinyan Zhao, Dan Tian, Wen Shi, Yue Tian, Kai Liu, Guangyong Sun, Hufeng Xu, Dong Zhang
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Research Article Hepatology Transplantation

OX40 expression in neutrophils promotes hepatic ischemia/reperfusion injury

Abstract

Neutrophils play critical roles during the initial phase of hepatic ischemia/reperfusion injury (HIRI). However, the regulation of neutrophil activation, infiltration, and proinflammatory cytokine secretion has not been fully elucidated. In this study, we revealed that OX40 was expressed by neutrophils, its expression in neutrophils was time-dependently upregulated following HIRI, and Ox40 knockout markedly alleviated liver injury. Compared with wild-type neutrophils, the adoptive transfer of Ox40–/– neutrophils decreased HIRI in neutrophil-depleted Rag2/Il2rg–/– or Ox40–/– mice. Moreover, consistently, the in vitro experiments showed that Ox40 not only prolonged neutrophil survival but also promoted proinflammatory cytokines, ROS production, and even neutrophil chemotaxis. Further investigation demonstrated that the knockout of Ox40 in neutrophils inhibited NF-κB signaling via the TRAF1/2/4 and IKKα/IKKβ/IκBα pathways. OX40L and OX86 stimulation could enhance neutrophil activation and survival in vitro and in vivo. In conclusion, our study provides a new understanding of OX40, which is expressed not only in adaptive immune cells but also in innate immune cells, i.e., neutrophils, contributing to the activation and survival of neutrophils. These findings provide a novel potential therapeutic target for the prevention of HIRI during liver transplantation or hepatic surgery.

Authors

Hua Jin, Chunpan Zhang, Chengyang Sun, Xinyan Zhao, Dan Tian, Wen Shi, Yue Tian, Kai Liu, Guangyong Sun, Hufeng Xu, Dong Zhang

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Figure 6

OX40L and OX40 agonist enhanced neutrophil activation and survival.

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OX40L and OX40 agonist enhanced neutrophil activation and survival. (A) ...
(A) Relative mRNA expression of Ox40l in the liver was measured by real-time PCR after 1 hour of reperfusion (n = 6–8/group). BM neutrophils (CD45+Ly6G+CD11b+ subsets) from WT mice were obtained by FACS and stimulated with or without OX40L-blocking antibody (2 μg/mL) for 3 hours followed by OX40L protein (0.5 μg/mL) for 6 hours. In other experiments, neutrophils were treated with OX40 agonist mAb OX86 (50 μg/mL) or control rat IgG1 (50 μg/mL) for 6 hours. (B) Relative MFI levels of ROS in neutrophils were measured by flow cytometry (n = 4/group). (C) Alamar Blue assay in each group (n = 10/group). Representative flow cytometric results (D) and statistical analyses (E) of annexin V+, 7-aminoactinomycin D–positive (7-AAD+) neutrophils measured by flow cytometry (n = 5/group). Relative MFI levels of TNF-α (F) and IL-1β (G) produced by neutrophils (n = 4/group). Rag2/Il2rg–/– mice were intraperitoneally injected with 0.25 mg rat IgG or OX40 agonist mAb (clone OX86, BioXCell) in 0.4 mL Dulbecco’s PBS (DPBS) at the beginning of reperfusion. (H) Representative flow cytometric results and statistical analyses of proportions of neutrophils in CD45+ gates (n = 5/group). (I) Relative MFI levels of ROS in neutrophils were measured by flow cytometry (n = 5/group). (J) Representative flow cytometric results and statistical analyses of annexin V+ neutrophils measured by flow cytometry (n = 5/group). Data represent mean ± SD. Significant differences were analyzed using 2-tailed Student’s t test and 1-way ANOVA. *P < 0.05; **P < 0.01; NS, nonsignificant.