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High‑dose X‑ray irradiation induces NETosis via the eCIRP/TREM‑1 axis in mouse neutrophils

  • Authors:
    • Satoshi Yamaga
    • Atsushi Murao
    • Monowar Aziz
    • Ping Wang
    • Max Brenner
  • View Affiliations / Copyright

    Affiliations: Center for Immunology and Inflammation, The Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA
    Copyright: © Yamaga et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 157
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    Published online on: July 28, 2025
       https://doi.org/10.3892/ijmm.2025.5598
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Abstract

High‑dose ionizing radiation induces multiple types of tissue injuries, including hematopoietic dysfunction characterized by neutropenia. Neutrophil extracellular traps (NETs) released during NETosis may contribute to the neutropenia, and subsequent infection and inflammation. Triggering receptor expressed on myeloid cells‑1 (TREM‑1) is one of receptors responsible for NET formation and extracellular cold‑inducible RNA‑binding protein (eCIRP) is a ligand for the TREM‑1 receptor. The present study aimed to investigate NET formation after exposure to high‑dose ionizing radiation and to explore the underlying role of the eCIRP/TREM‑1 axis as its mechanism. Bone marrow‑derived neutrophils (BMDNs) isolated from C57BL/6 mice were exposed to 5 to 15 Gy irradiation. C57BL/6 wild‑type (WT), CIRP‑/‑ and TREM‑1‑/‑ mice were exposed to 10 Gy total body irradiation (TBI). NET formation was analyzed 24 h after irradiation using flow cytometry and fluorescence microscopy, and also after treatment with eCIRP. TREM‑1 cell surface expression on neutrophils was assessed using flow cytometry. Peptidyl arginine deiminase 4 (PAD4) protein expression levels in BMDNs were evaluated using western blotting. TREM‑1 and PAD4 mRNA expression levels in BMDNs were assessed using reverse transcription‑quantitative PCR. In vitro irradiation of neutrophils resulted in a dose‑dependent increase in NET formation, as assessed using flow cytometry and validated using fluorescence microscopy, which demonstrated the characteristic long extracellular DNA structures of NETs in irradiated neutrophils. The in vivo mouse model of TBI exhibited similar results. Furthermore, TREM‑1 expression in BMDNs was significantly increased after irradiation. Protein and mRNA levels of PAD4 were significantly upregulated after irradiation. The addition of eCIRP to BMDNs further increased NET formation post‑irradiation in vitro. Conversely, knockout of CIRP and TREM‑1 in vivo significantly attenuated radiation‑induced NET formation compared with that of WT mice. High‑dose ionizing radiation induced NET formation through the eCIRP/TREM‑1 pathway and may contribute to early neutropenia post‑irradiation.
View Figures

Figure 1

Exposure to ionizing radiation
induces NETs. (A) BMDNs were exposed to 5, 10 and 15 Gy radiation.
NETs were assessed via flow cytometry 24 h after irradiation. (B)
The frequencies of NET positive neutrophils are shown. Groups were
compared using a one-way ANOVA and Tukey's multiple comparison
test. (C) Representative images of BMDNs that were exposed to 10 Gy
radiation. NET formation was assessed by fluorescence microscopy
using SYTOX Green 24 h after irradiation. White arrows indicate the
NET structures. Scale bar, 200 μm (Bright field and SYTOX
Green); scale bar, 100 μm [SYTOX Green (enlarged)]. (D)
Wild-type adult mice were exposed to 10 Gy TBI. Bone marrow cells
were isolated 24 h after TBI and NETs were assessed using flow
cytometry. (E) The frequencies of NET positive neutrophils. Groups
were compared using an unpaired two-tailed Student's t-test. Data
are expressed as the mean ± SEM (n=6/group). *P<0.05,
**P<0.01 and ****P<0.0001. ns, not
significant; cont, control; NET, neutrophil extracellular traps;
BMDNs, bone marrow-derived neutrophils; TBI, total body
irradiation.

Figure 2

Radiation upregulates PAD4 mRNA and
protein expression in neutrophils. BMDNs were exposed to 10 Gy
radiation. (A) PAD4 mRNA expression levels were assessed 4 h after
irradiation via reverse transcription-quantitative PCR. PAD4
protein expression levels were evaluated by western blotting 20 h
after irradiation. (B) Representative PAD4 western blot and (C)
quantitative bar diagrams are shown. Groups were compared using an
unpaired two-tailed Student's t-test. Data are expressed as the
mean ± SEM (n=6/group). *P<0.05 and
***P<0.001. Cont, control; Irrad, irradiation; NET,
neutrophil extracellular traps; BMDNs, bone marrow-derived
neutrophils; PAD4, peptidyl arginine deiminase 4.

Figure 3

eCIRP induces NET formation after
irradiation. (A) Adult WT and CIRP−/− mice were exposed
to 10 Gy TBI. Bone marrow cells were isolated 24 h after TBI and
NETs were assessed by flow cytometry. (B) Frequencies of NET
positive neutrophils. Data are expressed as the mean ± SEM
(n=6/group). Groups were compared using a one-way ANOVA and Tukey's
multiple comparison test. (C) BMDNs were exposed to 10 Gy radiation
and treated with 1 μg/ml eCIRP immediately after
irradiation. NETs were assessed via flow cytometry 24 h after
irradiation. (D) Frequencies of NET positive neutrophils. Groups
were compared using a one-way ANOVA and Tukey's multiple comparison
test. Data are expressed as the mean ± SEM (n=5-6/group).
*P<0.05, ***P<0.001 and
****P<0.0001. Cont, control; Irrad, irradiation; NET,
neutrophil extracellular traps; BMDNs, bone marrow-derived
neutrophils; CIRP, cold-inducible RNA-binding protein; eCIRP,
extracellular CIRP; WT, wild-type.

Figure 4

Radiation upregulates neutrophil
TREM-1. BMDNs were exposed to 10 Gy radiation. (A) TREM-1 cell
surface expression was assessed via flow cytometry 24 h after
irradiation. (B) MFI values of TREM-1. (C) TREM-1 mRNA expression
levels were assessed 4 h after irradiation via using reverse
transcription-quantitative PCR. Groups were compared using an
unpaired two-tailed Student's t-test. (D) WT adult mice were
exposed to 10 Gy TBI. Bone marrow cells were isolated 24 h after
TBI and TREM-1 cell surface expression were assessed via flow
cytometry. (E) MFI values were used to evaluate TREM-1 expression
levels. Groups were compared by unpaired two-tailed Student's
t-test. Data are expressed as the mean ± SEM (n=6/group).
**P<0.01 and ****P<0.0001. Cont,
control; Irrad, irradiation; NET, neutrophil extracellular traps;
BMDNs, bone marrow-derived neutrophils; TREM-1, triggering receptor
expressed on myeloid cells-1; TBI, total body irradiation; MFI,
mean fluorescence intensity; WT, wild-type.

Figure 5

TREM-1 positively regulates
radiation-induced NETs. (A) BMDNs were exposed to 10 Gy radiation.
BMDNs were classified into two populations with high and low TREM-1
expression and NETs were assessed via flow cytometry 24 h after
irradiation. (B) Frequencies of NET positive neutrophils. Groups
were compared using an unpaired two-tailed Student's t-test. (C) WT
adult mice were exposed to 10 Gy TBI. Bone marrow cells were
isolated 24 h after TBI and classified into two populations with
high and low TREM-1 expression. NETs were assessed via flow
cytometry. (D) Frequencies of NET positive neutrophils. Groups were
compared by unpaired two-tailed Student's t-test. (E) Adult WT and
TREM-1−/− mice were exposed to 10 Gy TBI. Bone marrow
cells were isolated 24 h after TBI and NETs were assessed via flow
cytometry. (F) Frequencies of NET positive neutrophils. Groups were
compared using a one-way ANOVA and Tukey's multiple comparison
test. Data are expressed as the mean ± SEM (n=6/group).
*P<0.05, **P<0.01 and
***P<0.001. ns, not significant; NET, neutrophil
extracellular traps; BMDNs, bone marrow-derived neutrophils;
TREM-1, triggering receptor expressed on myeloid cells-1; TBI,
total body irradiation; MFI, mean fluorescence intensity; WT,
wild-type.

Figure 6

Exposure to ionizing radiation
induces NETs via eCIRP/TREM-1 axis. Radiation-induced eCIRP
upregulates and activates TREM-1 on neutrophils, resulting in
increased expression of PAD4 and NET formation, causing
neutropenia. NETs, neutrophil extracellular traps; PAD4, peptidyl
arginine deiminase 4; eCIRP, extracellular cold-inducible
RNA-binding protein; TREM-1, triggering receptor expressed on
myeloid cells-1; MPO, myeloperoxidase; cit, citrulline; CitH3,
citrullinated H3. The schema was created in BioRender. Murao, A.
(2025) https://BioRender.com/qhaiby0
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Copy and paste a formatted citation
Spandidos Publications style
Yamaga S, Murao A, Aziz M, Wang P and Brenner M: High‑dose X‑ray irradiation induces NETosis via the eCIRP/TREM‑1 axis in mouse neutrophils. Int J Mol Med 56: 157, 2025.
APA
Yamaga, S., Murao, A., Aziz, M., Wang, P., & Brenner, M. (2025). High‑dose X‑ray irradiation induces NETosis via the eCIRP/TREM‑1 axis in mouse neutrophils. International Journal of Molecular Medicine, 56, 157. https://doi.org/10.3892/ijmm.2025.5598
MLA
Yamaga, S., Murao, A., Aziz, M., Wang, P., Brenner, M."High‑dose X‑ray irradiation induces NETosis via the eCIRP/TREM‑1 axis in mouse neutrophils". International Journal of Molecular Medicine 56.4 (2025): 157.
Chicago
Yamaga, S., Murao, A., Aziz, M., Wang, P., Brenner, M."High‑dose X‑ray irradiation induces NETosis via the eCIRP/TREM‑1 axis in mouse neutrophils". International Journal of Molecular Medicine 56, no. 4 (2025): 157. https://doi.org/10.3892/ijmm.2025.5598
Copy and paste a formatted citation
x
Spandidos Publications style
Yamaga S, Murao A, Aziz M, Wang P and Brenner M: High‑dose X‑ray irradiation induces NETosis via the eCIRP/TREM‑1 axis in mouse neutrophils. Int J Mol Med 56: 157, 2025.
APA
Yamaga, S., Murao, A., Aziz, M., Wang, P., & Brenner, M. (2025). High‑dose X‑ray irradiation induces NETosis via the eCIRP/TREM‑1 axis in mouse neutrophils. International Journal of Molecular Medicine, 56, 157. https://doi.org/10.3892/ijmm.2025.5598
MLA
Yamaga, S., Murao, A., Aziz, M., Wang, P., Brenner, M."High‑dose X‑ray irradiation induces NETosis via the eCIRP/TREM‑1 axis in mouse neutrophils". International Journal of Molecular Medicine 56.4 (2025): 157.
Chicago
Yamaga, S., Murao, A., Aziz, M., Wang, P., Brenner, M."High‑dose X‑ray irradiation induces NETosis via the eCIRP/TREM‑1 axis in mouse neutrophils". International Journal of Molecular Medicine 56, no. 4 (2025): 157. https://doi.org/10.3892/ijmm.2025.5598
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