Antigen-presenting aged neutrophils induce CD4+ T cells to exacerbate inflammation in sepsis
Hui Jin, … , Max Brenner, Ping Wang
Hui Jin, … , Max Brenner, Ping Wang
Published July 17, 2023
Citation Information: J Clin Invest. 2023;133(14):e164585. https://doi.org/10.1172/JCI164585.
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Research Article Cell biology Immunology

Antigen-presenting aged neutrophils induce CD4+ T cells to exacerbate inflammation in sepsis

Abstract

Extracellular cold-inducible RNA-binding protein (eCIRP) is a key mediator of severity and mortality in sepsis. We found that stimulation of mouse bone marrow–derived neutrophils (BMDNs) with eCIRP generated a distinct neutrophil subpopulation, characterized by cell surface markers of both antigen-presenting cells and aged neutrophils as well as expression of IL-12, which we named antigen-presenting aged neutrophils (APANs). The frequency of APANs was significantly increased in the blood, spleen, and lungs of WT mice subjected to cecal ligation and puncture–induced sepsis but not in CIRP–/– mice. Patients with sepsis had a significant increase in circulating APAN counts compared with healthy individuals. Compared with non–APAN-transfered mice, APAN-transferred septic mice had increased serum levels of injury and inflammatory markers, exacerbated acute lung injury (ALI), and worsened survival. APANs and CD4+ T cells colocalized in the spleen, suggesting an immune interaction between these cells. APANs cocultured with CD4+ T cells significantly induced the release of IFN-γ via IL-12. BMDNs stimulated with eCIRP and IFN-γ underwent hyper-NETosis. Stimulating human peripheral blood neutrophils with eCIRP also induced APANs, and stimulating human neutrophils with eCIRP and IFN-γ caused hyper-NETosis. Thus, eCIRP released during sepsis induced APANs to aggravate ALI and worsen the survival of septic animals via CD4+ T cell activation, Th1 polarization, and IFN-γ–mediated hyper-NETosis.

Authors

Hui Jin, Monowar Aziz, Atsushi Murao, Molly Kobritz, Andrew J. Shih, Robert P. Adelson, Max Brenner, Ping Wang

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Figure 5

APAN-activated CD4+ T cells promote NET formation via IFN-γ.

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APAN-activated CD4+ T cells promote NET formation via IFN-γ. (A) BMDNs (...
(A) BMDNs (1 × 107) were stimulated with eCIRP (1 μg/mL/106 cells) for 6 hours. FACS-isolated APANs, nAPANs, and naAPNs (1 × 106 cells/mL) were cultured with or without mouse splenic CD4+ T cells (1 × 106 cells/mL) in 48-well plates (200 μL, final volume) in the presence of IgG or IFN-γ–neutralizing Ab. After 4 hours, culture supernatant NETs were assessed by ELISA. Data reflecting ≥3 independent experiments are expressed as mean ± SEM and compared by ANOVA and SNK test. n = 6–15/group. *P < 0.05 vs. nAPAN only, #P < 0.05 vs. nAPAN+CD4+ T cells, †P < 0.05 vs. APAN+CD4+ T cells+IgG. (B) The mRNA expression of IFN-γR in APANs, nAPANs, and naAPNs was assessed by real-time qPCR. (C) APANs, nAPANs, naAPNs were stained with anti–IFN-γR Ab and then assessed IFN-γR expression by flow cytometry. Data reflecting ≥3 independent experiments are expressed as mean ± SEM and compared by ANOVA and SNK test. n = 6–12/group. *P < 0.05 vs. nAPAN, #P < 0.05 vs. naAPN. (D–F) Mouse BMDNs (1 × 106 cells/mL) were stimulated with IFN-γ (10 ng/mL) with or without eCIRP (1 μg/mL) and, 4 hours later, assessed for NET formation using 3 methods: (D) fluorescent microscopy by staining the cells with Sytox Green, where the white arrows indicate the NET-like structures (original magnification, ×200 [rows 1 and 2]; ×400 [row 3]; scale bar: 200 μm [rows 1 and 2]; 100 μm [row 3]) (n = 6–9/group); (E) flow cytometry by staining unpermeabilized cell for myeloperoxidase (MPO) and citH3 Ab (n = 9–11/group); and (F) ELISA using neutrophil elastase (NE) and anti-dsDNA Ab (n = 6/group). (G) Isolated human neutrophils (1 × 106 cells/mL) were stimulated with IFN-γ (10 ng/mL) with and without eCIRP (1 μg/mL), and, 4 hours later, NET formation was assessed by flow cytometry by staining unpermeabilized neutrophils with anti-human MPO and citH3 Ab. Data are expressed as mean ± SEM and compared by 1-way ANOVA and SNK test. n = 6/group. *P < 0.05 vs. PBS, #P < 0.05 vs. eCIRP.