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 3

APANs produce high levels of IL-12.

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APANs produce high levels of IL-12. (A and B) BMDNs (1 × 106) were treat...
(A and B) BMDNs (1 × 106) were treated with eCIRP (1 μg/mL) and brefeldin A (5 μg/ml). After 12 hours of stimulation with eCIRP, IL-12 expression in nAPANs (Ly6G+CXCR4–CD40–CD80–), naAPNs (Ly6G+CXCR4–CD40+CD80+), and APANs (Ly6G+CXCR4+CD62LloCD40+CD80+) was determined by flow cytometry. Data reflecting 3 experiments are expressed as mean ± SEM. n = 7/group. *P < 0.05 vs. nAPAN, #P < 0.05 vs. naAPN. (C and D) BMDNs (1 × 107) were stimulated with eCIRP (1 μg/mL/106 BMDNs) for 6 hours, and nAPANs, naAPNs, and APANs were sorted by flow cytometry. (C) IL12a and (D) IL12b mRNA was determined by real-time PCR. Data reflecting ≥3 experiments are expressed as mean ± SEM. n = 6/group. *P < 0.05 vs. nAPAN, P < 0.05 vs. naAPN. (E) BMDNs (1 × 107) were stimulated with eCIRP (1 μg/mL/106 BMDNs) for 6 hours, and nAPANs, naAPNs, and APANs sorted by flow cytometry were cocultured with CD4+ T cells (1:1 ratio) for 12 hours. IL-12p70 levels in the culture supernatants were assessed by ELISA. Data from ≥3 experiments are expressed as mean ± SEM. n = 5–6/group. *P < 0.05 vs. nAPAN without T cells, #P < 0.05 vs. nAPAN with T cells, †P < 0.05 vs. naAPN with T cells. (F and G) Mice were injected with eCIRP (5 mg/kg, i.v.), and 4 hours later, the frequencies of IL-12+ cells, PMNs, and APANs were assessed by flow cytometry. (F) Frequency of IL-12+ neutrophils and (G) of APANs in IL-12+ cells are shown. Data reflecting ≥3 experiments are expressed as mean ± SEM. n = 6/group. *P < 0.05 vs. PBS. (H) nAPANs or APANs (1 × 106) FACS-sorted from mouse BMDNs stimulated with eCIRP (1 μg/mL for 6 hours) were i.v. injected into mice immediately after CLP. 20 hours after CLP, the serum levels of IL-12p70 were assessed by ELISA. Data are expressed as mean ± SEM. n = 3/group. *P < 0.05 vs. sham, #P < 0.05 vs. PBS+CLP, †P < 0.05 vs. nAPAN+CLP. Data were compared by 1-way ANOVA and SNK test.