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 4

APANs promote CD4+ T cell proliferation and IFN-γ release.

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APANs promote CD4+ T cell proliferation and IFN-γ release. (A and B) Spl...
(A and B) Spleen tissue sections from WT septic mice (20-hour CLP) were stained with anti-Ly6G, CXCR4, CD86, and CD4 Abs. Representative images of immunohistochemical stains of spleen are shown. Original magnification, ×20 (A); ×200 (B). Scale bar: 50 μm (A); 5 μm (B). (C–E) WT mouse BMDNs (1 × 107) were stimulated with eCIRP (1 μg/mL/106 cells) for 6 hours. FACS-purified APANs, nAPANs, and naive splenic F4/80+ macrophage as antigen-presenting cell (APC) controls (1 × 105) were cocultured with splenic CD4+ T cells (1 × 105) isolated from naive OT-II transgenic mice. After 72 hours, the cells were stained with anti-CD4, and CD25 Ab, and CSFE dye. The proliferation of (C and D) total CD4+ T cells or (C and E) CD4+CD25+ T cells was assessed. (C) Representative flow cytometry gating of the CD4+ T cell proliferation. (D and E) Frequency of total CD4+ T cell or CD4+CD25+ T cell proliferation. Data reflecting ≥3 independent experiments are expressed as mean ± SEM and compared by 1-way ANOVA and SNK test. n = 6/group. *P < 0.05 vs. CD4+ T cells only, #P < 0.05 vs. nAPAN+CD4+ T cells. (F and G) APANs, nAPANs, or APCs (1 × 105) were cocultured with OT-II transgenic mouse splenic CD4+ T cells (1 × 105), and, 24 hours later, IFN-γ, IL-4, and IL-17 culture supernatant levels were determined by ELISA. Data reflecting ≥3 independent experiments are expressed as mean ± SEM and compared by ANOVA and SNK test. n = 6. *P < 0.05 vs. nAPAN, #P < 0.05 vs. APC. (H) APANs, nAPANs, or APCs (1 × 105) were cocultured with OT-II mouse splenic CD4+ T cells (1 × 105) in the presence of IgG or IL-12– neutralizing Ab. At 24 hours later, IFN-γ culture supernatant levels were determined by ELISA. 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. APC, †P < 0.05 vs. APAN+IgG.