Antigen-presenting aged neutrophils induce CD4+ T cells to exacerbate inflammation in sepsis
Hui Jin, Monowar Aziz, Atsushi Murao, Molly Kobritz, Andrew J. Shih, Robert P. Adelson, Max Brenner, Ping Wang
Hui Jin, Monowar Aziz, Atsushi Murao, Molly Kobritz, Andrew J. Shih, Robert P. Adelson, Max Brenner, Ping Wang
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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 6

APANs exaggerate sepsis by fueling inflammation, lung injury, and worsening survival.

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APANs exaggerate sepsis by fueling inflammation, lung injury, and worsen...
(A) BMDNs isolated from WT mice were stimulated with eCIRP for 6 hours. FACS-isolated APANs and nAPANs (1 × 106) were then adoptively transferred into mice via retro-orbital injection at the time of CLP. (B–I) Twenty hours later, the serum levels of (B) ALT, (C) AST, and (D) LDH were determined using specific colorimetric enzymatic assays; serum (E) TNF-α and (F) IL-6 levels were assessed by ELISA; and lung mRNA levels of (G) TNF-α, (H) IL-6, and (I) KC were assessed by real-time PCR. Data are expressed as mean ± SEM (n = 5–6 mice/group) and compared by ANOVA and SNK method. *P < 0.05 vs. sham, #P < 0.05 vs. CLP+PBS-treated mice, †P < 0.05 vs. CLP+nAPAN-injected mice. (J) Representative images of H&E-stained lung tissue. Original magnification, ×400. Scale bar: 100 μm. Enlarged images of the boxed areas are shown to the right. (K) Lung injury score. Average of 5 fields/slide/mouse. Data are expressed as mean ± SEM (n = 5 mice/group) and compared by ANOVA and SNK method. *P < 0.05 vs. sham, #P < 0.05 vs. CLP+PBS-treated mice, †P < 0.05 vs. CLP+nAPAN-injected mice. (L) Wet lung weight–to–body weight (BW) ratio 20 hours after CLP. Data are expressed as mean ± SEM (n = 4 mice/group) and compared by ANOVA and SNK method. *P < 0.05 vs. sham, #P < 0.05 vs. CLP+PBS-treated mice, †P < 0.05 vs. CLP+nAPAN-injected mice. (M) Kaplan-Meier 10-day survival curve generated from PBS-, APAN-, and nAPAN-treated CLP mice. n = 20 mice/group, *P < 0.05 vs. CLP+PBS, #P < 0.05 vs. CLP+nAPAN-injected mice determined by the log-rank test.