Neutrophils promote endothelial cell activation in pediatric Mycoplasma pneumoniae pneumonia
Xia Huang, Yifan Zhu, Yun Guo, Tian Lv, Haiyan Gu, Yingying Luo, Dan Li, Hang Sun, Deyu Zhao, Feng Liu
Xia Huang, Yifan Zhu, Yun Guo, Tian Lv, Haiyan Gu, Yingying Luo, Dan Li, Hang Sun, Deyu Zhao, Feng Liu
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Research Article Immunology Infectious disease

Neutrophils promote endothelial cell activation in pediatric Mycoplasma pneumoniae pneumonia

Abstract

Mycoplasma pneumoniae pneumonia (MPP) can cause serious extrapulmonary complications, including life-threatening thrombosis. This study aimed to elucidate the roles of neutrophils and neutrophil extracellular traps (NETs) in vascular endothelial cell (EC) activation in pediatric MPP-associated thrombosis. We analyzed the relationship between neutrophils and thrombosis in children with MPP and used mouse models of neutrophilia (Csf3 plasmid injection), neutropenia (Csf3 deficient, Csf3–/–), and defective NETs formation (Pad4 deficient, Pad4–/–). The effects of neutrophils and NETs on EC activation were further examined in vivo, in vitro, and in human samples. Elevated neutrophil count was observed in patients with thrombosis and functioned as a potential diagnostic marker as well as a risk factor for MPP-associated thrombosis. EC activation was enhanced in MPP mice with neutrophilia but attenuated in neutropenic or Pad4–/– mice. NETs activated ECs through TLR2 and JAK/STAT3 signaling, and inhibition of NETs formation (Cl-amidine), TLR2 (C29), and JAK (upadacitinib) each attenuated this response. Strong correlations among neutrophils, NETs, EC activation, and thrombosis were observed in pediatric patients. These findings suggest that neutrophils promoted thrombosis in MPP via NETs-mediated EC activation involving TLR2 and JAK/STAT3 signaling. This study provides mechanistic insights into the inflammatory-thrombotic processes in MPP-associated thrombosis and offers a rationale for further investigation of neutrophils, NETs, TLR2, and JAK/STAT3 signaling in this context.

Authors

Xia Huang, Yifan Zhu, Yun Guo, Tian Lv, Haiyan Gu, Yingying Luo, Dan Li, Hang Sun, Deyu Zhao, Feng Liu

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Figure 7

NETs and endothelial activation in blood samples and lung tissues from patients with thrombosis in the PSM MPP cohort.

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NETs and endothelial activation in blood samples and lung tissues from p...
(A) Plasma D-dimer levels in the PSM MPP cohort (n = 303). Data are presented as median (IQR) and were analyzed using the Mann-Whitney U test. (B) Plasma levels of MPO-DNA in the PSM MPP cohort (n = 303). Data are presented as median (IQR) and were analyzed using the Mann-Whitney U test. *P < 0.05 versus Con group; #P < 0.05 versus MP group. (C) Correlation between plasma MPO-DNA levels and D-dimer levels (n = 303). Spearman’s rank correlation analysis was performed. (D) Kaplan-Meier curves showing the cumulative probability of thrombosis stratified by plasma MPO-DNA count (high versus low) (n = 303). Differences between groups were assessed using the log-rank test. (E) Representative H&E staining of embolized lung tissue from a patient with MPP-associated thrombosis. Left panel, scale bar: 500 μm; right panel, scale bar: 50 μm. (F) Representative H&E staining of thrombus tissue from 2 patients with MPP-associated thrombosis. Left panel, scale bar: 2 mm; right panel, scale bar: 20 μm. (G) Representative scanning electron microscopy images of thrombus tissues from 2 patients with MPP-associated thrombosis showing extracellular web-like structures in close proximity to neutrophils. Scale bar: 20 μm. (H) Representative immunofluorescence images showing NETs formation in embolized lung tissue from 1 patient with MPP-associated thrombosis. Colocalization of CD11b (red), CitH3 (green), MPO (yellow), and DAPI (blue) indicates NETs formation. Left panel, scale bar: 500 μm; right panel, scale bar: 50 μm. (I) Representative immunofluorescence images of NETs in thrombus tissue from 2 patients with MPP-associated thrombosis. Left panel, scale bar: 100 μm; right panel, scale bar: 20 μm. (J–M) Representative immunofluorescence staining of pulmonary ECs for markers of endothelial activation: ICAM-1 (J), VCAM-1 (K), P-selectin (L), and vWF (M). ECs were labeled with CD31 (red), target activation markers (green), and DAPI (blue). Representative images from one patient with MPP-associated thrombosis are shown. Scale bar: 20 μm. ****P < 0.0001.