Myeloperoxidase-anchored ENO1 mediates neutrophil extracellular trap DNA to enhance Treg differentiation via IFITM2 during sepsis
Yi Jiang, Shenjia Gao, Xiya Li, Hao Sun, Xinyi Wu, Jiahui Gu, Zhaoyuan Chen, Han Wu, Xiaoqiang Zhao, Tongtong Zhang, Ronen Ben-Ami, Yuan Le, Timothy R. Billiar, Changhong Miao, Jie Zhang, Jun Wang, Wankun Chen
Yi Jiang, Shenjia Gao, Xiya Li, Hao Sun, Xinyi Wu, Jiahui Gu, Zhaoyuan Chen, Han Wu, Xiaoqiang Zhao, Tongtong Zhang, Ronen Ben-Ami, Yuan Le, Timothy R. Billiar, Changhong Miao, Jie Zhang, Jun Wang, Wankun Chen
View: Text | PDF
Research Article Immunology Infectious disease

Myeloperoxidase-anchored ENO1 mediates neutrophil extracellular trap DNA to enhance Treg differentiation via IFITM2 during sepsis

Abstract

Sepsis is a life-threatening disease caused by a dysfunctional host response to infection. During sepsis, inflammation-related immunosuppression is the critical factor causing secondary infection and multiple organ dysfunction syndrome. The regulatory mechanisms underlying Treg differentiation and function, which significantly contribute to septic immunosuppression, require further clarification. In this study, we found that neutrophil extracellular traps (NETs) participated in the development of sepsis-induced immunosuppression by enhancing Treg differentiation and function via direct interaction with CD4+ T cells. Briefly, NETs anchored enolase 1 (ENO1) on the membrane of CD4+ T cells through its key protein myeloperoxidase (MPO) and subsequently recruited interferon-induced transmembrane protein 2 (IFITM2). IFITM2 acted as a DNA receptor that sensed NET-DNA and activated intracellular RAS-associated protein 1B (RAP1B) and its downstream ERK signaling pathway to promote Treg differentiation and function. ENO1 inhibition significantly attenuated NET-induced Treg differentiation and alleviated sepsis in mice. Overall, we demonstrated the role of NETs in sepsis-induced immunosuppression by enhancing Treg differentiation, identified ENO1 as an anchor of NET-MPO, and elucidated the downstream molecular mechanism by which IFITM2-RAP1B-ERK regulates Treg differentiation. These findings improve our understanding of the immunopathogenesis of sepsis and provide potential therapeutic targets for sepsis-induced immunosuppression.

Authors

Yi Jiang, Shenjia Gao, Xiya Li, Hao Sun, Xinyi Wu, Jiahui Gu, Zhaoyuan Chen, Han Wu, Xiaoqiang Zhao, Tongtong Zhang, Ronen Ben-Ami, Yuan Le, Timothy R. Billiar, Changhong Miao, Jie Zhang, Jun Wang, Wankun Chen

×

Figure 4

MPO-ENO1 transmitted stimulatory signals for NET-DNA through upregulating IFITM2.

Options: View larger image (or click on image) Download as PowerPoint
MPO-ENO1 transmitted stimulatory signals for NET-DNA through upregulatin...
(A) ENO1 co-IP experiments were performed using lysates from in vitro–induced Tregs treated with NDMC (500 ng/mL), and nanoLC-MS/MS was used to identify the proteins bound to ENO1. ENO1-binding membrane proteins are shown in the heatmap (n = 2). (B) The expression of IFITM2 on Tregs in the spleens of mice 1 or 7 days after the CLP or sham procedure (n = 6). (C) The expression of IFITM2 on Tregs induced in vitro with or without NDMC treatment (100 ng/mL) was determined by flow cytometry (n = 3). (D) Immunoblot analysis of the lysates of in vitro–induced Tregs pulled down by biotinylated NET-DNA (500 ng) and detected with an IFITM2 antibody. (E) The membrane proteins of in vitro–induced Tregs (10 μg) and the biotinylated NET-DNA (1 ng) were incubated with or without a 200-fold excess of unbiotinylated NET-DNA, an IFITM2 antibody, or IgG control. EMSA demonstrated that the IFITM2 antibody supershifted the binding of NET-DNA to IFITM2. (F) Naive CD4+ T cells were transfected with LV-shRNA control or LV-shRNA Eno1 and then induced to Tregs in vitro with or without NDMC (100 ng/mL). The expression of IFITM2 on induced Tregs was determined by flow cytometry (n = 3). (G) IFITM2 expression of Tregs in the spleens of WT mice or Eno1fl/fl Cd4Cre mice 7 days after the CLP or sham procedure was determined by flow cytometry (n = 6). (H) Naive CD4+ T cells were transfected with control siRNA or Ifitm2 siRNA and then induced to differentiate into Tregs in vitro with or without the addition of NDMC (100 ng/mL). Flow cytometry was used to assess the proportion of Tregs (n = 3). Representative data are shown from 3 independent experiments (B–H). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA was used for B and F-H, and 1-way ANOVA was used for C.