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
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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

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Figure 3

Cell surface ENO1 interacted with MPO to navigate the effects of NDMC on Tregs.

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Cell surface ENO1 interacted with MPO to navigate the effects of NDMC on...
(A) Proteins interacting with MPO in the lysate of in vitro–induced Tregs treated with 500 ng/mL NETs were analyzed by nanoLC-MS/MS. The upregulated genes in CD4+ T cells collected from the spleens of CLP-operated mice versus sham-operated mice that overlapped with the purification of MPO-treated versus IgG control–treated pulled-down proteins are shown in the Venn diagram. (B) The expression of ENO1 on Tregs in the peripheral blood of healthy controls and patients during the immunosuppressive phase of sepsis was determined by flow cytometry (n = 10). (C) The expression of ENO1 on Tregs in the spleens of CLP or sham mice 1 or 7 days after operation was determined by flow cytometry (n = 6). (D) The expression of ENO1 on Tregs induced in vitro under NDMC treatment (100 ng/mL) for 24 or 48 hours was determined by flow cytometry (n = 3). (E) Immunofluorescence staining detected the colocalization of MPO and ENO1 on the cell membrane of induced Tregs with NDMC (100 ng/mL) or vehicle. Scale bars: 5 μm. Green, ENO1; red, MPO. (F) Immunoblot analysis of ENO1 immunoprecipitated with MPO from naive CD4+ T cells under Treg differentiation conditions treated with NDMC (500 ng/mL) or vehicle. (G) Immunoblot analysis of MPO immunoprecipitated with ENO1 from naive CD4+ T cells under Treg differentiation conditions with or without NDMC treatment (500 ng/mL). (H) Lentivirus-shRNA (LV-shRNA) Eno1 was used to knock down the Eno1 gene in naive CD4+ T cells. Immunoblot analysis of MPO immunoprecipitated with ENO1 from NDMC-treated (500 ng/mL) naive CD4+ T cells under Treg differentiation conditions treated with LV-shRNA control or LV-shRNA Eno1. (I) Naive CD4+ T cells transfected with LV-shRNA Eno1 or LV-shRNA control were induced to differentiate into Tregs in vitro with or without NDMC treatment (100 ng/mL). Flow cytometry was used to evaluate the proportion of Tregs (n = 3). (J) The proportion of Tregs was analyzed in the spleens of WT mice or Eno1fl/fl Cd4Cre mice 7 days after the CLP or sham procedure by flow cytometry (n = 6). (K) Flag-tagged full-length (FL) ENO1 or one of its truncated mutants (Δ1 to Δ2) was transfected into HEK293T cells. (L) Immunoblot analysis was conducted for MPO immunoprecipitated with beads coated with a Flag antibody. Representative data are shown from 3 independent experiments (B–J and L). Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Two-tailed Student’s t test was used for B; 2-way ANOVA was used for C, I, and J; and 1-way ANOVA was used for D.