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Eosinophil extracellular traps drive asthma progression through neuro-immune signals

Nature Cell Biology volume 23, pages 1060–1072 (2021)Cite this article

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Abstract

Eosinophilic inflammation is a feature of allergic asthma. Despite mounting evidence showing that chromatin filaments released from neutrophils mediate various diseases, the understanding of extracellular DNA from eosinophils is limited. Here we show that eosinophil extracellular traps (EETs) in bronchoalveolar lavage fluid are associated with the severity of asthma in patients. Functionally, we find that EETs augment goblet-cell hyperplasia, mucus production, infiltration of inflammatory cells and expressions of type 2 cytokines in experimental non-infection-related asthma using both pharmaceutical and genetic approaches. Multiple clinically relevant allergens trigger EET formation at least partially via thymic stromal lymphopoietin in vivo. Mechanically, EETs activate pulmonary neuroendocrine cells via the CCDC25–ILK–PKCα–CRTC1 pathway, which is potentiated by eosinophil peroxidase. Subsequently, the pulmonary neuroendocrine cells amplify allergic immune responses via neuropeptides and neurotransmitters. Therapeutically, inhibition of CCDC25 alleviates allergic inflammation. Together, our findings demonstrate a previously unknown role of EETs in integrating immunological and neurological cues to drive asthma progression.

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Fig. 1: EET levels correlate with asthma progression.
Fig. 2: Generation and components of EETs.
Fig. 3: DNase I treatment ameliorates asthma in mice.
Fig. 4: EETs amplify allergic asthma responses.
Fig. 5: EETs aggravate allergic inflammation by activating PNECs.
Fig. 6: EETs activate PNECs via CCDC25.
Fig. 7: EPX potentiates the interaction between EETs and PNECs.

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Data availability

Mass spectrometry data for EET-binding proteins have been deposited in ProteomeXchange with the identifier PXD027951 (https://www.ebi.ac.uk/pride/)122. Data of human CCDC25 RNA expression was derived from the FANTOM5 dataset included in the Human Protein Atlas database (https://www.proteinatlas.org/ENSG00000147419-CCDC25/tissue)37. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Key Research and Development Program of China (grant no. 2017YFA0106300 (S.S.)), Natural Science Foundation of China (grant nos 91942309 (S.S.), 92057210 (S.S.), 82071804 (S.J.) and 82002780 (Y.L.)), Science and Technology Program of Guangzhou (grant no. 202103000070 (S.S.)), Natural Science Foundation of Guangdong Province (grant no. 2020A1515010031 (Y.L.)) and Tencent Charity Foundation of China (S.J.).

Author information

Author notes

  1. These authors contributed equally: Yiwen Lu, Yijiao Huang, Jiang Li, Jingying Huang, Lizhi Zhang.

Authors and Affiliations

  1. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China

    Yiwen Lu, Yijiao Huang, Jiang Li, Jingying Huang, Lizhi Zhang, Jingwei Feng, Jiaqian Li, Qidong Xia, Linjie Huang, Shanping Jiang & Shicheng Su

  2. Breast Tumor Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China

    Yiwen Lu, Jiang Li, Jingying Huang, Jingwei Feng, Jiaqian Li, Qidong Xia & Shicheng Su

  3. Department of Pulmonary and Critical Care Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China

    Yijiao Huang, Lizhi Zhang, Linjie Huang & Shanping Jiang

  4. Department of Infectious Diseases, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China

    Qiyi Zhao & Shicheng Su

  5. Guangdong Provincial Key Laboratory of Liver Disease Research, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

    Qiyi Zhao

  6. Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education, Guangzhou, China

    Qiyi Zhao

  7. Institute of Pulmonary Diseases, Sun Yat-Sen University, Guangzhou, China

    Linjie Huang & Shanping Jiang

  8. Department of Immunology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China

    Shicheng Su

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  1. Yiwen Lu
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Contributions

S.S. conceived the study, designed the experiments and wrote the manuscript. S.J. and Y.H. provided the clinical samples. Y.L., Y.H., Jiang Li, J.H. and L.Z. performed the experiments, analysed the data and wrote the manuscript. J.F., Jiaqian Li., Q.X., Q.Z. and L.H. performed the experiments and analysed the data.

Corresponding authors

Correspondence to Shanping Jiang or Shicheng Su.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks Venizelos Papayannopoulos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 EETs are correlated with asthma progression.

a, Quantification of Fig. 1d. b, Levels of EETs were evaluated by the SYTOX Green, DAPI and ECP staining in BALF of mice immunized without or with OVA. c, Quantification of Fig. 1f. d, Quantification of SYTOX Green signals in ECP+ or MPO+ cells in BALF of mice immunized with OVA in Fig. 1e. a-d, Mean ± s.d.; n = 6 mice per group, representative of two independent experiments (a, c, d); Data are pooled from 3 independent experiments with 6-8 mice each (b); compared by two-tailed unpaired Student’s t-test.

Source data

Extended Data Fig. 2 Generation and components of EETs.

a, Venn diagram of overlapped proteins from three donors in Fig. 2g. The numbers of overlapped and unique proteins are shown in the corresponding area.

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Extended Data Fig. 3 DNase treatment ameliorates asthma in mice.

a, Quantification of Fig. 3f. b, Quantification of Fig. 3h. c, Protease activity of DNase I at indicated concentrations was examined by the protease assay. Trypsin was used as a positive control (n = 3). d-i, Wild-type mice were sensitized and challenged with PBS or OVA. OVA-sensitized mice were treated intratracheally with DNase before OVA challenge. d, Representative immunofluorescence images of cytokeratin (CK), TUNEL and DAPI staining in sections of lungs harvested at baseline (day 0) and 15 (day 15) and 17 (day 17) days after the first OVA sensitization. The arrow indicates CK+ TUNEL+ cells. Scale bar, 20 μm. e, Quantification of d. f, The area (%) of ECP+ SYTOX Green+ EETs in BALF smears was quantified at baseline (day 0) and 15 (day 15) and 17 (day 17) days after the first OVA sensitization. g, Eosinophils in the BALF at baseline (day 0) and 15 (day 15) and 17 (day 17) days after the first OVA sensitization were evaluated by flow cytometric analysis. Quantification are shown. h, i, F4/80−MHC-II+CD11c+ dendritic cells (DCs) in the BALF at baseline (day 0) and 15 (day 15) and 17 (day 17) days after the first OVA sensitization were evaluated by flow cytometric analysis. Quantification (h) and representative plots (i) are shown. a, b, e-h, Mean ± s.d.; n = 6 mice per group, representative of two independent experiments; compared by one-way ANOVA with Tukey’s multiple comparisons test (a, b, f-h) and two-way ANOVA with Sidak’s multiple comparisons test (e).

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Extended Data Fig. 4 EETs amplify allergic asthma responses.

a, Representative immunoblots of PAD4 and GAPDH in human eosinophils, neutrophils, monocytes, T cell, B cells and NK cells isolated from peripheral blood of healthy donors (n = 3). b, Representative immunofluorescence images of MPO, ECP, PAD4 and DAPI staining in human neutrophils and eosinophils isolated from peripheral blood of healthy donors. Scale bar, 50 μm. c, Quantification of Fig. 4b. d, Quantification of Fig. 4c. e, f, Wild-type and PAD4−/− mice were sensitized and challenged with PBS or OVA. PAD4−/− mice were adoptively transferred with 2×105 eosinophils or neutrophils isolated from wild-type or PAD4−/− mice. e, Quantification of Fig. 4e. f, Levels of IL-25, IL-33 and TSLP in BALF were evaluated by ELISA. c-f, Mean ± s.d.; n = 5 independent experiments (c, d); n = 6 mice per group, representative of two independent experiments (e, f); compared by one-way ANOVA with Tukey’s multiple comparisons test.

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Extended Data Fig. 5 EETs aggravate allergic inflammation by activating PNECs.

a, Expression profiles of CCDC25 in different tissues from the FANTOM5 dataset. b, ECP+ H3cit+DAPI+ spots (0.1-μm-diameter) in lung sections of OVA-immunized mice were defined as EETs as shown in Fig. 5a. The density of EETs (number of spots per mm2) within 50 μm of PNECs, randomly selected blood vessels and airway walls (from epithelium to serosa, excluding the cartilage and 50-μm area surrounding PNECs) was quantified. c, CCDC25 expression level of CGRP+ and CGRP− cells in mouse lung sections was quantified by immunofluorescence as shown in Fig. 5b. d, Representative immunoblots of CCDC25 of lung homogenate from C57BL/6 mice challenged with PBS or OVA (n = 3). e-g, C57BL/6 mice immunized with OVA were treated without or with BIBN4096 or CGP35348. e, The inflammatory score quantified from H&E staining of lung sections. f, Levels of indicated type 2 cytokines in BALF evaluated by ELISA. g, Percentages of PAS-stained goblet cells in total epithelial cells in lung sections. h, The relative expression level of CGRP in H146 cells with indicated treatment was measured by qRT-PCR. i, Quantification of Fig. 5e. j, The relative level of intracellular Fluo-4 immunofluorescence intensity in live lung slices with indicated treatment was quantified at indicated time point. k, Quantification of Fig. 5h. b, c, e-k, Mean ± s.d.; n = 6 mice per group, representative of two independent experiments (b, c, e-g, k); n = 4 (h, i) or 3 (j) independent experiments; compared by two-tailed unpaired Student’s t-test (c) and one-way ANOVA with Tukey’s multiple comparisons test (b, e-k).

Source data

Extended Data Fig. 6 EETs activate PNECs via CCDC25.

a, Representative immunoblots for CCDC25 in H146 cells with indicated treatment (n = 3). b, Representative immunoblots of CCDC25 in H146 cells that were transduced with sgControl or sgRNAs against CCDC25 (n = 3). c, Quantification of Fig. 6c. d-g, Wild-type and CCDC25−/− mice were immunized with OVA. d, Quantification of Fig. 6e. e, Quantification of indicated cells in immunofluorescence images of lung sections. f, The area (%) of EETs in BALF smear at baseline (day 0) and 15 (day 15) and 17 (day 17) days after the first OVA sensitization was quantified by immunofluorescence staining. g, The number of eosinophils in the BALF at indicated time point was quantified by flow cytometry. h, CRTC1 was silenced by shRNA in H146 cells. The efficiency was evaluated by western blotting (n = 3). i, Representative immunoblots of PKCα levels in membrane (Memb) and cytoplasmic (Cyto) fractions of H146 cells with indicated treatments (n = 3). j, k, ILK (j) and PKCα (k) were silenced by shRNAs in H146 cells. The efficiency was evaluated by western blotting (n = 3). l, m, H146 cells transduced with shRNAs against PKCα were treated with EETs. l, Cytosolic Ca2+ mobilization in H146 cells was examined using Fura-2/AM ratiometric fluorescence measurements. Fluorescence curves (left) and peak responses (right) are shown. Arrow indicates the time of stimulation. m, Representative immunoblots of CRTC1 in nuclear (Nuc) and cytoplasmic (Cyto) fractions of H146 cells (n = 3). c-g, l, Mean ± s.d.; n = 6 mice per group, representative of two independent experiments (d-g); n = 5 (c) or 4 (l) independent experiments; compared by one-way ANOVA with Tukey’s multiple comparisons test (d-g, l) and two-way ANOVA with Sidak’s multiple comparisons test (c).

Source data

Extended Data Fig. 7 EPX potentiates the interaction between EETs and PNECs.

a-c, Wild-type, EPX−/− and MBP−/− mice were sensitized and challenged with PBS or OVA. a, Quantification of Fig. 7d. b, Levels of indicated type 2 cytokines in BALF were evaluated by ELISA. c, The relative expression levels of CGRP in lung homogenates were examined by qRT-PCR. d, The area (%) of ECP+ SYTOX Green+ and EPX+ SYTOX Green+ signals in BALF smears as shown in Fig. 7f. e-f, PAD4−/− mice were adoptively transferred with eosinophils isolated from wild-type mice or EPX−/− mice. e, Percentages of PAS-stained cells in total epithelial cells in lung sections. f. The inflammatory score quantified from the H&E staining of lung sections. a-f, Mean ± s.d.; n = 6 (e, f) or 10 (a-d) mice per group, representative of two independent experiments; compared by one-way ANOVA with Tukey’s multiple comparisons test.

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Supplementary information

Reporting Summary

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Lu, Y., Huang, Y., Li, J. et al. Eosinophil extracellular traps drive asthma progression through neuro-immune signals. Nat Cell Biol 23, 1060–1072 (2021). https://doi.org/10.1038/s41556-021-00762-2

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