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Glutathione peroxidase 4–regulated neutrophil ferroptosis induces systemic autoimmunity

Nature Immunology volume 22, pages 1107–1117 (2021)Cite this article

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Abstract

The linkage between neutrophil death and the development of autoimmunity has not been thoroughly explored. Here, we show that neutrophils from either lupus-prone mice or patients with systemic lupus erythematosus (SLE) undergo ferroptosis. Mechanistically, autoantibodies and interferon-α present in the serum induce neutrophil ferroptosis through enhanced binding of the transcriptional repressor CREMα to the glutathione peroxidase 4 (Gpx4, the key ferroptosis regulator) promoter, which leads to suppressed expression of Gpx4 and subsequent elevation of lipid-reactive oxygen species. Moreover, the findings that mice with neutrophil-specific Gpx4 haploinsufficiency recapitulate key clinical features of human SLE, including autoantibodies, neutropenia, skin lesions and proteinuria, and that the treatment with a specific ferroptosis inhibitor significantly ameliorates disease severity in lupus-prone mice reveal the role of neutrophil ferroptosis in lupus pathogenesis. Together, our data demonstrate that neutrophil ferroptosis is an important driver of neutropenia in SLE and heavily contributes to disease manifestations.

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Fig. 1: SLE IgG and IFN-α modulate neutrophil viability.
Fig. 2: Neutrophil ferroptosis is prevalent in patients with SLE.
Fig. 3: Neutrophil ferroptosis, the main form of neutrophil death in SLE, is induced by autoantibodies and IFN-α.
Fig. 4: Ferroptosis inhibitors ameliorate disease progression in MRL/lpr mice.
Fig. 5: IFN-α and SLE IgG are the main drivers of neutropenia by reducing GPX4 in neutrophils.
Fig. 6: Mice with Gpx4 haploinsufficiency in neutrophils develop spontaneous lupus-like disease.
Fig. 7: IFN-α and SLE IgG suppress the transcription of GPX4 by promoting binding of CREM to the Gpx4 promoter.

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Data availability

All raw source data for all experiments included in this study are provided. RNA sequencing data that support the findings of this study have been deposited with the Gene Expression Omnibus (GEO) repository under accession number GSE153781. Correspondence and requests for materials should be addressed to zxpumch2003@sina.com. Source data are provided with this paper.

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Acknowledgements

This study was supported by National Natural Science Foundation of China grants no. 81788101 (to X.Z.) and no. 81630044 (to X.Z.); Chinese Academy of Medical Science Innovation Fund for Medical Sciences grants no. CIFMS2016-12M-1-003 (to X.Z.), no. 2017-12M-1-008 (to X.Z.), no. 2017-I2M-3-011 (to X.Z.) and no. 2016-12M-1-008 (to X.Z.); Capital’s Funds for Health Improvement and Research (grant no. 2020-2-4019) (to X.Z.); and NIH grants no. R01AR064350 (to G.C.T) and no. R37AI049954 (to G.C.T). We thank A. Davidson at Feinstein Institutes for Medical Research for providing the adenovirus IFN-α. We thank the staff of the Rheumatology and Immunology Laboratory and Medical Scientific Research Center in Peking Union Medical College Hospital (PUMCH) for providing experimental equipment. We thank the doctors at PUMCH, Anyang District Hospital of Henan Province, Xiangya Hospital, Huaian No.1 People’s Hospital and People’s Hospital of Xinjiang Uygur Autonomous Region for patient recruitment.

Author information

Author notes

  1. These authors contributed equally: Pengchong Li, Mengdi Jiang, Ketian Li, Hao Li.

Authors and Affiliations

  1. State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Pengchong Li, Mengdi Jiang, Ketian Li & Xinyue Xiao

  2. Clinical Immunology Centre, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Pengchong Li, Mengdi Jiang, Ketian Li & Yue Xu

  3. Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Hao Li, Suzanne Krishfield & George C. Tsokos

  4. Department of Internal Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Yangzhong Zhou

  5. RILITE Research Institute and AMPEL BioSolutions, Charlottesville, VA, USA

    Peter E. Lipsky

  6. Department of Rheumatology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing, China

    Xuan Zhang

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Contributions

X.Z. and P.L. conceived the project and designed the experiments. P.L., M.J., K.L. and H.L. performed most of the experiments with help from X.X., Y.X. and S.K. Y.Z. and H.L. contributed to discussions. P.L., H.L. and P.E.L. wrote the manuscript. G.C.T. and X.Z. supervised work and acquired funding.

Corresponding authors

Correspondence to Peter E. Lipsky, George C. Tsokos or Xuan Zhang.

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Competing interests

P.E.L. is an employee of AMPEL but has no competing interests with the content of this manuscript. All the authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks Marcus Conrad, Christian Lood and Chandra Mohan for their contribution to the peer review of this work. Peer reviewer reports are available. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 IgG and IFNα but not CXCL11 or IL12/23 p40 present in SLE sera contribute to neutropenia.

a-b. Flow cytometry quantification of cell viability of neutrophils (a: n = 3; b: n = 9) in vitro cultured with (a) 5%, 10%, or 20% SLE serum for 6, 16, 24 hours respectively, or with (b) 20 % HC, SLE or RA serum respectively for 16 hours. c. Detection of the inflammatory factors in the sera from RA (n = 16), BD (n = 20) and AS (n = 18) patients vs. HCs (n = 19). d-e. Flow cytometry quantification of cell viability and lipid ROS of HC neutrophils (d: n = 7; e: n = 7 for anti-CXCL11 or n = 4 for Ustekinumab) cultured in vitro with 20% SLE serum supplemented with anti-CXCL11 (0.1, 1, 5 µg ml-1) or Ustekinumab (0.1, 1, 10 µg ml-1) for 16 hours. f-g. The proportion of anti-dsDNA in total IgG correlated with SLE neutrophil counts and SLEDAI scores (n = 63). h. Western blot validation of purified IgG from serum and of serum with IgG depletion. i. Flow cytometry quantification of cell viability of neutrophils (n = 5) cultured in vitro with serum in the presence or absence of anti-IFNAR (10 µg ml-1), or IgG depletion, for 16 hours. j-k. Serum IgG was purified or depleted by Protein A/G. (j) Ponceau S staining (upper panel) and western blot (lower panel) detection of purified IgG and of serum with depleted IgG. (k) Flow cytometry quantification of cell viability of neutrophils (n = 4) with HC serum in the presence of HC or SLE IgG at different concentrations (1.2, 2.4, 3.6 g L-1), or SLE serum with/without IgG depletion, for 16 hours. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ns p > 0.05. Two-tailed paired or unpaired Student’s t-test was applied.

Source data

Extended Data Fig. 2 Ferroptosis is restricted in neutrophils but not other cells in SLE and this could be reverted by addition of Ferroptosis specific inhibitors.

a. Flow cytometry quantification of cell viability of HC lymphocytes, monocytes, and neutrophils cultured with 20% HC or SLE serum for 16 hours and the proportion of apoptotic (Annexin V + 7AAD-), necrotic (Annexin V + 7AAD + ) and live (Annexin V- 7AAD-) cells in each subset was analyzed (n = 6). b. HC neutrophils were cultured with 20% HC or SLE serum, and lipid-ROS productions at different time points were detected (n = 3). c. Dot plots show cell viability analyzed by lactate dehydrogenase (LDH) release. HC neutrophils (n = 9) were cultured with 20% HC serum supplemented with RSL-3 (10 μM) or SLE serum supplemented with LPX-1 (1 μM) for 16 hours before analysis. d-e. Dot plots show flow cytometry quantification of the percentage of apoptotic, necrotic, and live cells. HC neutrophils (n = 5) were cultured with 20% HC or SLE serum in the presence or absence of LPX-1 (1 μM) for 16 hours before analysis. f-g. HC B cells (n = 6) were cultured in 20% HC or SLE serum supplemented with LPX-1 for 72 hours, and plasmacytoid dendritic cells (pDC) (n = 3) were cultured for 24 hours, the level of IgG was assessed by ELISA and type I IFNs by flow cytometry individually. h-i. Dot plots show cell viability and lipid-ROS in HC neutrophils (n = 7) cultured with 20% HC or SLE serum supplemented with ß-ME (10/50 μM) for 16 hours. Data are shown as mean ± SD. ns p > 0.05. Two-tailed paired Student’s t-test was applied.

Source data

Extended Data Fig. 3 The cooperative effects between IFNα and SLE IgG on cell death.

a-c. HC neutrophils were cultured in the presence of HC or SLE serum with or without the addition of Cl-amidine (Cl) (peptidyl arginine deiminase 4 (PAD4) inhibitor, 100 μM), or LPX-1 (1 μM) for 4 or 16 hours and NETs were assessed in SYTOX Green+ cells based on morphology (n = 6). Neutrophils with DNA area greater than 400um2 were considered as NETs. Dot plots show the percentage of cells forming NETs in all dead neutrophils from the indicated group. d-e. Representative fluorescent images and related quantification of NETosis. HC neutrophils (n = 6) were stimulated by PMA (50 nM) with or without LPX-1(1 μM) for 4 hours. f-i. HC neutrophils were cultured with SLE IgG (3.6 g L-1) and/or IFN-α (10^5 U ml-1) for 4 or 16 hours and cells were stained with SYTOX Green for the detection of NETs. Dot plots show the immunofluorescence microscope quantification of NETosis in total dead neutrophils from the indicated group (4 h: n = 6; 16 h: n = 3). The scale bar represents 50 μm. Data are shown as mean ± SD. ns p > 0.05. Two-tailed paired Student’s t-test was applied.

Source data

Extended Data Fig. 4 The ferroptosis inhibitor ameliorates lupus progression with much better therapeutic effect compared to the NETosis inhibitor.

a-i. MRL/lpr mice (n = 6) were treated with DMSO (0.1 ml 10%), LPX-1(10 mg/kg) or CTX (20 mg/kg) every other day at week 12 for 6 weeks, DMSO (0.1 ml 10%) was applied to sex-matched MRL/Mpj mice (n = 5) as control. Mice were euthanized at 18 weeks of age for analysis. (a) Flow cytometry quantification of lipid ROS. (b) Representative immunofluorescent images of glomeruli stained with IgG (red), IgM (yellow), C1q (green), and DAPI (blue). (c-e) Flow cytometry quantification of plasma inflammatory factors and (f-i) plasma IgG. j-o. MRL/lpr mice (DMSO, LPX-1: n = 3; Cl, Cl+LPX-1: n = 4) were treated with DMSO, Cl, LPX-1, or Cl combined with LPX-1 every other day for 3 weeks starting at the age of week 12. Mice were euthanized at 15 weeks of age for analysis. (j-k) Representative images and related quantification of axillary spleens and lymph nodes. (l) Western blot analysis of cit-H3 in circulating neutrophils from mice subjected to the indicated treatment. (m) Dot plots show the ELISA assessment of serum complement 3. (n) Dot plots show the ELISA assessment of serum anti-dsDNA antibodies titers. (o) Dot plots shows the Bicinchoninic acid (BCA) assay of urine proteins. The scale bar represents 50 μm. Data are shown as mean ± SD. ns p > 0.05. Two-tailed unpaired Student’s t-test was applied.

Source data

Extended Data Fig. 5 The expression of cystine transporter SLC7A11 is not different between HC and SLE neutrophils.

a. Heatmap visualization of RNA-seq analysis on differentially expressed ferroptosis-related genes (Standardized with GAPDH) in neutrophils between new onset treatment-naïve SLE patients (n = 6) and HCs (n = 6). b. Western blot validation for SLC7A11 antibody. 293 T cells were transfected with Slc7a11 overexpression plasmid and cells without transfection were used as control. c. Western blot assay shows the expression of cystine transporter SLC7A11 in neutrophils from HCs (n = 8) and SLE patients (n = 8). Data are shown as mean ± SD. ns p > 0.05. Two-tailed unpaired Student’s t-test was applied.

Source data

Extended Data Fig. 6 GPX4 reduction was observed in neutrophils but not other immune cells in SLE.

a. Flow cytometry quantification of GPX4 expressions in HCs (n = 16) and SLE (n = 12) neutrophils. b. GPX4 expressions in neutrophils from treatment-naïve SLE patients correlated negatively with disease activities as measured by SLEDAI (n = 12). c. Flow cytometry quantification of GPX4 expressions in lymphocytes (including CD4 + T, CD8 + T, and B cells) and monocytes from HCs (n = 11) and SLE patients (n = 9). d-e. Western blot analysis of GPX4 expressions in lymphocytes and monocytes from HCs (n = 11) and SLE patients (n = 10). f-g. Western blot analysis of GPX4 expression in HC neutrophils, monocytes, and lymphocytes (n = 7). h-i. Western blot analysis of GPX4 expression in HC neutrophils, monocytes, and lymphocytes (n = 7) cultured with 20% HC or SLE serum for 30 hours. j-k. Western blot analysis of GPX4 expression in HC neutrophils (n = 3) when cultured with 20% HC serum or SLE serum supplemented with Cl-amidine (Cl, 100 μM), APX-115 (APX) (pan-NADPH oxidase (NOX) inhibitor, 20 μM), and GSK2795039 (GSK) (NOX2 inhibitor, 10 μM). Data are shown as mean ± SD. ns p > 0.05. Two-tailed unpaired Student’s t-test was applied.

Source data

Extended Data Fig. 7 FcγR3β is essential for the SLE IgG-mediated GPX4 downregulation in neutrophils.

a-b. Expression correlation analysis between different TLRs or FcRs with GPX4 based on RNA-seq data. In SLE neutrophils, (a) TLR signaling pathways are not associated with GPX4 reduction. (b) Fcγr3b but not other FcRs’ expression is negatively associated with GPX4 reduction. c. Different Fc receptor expressions in HCs (n = 6) and SLE (n = 6) analyzed by RNA-seq. d. Western blot analysis of FcγR3β expressions in neutrophils, monocytes and lymphocytes from HCs (n = 3). e. GPX4 expressions in HL60 cells after overexpression of FcγR3β (n=4). Control referred to cells without transfection. Data are presented as mean ± SD or median with interquartile range. ns p > 0.05. one-tailed or two-tailed unpaired Student’s t-test or Mann Whitney test was applied.

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Extended Data Fig. 8 Mice with Gpx4 haploinsufficiency in neutrophils developed spontaneous lupus-like disease, while Gpx4 fl/flLysMCre+ mice exhibited mild autoimmunity.

a-b. Flow cytometry quantification and western blot analysis of GPX4 in neutrophils (CD45+CD11b+Ly6G&Ly6C+) and non-neutrophils (including monocytes and lymphocytes) from Gpx4fl/fl (n = 6) and Gpx4fl/wtLysMCre+ (n = 9) mice. c-d. Flow cytometry analysis of peripheral neutrophils (CD45+CD11b+Ly6G&Ly6C+) and monocytes (CD45+CD11b+Ly6G&Ly6C-) from Gpx4fl/fl (c: n = 6, d: n = 10) and Gpx4fl/wtLysMCre+ (c: n = 9, d: n = 13) mice. e. Flow cytometry quantification of lipid-ROS and cell viability in neutrophils (n = 6) from Gpx4fl/wtLysMCre+ mice cultured in complete RPMI 1640 basic medium in the presence or absence of LPX-1 (1 μM). f. Skin lesions of Gpx4fl/wtLysMCre+ mice. g. Immunofluorescent images of glomeruli in Gpx4fl/fl mice and Gpx4fl/wtLysMCre+ mice. IgG (red), IgM (yellow), C1q (green), and DAPI (blue). h. Dot plots show the proteinuria of Gpx4fl/fl and Gpx4 fl/flLysMCre+ mice at 4 months of age assessed by BCA assay. i. ELISA assay shows the levels of serum complement 3 in Gpx4fl/fl (n = 8) and Gpx4 fl/flLysMCre+ (n = 8) mice at 6 months of age. j. ELISA assay shows the levels of serum anti-dsDNA antibodies in Gpx4fl/fl (n = 8) and Gpx4 fl/flLysMCre+ (n = 8) mice at 6 months of age. The scale bar represents 50 μm. Data are shown as mean ± SD, ns p > 0.05. Two-tailed unpaired or paired Student’s t-test was applied.

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Extended Data Fig. 9 IFNα and SLE IgG enhanced ferroptosis by promoting binding of CREM to the Gpx4 promoter.

a. Western blot analysis of CREMα and CaMKIV in cytoplasm and nucleus of neutrophils from HCs and SLE patients. b. Dot plots show the CHIP analysis results on CREMα binding to the promoter of Gpx4 from neutrophils (n = 6) with indicated treatment: IFN-α (10^5 U ml-1), anti-IFNAR (10 µg ml-1), SLE IgG (2.4 g L-1) or SLE sera with IgG depletion. c-d. Efficiency of CREMα knockdown by siRNA (n = 3) or CREMα over-expression (n = 4) in HL-60 cells validated by qPCR (c) and western blot (d). e. Effect of IFN-α or SLE IgG on GPX4 expressions in HL60 cells after knockdown or overexpression of CREMα. f. Efficiency of CREMα knockdown or overexpression on ferroptosis in HL60 cells (n = 4), assessed by flow cytometry using BODIPY C11. Data are shown as mean ± SD, ns p > 0.05. Two-tailed unpaired or paired Student’s t-test was applied.

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Extended Data Fig. 10 The hypothetical model for neutrophil ferroptosis in SLE pathogenesis.

Autoantibodies and interferon-α present in SLE sera enhance binding of the transcriptional repressor CREMα to Gpx4 promoter, which leads to suppressed expression of GPX4 and subsequent elevation of lipid-ROS. These lead to neutrophil ferroptosis and further promote SLE progression in patients. Moreover, mice with neutrophil-specific Gpx4 haploinsufficiency develop lupus phenotype and inhibition of neutrophil ferroptosis significantly mitigates disease development in lupus-prone mice.

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Li, P., Jiang, M., Li, K. et al. Glutathione peroxidase 4–regulated neutrophil ferroptosis induces systemic autoimmunity. Nat Immunol 22, 1107–1117 (2021). https://doi.org/10.1038/s41590-021-00993-3

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