Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Glutathione peroxidase 4–regulated neutrophil ferroptosis induces systemic autoimmunity

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

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.

References

  1. 1.

    Tsokos, G. C. Systemic lupus erythematosus. N. Engl. J. Med. 365, 2110–2121 (2011).

    CAS  PubMed  Google Scholar 

  2. 2.

    Lisnevskaia, L., Murphy, G. & Isenberg, D. Systemic lupus erythematosus. Lancet 384, 1878–1888 (2014).

    PubMed  Google Scholar 

  3. 3.

    Garcia-Romo, G. S. et al. Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci. Transl. Med. 3, 73ra20 (2011).

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Bosch, X. Systemic lupus erythematosus and the neutrophil. N. Engl. J. Med. 365, 758–760 (2011).

    CAS  PubMed  Google Scholar 

  5. 5.

    Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Yang, W. S. et al. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Alim, I. et al. Selenium drives a transcriptional adaptive program to block ferroptosis and treat stroke. Cell 177, 1262–1279.e25 (2019).

    CAS  PubMed  Google Scholar 

  8. 8.

    Ingold, I. et al. Selenium utilization by GPX4 is required to prevent hydroperoxide-induced ferroptosis. Cell 172, 409–422.e421 (2018).

    CAS  PubMed  Google Scholar 

  9. 9.

    Friedmann Angeli, J. P. et al. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat. Cell Biol. 16, 1180–1191 (2014).

    CAS  PubMed  Google Scholar 

  10. 10.

    Do Van, B. et al. Ferroptosis, a newly characterized form of cell death in Parkinson’s disease that is regulated by PKC. Neurobiol. Dis. 94, 169–178 (2016).

    PubMed  Google Scholar 

  11. 11.

    Matsushita, M. et al. T cell lipid peroxidation induces ferroptosis and prevents immunity to infection. J. Exp. Med. 212, 555–568 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    van Vollenhoven, R. F. et al. Efficacy and safety of ustekinumab, an IL-12 and IL-23 inhibitor, in patients with active systemic lupus erythematosus: results of a multicentre, double-blind, phase 2, randomised, controlled study. Lancet 392, 1330–1339 (2018).

    PubMed  Google Scholar 

  13. 13.

    ter Borg, E. J., Horst, G., Hummel, E. J., Limburg, P. C. & Kallenberg, C. G. Measurement of increases in anti-double-stranded DNA antibody levels as a predictor of disease exacerbation in systemic lupus erythematosus. A long-term, prospective study. Arthritis Rheum. 33, 634–643 (1990).

    PubMed  Google Scholar 

  14. 14.

    Pisetsky, D. S. Anti-DNA antibodies—quintessential biomarkers of SLE. Nat. Rev. Rheumatol. 12, 102–110 (2016).

    CAS  PubMed  Google Scholar 

  15. 15.

    Lood, C. et al. Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease. Nat. Med. 22, 146–153 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Papayannopoulos, V., Metzler, K. D., Hakkim, A. & Zychlinsky, A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J. Cell Biol. 191, 677–691 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Stockwell, B. R. et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell 171, 273–285 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Friedmann Angeli, J. P. et al. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat. Cell Biol. 16, 1180–1191 (2014).

    CAS  PubMed  Google Scholar 

  19. 19.

    Sun, Y., Zheng, Y., Wang, C. & Liu, Y. Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells. Cell Death Dis. 9, 753 (2018).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Cohen, P. L. & Eisenberg, R. A. Lpr and gld: single gene models of systemic autoimmunity and lymphoproliferative disease. Annu. Rev. Immunol. 9, 243–269 (1991).

    CAS  PubMed  Google Scholar 

  21. 21.

    Dubois, E. L., Horowitz, R. E., Demopoulos, H. B. & Teplitz, R. NZB/NZW mice as a model of systemic lupus erythematosus. JAMA 195, 285–289 (1966).

    CAS  PubMed  Google Scholar 

  22. 22.

    Ginzler, E. M. et al. Mycophenolate mofetil or intravenous cyclophosphamide for lupus nephritis. N. Engl. J. Med. 353, 2219–2228 (2005).

    CAS  PubMed  Google Scholar 

  23. 23.

    Alim, I. et al. Selenium drives a transcriptional adaptive program to block ferroptosis and treat stroke. Cell 177, 1262–1279.e25 (2019).

    CAS  PubMed  Google Scholar 

  24. 24.

    Ingold, I. et al. Selenium utilization by GPX4 is required to prevent hydroperoxide-induced ferroptosis. Cell 172, 409–422.e21 (2018).

    CAS  PubMed  Google Scholar 

  25. 25.

    Clark, S. R. et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat. Med. 13, 463–469 (2007).

    CAS  PubMed  Google Scholar 

  26. 26.

    Speckmann, B. et al. Induction of glutathione peroxidase 4 expression during enterocytic cell differentiation. J. Biol. Chem. 286, 10764–10772 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    De Cesare, D., Fimia, G. M. & Sassone-Corsi, P. Signaling routes to CREM and CREB: plasticity in transcriptional activation. Trends Biochem. Sci. 24, 281–285 (1999).

    PubMed  Google Scholar 

  28. 28.

    Hedrich, C. M. et al. cAMP response element modulator α controls IL2 and IL17A expression during CD4 lineage commitment and subset distribution in lupus. Proc. Natl Acad. Sci. USA 109, 16606–16611 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Hedrich, C. M., Rauen, T., Kis-Toth, K., Kyttaris, V. C. & Tsokos, G. C. cAMP-responsive element modulator α (CREMα) suppresses IL-17F protein expression in T lymphocytes from patients with systemic lupus erythematosus (SLE). J. Biol. Chem. 287, 4715–4725 (2012).

    CAS  PubMed  Google Scholar 

  30. 30.

    Juang, Y. T. et al. Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV. J. Clin. Invest. 115, 996–1005 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Triantafyllopoulou, A. et al. Proliferative lesions and metalloproteinase activity in murine lupus nephritis mediated by type I interferons and macrophages. Proc. Natl Acad. Sci. USA 107, 3012–3017 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Zhuang, H. et al. Toll-like receptor 7-stimulated tumor necrosis factor α causes bone marrow damage in systemic lupus erythematosus. Arthritis Rheumatol. 66, 140–151 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Kolb, J. P., Oguin, T. H. 3rd, Oberst, A. & Martinez, J. Programmed cell death and inflammation: winter is coming. Trends Immunol. 38, 705–718 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Tsokos, G. C., Lo, M. S., Costa Reis, P. & Sullivan, K. E. New insights into the immunopathogenesis of systemic lupus erythematosus. Nat. Rev. Rheumatol. 12, 716–730 (2016).

    CAS  PubMed  Google Scholar 

  35. 35.

    Ren, Y. et al. Increased apoptotic neutrophils and macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus erythematosus. Arthritis Rheum. 48, 2888–2897 (2003).

    PubMed  Google Scholar 

  36. 36.

    Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015).

    CAS  PubMed  Google Scholar 

  37. 37.

    Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012).

    CAS  PubMed  Google Scholar 

  38. 38.

    Stockwell, B. R. et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell 171, 273–285 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Maeshima, E., Liang, X. M., Goda, M., Otani, H. & Mune, M. The efficacy of vitamin E against oxidative damage and autoantibody production in systemic lupus erythematosus: a preliminary study. Clin. Rheumatol. 26, 401–404 (2007).

    PubMed  Google Scholar 

  40. 40.

    Legrand, A. J., Konstantinou, M., Goode, E. F. & Meier, P. The diversification of cell death and immunity: memento mori. Mol. Cell 76, 232–242 (2019).

    CAS  PubMed  Google Scholar 

  41. 41.

    Martin-Sanchez, D. et al. Ferroptosis, but not necroptosis, is important in nephrotoxic folic acid-induced AKI. J. Am. Soc. Nephrol. 28, 218–229 (2017).

    CAS  PubMed  Google Scholar 

  42. 42.

    Hu, C. L. et al. Reduced expression of the ferroptosis inhibitor glutathione peroxidase-4 in multiple sclerosis and experimental autoimmune encephalomyelitis. J. Neurochem. 148, 426–439 (2019).

    CAS  PubMed  Google Scholar 

  43. 43.

    Hochberg, M. C. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 40, 1725 (1997).

    CAS  PubMed  Google Scholar 

  44. 44.

    Uribe, A. G. et al. The Systemic Lupus Activity Measure-Revised, the Mexican Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), and a modified SLEDAI-2K are adequate instruments to measure disease activity in systemic lupus erythematosus. J. Rheumatol. 31, 1934–1940 (2004).

    PubMed  Google Scholar 

  45. 45.

    Kay, J. & Upchurch, K. S. ACR/EULAR 2010 rheumatoid arthritis classification criteria. Rheumatology (Oxford) 51, vi5–vi9 (2012).

    Google Scholar 

  46. 46.

    van der Linden, S., Valkenburg, H. A. & Cats, A. Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum. 27, 361–368 (1984).

    PubMed  Google Scholar 

  47. 47.

    International Team for the Revision of the International Criteria for Behçet’s Disease (ITR-ICBD). The International Criteria for Behçet’s Disease (ICBD): a collaborative study of 27 countries on the sensitivity and specificity of the new criteria. J. Eur. Acad. Dermatol. Venereol. 28, 338-347 (2014).

  48. 48.

    Knight, J. S. et al. Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice. Ann. Rheum. Dis. 74, 2199–2206 (2015).

    CAS  PubMed  Google Scholar 

  49. 49.

    Papayannopoulos, V., Metzler, K. D., Hakkim, A. & Zychlinsky, A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J. Cell Biol. 191, 677–691 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the \(2^{{-\Delta \Delta}{\rm {C}_T}}\) method. Methods 25, 402–408 (2001).

    CAS  Google Scholar 

Download references

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

Affiliations

Authors

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.

Ethics declarations

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.

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

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.

Source data

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.

Source data

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.

Source data

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.

Supplementary information

Supplementary Information

Supplementary Tables 1–7 and associated legends.

Reporting Summary

Peer Review Information

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 5

Unprocessed western blots and/or gels.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 7

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 5

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 6

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 7

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 8

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 9

Statistical source data.

Source Data Extended Data Fig. 9

Unprocessed western blots and/or gels.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing