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IRGM1 links mitochondrial quality control to autoimmunity

Nature Immunology volume 22, pages 312–321 (2021)Cite this article

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Abstract

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA–dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1–/– tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.

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Fig. 1: IRGM1 deficiency induces type I interferonopathy.
Fig. 2: mtDNA induces IFN-I response in Irgm1-null cells.
Fig. 3: IFN-I induction in Irgm1-null fibroblasts is cGAS/STING-dependent.
Fig. 4: Mitophagic flux deficit drives IFN-I response in Irgm1-null fibroblasts.
Fig. 5: Tissue-selective involvement of cGAS/STING in autoimmune pathology of Irgm1-null mice.
Fig. 6: IFN-I induction in Irgm1-null macrophages is TLR7 dependent.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. Thorlacius, G. E., Wahren-Herlenius, M. & Ronnblom, L. An update on the role of type I interferons in systemic lupus erythematosus and Sjogren’s syndrome. Curr. Opin. Rheumatol. 30, 471–481 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. Crow, Y. J. & Manel, N. Aicardi–Goutieres syndrome and the type I interferonopathies. Nat. Rev. Immunol. 15, 429–440 (2015).

    Article  CAS  PubMed  Google Scholar 

  3. Rodero, M. P. & Crow, Y. J. Type I interferon-mediated monogenic autoinflammation: the type I interferonopathies, a conceptual overview. J. Exp. Med. 213, 2527–2538 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. West, A. P. et al. Mitochondrial DNA stress primes the antiviral innate immune response. Nature 520, 553–557 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Rongvaux, A. et al. Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. Cell 159, 1563–1577 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gkirtzimanaki, K. et al. IFNα impairs autophagic degradation of mtDNA promoting autoreactivity of SLE monocytes in a STING-dependent fashion. Cell Rep. 25, 921–933.e5 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Monteith, A. J. et al. Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus. Proc. Natl Acad. Sci. USA 113, E2142–E2151 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Caza, T. N. et al. HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE. Ann. Rheum. Dis. 73, 1888–1897 (2014).

    Article  CAS  PubMed  Google Scholar 

  9. Perl, A., Gergely, P. Jr. & Banki, K. Mitochondrial dysfunction in T cells of patients with systemic lupus erythematosus. Int. Rev. Immunol. 23, 293–313 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Pilla-Moffett, D., Barber, M. F., Taylor, G. A. & Coers, J. Interferon-inducible GTPases in host resistance, inflammation and disease. J. Mol. Biol. 428, 3495–3513 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Haldar, A. K. et al. IRG and GBP host resistance factors target aberrant, ‘non-self’ vacuoles characterized by the missing of ‘self’ IRGM proteins. PLoS Pathog. 9, e1003414 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhao, Y. O., Könen-Waisman, S., Taylor, G. A., Martens, S. & Howard, J. C. Localisation and mislocalisation of the interferon-inducible immunity-related GTPase, Irgm1 (LRG-47) in mouse cells. PLoS ONE 5, e8648 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Maric-Biresev, J. et al. Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection. BMC Biol. 14, 33 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Traver, M. K. et al. Immunity-related GTPase M (IRGM) proteins influence the localization of guanylate-binding protein 2 (GBP2) by modulating macroautophagy. J. Biol. Chem. 286, 30471–30480 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Azzam, K. M. et al. Irgm1 coordinately regulates autoimmunity and host defense at select mucosal surfaces. JCI Insight 2, e91914 (2017).

    Article  PubMed Central  Google Scholar 

  16. Zhou, X. J. et al. Genetic association of PRDM1-ATG5 intergenic region and autophagy with systemic lupus erythematosus in a Chinese population. Ann. Rheum. Dis. 70, 1330–1337 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. Xia, Q. et al. Autophagy-related IRGM genes confer susceptibility to ankylosing spondylitis in a Chinese female population: a case–control study. Genes Immun. 18, 42–47 (2017).

    Article  CAS  PubMed  Google Scholar 

  18. Yao, Q. M. et al. Polymorphisms in autophagy-related gene IRGM are associated with susceptibility to autoimmune thyroid diseases. Biomed. Res. Int. 2018, 7959707 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Nocturne, G. & Mariette, X. Advances in understanding the pathogenesis of primary Sjogren’s syndrome. Nat. Rev. Rheumatol. 9, 544–556 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Feng, C. G., Weksberg, D. C., Taylor, G. A., Sher, A. & Goodell, M. A. The p47 GTPase Lrg-47 (Irgm1) links host defense and hematopoietic stem cell proliferation. Cell Stem Cell 2, 83–89 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Matsuzawa, T. et al. IFN-γ elicits macrophage autophagy via the p38 MAPK signaling pathway. J. Immunol. 189, 813–818 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. King, K. Y. et al. Irgm1 protects hematopoietic stem cells by negative regulation of IFN signaling. Blood 118, 1525–1533 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. West, A. P. & Shadel, G. S. Mitochondrial DNA in innate immune responses and inflammatory pathology. Nat. Rev. Immunol. 17, 363–375 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nakahira, K. et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat. Immunol. 12, 222–230 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Ma, F. et al. Positive feedback regulation of type I IFN production by the IFN-inducible DNA sensor cGAS. J. Immunol. 194, 1545–1554 (2015).

    Article  CAS  PubMed  Google Scholar 

  26. Hamacher-Brady, A. & Brady, N. R. Mitophagy programs: mechanisms and physiological implications of mitochondrial targeting by autophagy. Cell Mol. Life Sci. 73, 775–795 (2016).

    Article  CAS  PubMed  Google Scholar 

  27. Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T. & Miyawaki, A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem. Biol. 18, 1042–1052 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Singh, S. B., Davis, A. S., Taylor, G. A. & Deretic, V. Human IRGM induces autophagy to eliminate intracellular mycobacteria. Science 313, 1438–1441 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Georgakopoulos, N. D., Wells, G. & Campanella, M. The pharmacological regulation of cellular mitophagy. Nat. Chem. Biol. 13, 136–146 (2017).

    Article  CAS  PubMed  Google Scholar 

  30. McWilliams, T. G. et al. Phosphorylation of Parkin at serine 65 is essential for its activation in vivo. Open Biol. 8, 180108 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Di Malta, C., Cinque, L. & Settembre, C. Transcriptional regulation of autophagy: mechanisms and diseases. Front. Cell Dev. Biol. 7, 114 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Trudeau, K. M. et al. Lysosome acidification by photoactivated nanoparticles restores autophagy under lipotoxicity. J. Cell Biol. 214, 25–34 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. He, L., Weber, K. J., Diwan, A. & Schilling, J. D. Inhibition of mTOR reduces lipotoxic cell death in primary macrophages through an autophagy-independent mechanism. J. Leukoc. Biol. 100, 1113–1124 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Oka, T. et al. Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure. Nature 485, 251–255 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Rodríguez-Nuevo, A. et al. Mitochondrial DNA and TLR9 drive muscle inflammation upon Opa1 deficiency. EMBO J. 37, e96553 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Ewald, S. E. et al. The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 456, 658–662 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ewald, S. E. et al. Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase. J. Exp. Med. 208, 643–651 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kruger, A. et al. Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA. EMBO Rep. 16, 1656–1663 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Martinez, J. et al. Molecular characterization of LC3-associated phagocytosis reveals distinct roles for rubicon, NOX2 and autophagy proteins. Nat. Cell Biol. 17, 893–906 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Caielli, S. et al. Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus. J. Exp. Med. 213, 697–713 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lama, L. et al. Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression. Nat. Commun. 10, 2261 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Sharma, S. et al. Suppression of systemic autoimmunity by the innate immune adaptor STING. Proc. Natl Acad. Sci. USA 112, E710–E717 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Shi, B. et al. SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages. Autophagy 13, 285–301 (2017).

    Article  CAS  PubMed  Google Scholar 

  44. Vitner, E. B. et al. Induction of the type I interferon response in neurological forms of Gaucher disease. J. Neuroinflammation 13, 104 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Borralho, P. M., Rodrigues, C. M. & Steer, C. J. microRNAs in mitochondria: an unexplored niche. Adv. Exp. Med. Biol. 887, 31–51 (2015).

    Article  CAS  PubMed  Google Scholar 

  46. Gao, S. et al. Two novel lncRNAs discovered in human mitochondrial DNA using PacBio full-length transcriptome data. Mitochondrion 38, 41–47 (2018).

    Article  CAS  PubMed  Google Scholar 

  47. Chauhan, S., Mandell, M. A. & Deretic, V. IRGM governs the core autophagy machinery to conduct antimicrobial defense. Mol. Cell 58, 507–521 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kumar, S. et al. Mechanism of Stx17 recruitment to autophagosomes via IRGM and mammalian Atg8 proteins. J. Cell Biol. 217, 997–1013 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Sliter, D. A. et al. Parkin and PINK1 mitigate STING-induced inflammation. Nature 561, 258–262 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ajayi, T. A. et al. Crohn’s disease IRGM risk alleles are associated with altered gene expression in human tissues. Am. J. Physiol. Gastrointest. Liver Physiol. 316, G95–G105 (2019).

    Article  CAS  PubMed  Google Scholar 

  51. Collazo, C. M. et al. Inactivation of LRG-47 and IRG-47 reveals a family of interferon γ–inducible genes with essential, pathogen-specific roles in resistance to infection. J. Exp. Med. 194, 181–188 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).

    Article  CAS  PubMed  Google Scholar 

  53. Martinez, J. et al. Noncanonical autophagy inhibits the autoinflammatory, lupus-like response to dying cells. Nature 533, 115–119 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank P. West, D. Sliter, F. Zhao, and J. Santos for helpful discussions; N. Yan (UTSW) and R. Youle (NINDS/NIH) for reagents; G. Barber (University of Miami) for Tmem173–/– mice; C. Bosio (NIAID/NIH) for Tlr9–/– mice; L. Perrow for assistance with breeding; D. King for blood cell count analysis; the NIEHS Histology Core laboratory for assistance with processing, sectioning, and staining of tissues; K. Gerrish, B. Elgart and N. Clausen of the NIEHS Molecular Genomics Core; N. Martin and D. Chen of the NIEHS Viral Vector Core; C.J. Tucker, A.K. Janoshazi, and E. Scappini of the NIEHS Fluorescence Microscopy and Imaging Center; and C. Bortner and M. Sifre of the NIEHS Flow Cytometry Core Facility. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (grant no. Z01 ES102005 (M.B.F.) and grant no. ZIA ES103286 (J. Martinez)); and by grant nos. AI135398, AI145929, and AI148243 (G.A.T.); grant no. R21AG063373 (M.W.G.); and grant no. R21AG060456 (O.S.S.).

Author information

Authors and Affiliations

  1. Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA

    Prashant Rai, Julie Meacham, Jennifer H. Madenspacher, Wan-Chi Lin, Peer W. F. Karmaus, Jennifer Martinez & Michael B. Fessler

  2. Cellular & Molecular Pathology Branch, National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

    Kyathanahalli S. Janardhan

  3. Integrated Laboratory Systems, Inc., Research Triangle Park, NC, USA

    Kyathanahalli S. Janardhan

  4. Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Quan-Zhen Li & Mei Yan

  5. Microarray Core Facility, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Quan-Zhen Li & Mei Yan

  6. Department of Biomedical Engineering, Boston University, Boston, MA, USA

    Jialiu Zeng & Mark W. Grinstaff

  7. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Orian S. Shirihai

  8. Division of Endocrinology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Orian S. Shirihai

  9. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA

    Gregory A. Taylor

  10. Department of Immunology, Duke University Medical Center, Durham, NC, USA

    Gregory A. Taylor

  11. Division of Geriatrics, Department of Medicine, Center for the Study of Aging and Human Development, Duke University Medical Center, Durham, NC, USA

    Gregory A. Taylor

  12. Geriatric Research, Education, and Clinical Center, VA Medical Center, Durham, NC, USA

    Gregory A. Taylor

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  1. Prashant Rai
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Contributions

P.R. designed, conducted, and analyzed experiments and contributed to the writing of the manuscript. K.S.J., J. Meacham, J.H.M., W.-C.L., P.W.F.K., Q.-Z.L., and M.Y. all conducted analyses and contributed to the writing of the manuscript. J.Z., O.S.S., J. Martinez, M.W.G., and G.A.T. provided critical reagents and contributed to the writing of the manuscript. M.B.F. designed and analyzed experiments and contributed to the writing of the manuscript.

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Correspondence to Prashant Rai or Michael B. Fessler.

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Peer review information Nature Immunology thanks Søren Paludan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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 Role for type I IFN in disease phenotypes of Irgm1−/− mouse.

a, Expression of interferon-stimulated genes in bronchoalveolar lavage (BAL) cells, bone marrow cells, and spleen of wild type and Irgm1−/− animals (n = 3/genotype). b, Autoantibodies against full array of 124 antigens, measured in serum of animals (n = 3–4 mice/genotype; each column is an independent mouse). c, Total count of bone marrow cells in wild type (n = 8), Irgm1−/−(n = 8), and Irgm1−/−Ifnar−/− (n = 9) mice. d, Total leukocyte count (WBC), lymphocyte count, platelet count, hemoglobin concentration, and hematocrit in peripheral blood of wild-type (n = 5), Ifnar−/− (n = 3), Irgm1−/−(n = 8), and Irgm1−/−Ifnar−/− (n = 6) mice. Data are mean + /− s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Two-tailed unpaired t-test (a, d) and one-way ANOVA with Tukey’s adjustment (c).

Extended Data Fig. 2 Mitochondrial abnormalities in Irgm1−/− MEFs.

a,b, Interferon-β (Ifnb1) and interferon-stimulated gene (Ifit1) expression in mouse embryonic fibroblasts (MEFs) (n = 3) (a) and bone marrow-derived macrophages (n = 3) (b) treated with or without IFN-γ (20 ng/ml, 16 h). c, Colocalization of dsDNA and HSP60 immunostaining expressed as Mander’s coefficient (n = 4 for all conditions, except n = 3 for Irgm1+/++IFN-γ). d, Mitochondrial gene Dloop1 quantified by qPCR in total DNA isolates of MEFs, normalized to nuclear gene Tert (n = 7). e, Expression of mtDNA-encoded genes mt16S and mtND4 quantified by qPCR in MEFs (n = 3). f, Mitochondrial fractions evaluated for purity by immunoblotting for mitochondrial protein TIM23 and cytosolic protein tubulin. g,h, qPCR of mtDloop1 (n = 3) (g) and immunostaining for cytoplasmic dsDNA (h) confirming mitochondrial DNA depletion by EtBr (scale bar, 20 μm). i, Ifit1 expression in MEFs treated with mitochondria-specific antioxidant MitoTempo (50 μM) prior to IFN-γ (n = 3). a, b and e-i are representative of at least two independent experiments. d is combination of two independent experiments. Data are mean + /− s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Two-tailed unpaired t-test.

Source data

Extended Data Fig. 3 cGAS/STING/IRF3 axis in type I IFN response of Irgm1−/− MEFs.

a, Interferon-β (Ifnb1) and Ifit1 expression in murine embryonic fibroblasts (MEFs) transfected with two different siRNAs against cGAS or control siRNA and then treated as shown (n = 3). b, Mb21d1 qPCR confirmation of siRNA silencing (n = 3). c,d, Mb21d1 expression measured by qPCR (n = 3) (c) and cGAS by Western blot (d) in MEFs. e, Sting−/− (that is, Tmem173−/−; n = 53 for control, n = 62 for IFN-γ and n = 44 for Brefeldin A [BrefA] conditions) and Irgm1−/−Sting−/− (n = 49 for control, n = 70 for IFN-γ and n = 36 for BrefA conditions) MEFs transduced with Sting-GFP were analyzed for colocalization (Mander’s coefficient) of GFP with ER-Golgi intermediate complex (ERGIC)-53. Brefeldin A (2 μg/ml) treatment was used as negative control. f, Ifnb1 expression in MEFs treated with IFN-γ and then transfected with 2 μg/ml cyclic-GAMP or linear GpAp negative control (n = 3). g, qPCR confirmation of Irf3 silencing by siRNA (n = 3). a–d and f–g are representative of at least two independent experiments. e is combination of two independent experiments. Data are mean + /− s.e.m. *P < 0.05, **P < 0.01. Two-tailed unpaired t-test.

Source data

Extended Data Fig. 4 Deficient mitophagic flux in Irgm1−/− MEFs.

a, GFP-Parkin-expressing Irgm1−/− MEFs transduced with mt-mKeima and analyzed for mitolysosome signals. GFP vector served as transduction control. Oligomycin and Antimycin A (O + A; 10 μM each for 5–6 h) were used as positive control for mitophagy (n = 30 for Irgm1−/−-GFP, Irgm1−/−-Parkin treated with IFN-γ or O + A, n = 28 for Irgm1−/−-GFP treated with O + A, n = 31 for Irgm1−/−-GFP treated with IFN-γ and n = 27 for Irgm1−/−-Parkin control conditions). b, PARKIN-expressing (and GFP control) MEFs assessed for Ifnb1 expression by qPCR (n = 3). c, MEFs treated with or without IFN-γ were stained for mitochondria (HSP60) and endosomes (Rab5) and analyzed for colocalization (Mander’s coefficient shown at right) (n = 38 fields) (scale bar, 20 μm). d, Nuclear and cytoplasmic fractions were isolated and stained for TFEB. Nuclear Histone H3 and cytoplasmic GAPDH serve as fraction markers. e, Expression of lysosomal biogenesis genes in MEFs (n = 3). f, Immortalized MEFs of indicated genotypes were assessed by qPCR for expression of ISGs (Ifit1, Mx2, and Oas1a) (n = 3). g, LysoTracker fluorescence fold change in MEFs treated with Torin1 (1 μM, 8 h) (n = 3). h, Mitophagy measured in mt-mKeima-expressing MEFs treated with Torin1 (n = 11). i, Mitochondrial volume colocalizing with lysosomes analyzed by live-imaging of MitoTracker and LysoTracker-stained MEFs (n = 18 for all conditions, except n = 8 for IFN-γ + Torin). Surface rendering was performed in Imaris software for volumetric analysis. a, d, f and g are representative of two independent experiments. b, e and h are representative of three independent experiments. c and i are combination of three independent experiments. Data are mean + /− s.e.m. *P < 0.05, **P < 0.01, ****P < 0.0001, ns=not significant.Two-tailed unpaired t-test.

Source data

Extended Data Fig. 5 Mitophagy controls in Irgm1−/− MEFs.

a, YFP-Parkin expressing MEFs treated with Oligomycin and Antimycin A (O + A, 10 μM each for 6 h), stained for HSP60 and analyzed for colocalization (Mander’s coefficient; right panel) of HSP60 with Parkin (n = 85 for Irgm1+/+-Parkin and n = 100 for Irgm1−/−-Parkin cells) (scale bar, 20 μm). b,c, MEFs treated with O + A analyzed for mitochondrial fragmentation by HSP60 staining (b) and for mitochondrial protein expression (TIM23, COXIV, actin control) by Western blot (c) (scale bar, 20 µm). d, WT MEFs underwent Atg5 silencing with two siRNAs (or control siRNA), were untreated or treated with IFN-γ, and then analyzed by qPCR for Ifnb1, Ifit1, and Atg5 (n = 6). e, WT and Atg7−/− MEFs were treated as shown and then analyzed by qPCR for Ifnb1, Ifit1, and Atg7 (n = 6). f,g, Silencing efficiency for siRNAs against Pink1 (n = 3) (f) and Drp1 (n = 3) (g). b, c, f, and g are representative of at least two independent experiments. a, d, and e are combinations of two independent experiments. Data are mean + /− s.e.m. ***P < 0.001, ns = not significant. Two-tailed unpaired t-test.

Source data

Extended Data Fig. 6 Tissue-selective role of STING in autoimmune pathology of Irgm1-null mice.

a, Autoantibodies against full array of 124 antigens, measured in serum of animals (n = 3–4 mice/genotype; each column is an independent mouse). b-d, Expression of interferon-stimulated genes in lungs (n = 6 for all genotypes, except n = 4 for Irgm1−/−), salivary glands (n = 6 for all genotypes, except n = 4 for Irgm1−/−), and spleen of indicated genotypes (n = 4 for all genotypes). Data are mean + /− s.e.m. ***P < 0.001, ns = not significant. One-way ANOVA with Tukey’s adjustment.

Extended Data Fig. 7 Effects of cGAS, STING, and TLR9 silencing on type I IFN response of Irgm1−/− macrophages.

a,b, BMDMs of the indicated genotypes were treated as shown and then evaluated for expression of Ifnb1 and interferon-stimulated genes by qPCR. For (a) n = 12, and (b) n = 6. c, Cytosolic fractions of BMDMs were assayed for mitochondrial (mt)DNA (ND1) and nuclear DNA (β-actin) by digital droplet PCR (n = 3). d–f, WT and Irgm1−/− BMDMs were treated with three different lentiviral shRNAs targeting TLR9 (or control shRNA), treated as shown, and then analyzed by qPCR for Ifnb1 and Ifit1 (left) and Tlr9 (right) (n = 3). a is a combination of three independent experiments. b and c are combinations of two independent experiments. d and f are representative of three independent experiments. e is a representative of two independent experiments. Data are mean + /− s.e.m. ** p ≤ 0.01, ****p ≤ 0.0001, ns = not significant. Two-tailed unpaired t-test.

Extended Data Fig. 8 No impact of TLR9 deletion on histopathology of the Irgm1−/− mouse.

Representative H&E-stained sections of lungs, salivary glands (submandibular), lacrimal glands, and pancreas from the indicated genotypes (n = 5–7/genotype) (all scale bars are 100 μm, except for lacrimal glands of Tlr9−/−, Irgm1−/−, and Irgm1−/−Tlr9−/− [50 μm]).

Extended Data Fig. 9 Role of lysosome and mitochondrial cargo in type I IFN response of Irgm1−/− macrophages.

a, Lysosomal mass analyzed by LysoTracker staining (n = 9). b, Relative acidic pH measured by ratiometric Lysosensor yellow/blue dye (n = 9). c, Cathepsin B activity assessed by Magic red substrate (n = 3). Bafilomycin A1 (BafA1) is used as negative control. d,e, qPCR for indicated targets in BMDM pretreated prior to IFN-γ with BafA1 (100 nM, 2 h) (d) or co-treated with protease inhibitors (20 μM E64d, 50 μM pepstatin A) and IFN-γ (e) (n = 3). f-h, BMDM silenced for Tlr7 using four different lentiviral shRNAs (or control shRNA) and analyzed for Tlr7 expression (f); silenced for Mavs using two different shRNAs and analyzed for Ifnb1 (g) or Mavs expression (h) (n = 3). i, Mean fluorescence intensity (MFI) of LC3-GFP transgenic WT and Irgm1−/− BMDM after washing with 0.05% saponin (n = 3). Release of LC3-I was confirmed by microscopy showing only punctate LC3-II signal (not shown). j, BMDM stained for mitochondria (HSP60) and endogenous LC3, expressed as Mander’s coefficient of colocalization (n = 10). k, BMDM analyzed for colocalization of HSP60 and endosome (Rab5) (n = 30). l, mt-mKeima-expressing BMDM analyzed for mitophagy by flow cytometry using ratiometric measurements at 488 (pH 7) and 561 nm (pH 4) lasers with 610/20 nm emission and 600 nm long pass filters. m, BMDM analyzed for HSP60 and LAMP1 colocalization (n = 7). n, Evaluation of knockdown of Pink1 in BMDM (n = 3). a and k are combination of three independent experiments. b is combination of two independent experiments. c, d, i, j, l, and m are representative of three independent experiments. e-h and n are representative of two independent experiments. All scale bars are 10 μm. Data are mean + /− s.e.m. #P = 0.06, *P < 0.05, **P < 0.01, ****P < 0.0001, ns = not significant. One-way ANOVA or two-tailed unpaired t-test.

Extended Data Fig. 10 Deletion of ATG5, ATG7, and BECLIN1 does not induce type I IFN.

BMDMs from mice with myeloid-specific deficiency (LysM-Cre-targeted deletion) of Atg7 (a-b), Atg5, (c-d), and Beclin1 (e-f) were treated as shown and analyzed for expression of Ifnb1, Ifit1, and the respective deleted gene targets. Results are a combination of BMDM cultures from two animals (n = 6), except for (b) where n = 3. CCCP = carbonyl cyanide m-chlorophenyl hydrazine. g-i, Lungs and spleen from naïve mice from the three strains were harvested and analyzed by qPCR for the targets shown. n = 6, 58 week-old females for (g), n = 4 for LysMCre−Atg5Fx/Fx lungs, n = 5 for LysMCre−Atg5Fx/Fx spleen and n = 6 for LysMCre+Atg5Fx/Fx lungs and spleens, 9–14 week-old females for (h), and N = 3, 9–14 week-old females for (i). Data are mean + /− s.e.m, ns = not significant. Two-tailed unpaired t-test.

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Source Data Extended Data Fig. 3

Unprocessed western blots.

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Unprocessed western blots.

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Unprocessed western blots.

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Rai, P., Janardhan, K.S., Meacham, J. et al. IRGM1 links mitochondrial quality control to autoimmunity. Nat Immunol 22, 312–321 (2021). https://doi.org/10.1038/s41590-020-00859-0

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