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Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome

Abstract

Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. Here we demonstrate that depletion of the autophagic proteins LC3B and beclin 1 enhanced the activation of caspase-1 and secretion of interleukin 1β (IL-1β) and IL-18. Depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial reactive oxygen species (ROS). Cytosolic mtDNA contributed to the secretion of IL-1β and IL-18 in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity.

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Figure 1: Absence of LC3B and heterozygous disruption of beclin 1 enhance caspase-1-dependent cytokine secretion in macrophages.
Figure 2: Depletion of autophagic proteins alters mitochondrial phenotype.
Figure 3: EtBr abolishes caspase-1 activation.
Figure 4: Autophagic protein deficiency promotes mtDNA release into cytosol through increased MPT.
Figure 5: Cytosolic mtDNA is involved in caspase-1 activation.
Figure 6: NALP3 mediates the release of mtDNA.
Figure 7: Deficiency in autophagic proteins augments caspase-1-mediated inflammatory responses in vivo.

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References

  1. He, C. & Klionsky, D.J. Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 43, 67–93 (2009).

    Article  CAS  Google Scholar 

  2. Levine, B. & Kroemer, G. Autophagy in the pathogenesis of disease. Cell 132, 27–42 (2008).

    Article  CAS  Google Scholar 

  3. Virgin, H.W. & Levine, B. Autophagy genes in immunity. Nat. Immunol. 10, 461–470 (2009).

    Article  CAS  Google Scholar 

  4. Saitoh, T. et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1β production. Nature 456, 264–268 (2008).

    Article  CAS  Google Scholar 

  5. Stutz, A., Golenbock, D.T. & Latz, E. Inflammasomes: too big to miss. J. Clin. Invest. 119, 3502–3511 (2009).

    Article  CAS  Google Scholar 

  6. Schroder, K. & Tschopp, J. The inflammasomes. Cell 140, 821–832 (2010).

    Article  CAS  Google Scholar 

  7. Franchi, L., Eigenbrod, T., Munoz-Planillo, R. & Nunez, G. The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat. Immunol. 10, 241–247 (2009).

    Article  CAS  Google Scholar 

  8. Li, P. et al. Mice deficient in IL-1β-converting enzyme are defective in production of mature IL-1β and resistant to endotoxic shock. Cell 80, 401–411 (1995).

    Article  CAS  Google Scholar 

  9. Sutterwala, F.S. et al. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24, 317–327 (2006).

    Article  CAS  Google Scholar 

  10. Aoki, H. et al. Monitoring autophagy in glioblastoma with antibody against isoform B of human microtubule-associated protein 1 light chain 3. Autophagy 4, 467–475 (2008).

    Article  CAS  Google Scholar 

  11. Qu, X. et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J. Clin. Invest. 112, 1809–1820 (2003).

    Article  CAS  Google Scholar 

  12. Qu, Y., Franchi, L., Nunez, G. & Dubyak, G.R. Nonclassical IL-1β secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J. Immunol. 179, 1913–1925 (2007).

    Article  CAS  Google Scholar 

  13. Tal, M.C. et al. Absence of autophagy results in reactive oxygen species-dependent amplification of RLR signaling. Proc. Natl. Acad. Sci. USA 106, 2770–2775 (2009).

    Article  CAS  Google Scholar 

  14. Li, N. et al. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J. Biol. Chem. 278, 8516–8525 (2003).

    Article  CAS  Google Scholar 

  15. DiMauro, S. & Schon, E.A. Mitochondrial respiratory-chain diseases. N. Engl. J. Med. 348, 2656–2668 (2003).

    Article  CAS  Google Scholar 

  16. Liesa, M., Palacin, M. & Zorzano, A. Mitochondrial dynamics in mammalian health and disease. Physiol. Rev. 89, 799–845 (2009).

    Article  CAS  Google Scholar 

  17. Kroemer, G., Galluzzi, L. & Brenner, C. Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 87, 99–163 (2007).

    Article  CAS  Google Scholar 

  18. Hashiguchi, K. & Zhang-Akiyama, Q.M. Establishment of human cell lines lacking mitochondrial DNA. Methods Mol. Biol. 554, 383–391 (2009).

    Article  CAS  Google Scholar 

  19. King, M.P. & Attardi, G. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246, 500–503 (1989).

    Article  CAS  Google Scholar 

  20. Chandel, N.S. & Schumacker, P.T. Cells depleted of mitochondrial DNA (rho0) yield insight into physiological mechanisms. FEBS Lett. 454, 173–176 (1999).

    Article  CAS  Google Scholar 

  21. Trnka, J., Blaikie, F.H., Logan, A., Smith, R.A. & Murphy, M.P. Antioxidant properties of MitoTEMPOL and its hydroxylamine. Free Radic. Res. 43, 4–12 (2009).

    Article  CAS  Google Scholar 

  22. Jiang, J. et al. A mitochondria-targeted triphenylphosphonium-conjugated nitroxide functions as a radioprotector/mitigator. Radiat. Res. 172, 706–717 (2009).

    Article  CAS  Google Scholar 

  23. Rathinam, V.A. et al. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat. Immunol. 11, 395–402 (2010).

    Article  CAS  Google Scholar 

  24. Fernandes-Alnemri, T. et al. The AIM2 inflammasome is critical for innate immunity to Francisella tularensis. Nat. Immunol. 11, 385–393 (2010).

    Article  CAS  Google Scholar 

  25. Jones, J.W. et al. Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis. Proc. Natl. Acad. Sci. USA 107, 9771–9776 (2010).

    Article  CAS  Google Scholar 

  26. Lemasters, J.J., Theruvath, T.P., Zhong, Z. & Nieminen, A.L. Mitochondrial calcium and the permeability transition in cell death. Biochim. Biophys. Acta 1787, 1395–1401 (2009).

    Article  CAS  Google Scholar 

  27. Zhang, Q. et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 464, 104–107 (2010).

    Article  CAS  Google Scholar 

  28. Zhou, R., Tardivel, A., Thorens, B., Choi, I. & Tschopp, J. Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat. Immunol. 11, 136–140 (2010).

    Article  CAS  Google Scholar 

  29. Lamkanfi, M. et al. Glyburide inhibits the Cryopyrin/Nalp3 inflammasome. J. Cell Biol. 187, 61–70 (2009).

    Article  CAS  Google Scholar 

  30. Juliana, C. et al. Anti-inflammatory compounds parthenolide and Bay 11–7082 are direct inhibitors of the inflammasome. J. Biol. Chem. 285, 9792–9802 (2010).

    Article  CAS  Google Scholar 

  31. Fahy, R.J. et al. Inflammasome mRNA expression in human monocytes during early septic shock. Am. J. Respir. Crit. Care Med. 177, 983–988 (2008).

    Article  Google Scholar 

  32. Mortensen, M. et al. Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo. Proc. Natl. Acad. Sci. USA 107, 832–837 (2010).

    Article  CAS  Google Scholar 

  33. Cruz, C.M. et al. ATP activates a reactive oxygen species-dependent oxidative stress response and secretion of proinflammatory cytokines in macrophages. J. Biol. Chem. 282, 2871–2879 (2007).

    Article  CAS  Google Scholar 

  34. Adinolfi, E. et al. Basal activation of the P2X7 ATP receptor elevates mitochondrial calcium and potential, increases cellular ATP levels, and promotes serum-independent growth. Mol. Biol. Cell 16, 3260–3272 (2005).

    Article  CAS  Google Scholar 

  35. Garcia-Marcos, M. et al. Role of sodium in mitochondrial membrane depolarization induced by P2X7 receptor activation in submandibular glands. FEBS Lett. 579, 5407–5413 (2005).

    Article  CAS  Google Scholar 

  36. Dostert, C. et al. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 320, 674–677 (2008).

    Article  CAS  Google Scholar 

  37. Unterholzner, L. et al. IFI16 is an innate immune sensor for intracellular DNA. Nat. Immunol. 11, 997–1004 (2010).

    Article  CAS  Google Scholar 

  38. Hornung, V. & Latz, E. Intracellular DNA recognition. Nat. Rev. Immunol. 10, 123–130 (2010).

    Article  CAS  Google Scholar 

  39. Watanabe, E. et al. Sepsis induces extensive autophagic vacuolization in hepatocytes: a clinical and laboratory-based study. Lab. Invest. 89, 549–561 (2009).

    Article  Google Scholar 

  40. Fredriksson, K. et al. Dysregulation of mitochondrial dynamics and the muscle transcriptome in ICU patients suffering from sepsis induced multiple organ failure. PLoS ONE 3, e3686 (2008).

    Article  Google Scholar 

  41. d'Avila, J.C. et al. Sepsis induces brain mitochondrial dysfunction. Crit. Care Med. 36, 1925–1932 (2008).

    Article  Google Scholar 

  42. Cann, G.M. et al. Developmental expression of LC3α and β: absence of fibronectin or autophagy phenotype in LC3β knockout mice. Dev. Dyn. 237, 187–195 (2008).

    Article  CAS  Google Scholar 

  43. Wang, X.M., Kim, H.P., Nakahira, K., Ryter, S.W. & Choi, A.M. The heme oxygenase-1/carbon monoxide pathway suppresses TLR4 signaling by regulating the interaction of TLR4 with caveolin-1. J. Immunol. 182, 3809–3818 (2009).

    Article  CAS  Google Scholar 

  44. Meissner, F., Molawi, K. & Zychlinsky, A. Superoxide dismutase 1 regulates caspase-1 and endotoxic shock. Nat. Immunol. 9, 866–872 (2008).

    Article  CAS  Google Scholar 

  45. Chen, Z.H. et al. Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease. PLoS ONE 3, e3316 (2008).

    Article  Google Scholar 

  46. Eaton, J.S., Lin, Z.P., Sartorelli, A.C., Bonawitz, N.D. & Shadel, G.S. Ataxia-telangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis. J. Clin. Invest. 117, 2723–2734 (2007).

    Article  CAS  Google Scholar 

  47. Pelegrin, P., Barroso-Gutierrez, C. & Surprenant, A. P2X7 receptor differentially couples to distinct release pathways for IL-1β in mouse macrophage. J. Immunol. 180, 7147–7157 (2008).

    Article  CAS  Google Scholar 

  48. Chung, S.W., Liu, X., Macias, A.A., Baron, R.M. & Perrella, M.A. Heme oxygenase-1-derived carbon monoxide enhances the host defense response to microbial sepsis in mice. J. Clin. Invest. 118, 239–247 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank B. Levine (University of Texas Southwestern Medical Center) for Becn1+/− mice; and E. Ifedigbo for technical assistance. Supported by National Institutes of Health (HL079904-12, HL08554, HL060234-10 and HL097005 to A.M.K.C., and AI083713 and AI067497 to K.A.F.) and the New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (National Institute of Allergy and Infectious Diseases of the National Institutes of Health; AI057159 to V.A.K.R).

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K.N., H.P.K., J.A.H and A.M.K.C conceived of the study with assistance from S.W.R. and K.A.F.; M.R. supervised the generation of LC3B-deficent mice; K.A.F. supervised the generation of AIM-2 deficient mice; K.N., S.J.L. and V.A.K.R did the in vitro experiments; J.A.H., S.J.L. and J.A.E. did the in vivo experiments; H.C.L. did the transmission electron microscopy; M.C. and K.N. did flow cytometry; T.D. analyzed human samples; K.N., J.A.H., A.M.K.C. and S.W.R. wrote the paper; and A.M.K.C supervised the entire project.

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Correspondence to Augustine M K Choi.

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Nakahira, K., Haspel, J., Rathinam, V. 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). https://doi.org/10.1038/ni.1980

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