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.

The transcription factor IRF1 and guanylate-binding proteins target activation of the AIM2 inflammasome by Francisella infection

Nature Immunology volume 16, pages 467–475 (2015)Cite this article

Subjects

Abstract

Inflammasomes are critical for mounting host defense against pathogens. The molecular mechanisms that control activation of the AIM2 inflammasome in response to different cytosolic pathogens remain unclear. Here we found that the transcription factor IRF1 was required for activation of the AIM2 inflammasome during infection with the Francisella tularensis subspecies novicida (F. novicida), whereas engagement of the AIM2 inflammasome by mouse cytomegalovirus (MCMV) or transfected double-stranded DNA did not require IRF1. Infection of F. novicida detected by the DNA sensor cGAS and its adaptor STING induced type I interferon–dependent expression of IRF1, which drove the expression of guanylate-binding proteins (GBPs); this led to intracellular killing of bacteria and DNA release. Our results reveal a specific requirement for IRF1 and GBPs in the liberation of DNA for sensing by AIM2 depending on the pathogen encountered by the cell.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Infection with F. novicida induces IRF1 expression in a manner that requires signaling via type I interferons.
Figure 2: IRF1 is essential for activation of the AIM2 inflammasome by infection with F. novicida.
Figure 3: IRF1 is not required for activation of the canonical or non-canonical NLRP3 or NLRC4 inflammasome.
Figure 4: IRF1 controls the expression of GBPs for activation of the AIM2 inflammasome.
Figure 5: GBPs target bacteria to mediate killing during infection with F. novicida.
Figure 6: IRF1 provides host protection against infection with F. novicida in vivo.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Strowig, T., Henao-Mejia, J., Elinav, E. & Flavell, R. Inflammasomes in health and disease. Nature 481, 278–286 (2012).

    Article  CAS  PubMed  Google Scholar 

  2. Lamkanfi, M. & Dixit, V.M. Mechanisms and functions of inflammasomes. Cell 157, 1013–1022 (2014).

    Article  CAS  PubMed  Google Scholar 

  3. Rathinam, V.A.K., Vanaja, S.K. & Fitzgerald, K.A. Regulation of inflammasome signaling. Nat. Immunol. 13, 333–342 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bürckstümmer, T. et al. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat. Immunol. 10, 266–272 (2009).

    Article  PubMed  CAS  Google Scholar 

  5. Fernandes-Alnemri, T., Yu, J.W., Datta, P., Wu, J. & Alnemri, E.S. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 458, 509–513 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Hornung, V. et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458, 514–518 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Roberts, T.L. et al. HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA. Science 323, 1057–1060 (2009).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 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  PubMed  PubMed Central  Google Scholar 

  10. Dombrowski, Y. et al. Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions. Sci. Transl. Med. 3, 82ra38 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Jin, T. et al. Structures of the HIN domain:DNA complexes reveal ligand binding and activation mechanisms of the AIM2 inflammasome and IFI16 receptor. Immunity 36, 561–571 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Rathinam, V.A. et al. TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by Gram-negative bacteria. Cell 150, 606–619 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Broz, P. et al. Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1. Nature 490, 288–291 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sander, L.E. et al. Detection of prokaryotic mRNA signifies microbial viability and promotes immunity. Nature 474, 385–389 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Gurung, P. et al. Toll or interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-β (TRIF)-mediated caspase-11 protease production integrates Toll-like receptor 4 (TLR4) protein- and Nlrp3 inflammasome-mediated host defense against enteropathogens. J. Biol. Chem. 287, 34474–34483 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kayagaki, N. et al. Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341, 1246–1249 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Kayagaki, N. et al. Non-canonical inflammasome activation targets caspase-11. Nature 479, 117–121 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Hagar, J.A., Powell, D.A., Aachoui, Y., Ernst, R.K. & Miao, E.A. Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock. Science 341, 1250–1253 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Meunier, E. et al. Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases. Nature 509, 366–370 (2014).

    Article  CAS  PubMed  Google Scholar 

  20. Shi, J. et al. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514, 187–192 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Henry, T., Brotcke, A., Weiss, D.S., Thompson, L.J. & Monack, D.M. Type I interferon signaling is required for activation of the inflammasome during Francisella infection. J. Exp. Med. 204, 987–994 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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  PubMed  PubMed Central  Google Scholar 

  23. Man, S.M. et al. Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex. Proc. Natl. Acad. Sci. USA 111, 7403–7408 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yamamoto, M. et al. A cluster of interferon-γ-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii. Immunity 37, 302–313 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Staeheli, P., Prochazka, M., Steigmeier, P.A. & Haller, O. Genetic control of interferon action: mouse strain distribution and inheritance of an induced protein with guanylate-binding property. Virology 137, 135–142 (1984).

    Article  CAS  PubMed  Google Scholar 

  26. Kim, B.H. et al. A family of IFN-γ inducible 65-kD GTPases protects against bacterial infection. Science 332, 717–721 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. Santic, M., Molmeret, M., Klose, K.E., Jones, S. & Kwaik, Y.A. The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Cell. Microbiol. 7, 969–979 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Sauer, J.D. et al. The N-ethyl-N-nitrosourea-induced Goldenticket mouse mutant reveals an essential function of Sting in the in vivo interferon response to Listeria monocytogenes and cyclic dinucleotides. Infect. Immun. 79, 688–694 (2011).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  30. Ishikawa, H., Ma, Z. & Barber, G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Fujita, T. et al. Evidence for a nuclear factor(s), IRF-1, mediating induction and silencing properties to human IFN-β gene regulatory elements. EMBO J. 7, 3397–3405 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Miyamoto, M. et al. Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-beta gene regulatory elements. Cell 54, 903–913 (1988).

    Article  CAS  PubMed  Google Scholar 

  33. Mariathasan, S., Weiss, D.S., Dixit, V.M. & Monack, D.M. Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis. J. Exp. Med. 202, 1043–1049 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Checroun, C., Wehrly, T.D., Fischer, E.R., Hayes, S.F. & Celli, J. Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication. Proc. Natl. Acad. Sci. USA 103, 14578–14583 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Britzen-Laurent, N. et al. Intracellular trafficking of guanylate-binding proteins is regulated by heterodimerization in a hierarchical manner. PLoS ONE 5, e14246 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Kamijo, R. et al. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 263, 1612–1615 (1994).

    Article  CAS  PubMed  Google Scholar 

  37. Elkins, K.L., Cooper, A., Colombini, S.M., Cowley, S.C. & Kieffer, T.L. In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 subunit of interleukin-12 (IL-12) but not on IL-12 p70. Infect. Immun. 70, 1936–1948 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Shi, C.S. et al. Activation of autophagy by inflammatory signals limits IL-1β production by targeting ubiquitinated inflammasomes for destruction. Nat. Immunol. 13, 255–263 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Khare, S. et al. The PYRIN domain-only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA viruses. Nat. Immunol. 15, 343–353 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Connolly, D.J. & Bowie, A.G. The emerging role of human PYHIN proteins in innate immunity: implications for health and disease. Biochem. Pharmacol. 92, 405–414 (2014).

    Article  CAS  PubMed  Google Scholar 

  41. Matsuyama, T. et al. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75, 83–97 (1993).

    Article  CAS  PubMed  Google Scholar 

  42. Honda, K. et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434, 772–777 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Kimura, T. et al. Essential and non-redundant roles of p48 (ISGF3 γ) and IRF-1 in both type I and type II interferon responses, as revealed by gene targeting studies. Genes Cells 1, 115–124 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Müller, U. et al. Functional role of type I and type II interferons in antiviral defense. Science 264, 1918–1921 (1994).

    Article  PubMed  Google Scholar 

  45. Durbin, J.E., Hackenmiller, R., Simon, M.C. & Levy, D.E. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 84, 443–450 (1996).

    Article  CAS  PubMed  Google Scholar 

  46. Kanneganti, T.D. et al. Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature 440, 233–236 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Mariathasan, S. et al. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430, 213–218 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Yamamoto, M. et al. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301, 640–643 (2003).

    Article  CAS  PubMed  Google Scholar 

  49. Irizarry, R.A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).

    Article  PubMed  Google Scholar 

  50. Huang da, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank V.M. Dixit (Genentech) and N. Kayagaki (Genentech) for Aim2-deficient mice and Gbp5-deficient mice; K.A. Fitzgerald (University of Massachusetts Medical School) for femurs from cGAS-deficient mice; P. Broz (University of Basel) for femurs from Gbpchr3-deficient mice; K. Pfeffer (Heinrich-Heine-University Duesseldorf) for femurs from GBP2-deficient mice; P.G. Thomas (St. Jude Children's Research Hospital) for the MCMV Smith MSGV strain; and X. Qi and M. Barr for technical assistance. Supported by the US National Institutes of Health (AR056296, CA163507 and AI101935), the American Lebanese Syrian Associated Charities (T.-D.K.), European Research Council (281600), the Fund for Scientific Research-Flanders (G030212N to M.L.), St. Jude Children's Research Hospital and the National Health and Medical Research Council (Neoma Boadway Endowed Fellowship and R.G. Menzies Early Career Fellowship to S.M.M.).

Author information

Authors and Affiliations

  1. Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Si Ming Man, Rajendra Karki, R K Subbarao Malireddi & Thirumala-Devi Kanneganti

  2. School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia

    Si Ming Man

  3. Hartwell Center for Bioinformatics & Biotechnology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Geoffrey Neale

  4. Animal Resources Center and the Veterinary Pathology Core, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Peter Vogel

  5. Department of Microbiology and Immunology, Osaka University, Osaka, Japan

    Masahiro Yamamoto

  6. Department of Medical Protein Research, Vlaams Instituut voor Biotechnologie, Ghent, Belgium

    Mohamed Lamkanfi

  7. Department of Biochemistry, Ghent University, Ghent, Belgium

    Mohamed Lamkanfi

Authors
  1. Si Ming Man
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rajendra Karki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R K Subbarao Malireddi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Geoffrey Neale
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter Vogel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Masahiro Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mohamed Lamkanfi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Thirumala-Devi Kanneganti
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.M.M., R.K. and T.-D.K. designed the study; S.M.M., R.K., R.K.S.M., G.N. and P.V. performed experiments and analyzed the data; M.Y. contributed reagents; and S.M.M., M.L. and T.-D.K. wrote the manuscript.

Corresponding author

Correspondence to Thirumala-Devi Kanneganti.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 IRF1 is essential for the assembly of the AIM2 inflammasome by infection with F. novicida

(a,b) IL-1β release in unprimed bone marrow-derived macrophages (BMDMs) infected with F. novicida (MOI 100) for 20 h. (c) Induction of IRF1 expression in unprimed BMDMs stimulated with 25 U/ml of recombinant mouse IFN-β. (d,e) Confocal microscopy analysis of ASC and caspase-1 in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h or transfected with poly(dA:dT) for 5 h. Quantification of the prevalence of ASC inflammasome speck. F. novicida-infected cells: WT n=228; Irf1–/– n=550; Aim2–/– n=599; Poly(dA:dT)-transfected cells: WT n=470; Irf1–/– n=583; Aim2–/– n=838. Arrowheads indicate inflammasome specks. Scale bar indicates 10 μm. Graphs show mean and s.e.m. of two (b,c-e) or three (a) independent experiments. *P < 0.01; **P < 0.0001. NS, not significant. Two-tailed t-test (b) and One-way ANOVA with a Dunnett’s multiple comparisons test (a,e).

Supplementary Figure 2 The role of IRF1 in the induction of pro-inflammatory cytokines and IFN-β in response to infection with F. novicida.

(a) Relative gene expression of Aim2 and Il1β in BMDMs infected with F. novicida (MOI 100). (b) The levels of TNF-α, IL-6, and KC released by BMDMs infected with F. novicida (MOI 100) for 20 h. (c) Relative gene expression of Il12p40 in BMDMs infected with F. novicida (MOI 100). (d) Relative gene expression of Ifnβ in BMDMs infected with F. novicida (MOI 100) for 8 h. Graphs show mean of one experiment representative of three independent experiments (a,c,d) or mean and s.e.m. of three independent experiments (b). NS, not significant. One-way ANOVA with a Dunnett’s multiple comparisons test (b).

Supplementary Figure 3 GBP2 and GBP5 are needed to activate the AIM2 inflammasome by infection with F. novicida.

(a,b) WT BMDMs were transfected with siRNA for 48 h and infected with F. novicida (MOI 100) for 20 h. Levels of caspase-1 activation and IL-1β release were determined. (c) The knockdown efficiency of Gbp3 and Gbp7 by real time qRT-PCR in WT BMDMs. Graphs show mean and s.e.m of three independent experiments (b). *P < 0.05; **P < 0.001; NS, not significant (two-tailed t-test).

Supplementary Figure 4 IRF1 controls bacterial replication during infection with F. novicida.

Immunofluorescence staining of GBP5 in unprimed BMDMs infected with GFP-expressing F. novicida for 4, 8 and 16 h. Arrowheads indicate colocalization of GBP5 and GFP-expressing bacteria. Scale bar indicates 20 ÎĽm. Data are representative of two independent experiments.

Supplementary Figure 5 Escape from the vacuole is essential for IRF1-dependent activation of the AIM2 inflammasome by F. novicida.

(a) Confocal microscopy images of ASC, caspase-1 and DNA in unprimed WT BMDMs infected with F. novicida for 20 h. Arrowheads indicate inflammasome specks. (b) Induction of IRF1, GBP2 and GBP5 expression in WT BMDMs infected with WT F. novicida or F. novicida ΔmglA (MOI 50). (c) Caspase-1 activation, IL-1β and IL-18 release and (d) cell death in unprimed BMDMs infected with WT F. novicida or F. novicida ΔmglA (MOI 100) for 20 h. (e) The levels of TNF-α, KC and IL-6 released by WT BMDMs infected with WT F. novicida or F. novicida ΔmglA (MOI 100) for 20 h. Graphs show mean and s.e.m of two (a) or three (b-e) independent experiments. *P < 0.01; **P < 0.0001; NS, not significant (two-tailed t-test).

Supplementary Figure 6 Cytosolic recognition of infection with F. novicida is mediated by cGAS and STING.

(a) Caspase-1 activation by F. novicida-mediated delivery of Salmonella LPS. Caspase-1 activation in BMDMs infected with F. novicida (MOI 100) for 20 h in the presence of 500 ng/ml ultrapure LPS from Salmonella minnesota R595. (b) The levels of IL-18 and cell death in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h. (c) Caspase-1 activation and the levels of IL-1β and IL-18 release in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h. (d) Induction of IRF1 expression in unprimed BMDMs infected with F. novicida (MOI 50). (e) A model for the activation of the AIM2 inflammasome by F. novicida, transfected DNA and mCMV. Graphs show mean and s.e.m of two (b,d) or three (a,c) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. Two-tailed t-test (b) and One-way ANOVA with a Dunnett’s multiple comparisons test (c).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1 and 2 (PDF 1479 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Man, S., Karki, R., Malireddi, R. et al. The transcription factor IRF1 and guanylate-binding proteins target activation of the AIM2 inflammasome by Francisella infection. Nat Immunol 16, 467–475 (2015). https://doi.org/10.1038/ni.3118

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3118

This article is cited by

Search

Advanced 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