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

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.

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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.

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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.).

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Authors

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.

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Correspondence to Thirumala-Devi Kanneganti.

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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 μm. 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).

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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

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