Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 May;16(5):467-75.
doi: 10.1038/ni.3118. Epub 2015 Mar 16.

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

Si Ming Man  1 , Rajendra Karki  2 , R K Subbarao Malireddi  2 , Geoffrey Neale  3 , Peter Vogel  4 , Masahiro Yamamoto  5 , Mohamed Lamkanfi  6 , Thirumala-Devi Kanneganti  2
Affiliations

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

Si Ming Man et al. Nat Immunol. 2015 May.

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.

PubMed Disclaimer

Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. F. novicida infection induces IRF1 expression in a manner that requires type I interferon signaling
(a) Caspase-1 activation, IL-18 release and cell death in unprimed bone marrow-derived of the indicated strain macrophages (BMDMs) infected with F. novicida (MOI 100) for 20 h or transfected with poly(dA:dT) for 5 h. (b) IL-1β and IL-18 release and cell death in unprimed BMDMs infected with F. novicida for 20 h or transfected with poly(dA:dT) for 5 h. (c) Heat map of microarray analysis showing relative expression of interferon regulatory factors (IRFs) genes in Ifnar1−/− BMDMs compared to wildtype (WT) BMDMs infected with F. novicida for 8 h. (d) Induction of IRF1 expression in unprimed BMDMs infected with F. novicida (MOI 50). Graphs show mean and s.e.m. of two (b) or three (a,d) independent experiments. Microarray analysis was performed using duplicate samples of each genotype (c). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. One-way ANOVA with a Dunnett’s multiple comparisons test (a,b).
Figure 2
Figure 2. IRF1 is essential for AIM2 inflammasome activation by F. novicida infection
(a,b) Caspase-1 activation, IL-1β and IL-18 release in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h or transfected with poly(dA:dT) for 5 h. (c,d) Cell death in unprimed BMDMs infected with F. novicida for 20 h, transfected with poly(dA:dT) for 5 h or infected with mCMV (MOI 10) for 10 h. Arrowheads indicate dead cells. (e,f) Caspase-1 activation, IL-18 release and cell death in unprimed BMDMs transfected with pcDNA for 5 h. (g,h) Caspase-1 activation and IL-1β and IL-18 release in unprimed BMDMs infected with mCMV (MOI 10) for 10 h. (i) Caspase-1 activation in BMDMs infected with F. novicida with or without co-stimulation with recombinant mouse IFN-β (25, 250 and 500 U/ml). Graphs show mean and s.e.m. of two (g,h) or three (a–f, i) independent experiments. *P < 0.01; **P < 0.001; ***P < 0.0001; NS, not significant (two-tailed t-test).
Figure 3
Figure 3. IRF1 is not required for canonical or non-canonical NLRP3 or NLRC4 inflammasome activation
(a) Caspase-1 activation, (b) IL-1β and IL-18 release and cell death in LPS-primed BMDMs stimulated with ATP or nigericin or in unprimed BMDMs infected with Citrobacter rodentium (MOI 20) or ΔfliCΔfljB mutant S. Typhimurium (MOI 20) or wild-type S. Typhimurium (STm) for 20 h. (c) Caspase-1 activation, (d) IL-1β and IL-18 release and cell death in unprimed BMDMs infected with log-phase grown S. Typhimurium (MOI 1) for 4 h. Graphs show mean and s.e.m. of three independent experiments. NS, not significant (two-tailed t-test).
Figure 4
Figure 4. IRF1 controls expression of GBPs for AIM2 inflammasome activation
(a) Heat map of microarray data showing relative expression of macrophage-mediated immunity genes found significantly enriched in unprimed Irf1−/−, Ifnar1−/− or Aim2−/− BMDMs compared to unprimed WT BMDMs after 8 h of infection with F. novicida. (b) Gene expression of GBPs relative to β-actin in BMDMs infected with F. novicida for 8 h by real time qRT-PCR. (c–f) Caspase-1 activation, IL-1β and IL-18 release and cell death in unprimed WT, Gbpchr3-deleted (Gbpchr3-KO) or Aim2−/− BMDMs infected with F. novicida (MOI 100, 20 h), log-phase grown S. Typhimurium (MOI 1, 4 h), or transfected with poly(dA:dT) or stimulated with LPS+ATP. Microarray analysis was performed using duplicate samples of each genotype (a). Graphs show mean and s.e.m. of three (b–f) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. Two-tailed t-test (c) and One-way ANOVA with a Dunnett’s multiple comparisons test (f).
Figure 5
Figure 5. GBPs target bacteria to mediate killing during F. novicida infection
(a) Induction of IRF1, GBP2 and GBP5 expression in unprimed BMDMs infected with F. novicida (MOI 50). (b) Immunofluorescence staining of GBP5 in unprimed BMDMs infected with F. novicida for 16 h. Scale bar, 10 μm; insert scale bar, 1 μm. (c) The percentage of bacteria colocalized with GBP5 was quantified. n=3,575 bacteria in WT BMDMs and n=3,620 bacteria in Irf1−/− BMDMs. (d) CFU recovered from WT, Irf1−/− and Gbpchr3-deleted BMDMs infected with F. novicida (MOI 100). (e) Quantification of the number of F. novicida bacteria per BMDMs using confocal microscopy. n=758 WT BMDMs and n=679 Gbp5−/− BMDMs. (f) Quantification of the number of F. novicida bacteria per BMDMs using confocal microscopy. n=715 WT BMDMs, n=710 Irf1−/− BMDMs, n=706 Aim2−/− BMDMs. (g) Caspase-1 activation and IL-1β release in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h. Data from one experiment representative of two (d–f) or from three (a–c,g) independent experiments. *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001; NS, not significant. Two-tailed t-test (c,g) and One-way ANOVA with a Dunnett’s multiple comparisons test (d) or with a Tukey’s multiple comparisons test (e,f).
Figure 6
Figure 6. IRF1 provides host protection against F. novicida infection in vivo
(a) % Survival of 8 week-old WT (n=28), Irf1−/− (n=17), Aim2−/− (n=13) and Casp1 x Casp11 double-deficient (Casp1/11−/−) (n=5) mice subcutaneously infected with 7.5×104 CFU of F. novicida. (b) Body weight change of 8 week-old WT (n=16), Irf1−/− (n=9), Aim2−/− (n=9) and Casp1/11−/− (n=5) subcutaneously infected with 7.5×104 CFU of F. novicida. (c) Bacterial burden in the liver and spleen of 8 week-old male WT (n=8) and Irf1−/− (n=6) mice infected with 1×105 CFU of F. novicida. (d) Bacterial burden in the liver and spleen of 8-week old female WT (n=12) and Aim2−/− (n=9) mice infected with 1×105 CFU of F. novicida. (e) Concentrations of IL-18 in the serum of WT (n=15), Irf1−/− (n=11), Aim2−/− (n=11), and Casp1/11−/− (n=5) mice infected with 1×105 CFU of F. novicida for 24 h. (f) H&E and (g) Gram staining of F. novicida-infected livers collected on day 3. (h) Myeloperoxidase (MPO) and (i) TUNEL staining of F. novicida-infected livers collected on day 3. Top panels, 10× magnification; bottom panels 40× magnification. The dashed circle indicates a granuloma. Arrowheads indicate bacterial colonies. Data are from one experiment representative of two independent experiments (b) or pooled from two independent experiments (a,c–e). Error bars indicate s.e.m. *P < 0.05; **P < 0.001; ***P < 0.0001. Log-rank test (a), Two-tailed t-test (c,d) and One-way ANOVA with a Dunnett’s multiple comparisons test (e).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Strowig T, Henao-Mejia J, Elinav E, Flavell R. Inflammasomes in health and disease. Nature. 2012;481(7381):278–286. - PubMed
    1. Lamkanfi M, Dixit VM. Mechanisms and functions of inflammasomes. Cell. 2014;157(5):1013–1022. - PubMed
    1. Rathinam VAK, Vanaja SK, Fitzgerald KA. Regulation of inflammasome signaling. Nature immunology. 2012;14(4):333–342. - PMC - PubMed
    1. Burckstummer T, Baumann C, Bluml S, Dixit E, Durnberger G, Jahn H, et al. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nature immunology. 2009;10(3):266–272. - PubMed
    1. Fernandes-Alnemri T, Yu JW, Datta P, Wu J, Alnemri ES. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature. 2009;458(7237):509–513. - PMC - PubMed

Publication types

MeSH terms

Associated data