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The AIM2 inflammasome is critical for innate immunity to Francisella tularensis.

Fernandes-Alnemri T, Yu JW, Juliana C, Solorzano L, Kang S, Wu J, Datta P, McCormick M, Huang L, McDermott E, Eisenlohr L, Landel CP, Alnemri ES - Nat. Immunol. (2010)

Bottom Line: Francisella tularensis, the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18.We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response.Caspase-1 activation, IL-1beta secretion and cell death were absent in Aim2(-/-) macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

ABSTRACT
Francisella tularensis, the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18. We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response. Using mice deficient in the DNA-sensing inflammasome component AIM2, we demonstrate here that AIM2 is required for sensing F. tularensis. AIM2-deficient mice were extremely susceptible to F. tularensis infection, with greater mortality and bacterial burden than that of wild-type mice. Caspase-1 activation, IL-1beta secretion and cell death were absent in Aim2(-/-) macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA. Our study identifies AIM2 as a crucial sensor of F. tularensis infection and provides genetic proof of its critical role in host innate immunity to intracellular pathogens.

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IRF3 signaling is required for activation of the AIM2 inflammasome by F. novicida but not by liposome-delivered DNA. (a,b) Immunoblot analysis of mouse procaspase-1, caspase-1 and/or AIM2 in culture supernatants and lysates of mouse Irf3−/− and Irf3+/+ macrophages infected for 6 h with F. novicida (MOI, in parentheses above lanes), treated with LPS and nigericin as described in Figure 1b, or transfected with poly(dA:dT) (a), or infected with F. novicida (MOI, 250) in the presence or absence of IFN-β (b). (c) Immunoblot analysis of mouse procaspase-1, caspase-1 and/or AIM2 in culture supernatants and lysates of Ifnar1−/− and Ifnar1+/+ macrophages left untreated or treated for 2 h with IFN-β alone or followed by infection for 6 h with F. novicida (MOI, in parentheses above lanes). (d) Enzyme-linked immunosorbent assay of IFN-β in culture supernatants of Aim2−/− and Aim2+/+ macrophages left uninfected or infected for 6 h with F. novicida (MOI, 250). *P < 0.05 and **P < 0.005 (Student’s t-test). (e) Immunoblot analysis of mouse STAT1 phosphorylated at Tyr701 (p-STAT1), total STAT1 and AIM2 in lysates of mouse Aim2−/− and Aim2+/+ macrophages left uninfected or infected with F. novicida (MOI, 250). (f) Enzyme-linked immunosorbent assay of IFN-β in culture supernatants of Aim2+/+ macrophages left untreated or infected for 6 h with F. novicida (MOI, 250) in the presence (+ bafilo) or absence of bafilomycin (50 nM). *P < 0.01 (Student’s t-test). Data are representative of two (a,c) or three (b,d–f) experiments (mean and s.d. in d,f).
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Figure 4: IRF3 signaling is required for activation of the AIM2 inflammasome by F. novicida but not by liposome-delivered DNA. (a,b) Immunoblot analysis of mouse procaspase-1, caspase-1 and/or AIM2 in culture supernatants and lysates of mouse Irf3−/− and Irf3+/+ macrophages infected for 6 h with F. novicida (MOI, in parentheses above lanes), treated with LPS and nigericin as described in Figure 1b, or transfected with poly(dA:dT) (a), or infected with F. novicida (MOI, 250) in the presence or absence of IFN-β (b). (c) Immunoblot analysis of mouse procaspase-1, caspase-1 and/or AIM2 in culture supernatants and lysates of Ifnar1−/− and Ifnar1+/+ macrophages left untreated or treated for 2 h with IFN-β alone or followed by infection for 6 h with F. novicida (MOI, in parentheses above lanes). (d) Enzyme-linked immunosorbent assay of IFN-β in culture supernatants of Aim2−/− and Aim2+/+ macrophages left uninfected or infected for 6 h with F. novicida (MOI, 250). *P < 0.05 and **P < 0.005 (Student’s t-test). (e) Immunoblot analysis of mouse STAT1 phosphorylated at Tyr701 (p-STAT1), total STAT1 and AIM2 in lysates of mouse Aim2−/− and Aim2+/+ macrophages left uninfected or infected with F. novicida (MOI, 250). (f) Enzyme-linked immunosorbent assay of IFN-β in culture supernatants of Aim2+/+ macrophages left untreated or infected for 6 h with F. novicida (MOI, 250) in the presence (+ bafilo) or absence of bafilomycin (50 nM). *P < 0.01 (Student’s t-test). Data are representative of two (a,c) or three (b,d–f) experiments (mean and s.d. in d,f).

Mentions: Activation of the inflammasome by Francisella infection requires an intact type I interferon response for efficient inflammasome activation 10. Consistent with these studies, Francisella infection of Irf3−/− macrophages, which are defective in the secretion of type I interferons in response to cytosolic DNA, resulted in less efficient activation of the AIM2 inflammasome compared with wild-type macrophages (Fig. 4a, left panels). In contrast, liposome-delivered DNA induced similar activation of the AIM2 inflammasome in both WT and Irf3−/− macrophages (Fig. 4a, right panels). Similar expression levels of AIM2 in wild-type and Irf3−/− macrophages (Fig. 4a, lower panel), were observed ruling out the possibility that Irf3−/− macrophages express less AIM2. To determine if type I interferon signaling through its IFNAR1 could restore AIM2 activation by Francisella infection in the Irf3−/− macrophages, we treated these macrophages with IFN-β at the time of infection and assayed caspase-1 activation at 6 h post-infection. Simultaneous treatment of Irf3−/− macrophages with both IFN-β and F. novicida restored Francisella–induced caspase-1 activation in these cells (Fig. 4b, right panels). Concomitant treatment with IFN-β at the time of infection slightly enhanced Francisella–induced caspase-1 activation in WT Irf3+/+ macrophages (Fig. 4b, left panels). Restoration of Francisella–induced caspase-1 activation in Irf3−/− macrophages required concurrent treatment with IFNβ and Francisella, as no restoration was observed if IFN-β treatment was done 2 h post-infection (supplementary Fig. 7a, left panels). No activation of caspase-1 was observed by IFN-β treatment alone (supplementary Fig. 7a, right panel). Additionally, concurrent treatment of Aim2−/− macrophages with IFN-β and Francisella did not restore Francisella–induced caspase-1 activation in these cells (supplementary Fig. 7b), indicating that IFN-β is not sufficient for Francisella-induced caspase-1 activation in the absence of AIM2.


The AIM2 inflammasome is critical for innate immunity to Francisella tularensis.

Fernandes-Alnemri T, Yu JW, Juliana C, Solorzano L, Kang S, Wu J, Datta P, McCormick M, Huang L, McDermott E, Eisenlohr L, Landel CP, Alnemri ES - Nat. Immunol. (2010)

IRF3 signaling is required for activation of the AIM2 inflammasome by F. novicida but not by liposome-delivered DNA. (a,b) Immunoblot analysis of mouse procaspase-1, caspase-1 and/or AIM2 in culture supernatants and lysates of mouse Irf3−/− and Irf3+/+ macrophages infected for 6 h with F. novicida (MOI, in parentheses above lanes), treated with LPS and nigericin as described in Figure 1b, or transfected with poly(dA:dT) (a), or infected with F. novicida (MOI, 250) in the presence or absence of IFN-β (b). (c) Immunoblot analysis of mouse procaspase-1, caspase-1 and/or AIM2 in culture supernatants and lysates of Ifnar1−/− and Ifnar1+/+ macrophages left untreated or treated for 2 h with IFN-β alone or followed by infection for 6 h with F. novicida (MOI, in parentheses above lanes). (d) Enzyme-linked immunosorbent assay of IFN-β in culture supernatants of Aim2−/− and Aim2+/+ macrophages left uninfected or infected for 6 h with F. novicida (MOI, 250). *P < 0.05 and **P < 0.005 (Student’s t-test). (e) Immunoblot analysis of mouse STAT1 phosphorylated at Tyr701 (p-STAT1), total STAT1 and AIM2 in lysates of mouse Aim2−/− and Aim2+/+ macrophages left uninfected or infected with F. novicida (MOI, 250). (f) Enzyme-linked immunosorbent assay of IFN-β in culture supernatants of Aim2+/+ macrophages left untreated or infected for 6 h with F. novicida (MOI, 250) in the presence (+ bafilo) or absence of bafilomycin (50 nM). *P < 0.01 (Student’s t-test). Data are representative of two (a,c) or three (b,d–f) experiments (mean and s.d. in d,f).
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Figure 4: IRF3 signaling is required for activation of the AIM2 inflammasome by F. novicida but not by liposome-delivered DNA. (a,b) Immunoblot analysis of mouse procaspase-1, caspase-1 and/or AIM2 in culture supernatants and lysates of mouse Irf3−/− and Irf3+/+ macrophages infected for 6 h with F. novicida (MOI, in parentheses above lanes), treated with LPS and nigericin as described in Figure 1b, or transfected with poly(dA:dT) (a), or infected with F. novicida (MOI, 250) in the presence or absence of IFN-β (b). (c) Immunoblot analysis of mouse procaspase-1, caspase-1 and/or AIM2 in culture supernatants and lysates of Ifnar1−/− and Ifnar1+/+ macrophages left untreated or treated for 2 h with IFN-β alone or followed by infection for 6 h with F. novicida (MOI, in parentheses above lanes). (d) Enzyme-linked immunosorbent assay of IFN-β in culture supernatants of Aim2−/− and Aim2+/+ macrophages left uninfected or infected for 6 h with F. novicida (MOI, 250). *P < 0.05 and **P < 0.005 (Student’s t-test). (e) Immunoblot analysis of mouse STAT1 phosphorylated at Tyr701 (p-STAT1), total STAT1 and AIM2 in lysates of mouse Aim2−/− and Aim2+/+ macrophages left uninfected or infected with F. novicida (MOI, 250). (f) Enzyme-linked immunosorbent assay of IFN-β in culture supernatants of Aim2+/+ macrophages left untreated or infected for 6 h with F. novicida (MOI, 250) in the presence (+ bafilo) or absence of bafilomycin (50 nM). *P < 0.01 (Student’s t-test). Data are representative of two (a,c) or three (b,d–f) experiments (mean and s.d. in d,f).
Mentions: Activation of the inflammasome by Francisella infection requires an intact type I interferon response for efficient inflammasome activation 10. Consistent with these studies, Francisella infection of Irf3−/− macrophages, which are defective in the secretion of type I interferons in response to cytosolic DNA, resulted in less efficient activation of the AIM2 inflammasome compared with wild-type macrophages (Fig. 4a, left panels). In contrast, liposome-delivered DNA induced similar activation of the AIM2 inflammasome in both WT and Irf3−/− macrophages (Fig. 4a, right panels). Similar expression levels of AIM2 in wild-type and Irf3−/− macrophages (Fig. 4a, lower panel), were observed ruling out the possibility that Irf3−/− macrophages express less AIM2. To determine if type I interferon signaling through its IFNAR1 could restore AIM2 activation by Francisella infection in the Irf3−/− macrophages, we treated these macrophages with IFN-β at the time of infection and assayed caspase-1 activation at 6 h post-infection. Simultaneous treatment of Irf3−/− macrophages with both IFN-β and F. novicida restored Francisella–induced caspase-1 activation in these cells (Fig. 4b, right panels). Concomitant treatment with IFN-β at the time of infection slightly enhanced Francisella–induced caspase-1 activation in WT Irf3+/+ macrophages (Fig. 4b, left panels). Restoration of Francisella–induced caspase-1 activation in Irf3−/− macrophages required concurrent treatment with IFNβ and Francisella, as no restoration was observed if IFN-β treatment was done 2 h post-infection (supplementary Fig. 7a, left panels). No activation of caspase-1 was observed by IFN-β treatment alone (supplementary Fig. 7a, right panel). Additionally, concurrent treatment of Aim2−/− macrophages with IFN-β and Francisella did not restore Francisella–induced caspase-1 activation in these cells (supplementary Fig. 7b), indicating that IFN-β is not sufficient for Francisella-induced caspase-1 activation in the absence of AIM2.

Bottom Line: Francisella tularensis, the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18.We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response.Caspase-1 activation, IL-1beta secretion and cell death were absent in Aim2(-/-) macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

ABSTRACT
Francisella tularensis, the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18. We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response. Using mice deficient in the DNA-sensing inflammasome component AIM2, we demonstrate here that AIM2 is required for sensing F. tularensis. AIM2-deficient mice were extremely susceptible to F. tularensis infection, with greater mortality and bacterial burden than that of wild-type mice. Caspase-1 activation, IL-1beta secretion and cell death were absent in Aim2(-/-) macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA. Our study identifies AIM2 as a crucial sensor of F. tularensis infection and provides genetic proof of its critical role in host innate immunity to intracellular pathogens.

Show MeSH
Related in: MedlinePlus