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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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AIM2 is required for F. novicida–induced activation of the inflammasome. (a) Immunoblot analysis of mouse procaspase-1, caspase-1, IL-1β, AIM2 and/or pro-IL-1β in culture supernatants and lysates of mouse Aim2−/− and Aim2+/+ macrophages left untreated or infected for 6 h with F. novicida (FN; MOI in parentheses above lanes) or treated with LPS and nigericin as described in Figure 1b. (b) Release of LDH into culture supernatants of the macrophages in a. *P < 0.05, **P < 0.01 and ***P < 0.005, Aim2+/+ versus Aim2−/− (Student’s t-test). (c) Confocal live-cell microsopy of Aim2−/− and Aim2+/+ BMDMs left uninfected (UI) or infected for 6 h with F. novicida; nuclei were stained with Hoechst stain (blue). Images are merged differential interference contrast and Hoechst channels. Original magnification, x40. (d) Immunoblot analysis of mouse procaspase-1, caspase-1, ASC, AIM2 and/or Nlrp3 in culture supernatants and lysates of mouse wild-type, ASC-deficient (Pycard−/−; called ‘Asc−/−’ here) and Nlrp3−/− macrophages infected with F. novicida for 6 h or for 24 h (far right; MOI in parentheses above lanes). (e) Immunoblot analysis of mouse procaspase-1, caspase-1, IL-1β, pyrin and/or pro-IL-1β in culture supernatants and lysates of mouse pyrin-deficient (Mefv−/−) and pyrin-sufficient (Mefv+/+) macrophages infected for 6 h with F. novicida (MOI in parentheses above lanes) or treated with LPS and nigericin as described in a. (f) Release of LDH into culture supernatants of the macrophages in e. Data are representative of at least three experiments (mean and s.d. in b,f).
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Figure 2: AIM2 is required for F. novicida–induced activation of the inflammasome. (a) Immunoblot analysis of mouse procaspase-1, caspase-1, IL-1β, AIM2 and/or pro-IL-1β in culture supernatants and lysates of mouse Aim2−/− and Aim2+/+ macrophages left untreated or infected for 6 h with F. novicida (FN; MOI in parentheses above lanes) or treated with LPS and nigericin as described in Figure 1b. (b) Release of LDH into culture supernatants of the macrophages in a. *P < 0.05, **P < 0.01 and ***P < 0.005, Aim2+/+ versus Aim2−/− (Student’s t-test). (c) Confocal live-cell microsopy of Aim2−/− and Aim2+/+ BMDMs left uninfected (UI) or infected for 6 h with F. novicida; nuclei were stained with Hoechst stain (blue). Images are merged differential interference contrast and Hoechst channels. Original magnification, x40. (d) Immunoblot analysis of mouse procaspase-1, caspase-1, ASC, AIM2 and/or Nlrp3 in culture supernatants and lysates of mouse wild-type, ASC-deficient (Pycard−/−; called ‘Asc−/−’ here) and Nlrp3−/− macrophages infected with F. novicida for 6 h or for 24 h (far right; MOI in parentheses above lanes). (e) Immunoblot analysis of mouse procaspase-1, caspase-1, IL-1β, pyrin and/or pro-IL-1β in culture supernatants and lysates of mouse pyrin-deficient (Mefv−/−) and pyrin-sufficient (Mefv+/+) macrophages infected for 6 h with F. novicida (MOI in parentheses above lanes) or treated with LPS and nigericin as described in a. (f) Release of LDH into culture supernatants of the macrophages in e. Data are representative of at least three experiments (mean and s.d. in b,f).

Mentions: F. tularensis is a highly infectious bacterium that replicates in the cytoplasm of infected cells16, 17, leading to the activation of an IRF3-dependent and TLR-independent type I interferon response, as well as a Nlrp3-independent, but ASC-dependent inflammasome, that causes cell death 9, 10. These observations suggest that cytosolic DNA produced by Francisella during its escape from the phagosome might be the common ligand that activates both the type I interferon response, and possibly the AIM2 inflammasome pathways. To test this possibility, we analyzed inflammasome activation, and cell death by LDH release in macrophages from wild-type and Aim2−/− mice after infection with F. tularensis subspecies novicida (F. novicida). Indeed, processing of caspase-1, secretion of IL-1β, and LDH release were completely absent in Aim2−/− macrophages in response to F. novicida infection at 6 h post infection (Fig. 2a,b), even at high multiplicity of infection (Fig. 2a, 6th & 7th lane). Processing of caspase-1 and secretion of IL-1β were also absent in Aim2−/− macrophages at 24 h post infection, although there was LDH release at this time point (Supplementary Fig. 4a,b) likely due to caspase-1-independent cell death as previously reported in casp-1−/− macrophages 11. In contrast, normal processing of caspase-1, secretion of IL-1β, and LDH release were seen in wild-type macrophages (Fig. 2a,b, and Supplementary Fig. 4a,b). Processing of caspase-1 in response to Francisella infection or transfected DNA was also unimpaired in heterozygous Aim2+/− macrophages, although it was reduced compared to wild-type macrophages due to the reduced level of AIM2 in the Aim2+/− macrophages (supplementary Fig. 5a,b). Consistent with a critical role for AIM2 in the pyroptotic cell death pathway, morphological features of pyroptosis, including plasma membrane swelling and nuclear condensation, were very obvious in F. novicida-infected Aim2+/+, but not in Aim2−/− macrophages (Fig. 2c). Infection with Salmonella typhimurium which specifically activates the NLRC4 (Ipaf) inflammasome 18, resulted in normal processing of caspase-1 in both wild-type and Aim2−/− macrophages (Supplementary Fig. 4c), indicating that AIM2 is not involved in the pro-inflammatory response to Salmonella infection. Consistent with previous findings 10, the activation of caspase-1 by Francisella infection was dependent on ASC but not on Nlrp3 (Fig. 2d). Taken together, our results indicate that AIM2 is critical for the pro-inflammatory and cell death responses to Francisella infection, but dispensable for these processes in response to S. typhimurium infection.


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)

AIM2 is required for F. novicida–induced activation of the inflammasome. (a) Immunoblot analysis of mouse procaspase-1, caspase-1, IL-1β, AIM2 and/or pro-IL-1β in culture supernatants and lysates of mouse Aim2−/− and Aim2+/+ macrophages left untreated or infected for 6 h with F. novicida (FN; MOI in parentheses above lanes) or treated with LPS and nigericin as described in Figure 1b. (b) Release of LDH into culture supernatants of the macrophages in a. *P < 0.05, **P < 0.01 and ***P < 0.005, Aim2+/+ versus Aim2−/− (Student’s t-test). (c) Confocal live-cell microsopy of Aim2−/− and Aim2+/+ BMDMs left uninfected (UI) or infected for 6 h with F. novicida; nuclei were stained with Hoechst stain (blue). Images are merged differential interference contrast and Hoechst channels. Original magnification, x40. (d) Immunoblot analysis of mouse procaspase-1, caspase-1, ASC, AIM2 and/or Nlrp3 in culture supernatants and lysates of mouse wild-type, ASC-deficient (Pycard−/−; called ‘Asc−/−’ here) and Nlrp3−/− macrophages infected with F. novicida for 6 h or for 24 h (far right; MOI in parentheses above lanes). (e) Immunoblot analysis of mouse procaspase-1, caspase-1, IL-1β, pyrin and/or pro-IL-1β in culture supernatants and lysates of mouse pyrin-deficient (Mefv−/−) and pyrin-sufficient (Mefv+/+) macrophages infected for 6 h with F. novicida (MOI in parentheses above lanes) or treated with LPS and nigericin as described in a. (f) Release of LDH into culture supernatants of the macrophages in e. Data are representative of at least three experiments (mean and s.d. in b,f).
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Figure 2: AIM2 is required for F. novicida–induced activation of the inflammasome. (a) Immunoblot analysis of mouse procaspase-1, caspase-1, IL-1β, AIM2 and/or pro-IL-1β in culture supernatants and lysates of mouse Aim2−/− and Aim2+/+ macrophages left untreated or infected for 6 h with F. novicida (FN; MOI in parentheses above lanes) or treated with LPS and nigericin as described in Figure 1b. (b) Release of LDH into culture supernatants of the macrophages in a. *P < 0.05, **P < 0.01 and ***P < 0.005, Aim2+/+ versus Aim2−/− (Student’s t-test). (c) Confocal live-cell microsopy of Aim2−/− and Aim2+/+ BMDMs left uninfected (UI) or infected for 6 h with F. novicida; nuclei were stained with Hoechst stain (blue). Images are merged differential interference contrast and Hoechst channels. Original magnification, x40. (d) Immunoblot analysis of mouse procaspase-1, caspase-1, ASC, AIM2 and/or Nlrp3 in culture supernatants and lysates of mouse wild-type, ASC-deficient (Pycard−/−; called ‘Asc−/−’ here) and Nlrp3−/− macrophages infected with F. novicida for 6 h or for 24 h (far right; MOI in parentheses above lanes). (e) Immunoblot analysis of mouse procaspase-1, caspase-1, IL-1β, pyrin and/or pro-IL-1β in culture supernatants and lysates of mouse pyrin-deficient (Mefv−/−) and pyrin-sufficient (Mefv+/+) macrophages infected for 6 h with F. novicida (MOI in parentheses above lanes) or treated with LPS and nigericin as described in a. (f) Release of LDH into culture supernatants of the macrophages in e. Data are representative of at least three experiments (mean and s.d. in b,f).
Mentions: F. tularensis is a highly infectious bacterium that replicates in the cytoplasm of infected cells16, 17, leading to the activation of an IRF3-dependent and TLR-independent type I interferon response, as well as a Nlrp3-independent, but ASC-dependent inflammasome, that causes cell death 9, 10. These observations suggest that cytosolic DNA produced by Francisella during its escape from the phagosome might be the common ligand that activates both the type I interferon response, and possibly the AIM2 inflammasome pathways. To test this possibility, we analyzed inflammasome activation, and cell death by LDH release in macrophages from wild-type and Aim2−/− mice after infection with F. tularensis subspecies novicida (F. novicida). Indeed, processing of caspase-1, secretion of IL-1β, and LDH release were completely absent in Aim2−/− macrophages in response to F. novicida infection at 6 h post infection (Fig. 2a,b), even at high multiplicity of infection (Fig. 2a, 6th & 7th lane). Processing of caspase-1 and secretion of IL-1β were also absent in Aim2−/− macrophages at 24 h post infection, although there was LDH release at this time point (Supplementary Fig. 4a,b) likely due to caspase-1-independent cell death as previously reported in casp-1−/− macrophages 11. In contrast, normal processing of caspase-1, secretion of IL-1β, and LDH release were seen in wild-type macrophages (Fig. 2a,b, and Supplementary Fig. 4a,b). Processing of caspase-1 in response to Francisella infection or transfected DNA was also unimpaired in heterozygous Aim2+/− macrophages, although it was reduced compared to wild-type macrophages due to the reduced level of AIM2 in the Aim2+/− macrophages (supplementary Fig. 5a,b). Consistent with a critical role for AIM2 in the pyroptotic cell death pathway, morphological features of pyroptosis, including plasma membrane swelling and nuclear condensation, were very obvious in F. novicida-infected Aim2+/+, but not in Aim2−/− macrophages (Fig. 2c). Infection with Salmonella typhimurium which specifically activates the NLRC4 (Ipaf) inflammasome 18, resulted in normal processing of caspase-1 in both wild-type and Aim2−/− macrophages (Supplementary Fig. 4c), indicating that AIM2 is not involved in the pro-inflammatory response to Salmonella infection. Consistent with previous findings 10, the activation of caspase-1 by Francisella infection was dependent on ASC but not on Nlrp3 (Fig. 2d). Taken together, our results indicate that AIM2 is critical for the pro-inflammatory and cell death responses to Francisella infection, but dispensable for these processes in response to S. typhimurium infection.

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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Related in: MedlinePlus