Absent in melanoma 2 is required for innate immune recognition of Francisella tularensis

Abstract

Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1beta and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-alpha and -beta) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.

Fig. 1.

AIM2 is essential for inflammasome activation in response to cytosolic dsDNA. ■, asc^−/−; , nlrp3^−/− nlrc4^−/−; , aim2^−/−; □, wt. (A) IL-1β secretion by LPS-primed BMDMs treated with 5 mM ATP or transfected with 1 μg/mL of the indicated dsDNAs for 16 h. BMDMs were infected with S. typhimurium (multiplicity of infection = 100) without LPS priming. (B) IL-18 secretion by BMDMs treated as in A. LPS priming was used for ATP stimulation only. (C) Upper shows mature IL-1β and cleaved caspase-1 secreted from LPS-primed BMDMs after stimulation with ATP or transfection with dsDNA for 5 h. Lower shows procaspase-1 and pro–IL-1β in the cell lysate. (D) IFN-β secretion by BMDMs treated as in A but without LPS priming. Graphs show the mean ± SD of triplicate wells and are representative of three independent experiments.

Fig. 2.

AIM2 is required for inflammasome activation in response to F. tularensis. ■, asc^−/−; , casp-1^−/−; , aim2^−/−; □, wt. (A) IL-1β secretion by BMDMs infected with F. tularensis ssp. novidica strain U112 or isogenic mutant ΔFPI for 5 h. Moi, multiplicity of infection. BMDMs treated with 5 mM ATP for 4 h were primed with 500 ng/mL Pam3CSK4 for 16 h. (B) Cytotoxicity as measured by LDH release. Graphs show the mean ± SD of triplicate wells and are representative of three independent experiments.

Fig. 3.

AIM2 and ASC form a complex with F. tularensis DNA. (A) Immunofluorescence microscopy of F. novidica U112-infected BMDMs at 5.5 h postinfection. DIC, differential interference contrast. Arrows indicate colocalization of DNA, degraded bacteria, AIM2, and ASC. Asterisks label diffuse AIM2 accumulation with DNA. Images are representative of at least three independent biological replicates. (Scale bar: 10 μm.) (B) BMDMs were infected with F. novidica U112 prelabeled with Hoechst 33342 nucleic acid stain. Arrows and asterisks indicate colocalization of bacterial DNA and AIM2. (Scale bar: 10 μm for Upper; scale bar: 2 μm for Lower.)

Fig. 4.

AIM2 is required for the formation of an ASC focus. (A) 3D reconstruction of a confocal image taken of a wild-type BMDM from C. (Scale bar: 0.5 μm.) (B) , F. tularensis; □, ΔFPI. Graph showing the percentage of infected BMDMs containing an ASC focus from Fig. 3A. Bars represent the mean ± SD of two independent experiments. At least 300 cells of each genotype were examined per infection.

Fig. 5.

IFN-I increases AIM2 protein levels and inflammasome activity. , ifnar^−/−; , sting^−/−; □, wt. BMDMs were infected with S. typhimurium (moi = 100), F. tularensis ssp. novidica strain U112, or isogenic mutant ΔFPI for 5 h. (A) Western blot of AIM2 protein expression. (B) IFN-β mRNA expression quantified by RT-PCR. As a control, BMDMs were transfected with 1 μg/mL polyI:C. (C) IL-1β secretion into the culture supernatant. (D) Cytotoxicity as measured by LDH release. Where indicated, BMDMs were treated with 1,000 U/mL recombinant IFN-β at 1 h postinfection. Graphs show the mean ± SD of triplicate wells and are representative of three independent experiments.

Fig. 6.

AIM2 is required for host defense against F. tularensis. Mice were infected intradermally with 1 × 10⁵ cfu of F. tularensis ssp. novidica strain U112F. Organs were harvested after 36 h and homogenized, and cfu were determined by plating serially diluted tissue extracts on modified Mueller Hinton (MH) agar. Bars indicate the geometric mean cfu per genotype.