Francisella infection triggers activation of the AIM2 inflammasome in murine dendritic cells

Abstract

The intracellular bacterium Francisella tularensis is the causative agent of tularemia, a potentially fatal disease. In macrophages, Francisella escapes the initial phagosome and replicates in the cytosol, where it is detected by the cytosolic DNA sensor AIM2 leading to activation of the AIM2 inflammasome. However, during aerosol infection, Francisella is also taken up by dendritic cells. In this study, we show that Francisella novicida escapes into the cytosol of bone marrow-derived dendritic cells (BMDC) where it undergoes rapid replication. We show that F. novicida activates the AIM2 inflammasome in BMDC, causing release of large amounts of IL-1β and rapid host cell death. The Francisella Pathogenicity Island is required for bacterial escape and replication and for inflammasome activation in dendritic cells. In addition, we show that bacterial DNA is bound by AIM2, which leads to inflammasome assembly in infected dendritic cells. IFN-β is upregulated in BMDC following Francisella infection, and the IFN-β signalling pathway is partially required for inflammasome activation in this cell type. Taken together, our results demonstrate that F. novicida induces inflammasome activation in dendritic cells. The resulting inflammatory cell death may be beneficial to remove the bacterial replicative niche and protect the host.

Figure 1

F. novicida escapes into the cytosol of dendritic cells. Immunofluorescence microscopy of BMDCs stained for the lysosomal marker LAMP-1 at 30 minutes (left panels) and 4 hours (right panels) post-infection with wild-type F. novicida (U112, top panels) or the FPI deletion mutants (FPI, bottom panel). Scale bars, 10 μm.

Figure 2

F. novicida replicates in BMDCs. (A) BMDCs derived from either wild-type (WT) or Caspase-1-deficient mice (Casp1-/-) were infected with either WT F. novicida (U112) or the FPI deletion mutant (FPI) at an MOI of 1. Intracellular bacteria were enumerated by CFU counts. The graph shows mean ± standard deviation (SD) of triplicate wells and is representative of at least four independent experiments. (B) Immunofluorescence microscopy of F. novicida-infected BMDCs. BMDCs were infected at an MOI of 5. Bacteria were stained with an F. novicida-specific antibody (green), actin was stained with phalloidin (red) and DNA with DAPI (blue). Scale bars, 20 μm.

Figure 3

Rapid F. novicida-induced BMDC death and cytokine release depend on the AIM2 inflammasome. Unstimulated (A, C) or Pam3CSK4-stimulated (B, D) BMDCs from WT mice or mice deficient for inflammasome components AIM2, Asc or Caspase-1 were infected with F. novicida at an MOI of 10 (U112 10, FPI 10) or 100 (U112 100, FPI 100). Cell death was determined by measuring the release of LDH in the supernatant at 9 (A) or 7 hours (B) post-infection. Levels of IL-1β were measured by ELISA at 9 (C) or 7 hours (D) post-infection. Graphs show the mean ± SD of quadruplicate wells and are representative of at least three independent experiments. (E) Western blot analysis of infected cell supernatants probed with an antibody against the Caspase-1 p10 subunit. Unstimulated BMDCs were infected at an MOI of 10 and supernatants were collected 10 hours post-infection.

Figure 4

Inflammasome assembly in F. novicida-infected BMDCs. (A) Formation of AIM2 aggregates attached to bacterial DNA. Stimulated cells were infected with F. novicida pre-labeled with the Hoechst DNA stain at an MOI of 10 and analyzed by immunofluorescence microscopy 5 hours post-infection. Scale bars: 10 μm for upper, 1 μm for lower. (B) Formation of Asc aggregates in infected BMDCs derived from either WT or Caspase-1-, Asc-, or AIM2-deficient mice. Unstimulated cells were infected with F. novicida at an MOI of 10 and analyzed by immunofluorescence microscopy 10 hours post-infection. The graph bar on the right indicates quantitation of cells containing an ASC aggregate. DIC, differential interference contrast. Scale bars, 10 μm.

Figure 5

Role of type I interferon signaling in inflammasome activation in BMDCs. (A) IFN-β mRNA levels were determined by quantitative RT-PCR following infection of unstimulated WT, casp-1-/- or IFNR-/- BMDCs with F. novicida at an MOI of 100. The values represent increase from mRNA levels in uninfected cells. (B) and (C) Cell death and IL-1β release were measured 8 hours post-infection of unstimulated BMDCs with F. novicida.