Repression of inflammasome by Francisella tularensis during early stages of infection

Abstract

Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1β and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1β and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1β expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1β observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1β by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect.

Keywords: Bacterial Pathogenesis; Cytokine; Francisella; Il-1b; Immune Subversion; Immunosuppression; Inflammasome; Inflammation; Macrophages.

FIGURE 1.

Muted IL-1β and IL-18 responses were observed in naïve F. tularensis (Ft)-infected macrophages. Murine macrophages were infected with 100 m.o.i. of ΔiglC mutant of F. tularensis LVS, wild type F. tularensis LVS, F. tularensis SchuS4, and F. novicida strains. Levels of IL-1β (A) and IL-18 (B) were measured in the cell culture supernatants 24 h post-infection using IL-1β flex-set and IL-18 ELISA kit. Macrophages isolated from WT, TLR2^−/−, and caspase-1^−/− C57BL/6 mice were infected with 100 m.o.i. of F. tularensis LVS or F. novicida. IL-1β levels in culture supernatants were analyzed in F. tularensis LVS-infected (C) or F. novicida (D)-infected macrophages. A representative of one of the three experiments conducted with similar results is shown. The data were analyzed by ANOVA with a Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. Comparisons are shown with F. novicida. *, p < 0.05; ***, p < 0.001. The levels of IL-18 in the ΔiglC mutant, F. tularensis LVS and SchuS4 infected macrophages were below the level of detection (25 pg/ml).

FIGURE 2.

Differences in magnitude of inflammasome activation in F. tularensis (Ft) and F. novicida are due to differential activation of TLR2 signaling pathway. The proIL-1β (A) NLRP3 (B), and procaspase-1 and AIM2 (C) transcript levels were quantitated in macrophages infected with F. tularensis LVS and/or F. novicida at the indicated time points by qRT-PCR. The results are representative of at least two to three independent experiments. The data were analyzed by Student's t test, and a cut-off p value of 0.05 or less was considered significant. *, p < 0.05; **, p < 0.01. D, Western blot analysis shown is to visualize the activated forms of caspase-1, IL-1β, and IL-18 in the cell lysates from WT and TLR2^−/− macrophages 24 h post-infection.

FIGURE 3.

Stimulation through TLR4 but not IFN-β partially restores IL-1β suppression by F. tularensis (Ft) LVS. Macrophages were stimulated with E. coli LPS and infected with 100 m.o.i. of F. tularensis LVS (A) or F. novicida (B). The macrophages were treated with recombinant IFN-β and infected with F. tularensis LVS (C) or F. novicida (D). The IL-1β levels were measured in the culture supernatants at 6 and 24 h post-infection. The results are representative of at least two to three independent experiments. The data were analyzed by ANOVA with Tukey-Kramer post test, and a cut-off p value of 0.05 or less was considered significant. **, p < 0.01; ***, p < 0.001.

FIGURE 4.

FTL_0325 of F. tularensis (Ft) represses the expression of proIL-1β. Shown are Transcript levels of NLRP3, AIM2, procaspase-1, proIL-1β, and proIL-18 in IMCs derived from C57BL/6 mice. The IMCs were infected with either the FTL_0325 mutant or F. tularensis LVS at an m.o.i. of 100 for 2 h, and transcript levels were quantitated by qRT-PCR. The results are expressed as -fold change expression and are representative of three independent experiments performed using duplicate samples. The data were analyzed by ANOVA with Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. *, p < 0.05.

FIGURE 5.

FTL_0325 mutant of F. tularensis LVS induces an early activation of inflammasome in a TLR2-dependent fashion. Western blot analysis visualizes activated caspase-1 (A), IL-1β (B), and IL-18 (C). IMCs derived from WT C57BL/6 mice were infected with either the FTL_0325 mutant or F. tularensis LVS at an m.o.i. of 100. The cells were lysed 12 h post-infection and analyzed by Western blot analysis. The blots were stripped and re-probed for β-actin, which was used as a loading control. The Western blot images are representative of at least two-three independent experiments. The relative quantification of the bands from two-three blots is shown and expressed as relative intensity units (RIU). The data were analyzed by ANOVA with Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. **, p < 0.01; ***, p < 0.001. D, E, and F, shown is Western blot analysis using wild type and TLR2^−/− macrophages infected with the FTL_0325 mutant or F. tularensis LVS at 12 h post-infection.

FIGURE 6.

Early activation of inflammasome in the FTL_0325 mutant-infected macrophages is partially dependent on AIM2. WT or AIM2^−/− macrophages were infected with either the FTL_0325 mutant or F. tularensis (Ft) LVS at an m.o.i. of 100. The cells were lysed 12 h post-infection and analyzed by Western blot analysis for visualization of active caspase-1, IL-1β, and IL-18. The blots were stripped and re-probed for β-actin, which was used as a loading control. The Western blot images are representative of at least two-three independent experiments. The relative quantification of the bands from two-three blots is shown and expressed as relative intensity units (RIU). The data were analyzed by ANOVA with Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. **, p < 0.01; ***, p < 0.001.

FIGURE 7.

Early activation of inflammasome in the FTL_0325 mutant-infected macrophages is partially dependent on NLRP3. Ft, F. tularensis WT or NLRP3^−/− macrophages were infected with either the FTL_0325 mutant or Ft LVS at an m.o.i. of 100. The cells were lysed 12 h post-infection and analyzed by Western blot analysis for visualization of active caspase-1, IL-1β, and IL-18. The blots were stripped and re-probed for β-actin, which was used as a loading control. The Western blot images are representative of at least two-three independent experiments. The relative quantification of the bands from two-three blots is shown and expressed as relative intensity units (RIU). The data were analyzed by ANOVA with Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 8.

Activation of inflammasome in the FTL_0325 mutant-infected macrophages is independent of AIM2 during the later stages of infection. WT or AIM2^−/− macrophages were infected with either the FTL_0325 mutant or F. tularensis (Ft) LVS at an m.o.i. of 100. The cells were lysed 24 h post-infection and analyzed by Western blot analysis for visualization of active caspase-1, IL-1β, and IL-18. The blots were stripped and re-probed for β-actin, which was used as a loading control. The Western blot images are representative of at least two-three independent experiments. The relative quantification of the bands from two-three blots is shown and expressed as relative intensity units (RIU). The data were analyzed by ANOVA with Tukey-Kramer post-test and a cut-off p value of 0.05 or less was considered significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 9.

Activation of inflammasome in the FTL_0325 mutant-infected macrophages is independent of NLRP3 during the later stages of infection. WT or NLRP3^−/− macrophages were infected with either the FTL_0325 mutant or F. tularensis (Ft) LVS at an m.o.i. of 100. The cells were lysed 24 h post-infection and analyzed by Western blot analysis for visualization of active caspase-1, IL-1β, and IL-18. The blots were stripped and re-probed for β-actin, which was used as a loading control. The Western blot images are representative of at least two-three independent experiments. The relative quantification of the bands from two-three blots is shown and expressed as relative intensity units (RIU). The data were analyzed by ANOVA with Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. *, p < 0.05.

FIGURE 10.

FTL_0325 delays pyroptotic cell death in infected macrophages. Wild type uninfected macrophages (A) or infected with F. tularensis LVS (B), the FTL_0325 mutant (C), or the ΔiglC mutant (D) were stained with propidium iodide and analyzed by flow cytometry 12 h post-infection. E, WT, TLR2^−/−, ASC^−/−, caspase-1^−/−, NLRP3^−/−, and AIM2^−/− IMCs of C57BL/6 origin were infected with either the FTL_0325 mutant or F. tularensis (Ft) LVS at an m.o.i. of 100. At 12 h post-infection the lactate dehydrogenase release assay was performed using the Roche Applied Science cytotoxicity detection kit. The absorbance of the samples was read at 492 nm and adjusted using a background control before calculating the % cytotoxicity. The data are representative of three independent experiments, each conducted with three replicate samples. The data were analyzed by ANOVA with a Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 11.

FTL_0325-dependent early suppression of IL-1β facilitates the establishment of a fulminate infection by F. tularensis (Ft). C57BL/6 mice were inoculated intranasally with 1 × 10⁷ cfu of F. tularensis LVS, the FTL_0325 mutant, or the transcomplemented strain. The mice were sacrificed at days 1 and 3 post-infection. The IL-1β levels (A) and bacterial numbers (B) were determined at the indicated times in the lung homogenates. The data are cumulative of two independent experiments each conducted with three mice each (total n = 6). The data were analyzed by ANOVA with Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. *, p < 0.05; ***, p < 0.001.