Aim2 and Nlrp3 Are Dispensable for Vaccine-Induced Immunity against Francisella tularensis Live Vaccine Strain

Abstract

Francisella tularensis is a facultative, intracellular, Gram-negative bacterium that causes a fatal disease known as tularemia. Due to its extremely high virulence, ease of spread by aerosolization, and potential to be used as a bioterror agent, F. tularensis is classified by the CDC as a tier 1 category A select agent. Previous studies have demonstrated the roles of the inflammasome sensors absent in melanoma 2 (AIM2) and NLRP3 in the generation of innate immune responses to F. tularensis infection. However, contributions of both the AIM2 and NLRP3 to the development of vaccine-induced adaptive immune responses against F. tularensis are not known. This study determined the contributions of Aim2 and Nlrp3 inflammasome sensors to vaccine-induced immune responses in a mouse model of respiratory tularemia. We developed a model to vaccinate Aim2- and Nlrp3-deficient (Aim2^-/- and Nlrp3^-/-) mice using the emrA1 mutant of the F. tularensis live vaccine strain (LVS). The results demonstrate that the innate immune responses in Aim2^-/- and Nlrp3^-/- mice vaccinated with the emrA1 mutant differ from those of their wild-type counterparts. However, despite these differences in the innate immune responses, both Aim2^-/- and Nlrp3^-/- mice are fully protected against an intranasal lethal challenge dose of F. tularensis LVS. Moreover, the lack of both Aim2 and Nlrp3 inflammasome sensors does not affect the production of vaccination-induced antibody and cell-mediated responses. Overall, this study reports a novel finding that both Aim2 and Nlrp3 are dispensable for vaccination-induced immunity against respiratory tularemia caused by F. tularensis.

Keywords: AIM2; Francisella tularensis; NLRP3; adaptive immunity; immunization; inflammasome.

FIG 1

As for wild-type mice, the emrA1 mutant is attenuated for virulence in Aim2^−/− and Nlrp3^−/− mice. Wild-type and Aim2^−/− mice (n = 5 mice/group) (A) and wild-type and Nlrp3^−/− mice (n = 5 mice/group) (B) were infected intranasally with 1 × 10⁶ CFU of either the emrA1 mutant or F. tularensis LVS. Mice were observed for morbidity and mortality for 21 days. The survival results are plotted as Kaplan-Meier survival curves, and the body weights are expressed as percentages of the initial body weight. The P values were determined by the log rank test.

FIG 2

Aim2^−/− and Nlrp3^−/− mice infected with the emrA1 mutant clear the bacteria completely. Wild-type and Aim2^−/− mice (A) and wild-type and Nlrp3^−/− mice (B) (n = 3 per group/time point) infected intranasally with 1 × 10⁶ CFU of the emrA1 mutant were sacrificed at the indicated time points. Lungs, livers, and spleens were collected, homogenized, and plated on MH chocolate agar plates to enumerate the bacteria. Results are expressed as log₁₀ CFU. The P values were determined using one-way ANOVA. *, P < 0.05; **, P < 0.01. ND, not detected. The data shown are representative of results from two independent experiments.

FIG 3

The cytokine responses in Aim2^−/− and Nlrp3^−/− mice differ from those in wild-type mice following infection with the emrA1 mutant. Wild-type and Aim2^−/− mice (A) and wild-type and Nlrp3^−/− mice (B) (n = 3 per group/time point) infected intranasally with 1 × 10⁶ CFU of the emrA1 mutant were sacrificed at the indicated time points. Lungs were collected, homogenized, and assayed for the levels of IL-6, TNF-α, and IL-1β. Results are expressed as picograms per lung (means ± SEM). The P values were determined using one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The data shown are representative of results from two independent experiments.

FIG 4

Immunization with the emrA1 mutant protects both Aim2^−/− and Nlrp3^−/− mice against lethal intranasal challenge with F. tularensis LVS. Wild-type and Aim2^−/− mice (n = 5 mice/group) (A) and wild-type and Nlrp3^−/− mice (n = 5 mice/group) (B) vaccinated intranasally with 1 × 10⁶ CFU of the emrA1 mutant were challenged intranasally with 2 × 10⁷ CFU of F. tularensis LVS on day 28 after primary vaccination. Unvaccinated mice were used as controls. Mice were observed for 21 days for morbidity and mortality. The survival results are plotted as Kaplan-Meier survival curves, and body weight is expressed as a percentage of the initial body weight. P values were determined by the log rank test. The data shown are representative of results from two independent experiments.

FIG 5

Aim2 and Nlrp3 are dispensable for antibody responses following immunization and challenge. Wild-type and Aim2^−/− mice (n = 5 mice/group) (A and C) and wild-type and Nlrp3^−/− mice (n = 5 mice/group) (B and D) were immunized intranasally with 1 × 10⁶ CFU of the emrA1 mutant. The serum samples were collected on day 28 postimmunization (A and B) or 28 days postchallenge (C and D). The results are shown as endpoint titers of anti-Francisella-specific antibodies as determined by an ELISA and are represented as log₁₀ values ± SEM. Serum from naive mice was used as a control. The P values were determined using one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ND, not detected. The data shown are representative of results from two independent experiments.

FIG 6

F. tularensis-infected Aim2^−/− and Nlrp3^−/− BMDMs clear bacteria similarly to wild-type BMDMs when cocultured with splenocytes derived from emrA1 mutant-immunized mice. Wild-type (WT) and Aim2^−/− mice (A) and wild-type and Nlrp3^−/− mice (B) (n = 6 mice/group) were immunized intranasally with 1 × 10⁶ CFU of the emrA1 mutant. On day 28 postimmunization, mice were sacrificed, and splenocytes were isolated. The splenocytes obtained from naive mice served as controls. The coculture assays were performed as described in Materials and Methods. BMDMs were lysed and plated to enumerate intracellular bacteria at 24 h and 48 h postinfection. Results are expressed as log₁₀ CFU per milliliter. The P values were determined using one-way ANOVA. *, P < 0.05; ***, P < 0.001.

FIG 7

The immune responses responsible for producing IL-17 but not IFN-γ differ between wild-type and Aim2^−/− or Nlrp3^−/− mice immunized with the emrA1 mutant. Wild-type (WT) and Aim2^−/− mice (A and B) and wild-type and Nlrp3^−/− mice (C and D) (n = 6 mice/group) were immunized intranasally with 1 × 10⁶ CFU of the emrA1 mutant. On day 28 postimmunization, mice were sacrificed, and the splenocytes were isolated. The splenocytes obtained from naive mice served as controls. BMDMs isolated from wild-type, Aim2^−/−, or Nlrp3^−/− mice were infected with F. tularensis LVS (FtLVS) (MOI of 100) and then overlaid with splenocytes from immunized or naive mice. After 96 and 120 h, culture supernatants were collected and analyzed for the quantification of IFN-γ (A and C) and IL-17 (B and D) by a cytometric bead array. Results are expressed as picograms per milliliter (means ± SEM) from triplicate samples. The P values were determined using one-way ANOVA. **, P < 0.01.