Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection

Abstract

Invasive pulmonary aspergillosis is a leading cause of infection-associated mortality in immunocompromised individuals. Aspergillus fumigatus infection produces ligands that could activate inflammasomes, but the contribution of these host defenses remains unclear. We show that two inflammasome receptors, AIM2 and NLRP3, recognize intracellular A. fumigatus and collectively induce protective immune responses. Mice lacking both AIM2 and NLRP3 fail to confine Aspergillus hyphae to inflammatory foci, leading to widespread hyphal dissemination to lung blood vessels. These mice succumb to infection more rapidly than WT mice or mice lacking a single inflammasome receptor. AIM2 and NLRP3 activation initiates assembly of a single cytoplasmic inflammasome platform, composed of the adaptor protein ASC along with caspase-1 and caspase-8. Combined actions of caspase-1 and caspase-8 lead to processing of pro-inflammatory cytokines IL-1β and IL-18 that critically control the infection. Thus, AIM2 and NLRP3 form a dual cytoplasmic surveillance system that orchestrates responses against A. fumigatus infection.

Figure 1. NLRP3 and AIM2 inflammasomes provide resistance to Aspergillus-induced mortality

(A) Survival of mice infected with 1×10⁵ A. fumigatus conidia after immunosuppression with cyclophosphamide and cortisone acetate. (B) Gross pathology of A. fumigatus-infected lungs collected on day 3. (C) Gomori Methenamine Silver (GMS), hematoxylin and eosin (H&E) and myeloperoxidase (MPO) staining of A. fumigatus-infected lungs collected on day 3. Data represent two independent experiments. (A) Log-rank test, **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not statistically significant. See also Figure S1 and S6.

Figure 2. Bone marrow and stromal compartments contribute to the host defense against Aspergillus infection

(A) Survival of bone marrow chimeras infected with 1×10⁵ A. fumigatus conidia after immunosuppression with cyclophosphamide and cortisone acetate. (B) Gross pathology of A. fumigatus-infected lungs collected on day 3. (C-E) GMS, H&E and MPO staining of A. fumigatus-infected lungs collected on day 3. (A) Log-rank test, *, P<0.05. ns, not statistically significant. See also Figure S2.

Figure 3. Both NLRP3 and AIM2 are required for inflammasome activation in response to Aspergillus infection

(A-F) BMDCs from WT, Nlrp3^–/–, Aim2^–/–, Aim2^–/–Nlrp3^–/–, and Asc^–/– mice were infected with A. fumigatus (MOI 20) for 20 h. Caspase-1 activation was analyzed from the cell lysate and levels of IL-1β, IL-18 or IL-6 released were analyzed from the supernatant. Data represent means ± SEM of triplicate wells and are representative of three or more independent experiments. (B-D, and F) Tukey's multiple comparison test, **, P<0.01; ***, P<0.001; ns, not statistically significant. See also Figure S3-S5.

Figure 4. ROS and potassium efflux contributes to NLRP3 inflammasome activation in response to Aspergillus infection

(A-D) BMDCs from WT and Aim2^–/– mice were infected with A. fumigatus (MOI 20) for 20 h in the absence or presence of 10 mM N-acetyl-L-cysteine (NAC) or 50 mM KCl. Caspase-1 activation was analyzed from the cell lysate and levels of IL-1β released were analyzed from the supernatant. (E and F) WT BMDCs were infected with A. fumigatus (MOI 20), C. albicans (MOI 5) or stimulated with heat-killed (HK) or paraformaldehyde (PFA)-fixed A. fumigatus or C. albicans for 20 h. Caspase-1 activation was analyzed from the cell lysate and levels of IL-1β released were analyzed from the supernatant. Data represent means ± SEM of triplicate wells. Data are representative of two independent experiments. (B, D, and F) Tukey's multiple comparison test, *, P<0.05; **, P<0.01; ****, P<0.0001; ns, not statistically significant.

Figure 5. Activation of AIM2 and NLRP3 by Aspergillus infection induces the assembly of a single inflammasome composed of ASC, caspase-1 and caspase-8

(A) WT, Aim2^–/–, Nlrp3^–/–, and Aim2^–/– Nlrp3^–/– BMDCs were infected with A. fumigatus for 20 h and stained for ASC (red), Caspase-1 (magenta), Caspase-8 (green) and DNA (blue). Arrowheads indicate an inflammasome speck. (B) Percentage of BMDCs contained an ASC speck after A. fumigatus infection or transfected with 2.5 μg poly(dA:dT) for 5 h. At least 300 BMDCs from each genotype were counted. (C) Composition of the ASC specks. Data represent means ± SEM of one experiment representative of two independent experiments. (B) Dunnett's multiple comparison test, ***, P<0.001; ****, P<0.0001.

Figure 6. Aspergillus-induced IL-1β and IL-18 production is dependent on both caspase-1 and caspase-8

(A-F) BMDCs from WT, Casp1^–/–Casp11^–/–, Casp11^–/– and Casp1^–/–Casp11^Tg, Ripk3^–/–, Ripk3^–/–Casp8^–/+, Ripk3^–/–Casp8^–/–, Ripk3^–/–Fadd^–/+ and Ripk3^–/–Fadd^–/– mice were infected with A. fumigatus (MOI 20) for 20 h. Caspase-1 activation was analyzed from the cell lysate and levels of IL-1β and IL-18 released were analyzed from the supernatant. (G) Survival of mice infected with 1×10⁵ A. fumigatus conidia after immunosuppression with cyclophosphamide and cortisone acetate. (B-E) Data represent means ± SEM of triplicate wells. Data are representative of three or more independent experiments. Tukey's multiple comparison test, **, P<0.01; ***, P<0.001. (G) Log-rank test, ****, P<0.0001. ns, not statistically significant.

Figure 7. IL-1β and IL-18 are critical in controlling Aspergillus infection

WT and Aim2^–/–Nlrp3^–/– mice were infected with 1×10⁵ A. fumigatus conidia after immunosuppression with cyclophosphamide and cortisone acetate. (A, B and D) The levels of IL-1β, IL-18 and IFN-γ in lung homogenates after 3 days of A. fumigatus infection. (C) Caspase-1 activation in lung homogenates after 3 days of A. fumigatus infection. (E) Survival of WT, Il-1β^–/– and Il-18^–/– mice infected with 1×10⁵ A. fumigatus conidia after immunosuppression. (A, B, and D) Data represent means ± SEM. Unpaired t-test. (E) Log-rank test. *, P<0.05; **, P<0.01, ns, not statistically significant.