The inflammasome drives protective Th1 and Th17 cellular responses in disseminated candidiasis

Abstract

The Nlrp3 inflammasome has been proposed to play an important role in antifungal host defense. However, studies exploring the role of the inflammasome in antifungal host defense have been limited to the direct effects on IL-1β processing. Although IL-1β has important direct effects on the innate immune response, important effects of the caspase-1-dependent cytokines IL-1β and IL-18 are exerted on the initiation of the adaptive Th1 and Th17 cellular responses. No studies have been employed to assess the impact of the inflammasome on the Th1/Th17 defense mechanisms in vivo during candidiasis. In the present study, we demonstrate an essential role for caspase-1 and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) in disseminated candidiasis through regulating antifungal Th1 and Th17 responses. Caspase-1(-/-) and ASC(-/-) mice display diminished Th1/Th17 responses, followed by increased fungal outgrowth and lower survival. These observations identify a critical role for the inflammasome in controlling protective adaptive immune responses during invasive fungal infection.

Figure 1

Caspase-1−/− and ASC−/− are more susceptible to disseminated candidiasis. (A) Kaplan-Meier survival plots of WT (n=10), caspase-1−/− (n=6) (B) Kaplan-Meier survival plots of WT, ASC−/−, n=10 mice per group. (C) Fungal burden of kidneys of WT, Caspase-1−/−, and ASC−/− mice at day 3 and 7 after infection. n ≥ 5 mice per group.

Figure 2

Histopathologic assessment of the kidneys of WT, caspase-1 −/−and ASC−/− mice 3 and 7 days after intravenous injection with 2×10e5 CFU C. albicans. Kidneys from caspase-1−/− mice show C. albicans hyphae invading the tissue in the presence of little inflammation at day 7 of infection. Scale 200μm hematoxylin and eosin stainings.

Figure 3

(A) Lysates from bone marrow-derived dendritic cells (BMDCs) from WT and ASC−/− mice were collected 4 hours after exposure to 1×10⁴ CFU C. albicans/ml, and immunoblotted with anti-caspase-1 antibody. p45 indicates procaspase-1 and p20 processed caspase-1. (B) BMDCs are stimulated overnight live C. albicans 1×10e5/ml, IL18 and IL-1β were measured by ELISA. (C) Splenocytes from WT, IL-1β−/− and IL-18−/− mice were stimulated with RPMI (grey bars) or heat-killed 1×10⁶ C. albicans hyphae cells/ml. Cytokines were measured 5 days after stimulation. n=4 mice per group. (D) Splenocytes from WT, ASC−/−, and caspase-1−/− mice were re-stimulated with RPMI (grey bars) or heat-killed 1×10⁶ C. albicans hyphae cells/ml (black bars) 7 days after intravenous infection with C. albicans. Cytokines were measured 48 hours after stimulation with ELISA. *p<0.05. n=5 mice per group. (E) FACS analysis of splenocytes isolated 7 days after infection with intravenous infection with C. albicans gated for CD4, and stained for intracellular IL-17 and IFNγ. The data shown is representative for data observed in 2 separate experiments with a total of n=5 mice. (F) IFNγ, IL-17 and bioactive IL-1β were measured in the kidney homogenates from WT and caspase-1−/− mice 7 days after intravenous infection with C. albicans. n=5 mice per group.

Figure 4

ASC−/− mice have an increased inflammatory response during early infection. (A) Histopathologic assessment of the kidneys of WT, ASC−/− and caspase-1 −/− mice three days after intravenous injection with 2×10⁵ CFU C. albicans. Kidneys from ASC−/− mice show markedly more inflammatory granulomatous reaction. (B) Splenocytes from WT, ASC−/−, and caspase-1−/−mice were re-stimulated with RPMI (grey bars) or heat-killed 1×10⁶ C. albicans hyphae cells/ml (black bars) three days after intravenous infection with C. albicans. TNFα was measured 48 hours after stimulation with ELISA. *p<0.05. n=5 mice per group.