Cutting edge: Candida albicans hyphae formation triggers activation of the Nlrp3 inflammasome

Abstract

The proinflammatory cytokine IL-1beta plays an important role in antifungal immunity; however, the mechanisms by which fungal pathogens trigger IL-1beta secretion are unclear. In this study we show that infection with Candida albicans is sensed by the Nlrp3 inflammasome, resulting in the subsequent release of IL-1beta. The ability of C. albicans to switch from a unicellular yeast form into a filamentous form is essential for activation of the Nlrp3 inflammasome, as C. albicans mutants incapable of forming hyphae were defective in their ability to induce macrophage IL- 1beta secretion. Nlrp3-deficient mice also demonstrated increased susceptibility to infection with C. albicans, which is consistent with a key role for Nlrp3 in innate immune responses to the pathogen C. albicans.

FIGURE 1

C. albicans induces IL-1β secretion from LPS-primed Mϕ. A, BMM from WT mice were either primed with 50 ng/ml LPS or left untreated. Mϕ were infected with the C. albicans clinical isolate FC20 or ATCC strain UC820 and culture supernatants collected 6 h later; IL-1β was measured by ELISA. B, LPS-primed Mϕ were infected with C. albicans strain FC20 at the indicated MOI for 6 h; culture supernatants were collected and IL-1β release measured by ELISA. LPS-primed Mϕ were infected with the indicated C. albicans strains (C), and live, heat killed (HK) or UV killed FC20 and Zymosan (100 µg/ml) (D). IL-1β release into culture supernatants 6 h after challenge was measured by ELISA. Determinations were performed in triplicate and presented as the mean ± SEM. Results are representative of two (C, D) and three (A, B) separate experiments.

FIGURE 2

IL-1β secretion induced by C. albicans is dependent on the Nlrp3 inflammasome. A, LPS-primed BMM from WT, Nlrp3-, ASC-, caspase-1-, or Nlrc4-deficient mice were stimulated for 6 h with or without C. albicans strain FC20 at an MOI of 1:10. Culture supernatants were collected and IL-1β secretion quantified by ELISA. Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of two separate experiments. B, Lysates from LPS- primed Mϕ from WT, Nlrp3-, ASC-, caspase-1-, or Nlrc4-deficient mice were collected after 6h exposure to C. albicans strain FC20 and immunoblotted with antibodies against the p10 subunit of caspase-1, IL-1β and GAPDH. Results are representative of two separate experiments. C, WT (n=4) and Nlrp3−/− (n=4) mice were infected i.v. with 5 × 10⁵ CFU of C. albicans strain FC20. After 6 days C. albicans colonization was assessed in the left kidney, spleen and liver. Data are expressed as the mean ± SEM.

FIGURE 3

C. albicans bud-hyphae transition is essential for Nlrp3 inflammasome activation. A, IL-1β release from LPS-primed WT Mϕ was assessed by ELISA after 6 h exposure (MOI of 1:10) to C. albicans strain FC20 yeast and hyphae. B, LPS-primed WT Mϕ were infected with C. albicans strain SC5314 (wild-type) or the efg1Δ/Δcph1Δ/Δ̣ mutant at the indicated MOI. IL-1β release into culture supernatants 6 h after challenge was measured by ELISA. C, Lysates from LPS- primed WT Mϕ after a 6h infection with C. albicans strain SC5314 (wild-type) or the efg1Δ/Δcph1Δ/Δ̣ mutant at the indicated MOI were immunoblotted with antibodies against the p10 subunit of caspase-1 and GAPDH. D, WT mice were infected i.v. with 5 × 10⁶ CFU of C. albicans strain SC5314 (wild-type) or the efg1Δ/Δcph1Δ/Δ̣ mutant; 4 h post infection serum IL-1β was determined by ELISA. * p = 0.0159 by two-tailed Mann Whitney test. E, LPS-primed WT Mϕ were infected with C. albicans strains FC20 and WO-1 white and opaque phenotypes at an MOI of 1:10 for 6 h and IL-1β release was measured by ELISA. F, LPS-primed WT Mϕ were infected with three strains each of C. krusei (C.k., 1: ATCC6258, 2: P31, 3: 932638), C. tropicalis (C.t., 1: T4, 2: T362, 3: T5), and C. glabrata (C.g, 1: 932273, 2: 1480, 3: 932474) and C. albicans (C.a.) FC20 at an MOI of 1:10 for 6 h and IL-1β was measured by ELISA. Determinations were performed in triplicate and presented as the mean ± SEM (A, B, E). Determinations were performed in duplicate and presented as the mean (F). Results are representative of two (B, C, E, F) and three (A) separate experiments.