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, 5 (5), 487-97

An Essential Role for the NLRP3 Inflammasome in Host Defense Against the Human Fungal Pathogen Candida Albicans

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An Essential Role for the NLRP3 Inflammasome in Host Defense Against the Human Fungal Pathogen Candida Albicans

Amy G Hise et al. Cell Host Microbe.

Abstract

Candida albicans is an opportunistic fungal pathogen causing life-threatening mucosal and systemic infections in immunocompromised humans. Using a murine model of mucosal Candida infection, we investigated the role of the proinflammatory cytokine IL-1beta in host defense to Candida albicans. We find that the synthesis, processing, and release of IL-1beta in response to Candida are tightly controlled and first require transcriptional induction, followed by a second signal leading to caspase-1-mediated cleavage of the pro-IL-1beta cytokine. The known fungal pattern recognition receptors TLR2 and Dectin-1 regulate IL-1beta gene transcription, whereas the NLRP3-containing proinflammatory multiprotein complex, the NLRP3 inflammasome, controls caspase-1-mediated cleavage of pro-IL-1beta. Furthermore, we show that TLR2, Dectin-1, and NLRP3 are essential for defense against dissemination of mucosal infection and mortality in vivo. Therefore, in addition to sensing bacterial and viral pathogens, the NLRP3 inflammasome senses fungal pathogens and is critical in host defense against Candida.

Figures

Figure 1
Figure 1. In vitro IL-1β responses to Candida albicans are morphological stage dependent and blocked by caspase-1 inhibitor
(a) IL-1β responses of normal PBMC primed for 3 h with LPS then stimulated with fixed preparations of C. albicans of different morphological stages at ~ 106/ml; (b) Inhibition of IL-1β responses after the addition of caspase-1 specific inhibitor, Z-YVAD-FMK; (c) Western blot of supernatants from PBMC stimulation probed with anti-IL-1β antibody; (d) IL-1β responses of bone-marrow derived macrophages from wild-type mice primed for 3 h with LPS then stimulated with C. albicans at ~ 106/ml; (e) Western blot of supernatants from BMDM stimulation probed with anti-IL-1β antibody, (f) and anti-caspase-1 antibody. (*** P < 0.001; ** P < 0.01; * P < 0.05)
Figure 2
Figure 2. IL-1β responses to Candida albicans are dependent on TLR2 and dectin-1
(a) Up-regulation of IL-1β mRNA by qPCR of macrophages from Tlr2-/-, Dect1-/- and wild-type mice stimulated for 8 h with C. albicans of different morphological stages at ~ 106/ml; (b) IL-1β protein levels in supernatants of macrophages from Tlr2-/-, Dect1-/- and wild-type mice stimulated overnight with C. albicans of different morphological stages at ~ 106/ml; (c) IL-1β responses of wild-type macrophages stimulated O/N with LPS at 500 ng/ml, β-glucan at 10 μg/ml or zymosan at 1 μg/ml alone, or primed for 6 h then removed and stimulated for an additional 8 h by C. albicans at ~ 106/ml. (*** P < 0.001; ** P < 0.01; * P < 0.05)
Figure 3
Figure 3. IL-1β responses to Candida albicans are mediated by NLRP3, ASC and Caspase-1
(a) IL-1β responses of macrophages from Asc-/-, Nlrp3-/- and wild-type mice primed for 4 h with 500 ng/ml LPS then stimulated with C. albicans at ~ 106/ml; (b) Western blot of supernatants from wild-type or Asc-/- macrophages probed with anti-IL-1β antibody; (c) IL-1β responses of macrophages from Casp1-/- and wild-type mice stimulated overnight with C. albicans at ~ 106/ml; (d) IL-1β responses of macrophages from P2X7R-/- and wild-type mice stimulated overnight with C. albicans at ~ 106/ml. (*** P < 0.001; ** P < 0.01; * P < 0.05)
Figure 4
Figure 4. Protective role of IL-1β in a murine modal of oral infection with Candida albicans
(a) Fold induction of IL-1β mRNA measured by quantitative real-time PCR by oral buccal epithelium after infection with C. albicans; (b) IL-1β (pg/ml) protein production in homogenized whole tongues of mice after infection with C. albicans; (c) Quantitative fungal burden of tongues, and (d) kidneys of Il-1r1-/- and wild-type (WT) mice after oral infection with C. albicans; (e) Fungal burden of tongues, and (f) kidneys of Casp1-/- and WT mice after oral infection with C. albicans; (g) Kaplan-Meier survival plots of WT, Il-1r1-/- and Casp1-/- mice after infection (P <0.0001); (h) Mean clinical severity score of Il-1r1-/-, Casp1-/- and wild-type after 3, 7, 14 or 21d of infection; (*** P < 0.001; ** P < 0.01; * P < 0.05)
Figure 5
Figure 5. NLRP3 inflammasome plays a critical role in host defense to mucosal infection with Candida albicans
(a) Quantitative fungal burden of tongues, and (b) kidneys of Asc-/- and WT mice after oral infection with C. albicans; (c) Kaplan-Meier survival plots of WT and Asc-/- mice after infection (P = 0.0002); (d) Mean clinical severity score of Asc-/- and WT after 3, 7, 14 or 21d of infection; (e) Quantitative fungal burden of tongues, and (f) kidneys of Nlrp3-/- and WT mice after oral infection with C. albicans; (g) Mean clinical severity score of Nlrp3-/- and WT after 3 and 7d of infection. (*** P < 0.001; ** P < 0.01; * P < 0.05)
Figure 6
Figure 6. Innate PRRs enhance survival after oral infection with Candida albicans
(a) Quantitative fungal burden of tongues, and (b) kidneys of Dect1-/- and wild-type (WT) mice after oral infection with C. albicans; (c) Kaplan-Meier survival plots of WT and Dect1-/- mice after infection (P = 0.0009); (d) Mean clinical severity score of Dect1-/- and WT after 3 or 7d of infection; (e) Quantitative fungal burden of tongues and (f) kidneys of Tlr2-/- and wild-type (WT) mice after oral infection with C. albicans; (g) Kaplan-Meier survival plots of WT and Tlr2-/- mice after infection (P = 0.0002); (h) Mean clinical severity score of Tlr2-/- and WT after 3, 7, 14 or 21d of infection. (*** P < 0.001; ** P < 0.01; * P < 0.05)
Figure 7
Figure 7. Serum IL-1β responses to in vivo infection with C. albicans is dependent on TLR2, Dectin-1 and the NLRP3 inflammasome
(a) IL-1β responses in serum collected from wild-type, Nlrp3-/- or Asc-/- mice orally infected with C. albicans; (b) IL-1β responses in serum collected from orally infected wild-type, Tlr2-/- or double Tlr2/dect1-/- mice; (c) IL-1β responses in serum collected from orally infected wild-type or Dect1-/- mice (*** P < 0.001; * P < 0.05)

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