Figure 1.
Dectin-1 dependence is related to fungal strain and not mouse background.
Survival analysis of 129/Sv and C57BL/6 wild-type and Dectin-1−/− mice following infection with (A) C. albicans SC5314 or (B) ATCC18804. The data shown are representative of at least two independent experiments. Also shown are number of animals per group (n) and dosage used for infection. *; p<0.05. See also Table S1.
Figure 2.
Dectin-1 dependence is not related to the clade of C. albicans.
Survival analysis of 129/Sv wild-type and Dectin-1−/− mice following infection with strains from (A) several different clades of C. albicans, or (B) two additional isolates from clade 1 as indicated. The data shown are representative of at least two independent experiments. Also shown are number of animals per group (n) and dosage used for infection. *; p<0.05. See also Table S1.
Figure 3.
Loss of Dectin-1 is associated with increased fungal burdens and dysregulated cytokine responses with specific strains of C. albicans.
Fungal burdens in the kidneys of 129/Sv wild-type and Dectin-1−/− mice at day 9 post-infection with a (A) high or a (B) low dose of various C. albicans strains, as indicated. (C) Characterisation of cytokine levels in the kidneys of 129/Sv wild-type and Dectin-1−/− mice at day 9 post-infection with a high dose (as in A) of various C. albicans strains, as indicated. Shown are data from two pooled (A, B) and one representative (C) experiment. Each experiment involved 7–10 mice per group. Bar indicates the mean.*; p<0.05.
Figure 4.
The dependence of Dectin-1 cannot be recapitulated in vitro.
(A) Cell wall biochemical composition of in vitro grown C. albicans SC5314 and ATCC18804 yeast cells, as indicated. (B) Relative binding of fluorescently-labelled live C. albicans yeast cells or zymosan (Zy) to C57BL/6 wild-type and Clec7A−/− thioglycollate-elicited peritoneal macrophages, as indicated. (C) Measurement of TNF in culture supernatants from C57BL/6 wild type versus Clec7A−/− peritoneal macrophages after stimulation with live C. albicans yeast cells or zymosan (Zy), as indicated. (D) Measurement of TNF and IL-6 responses from homozygous Y238X patients after stimulation with C. albicans yeast cells or heat-killed yeast (HK; strain ATCC MYA-3573), as indicated. Results were normalized to the treated cells from normal individuals. (E) Measurement of TNF responses from 129S/Sv wild type versus Clec7A−/− peritoneal macrophages after stimulation with live C. albicans hyphae, as indicated. Data shown are means ± SEM of pooled data from at least two independent experiments, except for (E), which is the mean ± SD of a representative experiment.
Figure 5.
Dectin-1-responsiveness but not dependence is induced by hypoxia in vitro.
(A) Growth of various strains of C. albicans under normoxia or hypoxia, as indicated. (B) Measurement of TNF responses from C57BL/6 wild type versus Clec7A−/− peritoneal macrophages after stimulation with UV-irradiated C. albicans yeast or hyphae, grown under normoxic or hypoxic conditions, as indicated. (C) Quantitation of β-glucan exposure on UV-irradiated C. albicans hyphae, grown in vitro under conditions of normoxia or hypoxia, as indicated, and stained with soluble Dectin-1. (D) Fold change in FKS gene expression in C. albicans hyphae under normoxic versus hypoxic conditions, as indicated. Data shown (mean ± SD) are from one representative experiment.
Figure 6.
Dectin-1 dependence is not related to β-glucan exposure in vivo.
(A) Fungal burdens in the kidneys of 129/Sv wild-type and Dectin-1−/− mice at day 3 post-infection with various C. albicans strains, as indicated. (B) Characterisation of cytokine levels in the kidneys of 129/Sv wild-type and Dectin-1−/− mice at day 3 post-infection of various C. albicans strains, as indicated. Images and quantitation of β-glucan expression on fungal cells isolated from the infected kidneys of C57BL/6 mice at day 9 and stained with (C) anti-β-glucan antibodies or (D) soluble Dectin-1 (FcDectin-1). Control cells were stained with secondary antibody only. Data shown are from a representative experiment. Bar indicates the mean.*; p<0.05. ns, not significant.
Figure 7.
Dectin-1 dependence in vivo is related to changes in the fungal cell wall and chitin content.
(A) Box and whisker graph showing fungal burdens in the kidneys of 129/Sv wild-type and Dectin-1−/− mice at day 7 post-infection with ATCC18804, with and without caspofungin treatment, as indicated. (B) Comparative expression analysis of genes encoding selected cell wall associated and secreted proteins, as indicated, on fungal mRNA isolated from the kidneys of 129/Sv mice infected with SC5314 or ATCC18804 at day 7. See also Table S2. (C) Confocal images and quantitation of chitin levels in fungal cells isolated from the kidneys of 129/Sv mice infected with SC5314 and ATCC18804 at day 7, as indicated. (D) TEM images (left) and quantification of cell wall thickness (right) of fungal cells isolated from the kidneys of 129/Sv mice infected with SC5314 and ATCC18804 at day 7. Scale bar = 50 nm. (E) Confocal images and quantitation of chitin levels of in vitro cultured normal and high chitin SC5314 fungal cells, as indicated. Scale bar = 10 µm. (F) Flow cytometric analysis of exposed β-glucan on low and high-chitin containing in vitro grown C. albicans SC5314, using soluble Dectin-1 as a probe. The filled histograms represent secondary only control and the blue histogram indicates FcDectin-1 staining. (G) Survival analysis of 129/sv Dectin-1−/− or wt mice following infection with 3×104 CFU high-chitin or normal-chitin containing C. albicans SC5314. (H) Intraperitoneal inflammation, as measured by neutrophil influx, 4 hr after i.p. infection with 1×105 CFU high-chitin or normal-chitin containing C. albicans SC5314 in Dectin-1−/− or wt mice, as indicated. All data shown are from a representative experiment, except for (H) which is pooled data from two experiments. Bar indicates the mean.*; p<0.05. ns, not significant.