Fig 1.
Clinical C. albicans isolates show different inflammasome-dependent killing of macrophages, but broadly conserved killing by glucose starvation.
(A) Illustration of hyphal index calculation. For a given cell, the length from end-to-end (including all connecting septal junctions) was measured and divided by the width–defined as the widest measurement at the distal tip. True hyphae have a large hyphal index (great in length, narrow in width); pseudohyphae are reduced in length and greater in width; and yeast cells have a low hyphal index (similar proportions for both length and width). (B) Microscopy images of C. albicans clinical isolates 3 h post-phagocytosis in murine bone marrow-derived macrophages (BMDMs). The MOI was 6 Candida to 1 macrophage. Values in top-left corner of each image indicate the hyphal index, with the benchmark strain SC5314 normalized to 5. For each isolate, 200 cells were measured. Scale bar is 20 μm. These same images are also displayed in S1A Fig for direct comparison of hyphal index in the same tissue culture media but without macrophages. (C) BMDMs were infected with C. albicans strains at 6:1 MOI and assessed for cell death by live cell imaging over 24 h. Each graph compares a clinical isolate with the benchmark strain SC5314 and uninfected macrophages control, all assayed in the same live cell imaging experiment (note that P57055 and P78042 were assayed in the same experiment and therefore are being compared to the same control data (i.e. the data for strain SC5314 and the uninfected control is the same in these two graphs; the isolates are displayed in separate graphs for clarity in presentation). Data are the mean values and SEM from 2 independent experiments involving 4 different colonies (each colony was treated as a biological replicate) of the indicated Candida strain and at least 2000 macrophages surveyed for each strain, per experiment. The distinction between Phase I (inflammasome dependent killing) and Phase II (glucose starvation-dependent killing) is indicated in the first graph (labeled “I” and II”). The hyphal index (h.i.) is indicated for the clinical isolate being compared to SC5314 (SC5314 has an index of 5). A selection of clinical strains is shown here, and the entire set is displayed in S3 Fig. (D) Correlation plot of the time (in hours) until 50% macrophage death was reached versus hyphal index in macrophages, with the Pearson’s correlation coefficient (r) and p values.
Fig 2.
Distinct inflammasome and glucose-dependent killing by C. albicans isolates is recapitulated in human primary macrophages.
(A) Primary human monocyte-derived macrophages (hMDMs) infected with C. albicans strain SC5314 at 6:1 Candida:macrophage MOI. hMDMs were collected from two donors and were assayed on the same day in the same experiment. The two donors are plotted separately. Each graph shows the average and SEM of two technical repeats. At least 1500 macrophages were counted per condition, per donor. The NLRP3 inhibitor MCC950 was added at 10 μM to assess the involvement of NLRP3 inflammasome-dependent pyroptosis during Phase I death of Candida-infected hMDMs. (B) Same experiment as in A, this time comparing media with distinct glucose concentrations to assess glucose starvation during Phase II death of hMDMs. All of the conditions (+/-MCC950 shown in A and medium with increasing glucose concentrations shown in B) were assayed together in the same experiment but are plotted separately for clarity. Therefore, data for uninfected controls and infections in medium with 10 mM glucose are the same in panel A and panel B for each donor. (C) hMDMs infected with C. albicans strains SC5314, P57055 and P78042 at 6:1 Candida:macrophage MOI. hMDMs were collected from two different donors and are plotted separately. Each graph shows the average and SEM of two technical repeats. At least 1500 macrophages were counted per condition, per donor.
Fig 3.
Inflammasome activation in response to C. albicans occurs in two temporally distinct events.
(A) ASC-mCerulean-expressing immortalized macrophages following challenge with Candida strains SC5314 or P78042 at MOI 6:1. Left panel: representative images from live cell microscopy at 6 and 16 h post-phagocytosis, with ASC-mCerulean detection displayed in cyan. Scale bar is 50 μm. Right panel: graph showing quantification of ASC speck formation over time. Data are the mean values and SEM from 4 independent experiments. (B) Experimental conditions as in A. Shown are % macrophages containing an ASC speck, % macrophages with hyperpolarized mitochondria and % dead macrophages quantified in live cell imaging experiments. ASC speck data is the same as in panel A. Data are the mean values and SEM from 4 independent experiments (see S4 Fig for complete set of data involving all 21 clinical isolates). (C) Correlation plot of the time post-phagocytosis when peak ASC speck formation occurs versus hyphal index of the Candida isolates in macrophages, with the Pearson’s correlation coefficient (r) and p values as indicated. (D) Correlation plot of peak percentage of macrophages in the population that contain an ASC speck versus hyphal index in macrophages, with the Pearson’s correlation coefficient (r) and p values as indicated. (E) IL-1β detection following challenge with Candida strains SC5314 or P78042 at MOI 6:1. Primary murine BMDMs were primed with LPS (50 ng/ml) for 3 h, followed by challenge with Candida strains SC5314 or P78042 (MOI 6:1), nigericin (10 μM), or left uninfected. At 12 h post-challenge, supernatants were analysed by immunoblot for IL-1β. Shown is one representative immunoblot from two independent experiments involving different mice. Note that this immunoblot contains additional lanes that were spliced out (indicated by the black lines); see S5 Fig for the entire, uncropped immunoblot from this experiment. (F) BALBc mice were injected with 1x106 CFU of the indicated C. albicans strains: SC5314 (highly hyphal, triggers the early wave of inflammasome activation) or P78042 (poorly hyphal, triggers only the second wave of inflammasome activation). Mock injection was with PBS. Levels of IL-1β in kidney were measured by ELISA (n = 10 animals/group for C. albicans infection, and 5 for PBS injection). *** p < 0.001, **** p < 0.0001, Student’s two-tailed t test. (G) ASC speck formation over time in infections with the highly hyphal C. albicans strain SC5314 at high (6:1) and low (1:1) MOI. Left panel: representative images from live cell microscopy at 6 and 16 h during infection, with ASC-mCerulean detection displayed in cyan. Scale bar is 50 μm. Right panel: graph showing quantification of ASC speck formation over time. Data are the mean values and SEM from 5 independent experiments.
Fig 4.
The second mechanism of inflammasome activation depends on NLRP3, potassium efflux and release of the lysosomal protease cathepsin B.
(A) To assay the early event of inflammasome activation, ASC-mCerulean-expressing immortalized macrophages were challenged with Candida strain SC5314 (MOI 6:1). Uninfected macrophage controls were also included. The % macrophages containing an ASC speck, % macrophages with hyperpolarized mitochondria and % dead macrophages were quantified in live cell imaging experiments. Comparisons were assessed in the presence of the NLRP3 inhibitor MCC950 (10 μM) or the control inactive compound MCC6642 (10 μM), in the presence of cathepsin B inhibitor CA-074-Me (10 μM) or control (DMSO solvent) and in the presence of KCl (30 mM) or control (water solvent). Data are the mean values and SEM from 3 independent experiments involving at least 6000 macrophages surveyed for each condition, per experiment. (B) Same experimental setup as described in (A), but to assay the later event of inflammasome activation C. albicans strain P78042 was used (MOI 6:1). Data are the mean values and SEM from 3 independent experiments involving at least 6000 macrophages surveyed for each condition, per experiment. (C) Same experimental setup as described in (A). Macrophages were challenged with Candida strain SC5314 (MOI 6:1 and 1:1) or left uninfected, in the presence of the ROS scavenger N-acetyl-L-cysteine (NAC; 5 mM) or control (PBS solvent). Data are the mean values and SEM from 3 independent experiments involving at least 6000 macrophages surveyed for each condition, per experiment. Note that the 6:1 and 1:1 MOI infections were assayed in the same experiments alongside untreated controls; therefore, the data for “untreated, control” and “untreated, NAC” (black and grey lines) is the same in the left and right panels. The data for the two MOIs are displayed in separate graphs for clarity in presentation. (D) Quantification of ROS production in primary murine BMDMs. Macrophages were pre-loaded with 20 μM H2DCFDA (or no dye control) for 30 min and then infected with C. albicans SC5314 (MOI 6:1 or 1:1), or left uninfected, in media containing 5 mM antioxidant N-acetyl-L-cysteine (NAC) or control (water). NAC treatment significantly reducing ROS accumulation during C. albicans infections. Data are the mean values and SEM from 2 independent experiments.
Fig 5.
Activation of the inflammasome is triggered by macrophage glucose starvation.
(A) ASC-mCerulean-expressing immortalized macrophages following challenge with Candida strains P78042 at MOI 6:1 (to assay late activation), SC5314 at MOI 6:1 (to assay early activation), or left uninfected. Plotted on the left axes are % macrophages containing an ASC speck and % macrophages with hyperpolarized mitochondria as quantified in live cell imaging experiments (in cyan and red respectively). Plotted on the right axes are glucose concentration measurements at select time points (in green). Data are the mean values and SEM from 2 independent experiments, and in live cell imaging this involved at least 6000 macrophages surveyed for each condition, per experiment. (B) Comparison of the C. albicans glycolytic mutant tye7Δ/Δ gal4Δ/Δ versus the TYE7 GAL4 complemented strain in ASC-mCerulean-expressing immortalized macrophages, along with uninfected macrophages (“macrophages only” control). The MOI was 1:1 and starting tissue culture media contained 10 mM glucose. Displayed in each graph are % macrophages containing an ASC speck, % macrophages with hyperpolarized mitochondria and % dead macrophages as quantified in live cell imaging experiments. Data are the mean values and SEM from 3 independent experiments involving at least 10000 macrophages surveyed for each condition, per experiment. p values were calculated at the time points indicated by dashed lines (Student’s two-tailed t test).
Fig 6.
Restoring macrophage glucose homeostasis reduces inflammasome activation.
For all experiments in this Figure, the initial tissue culture media contained ~0.65 mM glucose, which was then supplemented with additional glucose as indicated in each panel. (A) ASC-mCerulean-expressing immortalized macrophages following infection with Candida hyphal strain SC5314 (MOI 6:1) or nigericin as positive control (10 μM) and with 0, 10, 40, or 100 mM glucose addition. Shown are % macrophages containing an ASC speck, % macrophages with hyperpolarized mitochondria and % dead macrophages, as quantified in live cell imaging experiments. Data are the mean values and SEM from 3 independent experiments. p values were calculated for % ASC speck at the time point indicated by dashed line (* p < 0.05, *** p < 0.001 and **** P < 0.0001, one-way ANOVA and Tukey’s multiple comparison test). (B) Representative images from live cell microscopy after 8 h of infection with hyphal strain SC5314 (MOI 6:1) in tissue culture media containing 0, 10, or 40 mM added glucose. ASC-mCerulean detection is displayed in cyan. Scale bar is 50 μm. (C) Primary murine BMDMs were primed with LPS (50 ng/ml) for 3 h, followed by challenge with Candida hyphal strain SC5314 (MOI 6:1), nigericin (10 μM), or uninfected. The starting tissue culture media contained 0, 10, 40, or 100 mM added glucose. At 3 h post-challenge, supernatants and lysates were analysed by immunoblot for IL-1β and actin (see S7 Fig for entire, uncropped immunoblots from three independent experiments and the Pounceau staining loading control). (D) The same supernatant samples from the Western blots (displayed in panel C of this Figure and S7 Fig) quantified by ELISA. Data are the mean values and SEM from 3 independent experiments. * p < 0.05, and **** p < 0.0001, one-way ANOVA and Tukey’s multiple comparison test (n.s.–not significant). (E) ASC-mCerulean-expressing immortalized macrophages following infection with Candida hyphal strain SC5314 (MOI 6:1), comparing tissue culture media containing 0, 3, 6, and 10 mM glucose addition. Displayed in each graph are % macrophages containing an ASC speck, % macrophages with hyperpolarized mitochondria and % dead macrophages as quantified in live cell imaging experiments. Data are the mean values and SEM from 2 independent experiments involving at least 9000 macrophages surveyed for each condition, per experiment.
Fig 7.
Glucose starvation is a broadly relevant mechanism of inflammasome activation across diverse C. albicans isolates.
ASC-mCerulean-expressing immortalized macrophages were infected with the indicated clinical isolates of C. albicans at MOI 6:1. Tissue culture media contained 10 or 40 mM glucose. Shown are % macrophages containing an ASC speck, % macrophages with hyperpolarized mitochondria and % dead macrophages as quantified in live cell imaging experiments. Data are the mean values and SEM from 2 independent experiments involving at least 8000 macrophages surveyed for each condition, per experiment. The hyphal index (h.i.) for the C. albicans strains is shown in the right top corner of the graphs. p values were calculated for % ASC speck at the time points indicated by dashed lines (Student’s two-tailed t test).