Specific pathways mediating inflammasome activation by Candida parapsilosis

Abstract

Candida albicans and C. parapsilosis are human pathogens causing severe infections. The NLRP3 inflammasome plays a crucial role in host defence against C. albicans, but it has been previously unknown whether C. parapsilosis activates this complex. Here we show that C. parapsilosis induces caspase-1 activation and interleukin-1β (IL-1β) secretion in THP-1, as well as primary, human macrophages. IL-1β secretion was dependent on NLRP3, K⁺-efflux, TLR4, IRAK, Syk, caspase-1, caspase-8 and NADPH-oxidase. Importantly, while C. albicans induced robust IL-1β release after 4 h, C. parapsilosis was not able to stimulate the production of IL-1β after this short incubation period. We also found that C. parapsilosis was phagocytosed to a lesser extent, and induced significantly lower ROS production and lysosomal cathepsin B release compared to C. albicans, suggesting that the low extent of inflammasome activation by C. parapsilosis may result from a delay in the so-called "signal 2". In conclusion, this is the first study to examine the molecular pathways responsible for the IL-1β production in response to a non-albicans Candida species, and these results enhance our understanding about the immune response against C. parapsilosis.

Figure 1. C. parapsilosis induces caspase-dependent IL-1β release in human macrophages.

THP-1 macrophages or human MDMs were stimulated with C. parapsilosis or C. albicans and the concentration of secreted IL-1β (in cell culture supernatants) or intracellular pro-IL-β (in cell lysates) was determined by ELISA or Western blot (cropped). (a,b) THP-1 macrophages were stimulated with C. parapsilosis at different MOIs for 24 h (a) or at MOI 5 for the indicated incubation periods (b). (c,d) THP-1 macrophages were stimulated with C. parapsilosis or C. albicans at MOI 0.01 for 24 h (c) or at MOI 5 for 4 h (d). (e) MDMs were co-incubated with C. parapsilosis at MOI 5 for 24 h. (f) THP-1 macrophages or MDMs were stimulated with C. parapsilosis at MOI 5 for 24 h. (g,h) THP-1 macrophages (g) or MDMs (h) were stimulated with C. parapsilosis (MOI 5, 24 h) or C. albicans (MOI 0.04, 24 h) in the presence or absence of pan-caspase inhibitor Z-VAD-FMK (10 μM). IL-1β production is expressed as % of control (IL-1β secretion measured in cells stimulated with Candida spp + vehicle). Results (mean ± SEM) are pooled data from at least three (a,c,d,e,g,h) or representative of two (b,f) independent experiments. Ctrl, control (medium-treated cells); Ca, C. albicans; Cp, C. parapsilosis; sup, supernatants; lys, cell lysates; MOI, multiplicity of infection. *p < 0.05, ***p < 0.001 (compared to control) as determined by paired t-test.

Figure 2. C. parapsilosis activates the NLRP3 inflammasome.

(a–d) THP-1 macrophages (a,b) or human MDMs (c,d) were stimulated with C. parapsilosis (MOI 5) for 24 h and caspase-1 activation was assessed by flow cytometry using the FAM-FLICA caspase-1 assay. (b–d) Values are expressed as MFI fold change compared to unstained samples. (e,f) THP-1 macrophages (control, ASC-deficient or NLRP3-deficient) were stimulated with C. parapsilosis (MOI 5) or C. albicans (MOI 0.04) for 24 h and secreted IL-1β or pro-IL-β were determined by Western blot (cropped) (e) or ELISA (f). (g) THP-1 macrophages were stimulated with C. parapsilosis (MOI 5) or C. albicans (MOI 0.04) for 24 h in the presence of 50 mM KCl and the concentration of secreted IL-1β was determined by ELISA. Cytokine levels are expressed as % of control values (cytokine levels of vehicle treated samples). Results (mean ± SEM) are pooled data from (b,d,f,g) or representative of (a,c,e) at least three independent experiments. Ctrl, control (medium-treated cells); Ca, C. albicans; Cp, C. parapsilosis; MFI, median fluorescence intensity. *p < 0.05, **p < 0.01, *** p < 0.001 (compared to control) as determined by paired (b,d,g) or unpaired (f) t-test.

Figure 3. C. paraspilosis induces inflammasome activation by a similar mechanism as C. albicans.

THP-1 macrophages were stimulated with C. parapsilosis (MOI 5) or C. albicans (MOI 0.04) for 24 h in the presence or absence of specific inhibitors (TLR2 mAb, 0.15 μg/ml; TLR4 inhibitor CLI-095, 5 μM; IRAK inhibitor 509093-47-4, 5 μM; Syk inhibitor ER 27319 maleate, 5 μM; caspase-8 inhibitor Z-IETD-FMK, 10 μM; NADPH-oxidase inhibitor apocynin, 600 μM). Secreted IL-1β was determined in cell culture supernatants by ELISA (a) and pro-IL-1β was measured by ELISA following the lysis of THP-1 cells by repeated freeze-thaw cycles (b). Results (mean ± SEM) are pooled data from at least three independent experiments. Ca, C. albicans; Cp, C. parapsilosis. *p < 0.05, **p < 0.01, ***p < 0.001 (compared to control) as determined by paired t-test. Cytokine levels are expressed as % of control values (cytokine levels of vehicle treated samples).

Figure 4. The lack of signal 2 results in delayed inflammasome activation upon C. parapsilosis stimulation.

(a) THP-1 macrophages were stimulated with different C. parapsilosis strains at MOI 0.02 or MOI 5 for 24 h and secreted IL-1β was determined in cell culture supernatants by ELISA. (b) THP-1 macrophages were stimulated with C. parapsilosis or C. albicans at MOI 5 for 4 h and the amount of intracellular pro-IL-1β was measured by ELISA. (c,d) Human MDMs were stimulated with C. parapsilosis or C. albicans at MOI 5 for 4 h and the amount of secreted IL-1β or intracellular pro-IL-1β was determined by ELISA. (e,g) THP-1 macrophages were stimulated with C. parapsilosis or C. albicans at MOI 1 for 4 h. Intracellular ROS was determined by DCFDA assay (e), cathepsin B release was measured by a fluorometric cathepsin B activity assay kit (g). (f) THP-1 macrophages were stimulated with C. parapsilosis or C. albicans at MOI 5 for 4 h in the presence or absence of phagocytosis inhibitor cytochalasin D (2.5 μM) and the amount of secreted IL-1β was determined by ELISA. (h) THP-1 macrophages were infected with AlexaFluor488-labeled C. albicans or C. parapsilosis (MOI 5) for 5, 15, 30, 60 and 90 min; phagocytosis was determined by flow cytometry. Results (mean ± SEM) are pooled data from at least three (a,b,e–h) or representative of two (c,d) independent experiments. Ctrl, control (medium-treated cells); Ca, C. albicans; Cp, C. parapsilosis. *p < 0.05, **p < 0.01, ***p < 0.001 (compared to control) as determined by paired t-test.

Figure 5. C. parapsilosis induces ASC- and NLRP3-dependent cell death in human macrophages.

THP-1 macrophages (control as well as ASC-deficient or NLRP3-deficient, where indicated) were stimulated with C. parapsilosis (MOI 5) or C. albicans (MOI 0.04) for 24 h (a,b,d) or for 16 and 24 h (c) and cell lysis was assessed by LDH assay. LDH release is expressed as % of positive control. (b,c) THP-1 macrophages were stimulated with Candida spp. in the presence of pyroptosis inhibitor glycine (5 mM; b) or pan-caspase inhibitor Z-VAD-FMK (10 μM; c). Results (mean ± SEM) are pooled data from at least three independent experiments. Ca, C. albicans; Cp, C. parapsilosis. *p < 0.05, ****p < 0.0001 (compared to control) as determined by paired t-test.