Fig 1.
E. histolytica activates caspase-4 and caspase-1 in a time-dependent manner.
The kinetics of Eh-induced caspase-4 activation was detected by incubating PMA-differentiated THP-1 macrophages for increasing amounts of time with 1:20 Eh to macrophage ratio. (A) Macrophages were incubated for increasing amounts of time with 1:20 Eh to macrophage ratio. Lipopolysaccharide (LPS) (50 ng/mL) and nigericin (NGC) (10 μM) stimulation for 60 min was used as a positive control. Immunoblot analysis was performed for caspase-4 and caspase-1 in supernatants (SN). (B) Quantifications of activated caspase-4 and caspase-1 were performed by densitometric analysis from three independent experiments and the negative control (cells only) acted as an internal control. Statistical significance was calculated between caspase-4 and caspase-1 at each time point. (C) Cell free supernatant was added to HEK-Blue IL-1β reporter cells to detect bioactive IL-1β via measurement of SEAP levels. (D) Cell death was quantified by lactate dehydrogenase (LDH) release into the culture supernatant and is shown as a percentage of LDH release compared to non-stimulated cells (control). (E, F) Macrophages were pre-incubated with the pan-caspase inhibitor Z-VAD-fmk (100 μM) and caspase-1 specific inhibitor Z-YVAD-fmk (100 μM) for 45 min followed by stimulation with Eh for 30 min. Caspase-4/1 activation as well as IL-1β secretion in the cell supernatant were assessed via immunoblotting. Cell free supernatant was added to HEK-Blue IL-1β reporter cells to detect bioactive IL-1β using the SEAP assay. Data and immunoblots are representative of at least three experiments (n = 3) and statistical significance was calculated with ANOVA and Bonferroni’s post-hoc test (*p < 0.05, **p < 0.01, ****p < 0.0001). Bars represent mean ± SEM.
Fig 2.
E. histolytica-macrophage contact is required for caspase-4 activation.
(A, B) Macrophages were incubated with live Eh: macrophage ratio (1:20), fixed Eh: macrophage ratio (1:20) for 30 min and with equivalent amount of freeze thawed whole lysates of Eh. Live Eh were fixed with 1.5% glutaraldehyde for 1 h at 4°C and washed 3 times with sterile cold PBS before use. LPS (50 ng/mL) and NGC (10 μM) stimulation for 60 min acted a positive control. Post incubation, the cell supernatant (SN) was TCA precipitated and equal amount was loaded onto the SDS-PAGE gel to detect caspase-4/1 activation with indicated antibodies. (B) Cell supernatant was added to HEK-Blue IL-1β reporter cells to detect bioactive IL-1β using the SEAP assay. Statistical significance was calculated between live Eh and fixed (dead) Eh, and between live Eh and Eh lysate (C) Macrophages were pretreated for 5 min with 55 mM D-galactose (Gal), or glucose (Glu) as an osmotic control and then incubated with Eh for 30 min at a 20:1 ratio. (D) Macrophages were incubated with Eh, and Eh deficient in CP5 (EhCP-A5-) for 60 min and 90 min, respectively. LPS (50 ng/mL) and NGC (10 μM) stimulation for 60 min were used as a positive control. (E) Cell supernatant was added to HEK-Blue IL-1β reporter cells to detect bioactive IL-1β using the SEAP assay. Statistical significance was calculated between WT Eh and EhCP-A5- Eh. Data and immunoblots are representative of at least three independent experiments (n = 3) and statistical significance was calculated with one-way ANOVA, followed by Bonferroni’s post-hoc test (***p < 0.001, ****p < 0.0001). Bars represent mean ± SEM.
Fig 3.
Mechanisms of E. histolytica-induced caspase-4 activation.
(A-C) Eh upregulated pro-caspase-4 expression and transcription in a time-dependent manner. (A) Quantitative PCR was conducted to investigate if Eh increased pro-caspase-4 transcription. (B, C) Macrophages were stimulated with Eh at 20:1 ratio for increasing amount of time. Equal amount of lysate (LYS) was resolved on SDS-PAGE and immunoblotted for pro-caspase-4 detection. Blots were reprobed for GAPDH. Densitometry was performed to assess pro-caspase-4 proteins and the negative (cells only) acted as an internal control. (D) Macrophages were treated with native Eh Gal-lectin (500 ng/mL) for 2 h and pro-caspase-4, pro-caspase-1 and GAPDH levels were determined by western blot. (E-H) Caspase-4 activation requires ATP signaling via the P2X7 receptor and pannexin-1 channels. (E) Macrophages were pretreated with oxidized ATP (oATP) for 2 h and then stimulated with Eh for 30 min. (F) Immunoblot analysis of active caspase-4 and caspase-1 in macrophages stimulated for 30 min with Eh with the addition of apyrase (20 U/mL). (G) Inhibition of caspase-4 with carbenoxolone (CBX), connexin/pannexin channel dual inhibitor, or pannexin channel inhibitor probenecid (PB). LPS and NGC were used as a positive control. Left: LPS (100 ng/ml) priming for 30 min and NGC (5 μM) stimulation for 30 min; right: LPS (50 ng/ml) priming for 30 min and NGC (10 μM) stimulation for 30 min. (H) Macrophages were incubated with CBX and PB for 30 min, prior to Eh stimulation for 30 min. Cell supernatant (SN) was TCA precipitated and cells were washed and lysed. Equal amount of supernatants and lysed cell lysates was loaded onto SDS-PAGE and immunoblot analysis was performed for caspase-4, caspase-1 and IL-1β. Data and immunoblots are representative of at least three independent experiments (n = 3) and statistical significance was calculated with one-way ANOVA, followed by Bonferroni’s post-hoc test (*p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001). Bars represent mean ± SEM.
Fig 4.
E. histolytica-induced caspase-4 activation is enhanced in the absence caspase-1.
(A, B) WT and CRISPR/Cas9 CASP1 KO macrophages were stimulated with Eh for 10 and 30 min and unstimulated cells were used as an internal control. Macrophages stimulated with LPS (50 ng/mL) and NGC (10 μM) was used as a positive control. Quantifications of active caspase-4 protein were performed by densitometric analysis and negative (cells only) acted as an internal control. Statistical significance was calculated between WT and CASP1 KO macrophages at each time point (C) Bioactive IL-1β secretion in the histogram was quantified by the SEAP assay and statistical significance was calculated between WT and CASP1 KO macrophages at each time point. (D, E) WT and CRISPR/Cas9 CASP4 KO macrophages were incubated with Eh (20:1) at increasing time points. Caspase-1 CARD densitometry was measured and statistical significance was calculated between WT and CASP4 KO macrophages at each time point. Cell supernatant (SN) was TCA precipitated and equal amount of cell supernatants was loaded onto SDS-PAGE and immunoblot analysis was performed for caspase-4 and caspase-1. Active caspase-1 was quantified by densitometric analysis, and negative (cells only) acted as an internal control. (F) Cell supernatant was added to HEK-Blue IL-1β reporter cells to detect bioactive IL-1β secretion via measuring the SEAP and statistical significance was calculated between WT and CASP4 KO macrophages at each time point. Data and immunoblots are representative of at least three separate experiments (n = 3) and statistical significance was calculated with with Student’s t-test between KO and WT, (*p < 0.05, **p < 0.01, ****p < 0.0001). Bars represent mean ± SEM.
Fig 5.
E. histolytica-induced caspase-4 activation does not require the recruitment of the canonical inflammasome components.
(A, B) WT and ASC deficient (ASC def) macrophages were treated with Eh (20:1) for 10 min and 30 min, respectively. Macrophages stimulated with LPS (50 ng/mL) and NGC (10 μM) acted as a positive control. Eh-induced caspase-4 activation was independent of ASC confirmed by aborted caspase-1 activation and IL-1β secretion. Quantifications of active caspase-4 were performed by densitometric analysis and statistical significance was calculated between WT and ASC def macrophages at each time point. (C, D) WT and CRISPR/Cas9 NLRP3 KO macrophages were stimulated with Eh (20:1) at 10 min and 30 min, respectively. Macrophages stimulated with LPS (50 ng/mL) and NGC (10 μM) were used as a positive control. Quantifications of activated caspase-4 were confirmed with densitometric analysis and statistical significance was calculated between WT and NLRP3 KO macrophages at each time point. Cell supernatant (SN) was TCA precipitated and equal amount of cell supernatants was loaded onto SDS-PAGE and immunoblot analysis was performed for caspase-4, caspase-1 and IL-1β. (E, F) Bioactive IL-1β secretion in the cell supernatant was quantified by the SEAP assay in HEK-Blue reporter cells and statistical significance was calculated between WT and ASC def, WT and NLRP3 KO macrophages at each time point. Data and immunoblots are representative of at least three independent experiments (n = 3) and statistical significance was calculated with Student’s t-test between KO and WT (****p < 0.0001). Bars represent mean ± SEM.
Fig 6.
E. histolytica activates caspase-4 and caspase-1 in a time-dependent manner and cleaves gasdermin D to regulate IL-1β secretion.
(A, C, E) WT, CRISPR/Cas9 CASP1 KO, and CRISPR/Cas9 CASP4 KO macrophages were incubated for increasing amounts of time with 1:20 Eh to macrophage ratio. LPS (50 ng/mL) and NGC (10 μM) for 60 min acted as a positive control. Cells were washed, lysed and equal amount of lysates (LYS) was loaded onto SDS-PAGE and immunoblot analysis was performed for GSDMD cleavage that present in cell lysates. (B, D, F) Quantifications of GSDMD p30 protein were performed by densitometric analysis and blots were reprobed for GAPDH. Negative cells only acted as an internal control. Data and immunoblots are representative of at least three independent experiments (n = 3) and statistical significance was calculated using with an ANOVA and Bonferroni’s post-hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Bars represent mean ± SEM.
Fig 7.
Dependency for E. histolytica-induced caspase-4 in gasdermin D cleavage and bioactive IL-1β release.
(A) WT, CRISPR/Cas9 CASP1 KO and CRISPR/Cas9 CASP4 KO macrophages were incubated with Eh for 30 min or the positive control LPS (50 ng/mL) and NGC (10 μM) for 60 min. Cell supernatant (SN) was TCA precipitated and cells were washed and lysed. Equal amount of supernatants and lysates (LYS) was loaded onto SDS-PAGE and immunoblot analysis was performed for caspase-4 and caspase-1 secreted to the supernatants and along with GSDMD p30 pore-forming fragment in lysates, and blots were reprobed for GAPDH. (B) Quantifications of cleaved GSDMD were performed by densitometric analysis from three independent experiments, and the negative (cells only) acted as an internal control. (C) Cell supernatant was added to HEK-Blue reporter cells to detect bioactive IL-1β by measuring SEAP. (D) THP-1 cell supernatant was assessed by the release of LDH following Eh stimulation and normalized to non-stimulated negative controls (basal cell death). Data and immunoblots are representative of at least three independent experiments (n = 3) and statistical significance was calculated with ANOVA and Bonferroni’s post-hoc test between WT and KO macrophages and between CASP1KO and CASP4 KO macrophages at each time point. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Bars represent mean ± SEM.
Fig 8.
E. histolytica-induced gasdermin D cleavage is not dependent on the canonical NLRP3 inflammasome components.
(A, C) ASC def and CRISPR/Cas9 NLRP3 KO macrophages were incubated with Eh from 5 min to 60 min or the positive control, LPS (50 ng/mL) and NGC (10 μM) for 60 min. Equal amount of cell lysates (LYS) was loaded onto SDS-PAGE and immunoblot analysis was performed for GSDMD p30 pore-forming fragment presented in lysates and blots were reprobed with GAPDH. (B, D) Quantification of cleaved GSDMD p30 fragment was performed by densitometric analysis from three independent experiments, and the negative (cells only) acted as an internal control. (E) Bioactive IL-1β levels were quantified by SEAP assay that detected in HEK-Blue reporter cells. (F) Cell death was quantified by LDH release into the culture supernatant and is shown as a percentage of LDH release compared to non-stimulated cells (control). (E, F) Statistical significance was calculated between WT and ASC def, WT and NLRP3 KO as well as between ASC def and NLRP3 KO macrophages at each time point. Data and immunoblots are representative of at least three independent experiments (n = 3) and statistical significance was calculated with ANOVA and Bonferroni’s post-hoc test (*p < 0.05, **p < 0.01, ****p < 0.0001). Bars represent mean ± SEM.
Fig 9.
E. histolytica triggered gasdermin D cleavage regulates bioactive IL-1β release.
(A) WT and CRISPR/Cas9 GSDMD KO macrophages were incubated with Eh for 10 and 30 min, respectively. LPS (50 ng/mL) and NGC (10 μM) stimulation for 60 min acted as the positive control. Cell supernatant (SN) was TCA precipitated and cells were washed and lysed. Equal amount of supernatants and lysates (LYS) was resolved on SDS-PAGE and immunoblot analysis was performed for GSDMD, caspase-4 and caspase-1 in both the cell lysates and supernatants, and blots were reprobed for GAPDH. (B) Quantifications of active caspase-4 proteins were performed by densitometric analysis from three independent experiments, and the negative (cells only) acted as an internal control. (C) Cell supernatant from stimulated macrophages was added to HEK-Blue reporter cells to detect bioactive IL-1β via the SEAP assay in both WT and CRISPR/ Cas9 GSDMD KO macrophages that were incubated with Eh for increasing amounts of time. (D) Cell death was determined by LDH assay using supernatant from stimulated macrophages, and is shown as a percentage of LDH release compared to non-stimulated cells (control). Data and immunoblots are representative of three separate experiments (n = 3) and a one-way ANOVA was used to determine statistical significance of differences between WT and GSDMD KO cells at each treatment time (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Bars represent mean ± SEM.
Fig 10.
Gasdermin D pores function as gatekeepers of IL-1β release in response to E. histolytica.
(A) Necrosulfonamide (NSA) as a direct chemical inhibitor of GSDMD, binds directly to GSDMD, inhibiting oligomerization of GSDMD p30 pore-forming fragment to inhibit pyroptosis. (B) NSA was added to macrophages 60 min prior to Eh stimulation. Since NSA was prepared in dimethyl sulfoxide (DMSO), DMSO, NSA only (20 μM) were used to detect if DMSO or NSA itself would have any effect on THP-1 cells, and unstimulated cells acted as the negative control. After Eh stimulation, cell free supernatant was added to HEK-Blue IL-1β reporter cells to detect bioactive IL-1β using the SEAP assay. (C) Pyroptotic pore formation and cell death were assessed through LDH release, cell free supernatants from the same experiments were used to quantify LDH released into the culture supernatant and is shown as a percentage of LDH release compared to non-stimulated cells. (B, C) Eh treatment only as a positive control and statistical significance was calculated between Eh 30 min and various concentration of NSA treatments. (D) Cell free supernatant was added to HEK-Blue IL-1β reporter cells to detect bioactive IL-1β using the SEAP assay to detect NSA inhibition in GSDMD pore formation in WT, CASP1 KO, CASP4 KO macrophage. LPS (50 ng/mL) and NGC (10 μM) stimulation for 60 min acted as the positive control. Statistical significance was calculated between WT and KO macrophages and between CASP1KO and CASP4 KO macrophages at each time point. (E-G) Immunoblot analysis was performed for GSDMD p30 cleavage in cell lysates (LYS), and blots were reprobed for GAPDH. WT, CRISPR/Cas9 CASP1 KO and CRISPR/Cas9 CASP4 KO macrophages were pre-incubated with NSA for 60 min before stimulation with LPS + NGC. Data and immunoblots are representative of six experiments (n = 6) and statistical significance was calculated with Student’s t-test and one-way ANOVA followed by post hoc Bonferroni test, (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Bars represent mean ± SEM.
Fig 11.
Full length gasdermin D is a proteolytic substrate for active caspase-4/1.
(A) GST and His-tagged recombinant GSDMD (rGSDMD) was incubated with 1U recombinant caspase-1 (rC-1) and recombinant caspase-4 (rC-4) at 37°C with indicated time points, following the detection by immunoblot analysis. (B) rGSDMD were incubated for 15 min at 37°C with active rC-1 and rC-4 in absence or presence of inhibitor Z-VAD-fmk and Z-YVAD-fmk (100 μM, 10 min, room temperature) and rGSDMD cleavage was assessed by western blot with anti-GSDMD, anti-GST and anti-His antibody. (C) Macrophages were immunoprecipitated with anti-GSDMD antibody and immunoprecipitants were incubated with active rC-1 and rC-4 for 16 h at 37°C and GSDMD cleavage was assessed by western blotting identified with anti-GSDMD antibody. Direct cell lysate was used as a control (Ctrl). (D) C terminal Myc-DDk-tagged human GSDMD plasmid was overexpressed in HEK 293T cells and, (E) Basal expression of GSDMD was detected via anti-GSDMD antibody. (F-H) HEK 293T cells transfected with Myc-DDk-tagged GSDMD plasmid were immunoprecipitated with anti-DYKDDDDK tag antibody. Immunoprecipitants were incubated with active rC-4 and rC-1 for 16 h at 37°C and GSDMD cleavage was assessed by western blot with anti-DYKDDDDK tag and anti-GSDMD antibody. Immunoblots are representative of at least three independent experiments (n = 3).
Fig 12.
Predicated cleavage site for caspase-4 on GSDMD.
The molecular weight of full length rGSDMD is 81 kDa as a GST tag is linked to NT of rGSDMD, whereas CT is tagged by His. (A) Cleavage of 5 μg purified recombinant GSDMD with 6U recombinant caspase-4 visualized by Coomassie blue staining of the protein bands that were excised (white dotted box) and sequenced by Edman degradation. Immunoblot is representative of at least three independent experiments (n = 3). (B) Schematic representation showed full length rGSDMD with tags. Red line indicated GST tag attached to the NT, while CT was linked by a His tag in grey, and full GSDMD sequence is marked in black. (C) Caspase-4 cleavage sequence logo was generated from “MEROPS” (https://www.ebi.ac.uk/merops/) based on the peptidase database. (D) After alignment of the calls that we obtained from Edman degradation, the predicated cleavage for caspase-4 on GSDMD marked the same cleavage site as caspase-1 (black arrow). (E) NT GST-tagged rGSDMD incubated with active rC-4 for 16 h at 37°C and degraded fragments generated from full length GSDMD were evaluated by immunoblot analysis followed by Coomassie blue staining. Edman degradation analysis of the 26 kDa band after alignment of amino acid calls suggested a cleavage site for caspase-4 on GSDMD at aspartic acid 275 (D275) position.
Fig 13.
Shotgun proteomics analysis of uninfected macrophages and E. histolytica-induced hyperactivated macrophages.
(A) Preparation and workflow for proteomic analysis. (B) Metascape analysis of different pathways within control and Eh-contacted hyperactivated macrophages. Some upregulated pathways in red and downregulated pathways in blue are what we considered most relevant and interesting. (C) Some interesting proteins involved in downregulated and upregulated pathways were characterized, and the common proteins were also indicated. (D) Macrophages were incubated with Eh (20:1) for 10 and 30 min to detect NINJ1 protein level and blots were reprobed for GAPDH. Immunoblots are representative of at least three independent experiments (n = 3). (E) STRING analysis of GSDMD protein-protein interaction, and NINJ1 protein-protein interaction with other top hits proteins were conducted.
Fig 14.
Proposed schematic representation of E. histolytica-macrophage interaction and induction of caspase-4/1 activation and IL-1β secretion.
The activation of caspase is initially triggered by Eh in contact with macrophage via the Gal-lectin to Gal/GalNAc residues on the surface of macrophage. EhCP-A5 is highly expressed on the surface of Eh and following Gal-lectin binding brings, EhCP-A5 RGD sequences ligate α5β1 integrin on the macrophage surface to induce the generation of ATP and release through the opening of pannexin-1 channel that subsequently signals back onto the P2X7 receptor to activate the NLRP3 inflammasome. Simultaneously, K+ efflux and the production of ROS collaborate to activate the NLRP3 inflammasome. The NLRP3 inflammasome in turn activates caspase-1, whereas, the activation of caspase-4 is independent of the inflammasome complex. Whereas both caspase-4/1 acted together to induce the cleavage of GSDMD, caspase-4 played a dominant role in this process. The cleaved GSDMD initiates pore formation allowing bioactive IL-1β release without causing significant cell pyroptosis.
Table 1.
Identification of the top-hit pathway proteins that were downregulated and upregulated in E. histolytica-induced hyperactivated macrophages.