Fig 1.
Egg-induced IL-1β and IL-17 production is dependent on both Caspase-1 and Caspase-8.
(A) BL/6 BMDCs were pretreated with caspase-1 inhibitor ZYVAD-FMK (50μM) for 1h before culturing for 24h with the indicated numbers of live eggs, or LPS plus nigericin (Nig). IL-1β (left) and TNFα (right) in supernatants were measured by ELISA. (B) BMDCs from BL/6 and Caspase-1-/- mice were cultured for 24h with 100 live (or no) eggs, or LPS plus Nig. IL-1β (left) and TNFα (right) in supernatants were measured by ELISA. (C) BL/6, RIP3-/- and RIP3-/-/Casp8-/- BMDCs were pretreated with ZYVAD-FMK (50μM) for 1h before culturing for 24h with 100 live (or no) eggs. IL-1β (left) and TNFα (middle) in supernatants were measured by ELISA. BL/6, RIP3-/- and RIP3-/-/Casp8-/- BMDCs were pretreated with ZYVAD-FMK (50μM) for 1h before establishing co-cultures with CD4 T cells in the presence and absence of 100 live eggs. IL-17A (right) in 72h supernatants was measured by ELISA. (D) Immunoblot analysis of pro-caspase-1 processing in whole cell lysates of BL/6, RIP3-/-, RIP3-/-/Casp8-/-, Casp1-/- and ASC-/- BMDCs left unstimulated or stimulated with 1000 eggs, LPS and Nig or LPS only. Bars represent the mean +/- SD cytokine levels of three biological replicates from one representative experiment of three with similar results. **p <0.005, ***p <0.0005.
Fig 2.
Eggs jointly coordinate a caspase-1 and caspase-8 dependent pyroptotic cell death.
(A) BL/6 BMDCs were cultured for 24h with the indicated number of live (or no) eggs, LPS plus Nig and Triton X-100 (Tri) for 10 min. (B) BL/6, RIP3-/- and RIP3-/-/Casp8-/- BMDCs were pretreated with ZYVAD-FMK (50μM) for 1h before culturing for 24h with 100 live (or no) eggs. Cell survival was measured using calcein AM. The medium was set at 100%. Bars represent the mean +/- SD cytokine levels of three biological replicates from one representative experiment of three with similar results. *p <0.05, **p <0.005.
Fig 3.
The loss of NLRP3 and AIM2 results in reduced Th17 and enhanced Th2 cytokine responses.
(A) BMDCs from BL/6, ASC-/- and NLRP3-/- mice were cultured for 24h with the indicated number of live (or no) eggs, or LPS plus Nig. IL-1β (left) and TNFα (right) in supernatants were measured by ELISA. (B) BMDCs from Aim2+/+ and Aim2-/- mice were cultured for 24h with the indicated number of live (or no) eggs, transfected with the indicated concentrations of Schistosoma mansoni (sm) genomic (g) DNA, LPS plus poly(dAdT), or LPS plus Nig. IL-1β in supernatants was measured by ELISA (left). Poly (dAdT) was used as an AIM2 inflammasome-activating control. (B) Confocal microscopy of LPS primed AIM2-citrine macrophages left untransfected or transfected with 100 ng/ml DAPI-labeled sm gDNA (middle). Scale bar: 20μm (top and bottom). The formation of AIM2 pyroptosomes was quantified using confocal microscopy (right). Data are representative of at least 10 fields of view and three independent experiments. (C) BL/6 and NLRP3-/- BMDCs were transduced with empty vector (EV), control shRNA against eGFP (shEGFP) and shRNA against AIM2 (shAIM2). Cells were cultured with 100 live eggs or LPS plus Nig or LPS plus poly (dAdT). IL-1β in 24h supernatants was measured by ELISA (left). The above-mentioned BMDCs were co-cultured with T cells and 100 live (or no) eggs. IL-17A (middle) and IL-4 (right) in 72h supernatants were measured by ELISA. Bars represent the mean +/- SD cytokine levels of three biological replicates from one representative experiment of three with similar results. *p <0.05, **p <0.005, ***p <0.0005.
Fig 4.
ROS, K+ efflux and cathepsin B are required for NLRP3 activation.
(A) BL/6 BMDCs were pretreated with the indicated concentrations of ROS inhibitor N-acetyl-l-cysteine (NAC) (left) or Diphenyleneiodonium (DPI) (right) for 1h before culturing for 24h with 100 live (or no) eggs, or LPS plus Nig. IL-1β in supernatants was measured by ELISA. (B) 106 BL/6 BMDCs were pretreated with 20 μM of piceatannol (pic) for 1h before culturing for 24h with 1000 eggs. Cells were then stained with CellRox Deep Red and ROS was measured using flow cytometry (C) BL/6 BMDCs were pretreated with indicated concentrations of potassium chloride (KCl), sodium chloride (NaCl) (left) or potassium channel blocker Glybenclamide (right) for 1h before culturing for 24h with 100 live (or no) eggs, or LPS plus Nig (D) BMDCs from BL/6 and Cathepsin B-/- mice were cultured for 24h with 100 live (or no) eggs or LPS plus Nig. IL-1β in supernatants was measured by ELISA (left). Immunoblot analysis of cathepsin B in whole cell lysates of BL/6 BMDCs left unstimulated or pretreated with 20 μM piceatannol for 1h and then stimulated with 1000 eggs for 24h, stimulated with eggs, or Piceatannol only (right). Bars represent the mean +/- SD cytokine levels of three biological replicates from one representative experiment of three with similar results. *p <0.05, **p <0.005, ***p <0.0005.
Fig 5.
Gsdmd deficiency reduces myeloid cell recruitment to the liver during schistosome infection.
(A) Weights of livers from uninfected and infected BL/6 and Gsdmd−/− mice (left). Granuloma size was determined by morphometric analysis. Each dot represents average granuloma size of 10–20 granulomas in two sections from individual mice (middle). Number of eggs per 2.4 mm2 field assessed on H&E-stained liver sections at 100× magnification. An average of 20 fields per liver section was assessed. Images are representative of three independent experiments (right). (B) Representative histopathology of liver granulomas of BL/6 and Gsdmd−/− mice (magnification, 100×). (C) UMAP of neutrophils, macrophages, CD4+T cells, and dendritic cells isolated from livers of C57BL/6 and Gsdmd-/- mice infected with Schistosoma mansoni for 7 weeks. (D) Percentages of liver- and granuloma-derived neutrophils, macrophages, dendritic cells and CD4+ T cells as a proportion of CD45+ cells: (top left) Ly6G+ cells, (top right) F4/80+ cells, (bottom left) CD11b+ CD11c+ cells, (bottom right) CD3+CD4+ cells. Data are representative of two independent experiments. Significance was determined using Student’s t-test *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005; ns, not significant.
Fig 6.
Blocking IFNAR leads to enhancement of IL-1β and IL-17 production.
(A) BMDCs from BL/6, NLRP3-/- and AIM2-/- BMDCs were cultured for 24h with 100 live (or no) eggs. IFNβ in supernatants was measured by ELISA. (B) BL/6, NLRP3-/- and AIM2-/- BMDCs were pretreated with IFN-I receptor blocking antibody (α-IFNAR1) (20μg/ml) or control IgG (20μg/ml) for 1h before culturing for 24h with 100 live (or no) eggs. IL-1β in supernatants was measured by ELISA. (C) The above mentioned BMDCs were co-cultured with T cells together with 100 live (or no) eggs. IL-17A in 72h supernatants were measured by ELISA. Bars represent the mean +/- SD cytokine levels of three biological replicates from one representative experiment of three with similar results. *p <0.05, **p <0.005, ***p <0.0005.