Fig 1: siCasp8 and caspase‐8 inhibitor (Casp8‐I) decreased PLF‐1 expression in vitro and in vivo. SMCs were seeded into 6‐well plates (2.5×105) and then treated with nontargeted siRNA (n‐siRNA) or siCasp8, respectively, under H2O2 condition at indicated concentrations for 2 days. Lysates were subjected to western blotting assays. Representative immunoblots and quantitative data exhibit changes in levels of PLF‐1 protein (A). CatK+/+ mice that had undergone double injury were injected subcutaneously with either DMSO or a synthetic specific Casp8‐I (5 mg/kg/day for each) on days −2, −1, 0, 1, 2, 3, and 4 postsurgery. On operative day 4, carotid arteries were isolated for western blotting. Representative images of western blots and quantitative show changes in levels of PLF‐1 (B) and caspase‐8 (C) proteins. SMCs were seeded into 6‐well plates (2.5×105) and then treated with siCasp8 or siTLR2, respectively, at H2O2 condition for 2 days. Lysates were subjected western blotting assays. Representative immunoblots and quantitative data exhibit changes in levels of procaspase‐8 (procasp8; D) and PLF‐1 (E) protein. CatK+/+ and CatK−/− SMCs were seeded into 6‐well plates (2.5×105) and then treated with H2O2 (750 μmol/L) for 24 hours. Lysates were subjected western blotting assays. Representative immunoblots and quantitative data exhibit changes in levels of PLF‐1 protein (F). On operative day 4, left and right carotid arteries of CatK+/+ and CatK−/− mice that had received a single or double injury were collected and subjected to western blotting assays. Representative immunoblots exhibit changes in levels of PLF‐1 protein (G). Data are means±SEM (n=3–5 for each group). P values calculated using Student t test or Tukey's post hoc tests. CatK indicates cathepsin K; PLF‐1, proliferin‐1; SMCs, smooth muscle cells.
Fig 2: Caspase-8 mediates late cell death and IL-1ß production in Carnivora cells infected with S. Typhimurium(A) Dog WT DH82 cells were primed with LPS (200 ng/mL) and stimulated with nigericin (20 µM for 1 h). Non-stimulated cells were left in cell culture medium. Live cells were stained for activated caspases (green, FLICA poly-caspase, FLICA caspase-1, or FLICA caspase-8). Following fixation, cells were stained for cytoplasmic ASC (red) and nuclei using DAPI staining (blue). White arrows point to ASC specks.(B) Dog WT, Casp8-/- bulk edited, and cells from 2 individual Casp8-/- DH82 clones were infected with S. Typhimurium MOI of 10, and the amount of LDH released in the supernatant was measured over time via a colorimetric assay.(C) Dog WT and Casp8-/- bulk-edited DH82 cells were infected with S. Typhimurium MOI of 10, and the amount of IL-1ß in the supernatant was measured over time by ELISA.(D and E) Dog WT and cells from 1 individual Ripk1-/- DH82 clone were infected with S. Typhimurium MOI of 10, and the amounts of LDH and IL-1ß were measured in the supernatant over time via colorimetric assay and ELISA, respectively.(F and G) Identical to (D) and (E), but with cells from a second individual Ripk1-/- DH82 clone; LDH and IL-1ß were measured in the supernatant at 24 h only.(H) Dog WT and cells from 1 individual Ripk1-/- DH82 clone were infected with S. Typhimurium MOI of 1 and total protein extracts from cell lysates were subject to western blot analysis against dog-specific IL-1ß. Uninfected controls (medium only) were also included for each cell line.Data are shown as means ± SEMs in (B)–(G). Data are pooled from 4 independent experiments in (C)–(E), from 1 representative of 3 independent experiments in (A), from 1 representative of 2 independent experiments in (B), and from 1 single experiment in (F)–(H). Statistical significance was calculated by the Mann-Whitney test in (C) and (D) and by 2-tailed unpaired t test assuming equal variances in (E); ns, not significant; *p < 0.05.See also Figure S4.
Fig 3: Role of TRAIL receptors and TRAIL-receptor-binding proteins in the triggering of apoptosis. (a) Percentage of cells with high levels of cleaved PARP in populations transfected with pooled siRNAs targeting DR4 (DR4), DR5 (DR5), DcR2 (DcR2) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (control) or challenged with TRAIL (6 h). (b) Percentage of cells with high levels of cleaved PARP in naïve (control) or TRAIL-challenged (6 h) DR5 knockout (DR5 KO) or DcR2 knockout (DcR2 KO) cells as compared with controls (ctrol). (c) Contour plot for cleaved PARP immunolabelling in DcR2 knockout (DcR2 KO) cells either unchallenged (control) or treated 6 h with TRAIL (TRAIL). Subpopulations displaying low (survivors, '1') and high (apoptotic, '2') levels of PARP cleavage in treated samples are depicted. Image shows one representative experiment out of three independent biological replicates. (d) Percentage of cells with high levels of cleaved PARP in control and DcR2 KO naïve cells (control) or control and DcR2 KO cells challenged with TRAIL for 16 h (TRAIL). (e) Percentage of cells with high levels of cleaved PARP in cells transfected with siRNAs targeting FADD (FADD), caspase-8 (casp-8), RIPK1 (RIPK1), TRAF2 (TRAF2) or cFlip (cFlip) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (control) or challenged for 6 h with TRAIL (TRAIL). (f) Percentage of cells with high levels of cleaved PARP in populations transfected with siRNAs targeting RIPK1 (RIPK1), TRAF2 (TRAF2) or RIPK1 and TRAF2 (RIPK1+TRAF2) mRNAs or non-targeting scramble siRNAs at concentrations corresponding to single (30 nM; scr 30) or double (60 nM; scr 60) transfections, further left either untreated (control) or challenged during 6 h with TRAIL (TRAIL). Results obtained in conditions of pan-caspase inhibition (control+ zVAD and TRAIL+zVAD) as compared with basal conditions of cell response (control and TRAIL) are shown. (g) Percentage of cells with high levels of cleaved PARP in cells transfected with siRNAs targeting RIPK1 (RIPK1), TRAF2 (TRAF2), DR5 (DR5), RIPK1 and DR5 (RIPK1+DR5), TRAF2 and DR5 (TRAF2+DR5) mRNAs or non-targeting scramble siRNAs ('scr 30' and 'scr 60') further left either untreated (control) or challenged 6 h with TRAIL (TRAIL). (a, b and d–g) Mean±standard deviation (S.D.) from three independent biological replicates is shown. ***P-value <0.0005, **P-value <0.005, *P-value <0.05. NS, not significant
Fig 4: Cholesterol crystals (CC)-mediated cell death is unrelated to mixed lineage kinase domain-like kinase (MLKL), gasdermin D (GSDMD), CASP8, Ca2+ influx, K+ efflux and SYK. (A) Left: MLKL was knocked down in iBMDM by shRNA. Gene expression was determined by Q-PCR. Right: iBMDM were treated with CC for 2 h or zVAD (10 µM) plus TNFα (50 ng/ml) and CHX (50 µg/ml) for 12 h and then the cell death was determined with PI staining. n = 6. N = 5. (B) Left: GSDMD was knocked down in iBMDM by shRNA as in (A). Right: iBMDM were primed then treated identical to Figure 1C except for the treatment duration (CC: 2 h, Silica: 1 h, ATP: 2 h, tLPS: 6 h). Cell death was determined with PI staining. n = 6. N = 4. (C) BMDM with Caspase 8 deficiency were treated and stained as in Figure 1G. n = 6. N = 3. (D) BMDM were primed then pretreated with 50 µM 2APB or 1 µM Thapsigargin for 0.5 h before stimulus treatment as in Figure 1C. 6 h later, IL1β production was determined by ELISA. n = 4. N = 3. (E) Identical to (D) except for the treatment duration (CC: 2 h, Silica: 1 h, ATP: 2 h). Cell death was determined with PI staining. n = 5. N = 6. (F) BMDM were primed and the media were exchanged into HBSS, 70 mM K+ HBSS, or HBSS containing 100 µM Calpeptin 15 min before stimulus treatment as in Figure 1C except for the treatment duration (CC: 2 h, Silica: 1 h, ATP: 2 h). Cell death was determined with PI staining. n = 6. N = 5. (G) The left panel shows the schematic of how attraction force between crystals and cells was measured by AFM. Right panel: BMDM were primed and pretreated with piceatannol (50 µM) or Cytochalasin B (1 µg/ml) for 20 min. The attraction force after 5 s contact time was measured by AFM. N = 3. (H) BMDM were pretreated as in (G) before being treated with stimuli as in Figure 1C except for the treatment duration (CC: 2 h, Silica: 1 h, ATP: 2 h). Cell death was determined by PI staining. n = 6. N = 3. (I) The design of Syk CKO. (J) Syk expression in Syk CKO BMDM was examined by western blot. N = 3. (K) Syk CKO and WT BMDM were primed and treated with stimuli as in Figure 1C except for the treatment duration (CC: 2 h, Silica: 1 h, ATP: 2 h). Cell death was determined by PI staining. n = 6. N = 7. (L) The attraction forces after 5 s contact time between Syk CKO BMDM and crystals were measured by AFM. N = 3.
Fig 5: Activation of the CASP8-JAK1/2-STAT1 cell death module in human colonic biopsies and organoids.A–C Colonic biopsies isolated from healthy (HL) individuals (n = 10) or patients with inactive/non-inflamed (n = 11) and active/inflamed (n = 11) Crohn’s disease (CD) were incubated ex vivo with anti-CD3/28 (5 µg/ml each) for 18 h. Relative mRNA expression of A IFNG, TNF and IL2, and B JAK1, JAK2, STAT1 and CASP8 measured by RT-qPCR. C Correlation between relative mRNA levels of the indicated genes or the JAK-STAT score in resting (left) and anti-CD3/28-stimulated biopsies (right). D Relative viability of primary human colonic organoids derived from HL (n = 3 lines) or CD (n = 3 lines) colonic biopsies, and stimulated with IFN-? and/or TNF-a (10 ng/ml each) for the indicated times. Coefficient of cytokine interaction (CCI) calculated per time-point (right). E–G Primary human colonic organoids derived from CD colonic biopsies (n = 3 lines) were pretreated for 1 h with vehicle (DMSO), JAK1-selective compound (upadacitinib and filgotinib), JAK1/2 inhibitor (baricitinib) or pan-JAK inhibitor (tofacitinib) at 0.1, 1 or 10 µM, followed by IFN-? and/or TNF-a (10 ng/ml each) for the indicated times. E STAT1 activity was measured by HTRF assay at 2 h (top) and relative viability at 8 h (bottom) of cytokine treatment. F SYTOX Green staining of a select organoid line at 8 h of cytokine treatment. G Pearson correlation between relative viability and STAT1 activity. Data were mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 (two-way, repeated-measures ANOVA with Bonferroni’s multiple comparisons test), #p < 0.05, ##p < 0.01 and ###p < 0.001 (two-way ANOVA with Tukey’s multiple comparisons test), /p < 0.05, ///p < 0.001 (one-way ANOVA with Dunnett’s multiple comparisons test vs. DMSO plus IFN-?/TNF-a). NT non-treated, JAKinib JAK inhibitor, UPA upadacitinib, BARI baricitinib, TOFA tofacitinib, FILGO filgotinib, org. organoids, CCI coefficient of cytokine interaction.
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