Fig 2: Co-deletion of GSDMD and GSDME protects from sHLH lethality.(A and B) Rectal temperatures of (A) WT (n = 15) and Il-1β−/− (n = 13) mice or (B) WT (n = 15) and Gsdmd−/− (n = 12) mice post-sHLH induction. Data are pooled from two (A) or three (B) independent experiments. (C and D) Serum concentration of IL-1β, IL-6, and TNF in (C) WT (n = 15) and IL-1β−/− (n = 13) mice or (D) WT (n = 10) and Gsdmd−/− (n = 11) mice 3 hours post-sHLH induction. (E) Rectal temperatures of WT (n = 11) and Ninj1−/− (n = 13) mice post-sHLH induction. Data are pooled from two independent experiments. (F) Serum concentration of IL-1β, IL-6, and TNF in WT (n = 6) and Ninj1−/− (n = 8) mice 3 hours post-sHLH induction. (G and H) Survival curve of (G) WT (n = 16), Il-18−/− (n = 11), and Il-18−/−Il-1β−/− (n = 10) mice or (H) WT (n = 19), Gsdmd−/− (n = 13), Gsdme−/− (n = 17), and Gsdmd−/− Gsdme−/− (n = 8) mice post-sHLH induction. Data are pooled from either two (G) or three (H) independent experiments. (A to D) Data represent the mean values ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. Mouse temperatures were analyzed using a two-way ANOVA, with a Bonferroni post hoc correction for multiple comparisons, while an unpaired nonparametric Mann-Whitney test was used for analyzing cytokine levels between genotypes. Survival graphs were analyzed using a log-rank (Mantel-Cox) test.

Fig 3: Caspase-8 and MLKL deletion protects from sHLH severity and increased inflammatory cytokines.(A) Temperatures of WT, Casp8−/−Mlkl−/−, Casp8−/+Mlkl−/−, and Mlkl−/− mice following sHLH induction. Pooled from two independent experiments involving WT, Casp8−/−Mlkl−/−, and Casp8−/+Mlkl−/− mice (one that reached the ethical endpoint after 3 hours and one after 4 hours) and one experiment using WT and Mlkl−/− animals. (B) Serum cytokine concentrations in WT (n = 9), Casp8−/−Mlkl−/− (n = 5 to 6), Casp8−/+Mlkl−/− (n = 5), and Mlkl−/− (n = 6) mice 3 to 4 hours post-sHLH induction. (C and D) Immunoblot analysis of spleen tissue from (C) WT healthy control (n = 4), WT HLH (n = 5), and Casp8−/−Mlkl−/− (n = 5) mice and (D) WT healthy control (n = 4), WT HLH (n = 5), and Mlkl−/− (n = 6) mice harvested 3 to 4 hours after sHLH disease induction. Gsdmd−/− Gsdme−/− HLH spleen, Casp1−/−Casp11−/− HLH spleen, and BMDMs treated with LPS (100 ng/ml) followed by nigericin (10 μM) were used as controls. (E) Model of cell death signaling in sHLH based on this study and other published research. IFN-γ and/or TLR/death receptor signaling activates caspase-8 transcription for optimal inflammasome priming. NLRP3 recruitment of caspase-1 and caspase-8 via ASC allows for GSDMD cleavage and, in part, caspase-3 activation of GSDME (also engaged directly downstream of TLRs/death receptors). Gasdermin pores cause pyroptosis to drive disease severity, and although they facilitate the release of NLRP3-activated IL-1β and IL-18, ultimately, gasdermin-mediated release of these two cytokines alone does not account for the full spectrum of disease protection resulting from GSDMD and GSDME deletion. Data in (A) and (B) represent the mean values ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. Temperatures were analyzed using a two-way ANOVA, with Tukey’s correction for multiple comparisons. An unpaired nonparametric Mann-Whitney test was used for analyzing cytokine levels. (E) created in BioRender. J. Vince (2025) https://BioRender.com/zhwmsiu.