ZBP1 promotes fungi-induced inflammasome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis)

Abstract

Candida albicans and Aspergillus fumigatus are dangerous fungal pathogens with high morbidity and mortality, particularly in immunocompromised patients. Innate immune-mediated programmed cell death (pyroptosis, apoptosis, necroptosis) is an integral part of host defense against pathogens. Inflammasomes, which are canonically formed upstream of pyroptosis, have been characterized as key mediators of fungal sensing and drivers of proinflammatory responses. However, the specific cell death pathways and key upstream sensors activated in the context of Candida and Aspergillus infections are unknown. Here, we report that C. albicans and A. fumigatus infection induced inflammatory programmed cell death in the form of pyroptosis, apoptosis, and necroptosis (PANoptosis). Further, we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as the apical sensor of fungal infection responsible for activating the inflammasome/pyroptosis, apoptosis, and necroptosis. The Zα2 domain of ZBP1 was required to promote this inflammasome activation and PANoptosis. Overall, our results demonstrate that C. albicans and A. fumigatus induce PANoptosis and that ZBP1 plays a vital role in inflammasome activation and PANoptosis in response to fungal pathogens.

Keywords: Aspergillus fumigatus; Candida albicans; MLKL; PANoptosis; PANoptosome; RIPK3; ZBP1; apoptosis; caspase-1; caspase-3; caspase-7; caspase-8; cell death; fungi; gasdermin D; host defense; infection; infectious disease; inflammasome; necroptosis; pyroptosis.

Figure 1

PANoptosis in response to C. albicans.A–C, Western blotting analysis of PANoptosis activation markers in bone marrow-derived macrophages (BMDMs) after C. albicans infection. A, Pyroptosis activation is assessed by immunoblotting of cleaved caspase-1 (CASP1) (p20) and gasdermin D (GSDMD) (p30). B, Apoptosis activation is determined by immunoblotting of active initiator CASP8 (p18) and executioner caspases CASP3 (p19/17) and CASP7 (p20). C, Necroptosis activation is indicated by the phosphorylation of mixed lineage kinase domain-like pseudokinase (pMLKL). Total MLKL (tMLKL) and GAPDH are used as loading controls. Molecular weight marker sizes are indicated on the right (kDa). D, Quantification of cell death in primary human peripheral blood mononuclear cells (hPBMCs) following C. albicans infection. Data presented are representative of three independent experiments. 2-way ANOVA was used to determine statistical significance. ***P < 0.001 moi, multiplicity of infection; UT, untreated. Black asterisks denote a nonspecific band.

Figure 2

Ablation of PANoptotic components inhibits C. albicans-induced PANoptosis and inflammation.A–C, Western blotting analysis of PANoptosis activation markers after C. albicans infection in the indicated bone marrow-derived macrophages (BMDMs). A, Pyroptosis activation is assessed by immunoblotting of cleaved caspase-1 (CASP1) (p20) and gasdermin D (GSDMD) (p30). B, Apoptosis activation is determined by immunoblotting of active initiator CASP8 (p18) and executioner caspases CASP3 (p19/17) and CASP7 (p20). C, Necroptosis activation is indicated by the phosphorylation of mixed lineage kinase domain-like pseudokinase (pMLKL). Total MLKL (tMLKL) and GAPDH are used as loading controls. ASC and RIPK3 proteins were probed to confirm their deletion in ASC and RIPK3-deficient BMDMs, respectively. Molecular weight marker sizes are indicated on the right (kDa). D, Inflammatory cytokine IL-18 release was evaluated in WT (WT) and Casp1/11^–/–Ripk3^–/–Casp8^–/– BMDMs following C. albicans infection for 20 h. Data shown are representative of at least three independent experiments (A–D). Unpaired t test with Welch's correction was used to determine statistical significance. ***P < 0.001 (D). UT, untreated. Black asterisks denote a nonspecific band.

Figure 3:

Zα2 domain of ZBP1 drives PANoptosis.A–C, Western blot analysis of PANoptosis activation markers after C. albicans infection in wildtype (WT), Zbp1^–/–, or Zbp1^{ΔZα2/ΔZα2} bone marrow-derived macrophages (BMDMs). A, Pyroptosis activation is assessed by immunoblotting of cleaved caspase-1 (CASP1) (p20) and gasdermin D (GSDMD) (p30). B, Apoptosis activation is determined by immunoblotting of active initiator CASP8 (p18) and executioner caspases CASP3 (p19/17) and CASP7 (p20). C, Necroptosis activation is indicated by the phosphorylation of mixed lineage kinase domain-like pseudokinase (pMLKL). ZBP1 protein was probed to confirm its deletion and MW shift in ZBP1-deficient and Zbp1^{ΔZα2/ΔZα2} BMDMs, respectively. Total MLKL (tMLKL) and GAPDH are used as loading controls. Molecular weight marker sizes are indicated on the right (kDa). D, Inflammatory cytokine IL-18 release was evaluated in WT, Zbp1^–/–, or Zbp1^{ΔZα2/ΔZα2} BMDMs following C. albicans infection for 20 h. E, Schematic summary of the regulation of PANoptosis by ZBP1 in response to the fungal pathogens C. albicans or A. fumigatus. ZBP1 senses the fungal pathogen and mediates PANoptosis possibly by engaging the ZBP1-PANoptosome to drive NLRP3-mediated CASP1 activation (pyroptosis), CASP3/CASP7 activation (apoptosis), and MLKL activation (necroptosis). Data shown are representative of at least three independent experiments (A–D). 2-way ANOVA was employed to determine statistical significance. *P < 0.05 (D). UT, untreated. Red asterisks denote a nonspecific band.