ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), continues to cause substantial morbidity and mortality in the ongoing global pandemic. Understanding the fundamental mechanisms that govern innate immune and inflammatory responses during SARS-CoV-2 infection is critical for developing effective therapeutic strategies. Whereas interferon (IFN)-based therapies are generally expected to be beneficial during viral infection, clinical trials in COVID-19 have shown limited efficacy and potential detrimental effects of IFN treatment during SARS-CoV-2 infection. However, the underlying mechanisms responsible for this failure remain unknown. In this study, we found that IFN induced Z-DNA-binding protein 1 (ZBP1)-mediated inflammatory cell death, PANoptosis, in human and murine macrophages and in the lungs of mice infected with β-coronaviruses, including SARS-CoV-2 and mouse hepatitis virus (MHV). In patients with COVID-19, expression of the innate immune sensor ZBP1 was increased in immune cells from those who succumbed to the disease compared with those who recovered, further suggesting a link between ZBP1 and pathology. In mice, IFN-β treatment after β-coronavirus infection increased lethality, and genetic deletion of Zbp1 or its Zα domain suppressed cell death and protected the mice from IFN-mediated lethality during β-coronavirus infection. Overall, our results identify that ZBP1 induced during coronavirus infection limits the efficacy of IFN therapy by driving inflammatory cell death and lethality. Therefore, inhibiting ZBP1 activity may improve the efficacy of IFN therapy, paving the way for the development of new and critically needed therapeutics for COVID-19 as well as other infections and inflammatory conditions where IFN-mediated cell death and pathology occur.

Fig. 1.. Delayed IFN responses during β-coronavirus infection.

(A) Heatmap depicting the altered pathways in whole blood cell transcriptomes of critically ill (CI) patients compared with non-critically ill (NCI) patients with COVID-19 ( 34 ). NES, normalized enrichment scores. (B) IFN-β release in the supernatant of wild type (WT) bone marrow-derived macrophages (BMDMs) infected with mouse hepatitis virus (MHV) or influenza A virus (IAV) (left) or THP-1 cells infected with SARS-CoV-2 or IAV (right) for the indicated time. (C) Immunoblot analysis of phosphorylated STAT1 (pSTAT1), total STAT1 (tSTAT1) and ISG15 in WT BMDMs infected with MHV or IAV (left) or THP-1 cells infected with SARS-CoV-2 or IAV (right) for the indicated time. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. (D) Kinetics of IFN-α and IFN-γ responses in SARS-CoV-2– or IAV-infected Calu-3 cells ( 41 , 104 ). Data are representative of at least three independent experiments (B, C). ****P < 0.0001. Analysis was performed using the two-way ANOVA (B). Data are shown as mean ± SEM.

Fig. 2.. IFN-β treatment drives lethality, cytokine storm and cell death during β-coronavirus infection.

(A) Survival of 6- to 12-week-old wild type (WT) mice treated with PBS (n = 11) or IFN-β (n = 18) on day 1 and 3 after intranasal infection with mouse hepatitis virus (MHV). (B) Survival of 6- to 12-week-old WT mice treated with PBS (n = 7) or IFN-β (n = 8) on day 2 and 4 after intranasal infection with SARS-CoV-2. (C) Analysis of IL-1β, IL-18, IL-6, TNF and IFN-γ levels in BALF of uninfected WT mice (mock, n = 6) or PBS- (n = 6) or IFN-β– (n = 6) treated WT mice 3 days after MHV infection. (D) Hematoxylin and eosin (H/E) staining of lung samples from WT mice 3 days after MHV infection (PBS- or IFN-β–treated) or no infection (mock). Arrows indicate infiltrating immune cells and dotted red outline represents a syncytial cell. (E) Real-time analysis of cell death in PBS- or IFN-β–treated WT bone marrow-derived macrophages (BMDMs) after infection with MHV or influenza A virus (IAV). (F) Representative images of cell death in PBS- or IFN-β–treated BMDMs are shown at 24 hours after MHV infection or 16 hours after IAV infection. Scale bar, 50 μm (D, F). Data are representative of two (A, C, D) or at least three independent experiments (E, F). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Analysis was performed using the one-way ANOVA (C, E) or log-rank test (Mantel-Cox) (A, B). Each symbol represents one mouse (C). Data are shown as mean ± SEM. Images are representative of an experiment containing at least 5 biologically independent samples in each group (D).

Fig. 3.. IFN-β promotes inflammatory cell death, PANoptosis, during β-coronavirus infection.

(A–C) Immunoblot analysis of (A) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); (B) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and (C) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from mock- or IFN-β–treated wild type (WT) mice with or without mouse hepatitis virus (MHV) infection 3 days post-infection. (D–I) Immunoblot analysis of (D, G) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; (E, H) pro- (P55) and cleaved CASP8 (P18), pro- (P35) and cleaved CASP3 (P19 and P17) and pro- (P35) and cleaved CASP7 (P20); (F) pMLKL, tMLKL, pRIPK3 and tRIPK3; and (I) pRIPK3 and tRIPK3 in mock- or IFN-β–treated bone marrow-derived macrophages (BMDMs) or THP-1 cells during MHV or SARS-CoV-2 infection, respectively. Actin was used as the internal control. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. Asterisk denotes non-specific bands (A, G). Data are representative of at least three independent experiments.

Fig. 4.. IFN-β–driven lethality, cytokine storm and cell death depend on ZBP1 during β-coronavirus infection.

(A) Volcano plot showing the genes that are enriched or depleted in immortalized bone marrow-derived macrophages (iBMDMs) following a genome-wide CRISPR/CAS9 knockout screen of cell death induced by mouse hepatitis virus (MHV) infection (MOI 0.2, 24 hours). (B) Survival of 6- to 12-week-old wild type (WT) and Zbp1 ^–/– mice treated with PBS (n = 15 for WT and n = 16 for Zbp1 ^–/– mice) or IFN-β (n = 21 for WT and n = 15 for Zbp1 ^–/– mice) on day 1 and 3 after intranasal infection with MHV. (C) Immunoblot analysis of ZBP1 in the lung samples from PBS- or IFN-β–treated WT mice 3 days after MHV infection. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. Actin was used as the internal control. (D) Real-time analysis of cell death in MHV-infected WT or Zbp1 ^–/– BMDMs in the presence or absence of IFN-β. (E) Representative images of cell death in media- or IFN-β–treated WT or Zbp1 ^–/– BMDMs are shown at 24 hours after MHV infection. Scale bar, 50 μm. (F) Real-time analysis of cell death in MHV-infected WT or Zbp1 ^∆Za2 BMDMs in the presence of IFN-β. (G) Real-time analysis of cell death in influenza A virus (IAV)-infected WT or Zbp1 ^–/– BMDMs in the presence and absence of IFN-β. (H) Survival of 6- to 12-week-old WT (n = 15) and Zbp1 ^–/– (n = 14) mice after intranasal infection of IAV. (I) Survival of 6- to 12-week-old WT and Zbp1 ^∆Za2 mice treated with PBS (n = 29 for WT and n = 21 for Zbp1 ^∆Za2 mice) or IFN-β (n = 28 for WT mice) on day 1 and 3 after intranasal infection of IAV. Survival data are pooled from 2 infection experiments (B, H, I). All other data are representative of at least three independent experiments. *P < 0.05, ***P < 0.001 and ****P < 0.0001. Analysis was performed using the one-way ANOVA (D, G), two-tailed t test (F) or log-rank test (Mantel-Cox) (B, H, I). Data are shown as mean ± SEM.

Fig. 5.. Zα domain of ZBP1 drives PANoptosis mediated by IFN-β during β-coronavirus infection.

(A–C) Immunoblot analysis of (A) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); (B) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and (C) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from PBS-treated mice or mouse hepatitis virus (MHV)-infected wild type (WT) and Zbp1 ^–/– mice treated with IFN-β harvested 3 days after infection. (D–F) Immunoblot analysis of (D) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; (E) pro- (P55) and cleaved (P18) CASP8, pro- (P35) and cleaved (P19 and P17) CASP3 and pro- (P35) and cleaved (P20) CASP7; and (F) pMLKL, tMLKL and ZBP1 in PBS- or IFN-β–treated WT, Zbp1 ^–/– and Zbp1 ^∆Za2 bone marrow-derived macrophages (BMDMs) during MHV infection. Actin was used as the internal control. Data are representative of at least three independent experiments.