ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways

Abstract

The interferon-inducible protein Z-DNA binding protein 1 (ZBP1, also known as DNA-dependent activator of IFN-regulatory factors (DAI) and DLM-1) was identified as a dsDNA sensor, which instigates innate immune responses. However, this classification has been disputed and whether ZBP1 functions as a pathogen sensor during an infection has remained unknown. Herein, we demonstrated ZBP1-mediated sensing of the influenza A virus (IAV) proteins NP and PB1, triggering cell death and inflammatory responses via the RIPK1-RIPK3-Caspase-8 axis. ZBP1 regulates NLRP3 inflammasome activation as well as induction of apoptosis, necroptosis and pyroptosis in IAV-infected cells. Importantly, ZBP1 deficiency protected mice from mortality during IAV infection owing to reduced inflammatory responses and epithelial damage. Overall, these findings indicate that ZBP1 is an innate immune sensor of IAV and highlight its importance in the pathogenesis of IAV infection.

Keywords: DAI; IFNs; NLRP3; RIPK3; ZBP1; apoptosis; caspase-1; caspase-8; inflammasome; influenza virus; necroptosis; pyroptosis.

Figure 1. The IFN-inducible protein ZBP1 mediates cell death in response to IAV infection

(A and B) Microscopic analysis and quantification of cell death by LDH release in BMDMs infected with IAV 16 h pi (p= 0.0131; n=4; two-tailed t test). (C and D) Microscopic analysis and quantification of cell death by LDH release in ear fibroblasts infected with IAV 16 h pi (p= 2.92096*10⁻⁶; n=4; two-tailed t test). (E) Microarray gene expression dataset enriched for nucleic acid sensing pathways with higher or lower expression in WT and Ifnar1^−/− BMDMs 9 h pi with IAV (one experiment with two biological replicates per genotype). (F) Real-time quantitative RT-PCR analysis of Zbp1 expression in WT and Ifnar1^−/− BMDMs 9 h pi with IAV (one experiment with two biological replicates per genotype). (G) Immunoblot analysis of ZBP1 and GAPDH (loading control) in unprimed BMDMs (0–12) h pi with IAV (n=3). (H) Immunostaining for ZBP1 in WT BMDMs infected with IAV for 16 h (n=2). (I and J) Microscopic analysis and quantification of cell death by LDH release in BMDMs infected with IAV 16 h pi (p= 0.0001; n=5; one-way ANOVA). (K and L) Microscopic analysis and quantification of cell death by LDH release in fibroblasts infected with IAV 16 h pi (p= 0.000460657; n=3; two-tailed t test). (M) Real time analysis of the kinetics of cell death in primary ear fibroblasts infected with IAV (MOI 10) (one experiment with three biological replicates per genotype).

Figure 2. ZBP1 regulates NLRP3 inflammasome activation and proinflammatory cytokine production during IAV infection

(A) Immunoblot analysis of pro-caspase-1 and caspase-1 subunit p20 in BMDMs 16 h pi with IAV (n=4)..(B and C) Levels of IL-1β and IL-18 in cell culture supernatants 16 h pi with IAV; (p= 0.0007 (IL-1β); p = 0.0015 (IL-18); n=3; one-way ANOVA)..(D and E) Immunoblot analysis of pro-caspase-1 and caspase-1 subunit p20 in BMDMs 16 h pi with VSV (Indiana strain) or IAV (n=3)..(F) Immunoprecipitation of ZBP1 from lysates of WT BMDMs infected with IAV for 16 h, and immunoblotted for ZBP1 and RIPK3 (n=3)..(G) Immunoblot analysis of pro-caspase-1 and caspase-1 subunit p20 in BMDMs 16 h pi with IAV (n=3)..(H) Levels of IL-1β and IL-18 in cell culture supernatants 16 h pi with IAV; (p=0.0003 (IL-1β); p=0.0033 (IL-18); n=3; one-way ANOVA)..(I and J) Levels of TNF and IL-6 in cell culture supernatants 16 h pi with IAV;.(I) (p=0.0003 (IL-6; one way ANOVA); p=0.04585299 (TNF; two-tailed t test) n=3);.(J) (p=0.00427068 (IL-6); p=0.01547619 (TNF;); n=3; two-tailed t test).

Figure 3. ZBP1 mediates RIPK3-dependent induction of apoptotic and necroptotic cell death pathways during IAV infection

(A and B) Microscopic analysis and quantification of cell death by LDH release in fibroblasts infected with IAV 16 h pi; (p=0.0016; n=3; one-way ANOVA)..(C) Quantification of cell death by LDH release in BMDMs infected with IAV 16 h pi (p=0.0001; n=4; one-way ANOVA)..(D) Immunoblot analysis of the pro- and cleaved-forms of caspase-8, caspase-3 and caspase-7 in BMDMs 16 h pi with IAV (n=3)..(E) Microscopic analysis of BMDMs infected with IAV for 16 h in the absence or presence of inhibitors (n=3).

Figure 4. ZBP1 regulates cell death in response to both mouse-adapted and human strains of IAV, but not in response to RNA viruses belonging to Paramyxoviriadae and Rhabdoviridae family

(A to F) Microscopic analysis and quantification of cell death by LDH release in fibroblasts infected with mouse adapted Influenza A/X31 (H3N2), Influenza A/Brisbane/59/2007 (H1N1) and A/Switzerland/9715293/2013 (H3N2) 16h pi (p= 4.5184*10⁻⁵ (X31); p=0.02523 (A/Brisbane); p=0.00671454 (A/Switzerland); n=4; two-tailed t test).(G) Quantification of cell death by LDH release in BMDMs infected with VSV (MOI 10), SeV (MOI 8) and RSV (MOI 10) 16 h pi (n=3).

Figure 5. ZBP1 functions as a sensor of IAV infection by interacting with IAV PB1 and NP proteins

(A) Immunoprecipitation of endogenous ZBP1 from lysates of WT BMDMs infected with IAV for 16 h, and immunoblotted for ZBP1, M1, NS1 and HA; (n= 4)..(B) Immunoprecipitation of endogenous ZBP1 from lysates of WT or Zbp1^−/− fibroblasts infected with IAV for 16 h, and immunoblotted for ZBP1, PB1 and NP; (n= 3)..(C) Immunoprecipitation of ZBP1, PB1 and NP from lysates of WT fibroblasts infected with IAV for 16 h, and immunoblotted for ZBP1, PB1 and NP; (n= 3)..(D) Immunoblot analysis of ZBP1, PB1, NP and GAPDH from whole cell lysates, nuclear and cytoplasmic fractions of WT fibroblasts infected with IAV for 8 h; (n=3)..(E) Immunoprecipitation of HA-tagged ZBP1 from lysates of 239T cells expressing HA-tagged ZBP1 transfected with plasmids expressing PB1 or NP, and immunoblotted for ZBP1, PB1 and NP; (n= 2)..(F) Domain architecture of murine ZBP1 and schematics of the constructs used in this study..(G and H) Immunoprecipitation of HA-tagged ZBP1 from lysates of 239T cells expressing HA-tagged ZBP1 constructs infected with IAV for 16 h, and immunoblotted for the IAV proteins PB1, HA and NP, and HA tag; (n= 2).

Figure 6. ZBP1 promotes inflammatory responses and epithelial damage during IAV infection in vivo.

(A) Survival analysis of female WT and Zbp1^−/− mice infected with 1000pfu (approximately one LD₅₀) of IAV; (p=0.0819; n = 15 (WT) and 9 (Zbp1^−/−) mice; Mantel-Cox test)..(B) Body weight (%) of WT and Zbp1^−/−mice 0–18 d after infection compared to pre-infection body weight (set as 100%); (p=0.02743813 (D5); 0.00103121 (D6); 0.00081354 (D7); 0.05424982 (D14); 0.0231683 (D15); 0.02152296 (D16); WT (n = 21) and Zbp1^−/− (n = 15); two-tailed t test)..(C) Lung viral titers in WT and Zbp1^−/− mice infected with IAV for 7 days; (p=0.0022; n=6; Mann Whitney test). H &E staining (D and E; E is an inset of D) and immunohistochemistry analysis for IAV NP protein (F and G; G is an inset of F) of lungs from WT and Zbp1^−/− mice infected with IAV for 7 days; (n=6).