ZBP1 Drives IAV-Induced NLRP3 Inflammasome Activation and Lytic Cell Death, PANoptosis, Independent of the Necroptosis Executioner MLKL

Abstract

Influenza A virus (IAV) continues to pose a significant global health threat, causing severe respiratory infections that result in substantial annual morbidity and mortality. Recent research highlights the pivotal role of innate immunity, cell death, and inflammation in exacerbating the severity of respiratory viral diseases. One key molecule in this process is ZBP1, a well-recognized innate immune sensor for IAV infection. Upon activation, ZBP1 triggers the formation of a PANoptosome complex containing ASC, caspase-8, and RIPK3, among other molecules, leading to inflammatory cell death, PANoptosis, and NLRP3 inflammasome activation for the maturation of IL-1β and IL-18. However, the role for other molecules in this process requires further evaluation. In this study, we investigated the role of MLKL in regulating IAV-induced cell death and NLRP3 inflammasome activation. Our data indicate IAV induced inflammatory cell death through the ZBP1-PANoptosome, where caspases and RIPKs serve as core components. However, IAV-induced lytic cell death was only partially dependent on RIPK3 at later timepoints and was fully independent of MLKL throughout all timepoints tested. Additionally, NLRP3 inflammasome activation was unaffected in MLKL-deficient cells, establishing that MLKL and MLKL-dependent necroptosis do not act upstream of NLRP3 inflammasome activation, IL-1β maturation, and lytic cell death during IAV infection.

Keywords: IAV; MLKL; NLRP3; PANoptosis; RIPK3; apoptosis; caspase; cell death; executioner; gasdermin; inflammasome; necroptosis; pyroptosis.

Figure 1

Loss of MLKL does not protect macrophages from IAV-induced cell death. (A–F) Analysis of cell death in IAV-infected wild-type (WT) and different gene-deficient bone marrow-derived macrophages (BMDM) as measured by IncuCyte-based image analyses. Quantification and time course analysis of cell death in WT, Zbp1^−/−, Mlkl^−/− and Ripk3^−/− BMDMs infected with IAV (A), infected with IAV plus treated with the MLKL inhibitor (MLKLi, GW806742X, 1 μM final concentration) (C), and infected with IAV plus treated with the RIPK1 inhibitor (Nec1s, 25 μM final concentration) (E). Representative images of cell death collected at 12 h post-infection from WT, Zbp1^−/−, Mlkl^−/−, and Ripk3^−/− BMDMs infected with IAV (B), infected with IAV plus treated with the MLKL inhibitor (MLKLi, GW806742X, 1 μM final concentration) (D), and infected with IAV plus treated with the RIPK1 inhibitor (Nec1s, 25 μM final concentration) (F). The RIPK1 and MLKL inhibitors were added 1 h post-infection (C–F). The data are representative of at least three independent experiments (A–F). The red mask denotes the dead cells, and the scale bar represents 50 μM (B,D,F). ** p < 0.01; **** p < 0.0001. A one-way ANOVA (Dunnett’s multiple comparisons test) was used (A,C,E). The data are represented as mean ± SEM (A,C,E).

Figure 2

Loss of MLKL does not protect macrophages from IAV-induced activation of PANoptosis molecules. (A,B) Western blot analysis of PANoptosis in IAV-infected wild-type (WT) and different gene-deficient bone marrow-derived macrophages (BMDMs). Western blot analysis of pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME), pro- (P55) and cleaved (P43 and P18) caspase-8 (CASP8), pro- (P35) and cleaved (P20) caspase-7 (CASP7), pro- (P35) and cleaved (P17) caspase-3 (CASP3), phosphorylated MLKL (pMLKL) and total MLKL (tMLKL) in WT, Zbp1^−/−, Nlrp3^−/−, Mlkl^−/−, Ripk3^−/−Casp8^−/−, and Ripk3^−/− BMDMs at 12 h (A) and 24 h (B) post-infection with IAV. The data are representative of at least three independent experiments (A,B). The uppercase ’P’ in the Western blots represents the molecular weight of the proteins. The lowercase letters ‘p’ and ‘t’ represent the phospho- and total-forms of the proteins, respectively.

Figure 3

MLKL is not required for IAV-induced inflammatory or inflammasome-dependent cytokine production. (A–D) ELISA-based analysis of cytokines in IAV-infected wild-type (WT) and different gene-deficient bone marrow-derived macrophages (BMDMs). Quantification of cytokines IL-1β (A), IL-6 (B), TNF (C), and KC (D) in supernatants collected from IAV-infected WT, Zbp1^−/−, Nlrp3^−/−, Mlkl^−/−, Ripk3^−/−Casp8^−/−, and Ripk3^−/− BMDMs at 24 h post-infection. The data are representative of at least three independent experiments (A–D). ns, not significant, * p < 0.05; ** p < 0.01. The Student’s t-test was used (A–D). The data are represented as mean ± SEM (A–D).