Impaired NLRP3 inflammasome activation/pyroptosis leads to robust inflammatory cell death via caspase-8/RIPK3 during coronavirus infection

Abstract

Coronaviruses have caused several zoonotic infections in the past two decades, leading to significant morbidity and mortality globally. Balanced regulation of cell death and inflammatory immune responses is essential to promote protection against coronavirus infection; however, the underlying mechanisms that control these processes remain to be resolved. Here we demonstrate that infection with the murine coronavirus mouse hepatitis virus (MHV) activated the NLRP3 inflammasome and inflammatory cell death in the form of PANoptosis. Deleting NLRP3 inflammasome components or the downstream cell death executioner gasdermin D (GSDMD) led to an initial reduction in cell death followed by a robust increase in the incidence of caspase-8- and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated inflammatory cell deathafter coronavirus infection. Additionally, loss of GSDMD promoted robust NLRP3 inflammasome activation. Moreover, the amounts of some cytokines released during coronavirus infection were significantly altered in the absence of GSDMD. Altogether, our findings show that inflammatory cell death, PANoptosis, is induced by coronavirus infection and that impaired NLRP3 inflammasome function or pyroptosis can lead to negative consequences for the host. These findings may have important implications for studies of coronavirus-induced disease.

Keywords: NLRP3; PANoptosis; RIPK3; RNA virus; apoptosis; caspase-1; caspase-8; coronavirus; gasdermin D; host defense; infection; infectious disease; inflammasome; mouse hepatitis virus (MHV); necroptosis; plus-stranded RNA virus; pyroptosis.

Figure 1.

Coronavirus infection induces PANoptosis (pyroptosis, apoptosis, and necroptosis). A, immunoblot analysis of pro- (p45) and cleaved CASP1 (p20), pro- (p53), activated (p30), and inactivated (p20) GSDMD, pro- (p55) and cleaved CASP8 (p18), pro- (p35) and cleaved CASP7 (p19), pro- (p35) and cleaved CASP3 (p17), and phosphorylated MLKL (pMLKL) in BMDMs after MHV infection for 12 h. Actin was used as the internal control. B, real-time analysis of cell death in BMDMs using the IncuCyte imaging system and SYTOX Green nucleic acid staining after infection with MHV. Results at the indicated time points are shown. The red denotes the dead cells counted during the analysis. Scale bar, 50 µm. C, quantification of the cell death observed in (B). D, IL-1β, IL-18, IL-6, and TNF release from BMDMs infected with MHV at the indicated time points. Data are representative of at least three independent experiments. Data are shown as mean ± S.E. (error bars) (C and D). *p < 0.05; ***p < 0.001; and ****p < 0.0001 (one-way ANOVA); ns, not significant; hpi, hours post-infection.

Figure 2.

Pyroptosis deficiency leads to increased cell death after coronavirus infection. A, real-time analysis of cell death in BMDMs using the IncuCyte imaging system and SYTOX Green nucleic acid staining after infection with MHV. Quantification (left) and images (right) of the cell death at the indicated time points are shown. The red in cell death images denotes the dead cells counted during the analysis. Scale bar, 50 µm. B and C, real-time analysis of cell death in BMDMs using the IncuCyte imaging system and SYTOX Green nucleic acid staining after infection with IAV (B) or VSV (C). D, immunoblot analysis of pro- (p45) and cleaved CASP1 (p20), pro- (p53), activated (p30), and inactivated (p20) GSDMD, pro- (p55) and cleaved CASP8 (p18), pro- (p35) and cleaved CASP7 (p19), pro- (p35) and cleaved CASP3 (p17), and pMLKL in BMDMs after MHV infection for 12 h. GAPDH was used as the internal control. Data are shown as mean ± S.E. (error bars) (A–C). *p < 0.05 and ****p < 0.0001 (one-way ANOVA). Data are representative of at least three independent experiments. ns, not significant; hpi, hours post-infection.

Figure 3.

Loss of the NLRP3 inflammasome leads to increased apoptosis and necroptosis. A, immunoblot analysis of pro- (p45) and cleaved CASP1 (p20), pro- (p53), activated (p30), and inactivated (p20) GSDMD, pro- (p55) and cleaved CASP8 (p18), pro- (p35) and cleaved CASP7 (p19), pro- (p35) and cleaved CASP3 (p17), and pMLKL in BMDMs after MHV infection for 12 h. GAPDH was used as the internal control. B, real-time analysis of cell death in BMDMs using the IncuCyte imaging system and SYTOX Green nucleic acid staining after infection with MHV. Quantification of the cell death at the indicated time points is shown. C, representative cell death images from (B) are shown. The red denotes the dead cells counted during the analysis. Scale bar, 50 µm. Data are shown as mean ± S.E. (error bars) (B). Significant differences compared with WT are denoted as ***p < 0.001 and ****p < 0.0001 (one-way ANOVA). Data are representative of three independent experiments. hpi, hours post-infection.

Figure 4.

Increased cell death in the absence of NLRP3/pyroptosis depends on caspase-8 and RIPK3. A, real-time analysis of cell death in BMDMs using the IncuCyte imaging system and SYTOX Green nucleic acid staining after infection with MHV. Quantification of the cell death at the indicated time points is shown. B, representative cell death images from (A) are shown. The red denotes the dead cells counted during the analysis. Scale bar, 50 µm. C, immunoblot analysis of pro- (p45) and cleaved CASP1 (p20), pro- (p53), activated (p30), and inactivated (p20) GSDMD, pro- (p55) and cleaved CASP8 (p18), pro- (p35) and cleaved CASP7 (p19), pro- (p35) and cleaved CASP3 (p17), and pMLKL in BMDMs after MHV infection for 12 h. GAPDH was used as the internal control. Data are shown as mean ± S.E. (error bars) (A). Significant differences compared with WT are denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (one-way ANOVA). Data are representative of at least three independent experiments. hpi, hours post-infection.

Figure 5.

GSDMD deficiency leads to increased cytokine release after MHV infection. IL-1β (A), IL-18 (B), IL-6 (C), TNF (D), G-CSF (E), GM-CSF (F), IP-10 (G), and MIP-1α (H) release from BMDMs infected with MHV at the indicated time points. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (one-way ANOVA). Data are representative of three independent experiments. Data are shown as mean ± S.E. (error bars) (A–H). hpi, hours post-infection