Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: Pro-inflammatory Cytokines Are Increased in COVID-19, and Co-treatment of TNF-α and IFN-γ Induces Cell Death (A) Heatmap depicting the levels of pro-inflammatory cytokines in serum of patients with COVID-19 and healthy people ( Lucas et al., 2020 ). (B) Pro-inflammatory cytokines released from PBMCs infected with SARS-CoV-2. (C) Percent of BMDMs that are dead 48 h after cytokine treatment using the IncuCyte imaging system and propidium iodide (PI) staining. “Cocktail-1” contained all 8 cytokines (IL-6, IL-18, IFN-γ, IL-15, TNF-α, IL-1α, IL-1β, and IL-2). (D) Percent of BMDMs that are dead 48 h after treatment with the indicated combination of cytokines. (E) Real-time analysis of cell death in BMDMs treated with the indicated cytokines. (F and G) Representative images of cell death in BMDMs (F) and THP-1 cells (G) after 48 h of the indicated treatments. Scale bar, 50 μm. Data are representative of at least three independent experiments. ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. Analysis was performed using the one-way ANOVA (B–D) or the two-way ANOVA (E). Significance asterisks in (C) and (D) indicate the comparison to the media-treated control. Data are shown as mean ± SEM (B–E). See also Figure S1 .

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Infection, Imaging, Staining, Comparison, Control

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: Co-treatment of TNF-α and IFN-γ Induces Cell Death, Related to Figure 1 (A) Percent of bone marrow-derived macrophages (BMDMs) that are dead 48 h after cytokine treatment using the IncuCyte imaging system and propidium iodide (PI) staining. BMDMs were stimulated with the indicated cytokines, “Cocktail-1” (IL-6, IL-18, IFN-γ, IL-15, TNF-α, IL-1α, IL-1β, and IL-2), “Cocktail-2” (IL-6, IL-18, IL-15, IL-1α, IL-1β, and IL-2), Cocktail-2+TNF-α, or Cocktail-2+IFN-γ. (B) Percent of BMDMs that are dead 48 h after treatment with the indicated cytokines using the IncuCyte imaging system and PI staining. (C) Percent of BMDMs that are dead 48 h after treatment with increasing concentration of cytokines in Cocktail-2 or TNF-α and IFN-γ using the IncuCyte imaging system and PI staining. (D) Real-time analysis of cell death in BMDMs using the IncuCyte imaging system and PI staining after treatment with increasing concentrations of TNF-α and IFN-γ. (E) Real-time analysis of cell death in primary human umbilical vein endothelial cells (HUVEC) treated with the indicated cytokines using the IncuCyte imaging system and PI staining. (F) Circulating amounts of TNF-α and IFN-γ in healthy volunteers and patients with mild, moderate, or severe COVID-19 ( Silvin et al., 2020 ). (G) Expression of pro-inflammatory cytokines in macrophages, NK cells, CD8 + T cells, and B cells based on publicly available single-cell RNA-seq data using peripheral blood mononuclear cells obtained from healthy donors and patients with mild and severe COVID-19 ( Lee et al., 2020b ). Data are representative of at least three independent experiments. ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. Analysis was performed using the one-way ANOVA (A–C) or the two-way ANOVA (E and G). Data are shown as mean ± SEM.

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Derivative Assay, Imaging, Staining, Concentration Assay, Expressing, RNA Sequencing

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: Cytokine Shock by TNF-α and IFN-γ Mirrors COVID-19 Symptoms (A) Survival of 6- to 8-week-old WT mice after intraperitoneal (i.p.) injection of PBS (n = 10), IFN-γ (n = 12), TNF-α (n = 15), or TNF-α+IFN-γ (n = 15). (B) H/E staining, TUNEL, and cleaved caspase-3 (Clvd CASP3) immuno-staining of colon samples from mice injected with PBS or TNF-α+IFN-γ after 5 h. Red arrows indicate stained cells. (C–E) Analysis of (C) serum levels of LDH, ALT, AST, blood urea nitrogen (BUN), and ferritin; (D) the number of thrombocytes, plateletcrit (PCT), RBC count, hematocrit (HCT), and hemoglobin (Hb) concentration in the blood; and (E) the percentage of macrophages, neutrophils, T cells, and B cells and the neutrophil-to-lymphocyte ratio (NLR) in the blood of mice injected with PBS or TNF-α and IFN-γ after 5 h. Data are representative of at least three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Analysis was performed using the survival curve comparison (log-Rank [Mantel-Cox] test) (A) or the t test (C–E). Data are shown as mean ± SEM (C–E). See also Figure S2 .

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Injection, Staining, TUNEL Assay, Immunostaining, Concentration Assay, Comparison

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: TNF-α and IFN-γ Shock Induces Inflammatory Responses and Intestinal and Lung Damage, Related to Figure 2 (Α) CD45 immuno-staining in the intestine collected from mice injected intraperitoneally with PBS or TNF-α and IFN-γ at 5 h post-treatment. (B) Hematoxylin and eosin staining (H/E), cleaved caspase-3 (Clvd CASP3), and CD45 immuno-staining in the lungs collected from mice injected intraperitoneally with PBS or TNF-α and IFN-γ at 5 h post-treatment. Red arrows indicate stained cells for Clvd CASP3. (C) Quantitative analysis of Clvd CASP3-positive and TUNEL-positive cells in the intestine collected from mice injected intraperitoneally with PBS or TNF-α and IFN-γ at 5 h post-treatment. Fifty fields were analyzed under the microscope. (D) Quantitative analysis of Clvd CASP3-positive cells in the lungs collected from mice injected intraperitoneally with PBS or TNF-α and IFN-γ at 5 h post-treatment. Fifty fields were analyzed under the microscope. Data are representative of at least three independent experiments. Data are shown as mean ± SEM (C and D). ∗∗∗∗ p < 0.0001. Analysis was performed using the t test (C and D).

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Immunostaining, Injection, Staining, TUNEL Assay, Microscopy

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: Co-treatment of TNF-α and IFN-γ Induces PANoptosis (A–C) Immunoblot analysis of (A) pro- (P53), activated (P30), and inactivated (P20) GSDMD, pro- (P53) and activated (P34) GSDME, pro- (P45) and activated (P20) CASP1, and pro- (P43) and cleaved (P36 and P26) CASP11; (B) pro- (P35) and cleaved (P19 and P17) CASP3, pro- (P35) and cleaved (P20) CASP7, pro- (P55) and cleaved (P18) CASP8, and pro- (P49) and cleaved (P18) CASP9; and (C) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK1 (pRIPK1), and pro- (P75) and cleaved (P30) RIPK1 in BMDMs co-treated with TNF-α and IFN-γ. (D–F) Immunoblot analysis of (D) GSDMD and GSDME; (E) CASP3, CASP7, and CASP8; and (F) pMLKL and tMLKL in BMDMs after treatment with TNF-α alone, IFN-γ alone, or co-treatment with TNF-α and IFN-γ for 36 h. GAPDH was used as the internal control. Asterisks denote a nonspecific band. Data are representative of at least three independent experiments.

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Western Blot, Control

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: IRF1 and NOS2 Mediate TNF-α and IFN-γ-Induced Inflammatory Cell Death (A) Heatmap depicting the expression levels of type II IFN-responsive genes in WT BMDMs treated with IFN-γ alone or co-treated with TNF-α and IFN-γ for 16 h relative to their expression in untreated (Mock) BMDMs. (B) Heatmap depicting the expression levels of type II IFN-responsive genes in patients with moderate, severe, and critical COVID-19 relative to their expression in healthy patients ( Hadjadj et al., 2020 ). (C) Real-time analysis of cell death in TNF-α and IFN-γ co-treated WT and Irf1 –/– BMDMs. Representative images of cell death are shown at 0 h and after 48 h of TNF-α and IFN-γ treatment. (D) Heatmap depicting the expression levels of the most downregulated genes in Irf1 –/– BMDMs co-treated with TNF-α and IFN-γ for 16 h relative to their expression in WT treated BMDMs. (E) Immunoblot analysis of iNOS in WT and Irf1 –/– BMDMs co-treated with TNF-α and IFN-γ. Actin was used as the internal control. (F) Expression analysis of NOS2 in patients with moderate, severe, and critical COVID-19 relative to the expression in healthy patients ( Hadjadj et al., 2020 ). (G) Real-time analysis of cell death in WT, Irf1 –/– , and Nos2 –/– BMDMs during co-treatment with TNF-α and IFN-γ. Representative images of cell death are shown at 0 h and after 48 h of TNF-α and IFN-γ treatment. Scale bar, 50 μm. Data are representative of at least three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Analysis was performed using the one-way ANOVA (F) or two-way ANOVA (C and G). Data are shown as mean ± SEM (C, F, and G). See also , , and .

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Expressing, Western Blot, Control

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: IRF1 and STAT1 Are Required for Cell Death Downstream of TNF-α and IFN-γ Co-treatment, Related to Figure 4 (A) Percent of bone marrow-derived macrophages (BMDMs) that are dead 48 h after TNF-α and IFN-γ co-treatment using the IncuCyte imaging system and propidium iodide (PI) staining. (B) Real-time analysis of cell death in wild type (WT), Irf1 –/– , and Stat1 –/– BMDMs using the IncuCyte imaging system and PI staining after treatment with TNF-α and IFN-γ. (C) Representative images of cell death in WT, Irf1 –/– , and Stat1 –/– BMDMs are shown at 0 h and after 48 h of TNF-α and IFN-γ treatment. Scale bar, 50 μm. (D and E) Immunoblot analysis of (D) pro- (P35) and cleaved caspase-3 (P19 and P17; CASP3), pro- (P35) and cleaved caspase-7 (P20; CASP7), and pro- (P55) and cleaved caspase-8 (P18; CASP8) and (E) pro- (P53) and activated (P34) gasdermin E (GSDME), phosphorylated MLKL (pMLKL), and total MLKL (tMLKL) in WT and Irf1 –/– BMDMs co-treated with TNF-α and IFN-γ for the indicated time. GAPDH was used as the internal control. Asterisks denote a nonspecific band. Data are representative of at least three independent experiments. ∗∗∗∗ p < 0.0001. Analysis was performed using the one-way ANOVA (A) or two-way ANOVA (B). Data are shown as mean ± SEM (A and B).

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Derivative Assay, Imaging, Staining, Western Blot, Control

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: Nitric Oxide Produced Downstream of IRF1 and STAT1 Is Required for Cell Death Triggered by TNF-α and IFN-γ Co-treatment, Related to Figure 4 (A) Immunoblot analysis of iNOS in wild type (WT) and Stat1 –/– bone marrow-derived macrophages (BMDMs) co-treated with TNF-α and IFN-γ for the indicated time. GAPDH was used as the internal control. (Β) Nitric oxide production in WT, Irf1 –/– , and Stat1 –/– BMDMs co-treated with TNF-α and IFN-γ for the indicated time. (C) Immunoblot analysis of iNOS in WT and Nos2 –/– BMDMs co-treated with TNF-α and IFN-γ for the indicated time. GAPDH was used as the internal control. (D) Production of nitric oxide by WT BMDMs treated with nitric oxide inhibitors, 1400W (100 μΜ) or L-NAME (1 mM), together with TNF-α and IFN-γ for 36 h. (E) Real-time analysis of cell death in PBS-, 1400W-, or L-NAME–treated WT BMDMs using the IncuCyte imaging system and propidium iodide (PI) staining after stimulation with TNF-α and IFN-γ. (F) Representative images of cell death in PBS-, 1400W-, or L-NAME–treated WT BMDMs are shown at 0 h and after 48 h of TNF-α and IFN-γ treatment. Scale bar, 50 μm. (G and H) Immunoblot analysis of (G) pro- (P35) and cleaved caspase-3 (P19 and P17; CASP3), pro- (P35) and cleaved caspase-7 (P20; CASP7), and pro- (P55) and cleaved caspase-8 (P18; CASP8) and (H) pro- (P53) and activated (P34) gasdermin E (GSDME), phosphorylated MLKL (pMLKL), and total MLKL (tMLKL) in WT and Nos2 –/– BMDMs co-treated with TNF-α and IFN-γ for the indicated time. GAPDH was used as the internal control. Asterisk denotes a nonspecific band. Data are representative of at least three independent experiments. ∗∗∗∗ p < 0.0001. Analysis was performed using the one-way ANOVA (D) or two-way ANOVA (B and E). Data are shown as mean ± SEM (B, D, and E). n.d., not detected.

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Produced, Western Blot, Derivative Assay, Control, Imaging, Staining

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: Concentration of Nitric Oxide Is Critical to Induce Cell Death, and IFN-γ Does Not Suppress TNF-α-Mediated NF-κB Signaling, Related to Figure 4 (A) Immunoblot analysis of iNOS in wild type (WT) bone marrow-derived macrophages (BMDMs) treated with TNF-α alone, IFN-γ alone, or TNF-α and IFN-γ together for 24 h. GAPDH was used as the internal control. (Β) Nitric oxide production in WT BMDMs treated with TNF-α alone, IFN-γ alone, or TNF-α and IFN-γ together for the indicated time. (C) Real-time analysis of cell death in PBS- and nitric oxide donor SIN-1–treated WT BMDMs using the IncuCyte imaging system and propidium iodide (PI) staining. (D and E) Heatmap depicting the expression levels of NF-κB target genes for (D) inflammatory cytokines/chemokines and (E) apoptosis regulators in WT BMDMs treated with TNF-α alone or co-treated with TNF-α and IFN-γ for 16 h relative to their expression in untreated (Mock) BMDMs. Data are representative of at least three independent experiments. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Analysis was performed using the two-way ANOVA (B and C).

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Concentration Assay, Western Blot, Derivative Assay, Control, Imaging, Staining, Expressing

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: Caspase-8 Drives PANoptosis Induced by Co-treatment with TNF-α and IFN-γ (A) Real-time analysis of cell death in WT, Ripk3 –/– , Ripk3 –/– Casp8 –/– , and Apaf1 –/– BMDMs co-treated with TNF-α and IFN-γ. (B) Representative images of cell death in WT, Ripk3 –/– , Ripk3 –/– Casp8 –/– , and Apaf1 –/– BMDMs. Scale bar, 50 μm. (C and D) Immunoblot analysis of (C) pro- (P55) and cleaved (P18) CASP8, pro- (P35) and cleaved (P19 and P17) CASP3, and pro- (P35) and cleaved (P20) CASP7 and (D) pro- (P53) and activated (P34) GSDME, phosphorylated MLKL (pMLKL), and total MLKL (tMLKL) in WT and Ripk3 –/– Casp8 –/– BMDMs co-treated with TNF-α and IFN-γ. GAPDH was used as the internal control. Asterisks denote a nonspecific band. Data are representative of at least three independent experiments. ∗∗∗∗ p < 0.0001. Analysis was performed using the two-way ANOVA. Data are shown as mean ± SEM (A). See also and .

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Western Blot, Control

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: FADD Regulates Cell Death Induced by TNF-α and IFN-γ Co-treatment, Related to Figure 5 (A) Real-time analysis of cell death in wild type (WT), Ripk3 –/– , and Ripk3 –/– Fadd –/– bone marrow-derived macrophages (BMDMs) using the IncuCyte imaging system and propidium iodide (PI) staining after co-treatment with TNF-α and IFN-γ. (B) Representative images of cell death in WT, Ripk3 –/– , and Ripk3 –/– Fadd –/– BMDMs are shown at 0 h and after 48 h of TNF-α and IFN-γ treatment. Scale bar, 50 μm. (C) Immunoblot analysis of pro- (P55) and cleaved caspase-8 (P18; CASP8), pro- (P35) and cleaved caspase-3 (P19 and P17; CASP3), and pro- (P35) and cleaved caspase-7 (P20; CASP7) in WT and Ripk3 –/– Fadd –/– BMDMs co-treated with TNF-α and IFN-γ for the indicated time. GAPDH was used as the internal control. (D) Immunoblot analysis of pro- (P53) and activated (P34) gasdermin E (GSDME), phosphorylated MLKL (pMLKL), and total MLKL (tMLKL) in WT and Ripk3 –/– Fadd –/– BMDMs co-treated with TNF-α and IFN-γ for the indicated time. GAPDH was used as the internal control. Asterisk denotes a nonspecific band. Data are representative of at least three independent experiments. ∗∗∗∗ p < 0.0001. Analysis was performed using the two-way ANOVA (A). Data are shown as mean ± SEM (A).

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Derivative Assay, Imaging, Staining, Western Blot, Control

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: Deletion of Individual Cell Death Pathways Is Not Sufficient to Protect Cells from Death Induced by TNF-α and IFN-γ, Related to Figure 5 (A–C) Real-time analysis of cell death in wild type (WT), Casp7 –/– , and Casp3 –/– bone marrow-derived macrophages (BMDMs) (A); WT, Gsdmd –/– , Gsdme –/– , Mlkl –/– , and Gsdmd –/– Gsdm e –/– ( Gsdmd /e –/– ) Mlkl –/– BMDMs (B); or WT, Casp1 –/– , Casp11 –/– , and Casp1 / 11 –/– BMDMs (C) using the IncuCyte imaging system and propidium iodide (PI) staining after co-treatment with TNF-α and IFN-γ. Representative images of cell death are shown at 0 h and after 48 h of TNF-α and IFN-γ treatment. Scale bar, 50 μm. ∗∗∗∗ p < 0.0001. Analysis was performed using the two-way ANOVA. Data are representative of at least three independent experiments. Data are shown as mean ± SEM.

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Derivative Assay, Imaging, Staining

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: Inhibition of PANoptosis Provides Protection against TNF-α and IFN-γ-Driven Lethality in Mice (A and B) Survival of 6- to 8-week-old (A) WT (n = 10) and Stat1 –/– (n = 10) mice and (B) WT (n = 10), Ripk3 –/– (n = 12), and Ripk3 –/– Casp8 –/– (n = 15) mice after i.p. injection of TNF-α and IFN-γ. (C–E) Analysis of (C) serum levels of LDH, ALT, and AST; (D) percentage of T cells in blood; (E) the number of thrombocytes and plateletcrit (PCT) in the blood; and (F) RBC count, hematocrit (HCT), and hemoglobin (Hb) concentration in the blood of WT, Stat1 –/– , and Ripk3 –/– Casp8 –/– mice injected i.p. with PBS or TNF-α and IFN-γ at 5 h post-treatment. Data are representative of two independent experiments. Data are shown as mean ± SEM (C–F). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. Analysis was performed using the survival curve comparison (log-Rank [Mantel-Cox] test) (A and B) or the two-way ANOVA (C–F).

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Inhibition, Injection, Concentration Assay, Comparison

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet: Blocking TNF-α and IFN-γ Provides Protection in Disease Models Associated with Cytokine Storm: SARS-CoV-2 Infection, Cytokine Shock, Sepsis, and HLH (A) Survival of 6- to 8-week-old WT mice injected with isotype control (n = 10) or neutralizing antibodies against TNF-α and IFN-γ (n = 14) 12 h before i.p. injection of TNF-α and IFN-γ. (B) Survival of 7- to 8-week-old K18-hACE2 transgenic mice injected with isotype control (n = 12) or neutralizing antibodies against TNF-α and IFN-γ (n = 13) on day 1, 3, and 4 after infection with SARS-CoV-2 (2 × 10 4 pfu/mouse). (C) Survival of 7- to 8-week-old WT mice injected i.p. with poly I:C (10 mg/kg body weight) followed 24 h later by i.p. injection of PBS (n = 15), isotype control (n = 15), neutralizing antibody against TNF-α (n = 15), neutralizing antibody against IFN-γ (n = 15), or neutralizing antibodies against both TNF-α and IFN-γ (n = 15). Mice were then challenged with LPS (5 mg/kg body weight) 1 h after the treatments. (D) Survival of 7- to 8-week-old WT mice injected with PBS (n = 17), isotype control (n = 10), neutralizing antibody against TNF-α (n = 17), neutralizing antibody against IFN-γ (n = 17), or neutralizing antibodies against both TNF-α and IFN-γ (n = 18) 30 min and 6 h after i.p. injection of a lethal dose of LPS (20 mg/kg body weight). (E) Schematic overview of the mechanism of TNF-α and IFN-γ-induced pathology and the inflammatory cell death pathway with strategies for potential therapeutics. Data are pooled from two independent experiments (A–D). ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Analysis was performed using the survival curve comparison (log-Rank [Mantel-Cox] test).

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Blocking Assay, Infection, Injection, Control, Transgenic Assay, Comparison

Journal: Cell

Article Title: Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

doi: 10.1016/j.cell.2020.11.025

Figure Lengend Snippet:

Article Snippet: Infected mice were administered intraperitoneally 200 μL of DPBS containing 500 μg of isotype control (Leinco Technologies, Inc., I-536) (n = 12) or 500 μg of neutralizing antibody against TNF-α (Leinco Technologies, Inc., T-703) plus 500 μg of neutralizing antibody against IFN-γ (Leinco Technologies, Inc., I-1190) (n = 13) on days 1, 3, and 4 post-infection.

Techniques: Control, Virus, Recombinant, Protease Inhibitor, Western Blot, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Gene Expression, Microarray, Software

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Staphylococcal entertotoxins of the enterotoxin gene cluster (egcSEs) induce nitric oxide- and cytokine dependent tumor cell apoptosis in a broad panel of human tumor cells

doi: 10.3389/fcimb.2013.00038

Figure Lengend Snippet: Anti- TNF-α and NO inhibitor, alone and in combination inhibit Hep-2 tumor cells cytotoxicity induced by 10% supernatants from SEA and egcSE-stimulated PBMCs . Hep-2 tumor cells cytotoxicity induced by 10% supernatants from SEA and egcSE-stimulated PBMCs before (black) or after treatment with L-NMMA (dark gray) or anti-TNF-α antibody (light gray) or a combination of L-NMMA plus anti-TNF-α antibody was analyzed by flow cytomerter using FITC-conjugated annexin-V and propidium iodide (IP) staining. The results are expressed as the mean ± SEM ( n = 3 independent experiments). Asterisks indicate statistical significance compared to values obtained before treatment with L-NMMA or anti-TNF-α antibody.

Article Snippet: PBMC supernatants were incubated with and without 30 μg/mL of mouse neutralizing polyclonal anti-TNF-α antibody (Abcys, Paris, France) for 18 h at 22°C before carrying out the cytotoxic assays described above.

Techniques: Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Staphylococcal entertotoxins of the enterotoxin gene cluster (egcSEs) induce nitric oxide- and cytokine dependent tumor cell apoptosis in a broad panel of human tumor cells

doi: 10.3389/fcimb.2013.00038

Figure Lengend Snippet: Level of cytokines in supernatants of PBMC stimulated by SEA, SEG, SEI, SElM, SElN, and SElO .

Article Snippet: PBMC supernatants were incubated with and without 30 μg/mL of mouse neutralizing polyclonal anti-TNF-α antibody (Abcys, Paris, France) for 18 h at 22°C before carrying out the cytotoxic assays described above.

Techniques: