Journal: F1000Research

Article Title: Recent advances in understanding inhibitor of apoptosis proteins

doi: 10.12688/f1000research.16439.1

Figure Lengend Snippet: Tumor necrosis factor (TNF) binding to TNF receptor 1 (TNFR1) triggers complex I formation, in which cIAP1 and 2 ubiquitylate RIPK1. This leads to the induction of canonical (canon.) nuclear factor kappa B (NFκB)- and mitogen-activated protein kinase (MAPK)-dependent genes, including cFlar encoding cFLIP. Subsequently, cytosolic complex II containing FADD, caspase-8, RIPK1, RIPK3, and cFLIP is formed. In this complex, cFLIP inhibits caspase-8 activation to block apoptosis and necroptosis. Inhibition of cIAP1 and 2 by Smac-mimetic drugs impairs canonical NFκB activation and accelerates the formation of complex II, which leads to apoptosis. When caspase-8 activation is blocked within complex II, RIPK1 and 3 are not cleaved and necroptosis is activated. Stimulation of nucleotide-binding oligomerization domain 1/2 (NOD1/2) receptors induces RIPK2 ubiquitylation by XIAP and activates the transcription of NFκB- and MAPK-dependent cytokines such as TNF, which amplifies the inflammatory signal. Binding of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to Toll-like receptors (TLRs) leads to the recruitment of the Myd88/TRAF3/6/cIAP1/2 complex. Within this complex, cIAP1 and 2 ubiquitylate TRAF3, inducing its degradation and increasing the expression of cytokines and chemokines. The other TLR adaptor, TRIF, recruits RIPK1 via its RIP homotypic interaction motif (RHIM) domain (yellow). Upon TLR activation, inhibition of IAPs by Smac-mimetics promotes the formation of the ripoptosome, which has a composition similar to that of complex II. TLR-induced expression of TNF and TNFR2 triggers cIAP1/2 degradation and a subsequent accumulation of NFκB-inducing kinase (NIK), which activates non-canonical (non canon.) NFκB-dependent genes. In the context of XIAP deficiency, the degradation of cIAP1 and 2 by TNFR2 leads to the formation of complex II. Activation of complex II or the ripoptosome can activate pyroptosis after TLR priming. TRAF, tumor necrosis factor receptor-associated factor.

Article Snippet: In this line, GlaxoSmithKline (Brentford, UK) has an ongoing clinical trial testing RIPK1 inhibitors for the treatment of psoriasis (ClinicalTrials.gov Identifier: NCT02776033).

Techniques: Binding Assay, Activation Assay, Blocking Assay, Inhibition, Expressing

Journal: Cancer Science

Article Title: Immunostimulatory oncolytic activity of coxsackievirus A11 in human malignant pleural mesothelioma

doi: 10.1111/cas.15645

Figure Lengend Snippet: CVA11 induces apoptosis, pyroptosis, and necroptosis in human MPM cell lines. (A) Flow cytometric analysis of apoptosis in human MPM cells at 24 h after infection with CVA11 (MOI = 10). Annexin V + PI − and annexin V + PI + cells represent early and late apoptotic cells, respectively. (B) H2052 cells infected (or not) with CVA11 (MOI = 10) for 36 h were subjected to immunoblot analysis with antibodies to cleaved caspase‐3 and to PARP. (C) Human MPM cells were infected with CVA11 or mock for 24 h, after which the concentration of IL‐1β in the culture supernatant was measured with an ELISA. (D–G) H2052 cells were incubated in the absence or presence of various concentrations of caspase inhibitors for 1 h and then infected with CVA11 (MOI = 10) for 36 h, after which cell viability was assessed with the MTS assay (D, F) and the CVA11 titer in the supernatant was measured (E, G). (H) H2052 cells infected (or not) with CVA11 (MOI = 10) for 36 h were subjected to immunoblot analysis with antibodies to S358‐phosphorylated (P‐) or total forms of MLKL. (I, J) H2052 cells were incubated with the indicated concentrations of necrostatin‐1 (RIPK1 inhibitor) for 1 h and then infected with CVA11 (MOI = 10) for 36 h, after which cell viability was assessed with the MTS assay (I) and the CVA11 titer in the supernatant was measured (J). Data are representative of three independent experiments (A, B, H). Quantitative data are expressed relative to the data of mock‐infected cells (C) or nontreated infected cells (D–F, I), and are means + SD from three independent experiments.

Article Snippet: Cells were exposed for 1 h before CVA11 infection to the PI3K inhibitor LY294002 (Santa Cruz Biotechnology), the MEK inhibitor PD0325901 (Wako), the pan‐caspase inhibitor Z‐VAD‐FMK (R&D Systems), the caspase‐3 inhibitor Z‐DEVD‐FMK (Selleck, Houston, TX), the caspase‐8 inhibitor Z‐IETD‐FMK (Selleck), the caspase‐1 inhibitor Ac‐YVAD‐CMK (Bachem), the caspase‐12 inhibitor Z‐ATAD‐FMK (R&D Systems), or the RIPK1 inhibitor necrostatin‐1 (Chemscene).

Techniques: Infection, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, MTS Assay

Journal: Cell Research

Article Title: SARS-CoV-2 promotes RIPK1 activation to facilitate viral propagation

doi: 10.1038/s41422-021-00578-7

Figure Lengend Snippet: a IHC staining of p-S166 RIPK1 in the lungs of a COVID-19 patient (middle and right) and an age-matched control (left). Scale bars, 1 inch. The enlarged image (right) shows the nuclear morphology of p-S166 RIPK1 + cells. b Activated RIPK1 in the bronchial ciliated epithelial cells of patients with severe COVID-19. IHC staining of viral nucleocapsid protein (NP) and p-S166 RIPK1 in the bronchial region of the COVID-19 patient and control was shown. The lower row shows the enlarged images of specified areas above. Scale bars, 100 μm. c IHC staining of c-casp3 in the lungs of patients with severe COVID-19 (bottom) and control (top). Scale bars, 20 μm.

Article Snippet: Additional four RIPK1 inhibitors have been tested for anti-SARS-CoV-2 ability, including CMP21 (Takeda), GSK2982772 (GlaxoSmithKline), GSK963 (GlaxoSmithKline), and QY10-40, using the same protocol.

Techniques: Immunohistochemistry, Control

Journal: Cell Research

Article Title: SARS-CoV-2 promotes RIPK1 activation to facilitate viral propagation

doi: 10.1038/s41422-021-00578-7

Figure Lengend Snippet: a – c Treatment with Nec-1s inhibits SARS-CoV-2-induced inflammation in the human lung organoids. Human lung organoids were first infected with SARS-CoV-2 at MOI of 1 for 2 h, washed with PBS and replaced with fresh media with 10 μM Nec-1s or vehicle, and incubated for 24 or 48 h. Activated RIPK1 in the lung organoids was determined by western blotting using anti-p-S166 RIPK1 and anti-RIPK1 antibodies. Viral RNA was quantified by RT-qPCR ( b ), and western blotting for NP ( c ). d – f The lung organoids were treated as in a and total RNA was extracted for RNA-seq. The heatmap shows inflammation-related genes that were significantly downregulated after the treatment with Nec-1s (adjusted P value < 0.05) ( d ). The levels of ACE2 in the lung organoids were determined by RT-qPCR ( e ) or western blotting ( a ). Actin was used as a loading control. The levels of EGFR in the lung organoids were determined by RT-qPCR ( f ) or western blotting ( a ). g The levels of indicated proinflammatory cytokines in the lung organoids treated as in a were measured by RT-qPCR. Paired t -test was used in RT-qPCR analysis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Additional four RIPK1 inhibitors have been tested for anti-SARS-CoV-2 ability, including CMP21 (Takeda), GSK2982772 (GlaxoSmithKline), GSK963 (GlaxoSmithKline), and QY10-40, using the same protocol.

Techniques: Infection, Incubation, Western Blot, Quantitative RT-PCR, RNA Sequencing, Control

Journal: Cell Research

Article Title: SARS-CoV-2 promotes RIPK1 activation to facilitate viral propagation

doi: 10.1038/s41422-021-00578-7

Figure Lengend Snippet: a RIPK1 KO 293T cells were co-transfected with Myc-RIPK1 and Flag-NSP12 expression vectors as indicated for 20 h. The cell lysates in 1% NP40 lysis buffer were analyzed by immunoprecipitation using anti-Flag M2-affinity gel and followed by western blotting using indicated antibodies. b Calu3 cells were infected with SARS-CoV-2 at MOI of 1 for 36 h. The cell lysates in 1% NP40 lysis buffer were analyzed by immunoprecipitation using anti-RIPK1 antibody and followed by western blotting using indicated antibodies. c HeLa cells were transfected with Strep tag II-NSP12 expression vector in the absence or presence of Nec-1s for 24 h. The cell lysates were analyzed by western blotting using indicated antibodies. The arrow points to the band of p-S166 RIPK1. d HeLa cells were transfected with vector control or expression vectors of NSP12 variants (323P or 323L). The cell lysates were analyzed by western blotting using p-S166 RIPK1 antibody for detection of activated RIPK1 kinase. Actin served as a loading control. The arrow points to the band of p-S166 RIPK1. e RIPK1 KO 293T cells were co-transfected with 0.1 μg of Myc-RIPK1 expression vector and different amounts of Flag-NSP12-323P and Flag-NSP12-323L expression vectors as indicated. The cells were lysed with 2× Laemmli sample buffer and analyzed by western blotting using indicated antibodies as shown on the left. Densitometric analysis for the levels of RIPK1 activation (p-S166 RIPK1)/actin is shown on the right. Fold changes were calculated compared to expression of Myc-RIPK1 alone.

Article Snippet: Additional four RIPK1 inhibitors have been tested for anti-SARS-CoV-2 ability, including CMP21 (Takeda), GSK2982772 (GlaxoSmithKline), GSK963 (GlaxoSmithKline), and QY10-40, using the same protocol.

Techniques: Transfection, Expressing, Lysis, Immunoprecipitation, Western Blot, Infection, Strep-tag, Plasmid Preparation, Control, Activation Assay

Journal: Cell Research

Article Title: SARS-CoV-2 promotes RIPK1 activation to facilitate viral propagation

doi: 10.1038/s41422-021-00578-7

Figure Lengend Snippet: a Experimental design of in vivo study. AC70 transgenic mice (6–8 weeks of age, male) were intranasally infected with 20 μL 1 × 10 4 TCID 50 SARS-CoV-2. Two hours after infection, the mice were intragastrically administered with Nec-1s ( n = 13) (50 mg/kg, dissolved in 0.5% Carboxymethyl Cellulose (CMC)) or 0.5% CMC vehicle alone ( n = 9), and the dosing was repeated every 12 h. On the day 4 post-infection, 3 mice in each group were sacrificed and their lungs and brains were dissected. The remaining 10 mice in the treatment group (Nec-1s treated for 4 days) and 6 mice in the vehicle group stopped dosing after day 4 and were used to observe the survival rate. b Survival of AC70 transgenic mice after intranasal SARS-CoV-2 infection with intragastric administration of Nec-1s or vehicle. The statistical significance of the survival curve was estimated according to the method of Kaplan and Meier, and the curve was compared with the generalized Wilcoxon test (* P < 0.05). c , d Treatment with Nec-1s inhibits SARS-CoV-2 infection in AC70 transgenic mice. The lungs of mice in the control group and Nec-1s-treated group were fixed and stained for SARS-CoV-2 NP by IHC ( c ). Scale bars, 900 μm. The lower row shows the enlarged images of specified areas above. Scale bars, 100 μm. Freshly isolated mouse lungs were extracted with Trizol for isolating RNA. After reverse transcription, the virus RNA was analyzed by RT-qPCR ( d ). Paired t -test was used in RT-qPCR analysis (* P < 0.05). e Treatment with Nec-1s inhibits inflammation in the lungs of AC70 transgenic mice infected with SARS-CoV-2. The lungs of mice in the control group ( n = 3) and Nec-1s-treated group ( n = 3) were extracted with Trizol, and the expression of cytokines was analyzed by RT-qPCR. Paired t -test was used in RT-qPCR analysis (* P < 0.05, ** P < 0.01). f RIPK1 was activated in the lungs of AC70 transgenic mice with SARS-CoV-2 infection. IHC staining of p-S166 RIPK1 in the lungs of control or Nec-1s-administrated AC70 transgenic mice infected with SARS-CoV-2 was shown. Scale bars, 900 μm. The lower row shows the enlarged images of specified areas above. Scale bars, 100 μm.

Article Snippet: Additional four RIPK1 inhibitors have been tested for anti-SARS-CoV-2 ability, including CMP21 (Takeda), GSK2982772 (GlaxoSmithKline), GSK963 (GlaxoSmithKline), and QY10-40, using the same protocol.

Techniques: In Vivo, Transgenic Assay, Infection, Control, Staining, Isolation, Reverse Transcription, Virus, Quantitative RT-PCR, Expressing, Immunohistochemistry

Journal: Cell Research

Article Title: SARS-CoV-2 promotes RIPK1 activation to facilitate viral propagation

doi: 10.1038/s41422-021-00578-7

Figure Lengend Snippet: a IHC staining with antibody against viral NP on the saggital whole brain sections of the SARS-CoV-2-infected AC70 transgenic mice with or without Nec-1s treatments. Scale bars, 1000 μm. The lower row shows the enlarged images of specified areas above. Scale bars, 100 μm. Viral NP was robustly detected in multiple brain regions, including the olfactory bulb, the deep cortical layers, the connected hippocampal subiculum, the hindbrain/medulla and the cerebellar dentate nucleus. b Freshly isolated mouse brains were extracted with Trizol for isolating RNA. After reverse transcription, the virus RNA was analyzed by RT-qPCR. Paired t -test was used in RT-qPCR analysis (**** P < 0.0001). c , d The brain tissues of viral-infected AC70 mice with or without Nec-1s treatment (1 g) were cryogenically ground in 1 mL DMEM medium. After fully grinding and centrifugation, the supernatant was collected and stored at –80 °C. 4 × 10 4 Vero-E6 cells were seeded in 96-well plates for 23 h. The supernatant (50 μL) was added to the cells and incubated for 24 h. The SARS-CoV-2 infection was detected by immunofluorescence using COVID-19 convalescent sera ( c ). Scale bars, 1000 μm. Infectious clones are automatically quantified by Cytation 5 ( d ). Paired t -test was used in viral titer analysis (** P < 0.01). e The brains of mice in the control group ( n = 3) and Nec-1s-treated group ( n = 3) were extracted with Trizol for total RNA, and the expression of cytokines was analyzed by RT-qPCR. Paired t -test was used in RT-qPCR analysis (* P < 0.05, ** P < 0.01). f RIPK1 was activated in the brains of AC70 transgenic mice with SARS-CoV-2 infection. IHC staining of p-S166 RIPK1 in the brains of control or Nec-1s-administrated AC70 transgenic mice infected with SARS-CoV-2 was shown. Scale bars, 1000 μm. The lower row shows the enlarged images of specified areas above. Scale bars, 50 μm.

Article Snippet: Additional four RIPK1 inhibitors have been tested for anti-SARS-CoV-2 ability, including CMP21 (Takeda), GSK2982772 (GlaxoSmithKline), GSK963 (GlaxoSmithKline), and QY10-40, using the same protocol.

Techniques: Immunohistochemistry, Infection, Transgenic Assay, Isolation, Reverse Transcription, Virus, Quantitative RT-PCR, Centrifugation, Incubation, Immunofluorescence, Clone Assay, Control, Expressing

Journal: Frontiers in Medicine

Article Title: Fractalkine Is Linked to the Necrosome Pathway in Acute Pulmonary Inflammation

doi: 10.3389/fmed.2021.591790

Figure Lengend Snippet: List of murine and human primer sequences used for RT-PCR analysis.

Article Snippet: Furthermore, we evaluated the effects of the specific inhibition of RIPK1 (300 nM; GSK'481; 2608; Axon Medchem), RIPK3 (3 μM; GSK'872; 530389; Merck) and MLKL (1 μM; Necrosulfonamide; NSA; S8251; Selleckchem) on murine and human PMNs.

Techniques: Sequencing

Journal: Frontiers in Medicine

Article Title: Fractalkine Is Linked to the Necrosome Pathway in Acute Pulmonary Inflammation

doi: 10.3389/fmed.2021.591790

Figure Lengend Snippet: In vivo detection of necrosome activation after the onset of inflammation. Expression of the necrosome-related receptor-interacting serine/threonine-protein kinase (RIPK)1 and RIPK3 at the gene (A) ( n = 8) and protein (B) levels was detected in the lungs of C57BL/6J and CX 3 CR1 −/− mice 24 h after LPS exposure. The protein expression was further analyzed by measuring the phosphorylation and therefore activation of RIPK1, RIPK3, and mixed lineage kinase domain-like protein (MLKL). Representative blots are shown, and the density was assessed ( n = 3–4). The intensity of the blots was evaluated by ImageJ. (C) The phosphorylated form of MLKL was also detected by immunofluorescence in the lungs of C57BL/6J and CX 3 CR1 −/− mice (pMLKL appears green; original magnification 63x; one representative image of six is shown; n = 4). (D) Gene expression of IL-33 was determined in the lung tissue of C57BL/6J and CX 3 CR1 −/− mice 24 h after LPS exposure ( n = 6–8). The release of IL-33 and HMGB1 was evaluated in the bronchoalveolar lavage of C57BL/6J and CX 3 CR1 −/− mice ( n = 6–8). (E) Schematic diagram of LPS-dependent necrosome formation in murine lungs. Transmembrane toll-like receptor (TLR) 4 activates (toll/interleukin-1 receptor) domain-containing adaptor protein (TRIF), leading to the phosphorylation of RIPK1 and RIPK3. Necrosome activation induces the phosphorylation of MLKL, resulting in necroptosis and the release of damage-associated patterns (DAMPs). All the data are presented as the mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; statistical analyses were performed by Student's t -test to compare two groups or one-way ANOVA + Bonferroni test to compare multiple groups.

Article Snippet: Furthermore, we evaluated the effects of the specific inhibition of RIPK1 (300 nM; GSK'481; 2608; Axon Medchem), RIPK3 (3 μM; GSK'872; 530389; Merck) and MLKL (1 μM; Necrosulfonamide; NSA; S8251; Selleckchem) on murine and human PMNs.

Techniques: In Vivo, Activation Assay, Expressing, Phospho-proteomics, Immunofluorescence, Gene Expression

Journal: Frontiers in Medicine

Article Title: Fractalkine Is Linked to the Necrosome Pathway in Acute Pulmonary Inflammation

doi: 10.3389/fmed.2021.591790

Figure Lengend Snippet: In vitro necrosome activation on human PMNs. (A) Necrosome-related protein kinase RIPK1 and RIPK3 expression in human PMNs after LPS stimulation ( n = 8) was detected by RT-PCR. (B) Immunoblots of RIPK1, RIPK3, MLKL and their phosphorylated forms were obtained from cell lysates of human PMNs after LPS stimulation (representative blots of n = 4–5 are shown). The intensity of the blots was evaluated by ImageJ. (C) Phosphorylation of RIPK1, RIPK3, and MLKL was detected in human PMNs after LPS (original magnification 63x; one representative image of five is shown; n = 4). (D) Effects of specific RIPK1 and RIPK3 inhibition on MLKL phosphorylation in human PMNs after LPS exposure were evaluated (representative blots from 3 independent experiments; n = 3). (E) Human IL-33 and HMGB1 were determined in the supernatant of stimulated and treated PMNs as indicated ( n = 6–8). (F) Effects of fractalkine depletion on necrosome-related RIPK1 and RIPK3 expression in human PMNs ( n = 6–8) and on the release of human alarmins IL-33 and HMGB1 (G) were evaluated ( n = 8–12). All the data are presented as the mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; Student's t -test to compare two groups or one-way ANOVA + Bonferroni test to compare multiple groups. (H) Schematic diagram of LPS-dependent necrosome formation in human PMNs. Transmembrane toll-like receptor (TLR) 4 activates (toll/interleukin-1 receptor) domain-containing adaptor protein (TRIF), leading to the phosphorylation of RIPK1 and RIPK3. Necrosome activation induces the phosphorylation of MLKL, resulting in necroptosis and the release of damage-associated patterns (DAMPs) (representative blots from 3 independent experiments; n = 4). **** p < 0.0001.

Article Snippet: Furthermore, we evaluated the effects of the specific inhibition of RIPK1 (300 nM; GSK'481; 2608; Axon Medchem), RIPK3 (3 μM; GSK'872; 530389; Merck) and MLKL (1 μM; Necrosulfonamide; NSA; S8251; Selleckchem) on murine and human PMNs.

Techniques: In Vitro, Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Inhibition

Journal: Frontiers in Medicine

Article Title: Fractalkine Is Linked to the Necrosome Pathway in Acute Pulmonary Inflammation

doi: 10.3389/fmed.2021.591790

Figure Lengend Snippet: Depletion of CX 3 CR1 leads to increased activation of the necrosome in PMNs. Activation of TLR4 in PMNs by lipopolysaccharide leads to the phosphorylation of the necrosome-related RIPK1, RIPK3, and MLKL. The activated MLKL induces the full necroptotic response by disrupting the cellular membrane, releasing DAMPs (IL-33 and HMGB1) and therefore further increasing inflammation.

Article Snippet: Furthermore, we evaluated the effects of the specific inhibition of RIPK1 (300 nM; GSK'481; 2608; Axon Medchem), RIPK3 (3 μM; GSK'872; 530389; Merck) and MLKL (1 μM; Necrosulfonamide; NSA; S8251; Selleckchem) on murine and human PMNs.

Techniques: Activation Assay, Phospho-proteomics, Membrane

Journal: Cell Death and Differentiation

Article Title: Type I IFN operates pyroptosis and necroptosis during multidrug-resistant A. baumannii infection

doi: 10.1038/s41418-017-0041-z

Figure Lengend Snippet: A. baumannii infection-induced apoptosis, necroptosis, and pyroptosis.a Immunoblot analysis of caspase-3 preform (Casp-3), and cleaved caspase-3 in WT BMDMs infected with A. baumannii (MOI 50) in the presence of zVAD (20 μM), RIPK1 inhibitor (10 μM), RIPK3 inhibitor (20 μM), MLKL inhibitor (20 μM), or caspase-1 inhibitor (25 μM) for 5 h. b Immunoblot analysis of caspase-1 preform and cleaved caspase-1 (P20) in WT BMDMs infected with A. baumannii (MOI 50) in the presence of caspase-1 inhibitor (25 μM), RIPK1 inhibitor (10 μM), RIPK3 inhibitor (20 μM), or MLKL inhibitor (20 μM) for 12 h. c IL-1β ELISA analysis for samples in b. d Flow cytometry analysis of PI and annexin V staining in WT BMDMs infected with A. baumannii (MOI 50) in the presence of RIPK1 inhibitor (10 μM), RIPK3 inhibitor (20 μM), or MLKL inhibitor (20 μM) for 5 h. e The quantification analysis of double positive cells with PI and annexin V staining for d. f LDH release analysis of WT BMDMs infected with A. baumannii (MOI 50) in the presence of RIPK1 inhibitor (10 μM), RIPK3 inhibitor (20 μM), or MLKL inhibitor (20 μM) for 5 h. Data are representative of three independent experiments. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. SN supernatant, CL cell lysate

Article Snippet: Caspase inhibitor (Z-VAD (OMe)-FMK), caspase-1 inhibitor (Ac-YVAD-CMK), RIPK1 inhibitor (Necrostatin-1), RIPK3 inhibitor (GSKʹ872), and MLKL inhibitor (GW806742X) were purchased from MERCK and used for blocking cell death pathway.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining