Inhibiting NINJ1-dependent plasma membrane rupture protects against inflammasome-induced blood coagulation and inflammation

  1. Jian Cui
  2. Hua Li
  3. Dien Ye
  4. Guoying Zhang
  5. Yan Zhang
  6. Ling Yang
  7. Martha MS Sim
  8. Jeremy P Wood
  9. Yinan Wei
  10. Zhenyu Li
  11. Congqing Wu  Is a corresponding author
  1. Saha Cardiovascular Research Center, College of Medicine, University of Kentucky, United States
  2. Department of Pharmaceutical Sciences, Irma Lerma Rangel School of Pharmacy, Texas A&M University, United States
  3. Department of Molecular and Cellular Biochemistry, University of Kentucky, United States
  4. The Gill Heart and Vascular Institute, College of Medicine, University of Kentucky, United States
  5. Department of Microbiology, Immunology, and Molecular Genetics, College of Medicine, University of Kentucky, United States
  6. Department of Surgery, College of Medicine, University of Kentucky, United States
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements
NINJ1 is critical for flagellin-induced systemic coagulation, inflammation, and lethality.

(A–E) Mice were injected intravenously with Ctrl (PBS) or Fla (500 ng LFn-Fla plus 3 μg protective agent [PA]). Blood was collected 90 min after Ctrl or Fla injection. Prothrombin time (A), plasma thrombin-antithrombin (TAT) (B), and plasma cytokines (C–E) were measured. Circles represent individual mice, with bars denoting means. **p<0.01 (two-way ANOVA with Holm-Sidak multiple comparisons). (F–G) Mice were injected intravenously with Ctrl or Fla. After 90 min, mice were euthanized and perfused with PBS, and tissues were isolated. (F) Lung sections were stained with the anti-fibrin monoclonal antibody (59D8). Scale bar denotes 20 μm. (G) Fibrin in the liver and lungs was detected by immunoblot with the anti-fibrin monoclonal antibody (59D8). (H) Mice were injected intravenously with a lethal dose of Fla (2.5 μg LFn-Fla plus 6 μg PA). Kaplan-Meier survival plots for mice challenged with Fla are shown. n=7–9. ****p<0.0001 versus WT (log rank test [Mantel-Cox]).

Figure 1—source data 1

Excel file containing numeric values for panels A-E and H.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig1-data1-v1.xlsx
Figure 1—source data 2

PDF file containing uncropped western blots with labeling for panel G.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig1-data2-v1.zip
Figure 1—source data 3

Original tiff files of western blots for panel G.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig1-data3-v1.zip
Figure 1—figure supplement 1
NINJ1 protein abundance at baseline in different tissues.

Protein was extracted from fresh frozen tissues and detected by immunoblot. Band intensities are not comparable between different tissues.

Figure 1—figure supplement 1—source data 1

PDF file containing uncropped western blots with labeling.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig1-figsupp1-data1-v1.zip
Figure 1—figure supplement 1—source data 2

Original tiff files of western blots.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
Flagellin-induced inflammasome activation and pyroptosis.

Bone marrow-derived macrophages (BMDMs) from Ninj1+/+ mice were incubated with LFn-Fla (1 μg/mL) and/or protective agent (PA) (1 μg/mL) for 90 min. (A) Plasma membrane rupture (PMR) was measured by lactate dehydrogenase (LDH) release. (B) Caspase-1, IL-1β, and gasdermin-D (GSDMD) in the cell lysates and culture supernatant was detected by immunoblot. Circles represent individual mice, with bars denoting means. **p<0.01 versus each of the other three groups (Student’s t-test; unpaired).

Figure 1—figure supplement 2—source data 1

Excel file containing numeric values for panel A.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig1-figsupp2-data1-v1.xlsx
Figure 1—figure supplement 2—source data 2

PDF file containing uncropped western blots with labeling for panel B.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig1-figsupp2-data2-v1.zip
Figure 1—figure supplement 2—source data 3

Original tiff files of western blots for panel B.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig1-figsupp2-data3-v1.zip
Figure 1—figure supplement 3
Flagellin-induced tissue fibrin deposition (H&E staining) in Ninj1+/+ and Ninj1+/- mice.

Mice were injected intravenously with Ctrl (PBS) or Fla (500 ng LFn-Fla plus 3μg protective agent [PA]). Tissues was collected 90 min after Ctrl or Fla injection. H&E staining was performed on PBS-perfused and paraffin-embedded lung tissue sections.

E. coli infection-induced blood coagulation is limited in Ninj1+/- mice.

(A–E) Mice were injected intraperitoneally with Ctrl (saline) or E. coli (2×108 cfu per mouse). Blood was collected 6 hr afterward. Prothrombin time (A), plasma thrombin-antithrombin (TAT) (B), and plasma cytokines (C–E) were measured. Circles represent individual mice, with bars denoting means. *p<0.05, **p<0.01 (two-way ANOVA with Holm-Sidak multiple comparisons).

Figure 2—source data 1

Excel file containing numeric values for panel A-E.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
Plasma membrane rupture (PMR) promotes the release of procoagulant microvesicles (MVs).

(A–B, E) Bone marrow-derived macrophages (BMDMs) were incubated with Ctrl (PBS) or Fla (1 μg/mL LFn-Fla plus 1 μg/mL protective agent [PA]). Cell culture supernatant and MVs were collected after 90 min of incubation. (A) NINJ1 in cell lysates and MVs was detected by immunoblot. (B) BMDM MVs were counted with NanoSight. (E) BMDM MV tissue factor (TF) activity. Circles represent individual mouse, with bars denoting means. **p<0.01 (two-way ANOVA with Holm-Sidak multiple comparisons). (C, D, F) Mice were injected intravenously with Ctrl (PBS) or Fla (500 ng LFn-Fla plus 3 μg PA). Blood was collected 90 min after Ctrl or Fla injection. (C) Plasma MVs were counted with NanoSight. (D) NINJ1 in plasma MVs isolated from equal volume of plasma was detected by immunoblot. (F) Plasma MV TF activity. Circles represent individual mice, with bars denoting means. **p<0.01 (two-way ANOVA with Holm-Sidak multiple comparisons).

Figure 3—source data 1

PDF file containing uncropped western blots with labeling for panels A and D.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig3-data1-v1.zip
Figure 3—source data 2

Original tiff files of western blots for panels A and D.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig3-data2-v1.zip
Figure 3—source data 3

Excel file containing numeric values for panels B, C, E, and F.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Flagellin-induced pyroptosis and microvesicle (MV) release in Ninj1+/+ and Ninj1+/- bone marrow-derived macrophages (BMDMs).

BMDMs from Ninj1+/+ and Ninj1+/- mice were incubated with Ctrl (PBS) or Fla (1 μg/mL LFn-Fla plus 1 μg/mL protective agent [PA]) for 90 min. Lactate dehydrogenase (LDH) release (A) and ATP (B) were measured. Circles represent individual mice, with bars denoting means. **p<0.01, n.s. denotes not significant (two-way ANOVA with Holm-Sidak multiple comparisons). (C) BMDM MV quantity and size were analyzed with NanoSight.

Figure 3—figure supplement 1—source data 1

Excel file containing numeric values for panels A-C.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
Glycine inhibition of NINJ1 blocks pyroptosis-induced blood coagulation.

Mice were injected intravenously 50 μL of 0.5 M glycine 2 hr before administrating Ctrl (PBS) or Fla (500 ng LFn-Fla plus 3 μg protective agent [PA]). Blood was collected 90 min after Ctrl or Fla injection. Prothrombin time (A), plasma thrombin-antithrombin (TAT) (B), plasma tissue factor (TF) microvesicle (MV) activity (C), plasma MV counts (D), and plasma cytokines (E–G) were measured. Circles represent individual mice, with bars denoting means. *p<0.05, **p<0.01, n.s. denotes not significant (two-way ANOVA with Holm-Sidak multiple comparisons).

Figure 4—source data 1

Excel file containing numeric values for panels A-G.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Glycine treatment inhibits bone marrow-derived macrophage (BMDM) microvesicle (MV) release.

(A–C) BMDMs from Ninj1+/+ mice were treated with 5 mM glycine for 30 min before incubation with Ctrl (PBS) or Fla (1 μg/mL LFn-Fla plus 1 μg/mL protective agent [PA]). Cell culture supernatant was collected after 90 min incubation. Lactate dehydrogenase (LDH) release (A), BMDM MV tissue factor (TF) activity (B), and MV counts (C) were measured. Circles represent individual mouse, with bars denoting means. **p<0.01, n.s. denotes not significant (two-way ANOVA with Holm-Sidak multiple comparisons).

Figure 4—figure supplement 1—source data 1

Excel file containing numeric values for panels A-G.

https://cdn.elifesciences.org/articles/91329/elife-91329-fig4-figsupp1-data1-v1.xlsx
Author response image 1
Mice were injected with Fla (500 ng lFn-Fla plug3 ugPA).

Blood was collected 6 hours after Fla injection. Prothrombin time (A), plasma TAT (B), and plasma IL-6 (C) were measured. Mann-Whitney test were performed.

Author response image 2

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)Ninj1 knockoutGenentech, Inc;
Kayagaki et al., 2021
C57Bl/6 strain background
Biological sample (Mus musculus)Primary bone marrow-derived macrophages (Ninj1+/+ BMDMs)This paperFreshly prepared from Ninj1+/+ male mice at 8–12 weeks of age, described in the Materials and methods section
Biological sample (Mus musculus)Primary bone marrow-derived macrophages (Ninj1+/- BMDMs)This paperFreshly prepared from Ninj1+/- male mice at 8–12 weeks of age, described in the Materials and methods section
Strain and strain background (Escherichia coli)E. coli strain (EC10)Dr. Kwang Sik Kim
Division of Pediatric Infectious Diseases, Department of Pediatrics, Johns Hopkins University School of Medicine
Strain and strain background (Escherichia coli)E. coli strain ClearColi BL21(DE3)Lucigen Corporation (now Bioresearch Technologies)60810
AntibodyAnti-mouse NINJ1 (rabbit monoclonal)Genentech, Inc;
Kayagaki et al., 2021
Ninj1-rbIgG-25:10363Western blot, 1 µg/mL
AntibodyAnti-TF antibody (rat monoclonal)Genentech, Inc1H1MV TF activity, 100 µg/mL
AntibodyAnti-β-Actin (mouse monoclonal)Bio-RadMCA5775Western blot, 1:1000
AntibodyAnti-Caspase-1 (mouse monoclonal)AdipogenAG-20B-0042-C100Western blot, 1:1000
AntibodyAnti-GSDMD (rabbit monoclonal)Abcamab219800Western blot, 1:1000
AntibodyAnti-IL-1β (rabbit polyclonal)GeneTexGTX74034Western blot, 1:1000
AntibodyAnti-fibrin (mouse monoclonal)Gift from Dr. Hartmut Weiler (Medical College of Wisconsin) and Dr. Rodney M Camire (University of Pennsylvania)59D8Western blot, 1:2000;
Immunohistochemistry, 1:250
Commercial assay or kitCellTiter-Glo Luminescent Cell Viability AssayPromegaG7572
Commercial assay or kitCytoTox 96 Non-Radioactive Cytotoxicity AssayPromegaG1780
Commercial assay or kitMouse TAT ELISA kitAbcamab137994
Commercial assay or kitMouse IL-1β ELISA kitThermo Fisher Scientific88-7013A-88
Commercial assay or kitMouse IL-6 ELISA kitThermo Fisher Scientific88-7064-22
Commercial assay or kitMouse TNFα ELISA kitThermo Fisher Scientific88-7324-22
Peptide, recombinant proteinProtective agent (PA)Zhenyu Li Laboratory
Yinan Wei Laboratory
Described in the Materials and methods section
Peptide, recombinant proteinLFn-flagellinZhenyu Li Laboratory
Yinan Wei Laboratory
Described in the Materials and methods section
Chemical compound and drugHisPur Ni-NTA resinThermo Fisher Scientific88222
Chemical compound and drugThromboplastin-DPacific Hemostasis100357
Chemical compound and drugRGR-XaChromEnzyme Research Laboratories100-03
Chemical compound and drugGlycineSigmaG7126
Chemical compound and drugLPS (E. coli O111:B4)SigmaL4130
Chemical compound and drugT-PER tissue protein extraction reagentThermo Fisher Scientific78510
Chemical compound and drugCocktail inhibitorSigmaP8340
Software and algorithmImageStudio 5.0Li-CORWestern blot image
Software and algorithmPrism 9GraphPad Software Inc
OtherM.O.M. (Mouse on Mouse) ImmPRESS HRP (Peroxidase) Polymer KitVector LaboratoriesMP-2400Immunohistochemistry

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  1. Jian Cui
  2. Hua Li
  3. Dien Ye
  4. Guoying Zhang
  5. Yan Zhang
  6. Ling Yang
  7. Martha MS Sim
  8. Jeremy P Wood
  9. Yinan Wei
  10. Zhenyu Li
  11. Congqing Wu
(2025)
Inhibiting NINJ1-dependent plasma membrane rupture protects against inflammasome-induced blood coagulation and inflammation
eLife 12:RP91329.
https://doi.org/10.7554/eLife.91329.3