NINJ1 is critical for flagellin-induced systemic coagulation, inflammation, and lethality.

(A–E) Mice were injected intravenously with Ctrl (saline) or Fla (500 ng LFn-Fla plus 3 μg PA). Blood was collected 90 minutes after Ctrl or Fla injection. Prothrombin time (A), plasma TAT (B), and plasma cytokines (C-E) were measured. Circles represent individual mouse, with bar donating mean. ** p < 0.01 (two-way ANOVA with Holm-Sidak multiple comparisons).

(F-G) Mice were injected intravenously with Ctrl or Fla. After 90 minutes, 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. Data are representative of 3 independent experiments (biological replicates). (G) Fibrin in the liver and lungs was detected by immunoblot with the anti-fibrin monoclonal antibody (59D8). Data are representative of 3 independent experiments (biological replicates).

(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]).

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

(A–E) Mice were injected intravenously with Ctrl (saline) or E. coli (2 x 108 cfu per mouse). Blood was collected 6 hours afterwards. Prothrombin time (A), plasma TAT (B), and plasma cytokines (C-E) were measured. Circles represent individual mouse, with bars donating mean. * p < 0.05, ** p < 0.01 (two-way ANOVA with Holm-Sidak multiple comparisons).

PMR promotes the release of procoagulant MVs.

(A-B, E) BMDMs were incubated with Ctrl (PBS) or Fla (1 μg/mL LFn-Fla plus 1 μg/mL PA). Cell culture supernatant and MVs were collected after 90 minutes of incubation. (A) NINJ1 in the cell lysate and MVs was detected by immunoblot. (B) BMDM MVs were counted with Nanosight. (E) BMDM MV TF activity. Circles represent individual mouse, with bars donating mean. ** p < 0.01 (two-way ANOVA with Holm-Sidak multiple comparisons).

(C, D, F) Mice were injected intravenously with Ctrl (saline) or Fla (500 ng LFn-Fla plus 3 μg PA). Blood was collected 90 minutes after Ctrl or Fla injection. (C) NINJ1 in plasma MVs was detected with equal plasma volumes by immunoblot. (D) Plasma MVs were counted with NanoSight. (F) Plasma MV TF activity. Circles represent individual mouse, with bars donating mean. ** p < 0.01 (two-way ANOVA with Holm-Sidak multiple comparisons).

Glycine inhibition of NINJ1 blocks pyroptosis-induced blood coagulation.

Mice were injected intravenously 50 μl of 0.5 M glycine 2 hours before administrating Ctrl (saline) or Fla (500 ng LFn-Fla plus 3 μg PA). Blood was collected 90 minutes afterwards. Prothrombin time (A), plasma TAT (B), plasma TF MV activity (C), plasma MV counts (D), and plasma cytokines (E-G) were measured. Circles represent individual mouse, with bars donating mean. * p < 0.05, ** p < 0.01, n.s. donates not significant (two-way ANOVA with Holm-Sidak multiple comparisons).

NINJ1 protein abundance in different tissues.

Protein was extracted from fresh frozen tissues and detected by immunoblot.

Flagellin-induced inflammasome activation and pyroptosis.

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

Flagellin-induced pyroptosis in Ninj1+/+ and Ninj1+/- BMDMs.

BMDMs from Ninj1+/+ and Ninj1+/- mice were incubated with Ctrl (PBS) or Fla (1 μg/mL LFn-Fla plus 1 μg/mL PA) for 90 minutes. LDH release (A) and ATP (B) were measured. Circles represent individual mouse, with bars donating mean. ** p < 0.01, n.s. donates not significant (two-way ANOVA with Holm-Sidak multiple comparisons).

Glycine treatment inhibits BMDM MV release.

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