The role of type 1 interferons in coagulation induced by gram-negative bacteria

Abstract

Bacterial infection not only stimulates innate immune responses but also activates coagulation cascades. Overactivation of the coagulation system in bacterial sepsis leads to disseminated intravascular coagulation (DIC), a life-threatening condition. However, the mechanisms by which bacterial infection activates the coagulation cascade are not fully understood. Here we show that type 1 interferons (IFNs), a widely expressed family of cytokines that orchestrate innate antiviral and antibacterial immunity, mediate bacterial infection-induced DIC by amplifying the release of high-mobility group box 1 (HMGB1) into the bloodstream. Inhibition of the expression of type 1 IFNs and disruption of their receptor IFN-α/βR or downstream effector (eg, HMGB1) uniformly decreased gram-negative bacteria-induced DIC. Mechanistically, extracellular HMGB1 markedly increased the procoagulant activity of tissue factor by promoting the externalization of phosphatidylserine to the outer cell surface, where phosphatidylserine assembles a complex of cofactor-proteases of the coagulation cascades. These findings not only provide novel insights into the link between innate immune responses and coagulation, but they also open a new avenue for developing novel therapeutic strategies to prevent DIC in sepsis.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

© 2020 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Type 1 IFN signaling mediates the activation of coagulation cascades in endotoxemia. (A-D) WT and IFN-α/βR1 KO mice were administered LPS intraperitoneally (4 mg/kg) for 6 hours. Heparin (200 IU/kg) was injected subcutaneously 30 minutes before LPS injection. (A) Representative SD-IVM images of thrombin generation (green), platelet aggregation (blue), fibrin deposition (dark red), and albumin (red) within the liver microvasculature or representative multiphoton microscopy images of albumin (red) within lung microvasculature. AF647-albumin (red) was represented as a contrast material to identify perfused vessels; the occluded vessels exhibited weak fluorescent signals. Quantitative analysis was conducted of thrombin and platelets (B), occluded vessels (C), and fibrin deposition (D) within the liver microcirculation by using ImageJ software. (E-H) Mice were primed with 0.4 mg/kg of LPS for 7 hours and then challenged with 10 mg/kg of LPS for 8 hours. (E) Representative images of hematoxylin and eosin and immunohistochemical staining of fibrin in livers and lungs of WT mice vs IFN-α/βR1 KO mice (400×). The black arrow indicates thrombus in liver and lung capillaries from WT mice challenged with LPS. (F) Plasma levels of TAT complexes, PAI-1, fibrinogen (Fib), and D-dimer were detected in WT mice vs IFN-α/βR1 KO mice. (G) Time course of thrombocytopenia in WT mice vs IFN-α/βR1 KO mice after administration of 10 mg/kg of LPS treated at time 0, 8, and 14 hours. (H) Kaplan-Meier survival plots for WT mice vs IFN-α/βR1 KO mice treated with saline or LPS or LPS plus heparin (n = 11 mice per group). (I) Representative images of FeCl3-induced mesenteric arteriole thrombosis in WT mice and IFN-α/βR1 KO mice (left). Occlusion time of the mesenteric arteriole was analyzed (right). All data are shown as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001. N = 3 to 11 mice per group. Scale bar, 50 μm. NS, not significant.

Figure 2.

TRIF is critical for the activation of coagulation cascades in endotoxemia. (A-B) IFN-β messenger RNA (mRNA) expression in lungs, spleens (A), and guts (B) from WT mice vs TRIF KO mice as detected by quantitative polymerase chain reaction after LPS challenge for 2 hours. (C) Plasma levels of IFN-β detected by enzyme-linked immunosorbent assay in WT mice vs TRIF KO mice after LPS treatment (0.4 mg/kg of LPS for 7 hours + 10 mg/kg of LPS for 8 hours). (D) Representative SD-IVM images of thrombin (green), platelet adhesion (blue), fibrin (dark red), and albumin (red) within the liver microvasculature in endotoxemic WT and TRIF KO mice (4 mg/kg of LPS for 6 hours). (E-F) Quantitative analysis of thrombin, platelets, and fibrin probe fluorescence intensity and occluded vessels within the liver microcirculation by using ImageJ software. (G-I) WT and TRIF KO mice were injected with 0.4 mg/kg of LPS for 7 hours followed by 10 mg/kg of LPS for 8 hours. (G) Representative images of immunohistochemical staining of fibrin in livers and lungs are shown (×400). (H) Plasma levels of TAT complexes, PAI-1, fibrinogen (Fib), and D-dimer were measured. (I) Platelet counts were detected. (J) Kaplan-Meier survival plots for WT mice vs TRIF KO mice (n = 11 mice per group). Data are shown as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001. N = 4 to 11 mice per group. Scale bar, 50 μm.

Figure 3.

Inhibition of NF-kB activation enhanced LPS-induced coagulation. (A) Plasma concentration of TNF-α was measured at 1 hour after Myd88-deficient and WT mice were injected with 0.4 mg/kg of LPS. (B) Plasma concentration of TAT was measured in Myd88-deficient and WT mice primed with 0.4 mg/kg of LPS and then challenged with 10 mg/kg of LPS for 8 hours. (C-F) WT mice were treated with or without the IKKβ inhibitor ML120B (400 mg/kg by oral gavage twice daily for 4 days). Plasma concentration of TNF-α was measured at 1 hour after 0.4 mg/kg of LPS was injected into mice pretreated with or without ML120B (C). Representative SD-IVM images of thrombin (green), platelet adhesion (blue), and albumin (red) within the liver microvasculature in mice pretreated with or without ML120B, and then challenged with 4 mg/kg of LPS for 6 hours (D). Quantitative analysis of thrombin, platelets, and occluded vessels within the liver microcirculation by using ImageJ software (E). Plasma concentration of TAT complexes and PAI-1 were detected in mice pretreated with or without ML120B, and primed with 0.4 mg/kg of LPS for 7 hours followed by10 mg/kg of LPS for 8 hours (F). *P < .05; ***P < .001. NS, not significant.

Figure 4.

Type 1 IFN signaling mediates the activation of coagulation cascades in bacterial sepsis. (A-D) WT, IFN-α/βR1 KO, and TRIF KO mice subjected to either CLP or sham operation for 16 hours (WT group is the mixture of WT littermates of IFN-α/βR1 KO and TRIF KO mice). Thrombin and platelet fluorescence intensity in the liver microcirculation was quantified by using ImageJ software (A). Plasma levels of TAT complexes, PAI-1, fibrinogen (Fib), and D-dimer were measured (B). Representative images of immunohistochemical staining of fibrin in livers and lungs (×400) (C). Platelet counts in 16 hours after CLP- or sham-treated mice of indicated genotypes (D). (E) Kaplan-Meier survival plots for mice subjected to either CLP or sham operation (N = 11 mice per group). Data are shown as mean ± standard error of the mean. *P < .05; **P < .01. N = 4 to 11 mice per group.

Figure 5.

Type 1 IFN signaling mediates the activation of coagulation cascades by amplifying the release of HMGB1 into the bloodstream. (A-B) Plasma HMGB1 concentration was detected in mice of indicated genotypes primed with 0.4 mg/kg of LPS for 7 hours and then challenged with 10 mg/kg of LPS for 8 hours. (C) Quantitative analysis of fibrin in the liver microcirculation in SD-IVM images. (D) Representative SD-IVM images of thrombin (green), platelets (blue), and fibrin (dark red, AF594) in the liver microvasculature. (E) Quantitative analysis of thrombin generation and platelet activation in the liver microvasculature. (F-G) Mice of indicated genotypes were primed with 0.4 mg/kg of LPS for 7 hours and then challenged with 10 mg/kg of LPS for 8 hours. Plasma TAT and PAI-1 concentrations were detected (F). Representative images of immunohistochemical staining of fibrin in livers and lungs are shown (G) (×400). (H-I) WT mice were injected with or without monoclonal HMGB1 neutralizing antibody (2G7, 160 μg/mouse) or the isotype control IgG (160 μg/mouse) 30 minutes before administration of 10 mg/kg of LPS (TAT and PAI-1) or 4 mg/kg of LPS (SD-IVM). Quantitative analysis of thrombin generation and platelet activation within the liver microvasculature (H) and plasma levels of TAT complexes and PAI-1 (I) are shown. Data are shown as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001. N = 3 to 7 mice per group. Scale bar, 50 μm. NS, no significant.

Figure 6.

Type 1 IFN signaling and HMGB1 mediates TF-dependent coagulopathy in endotoxemia through PS exposure. (A) Quantitative analysis SD-IVM images of thrombin and platelet fluorescence intensity within the liver microcirculation from endotoxemic mice expressing normal or low levels of TF (4 mg/kg of LPS for 6 hours). (B) Plasma levels of TAT complexes and PAI-1 in saline- and LPS-challenged normal or low levels of TF mice (0.4 mg/kg of LPS for 7 hours + 10 mg/kg of LPS for 8 hours). (C) Mice were injected with liposome-clodronate and liposome–phosphate-buffered saline before LPS injection (0.4 mg/kg of LPS for 7 hours + 10 mg/kg of LPS for 8 hours). Plasma concentrations of TAT and PAI-1 were measured. (D-E) TF protein detected by western blot in the lungs, livers, and spleens from WT, IFN-α/βR1 KO, TRIF KO, Hmgb1fl/fl, and Hmgb1fl/fl Alb-cre+ mice that were challenged with LPS. (F-G) Quantitative analysis of TF protein and messenger RNA (mRNA) (liver) expression. Values are given as fold increase over unstimulated controls. (H) The correction between TF protein in liver detected by using enzyme-linked immunosorbent assay and plasma concentrations of TAT. (I) Quantitative analysis of PS exposure in peripheral leukocytes and splenocytes from endotoxemic WT mice by using FlowJo software. (J-K) WT mice were injected with or without recombinant MFG-E8 (rMFG-E8; 160 μg/kg) 2 hours before administration of 10 mg/kg of LPS (TAT and PAI-1) or 4 mg/kg of LPS (SD-IVM). Plasma levels of TAT complexes and PAI-1 (J) and quantitative analysis of thrombin and platelet fluorescence intensity within the liver microcirculation (K). (L-M) Flow cytometric analysis of PS exposure labeled by fluorescein isothiocyanate (FITC)–AnnexinV in peripheral leukocytes and splenocytes from mice of indicated genotypes. Representative images of PS exposure in splenocytes (L). Quantitative analysis of PS exposure in peripheral leukocytes and splenocytes (M). Data are shown as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001. N = 3 to 12 mice per group. NS, not significant.

Figure 7.

Extracellular HMGB1 induces PS exposure and TF activation through caspase-11. (A-C, E, G-H) WT, caspase-11 KO, and GSDMD KO macrophages stimulated with ultrapure LPS (L, 1 μg/mL) or HMGB1 (H, 400 ng/mL) or luteinizing hormone (LH) for 10 hours. Representative confocal images of PS exposure labeled by Annexin V–fluorescein isothiocyanate are shown (A), PS exposure was quantified by using ImageJ software for the area percentage of cells that are positive (B and E), and TF activity was detected (C). (D) TF activity was measured at different time points after WT and caspase-11 KO macrophages were treated with ultrapure LPS plus HMGB1. (F) WT, caspase-11 KO, and GSDMD KO macrophages stimulated with ultrapure LPS (L, 1 μg/mL) or HMGB1 (H, 400 ng/mL), or LH for 2 hours. TF messenger RNA (mRNA) expression was detected by using quantitative polymerase chain reaction. Values are given as fold increase over unstimulated controls. (G) TF protein expression was detected by using western blot. (H) Quantitative analysis of TF protein expression. (I) TF activity was measured in HCV macrophages stimulated with HMGB1 plus ultrapure LPS with or without MFG-E8. PS exposure (J) and TF activity (K) were detected in LH-treated HCV macrophages transfected with control small interfering RNA (siRNA) or TMEM16F-specific siRNA. si1 and si2 represent 2 sequences of Tmem16f-specific siRNAs. (L) WT and GSDMD KO macrophages stimulated with ultrapure LPS (1 μg/mL) or crude LPS (1 μg/mL) for 10 hours. PS exposure was quantified (left) and TF activity was detected (right). HCV mice were used for the aforementioned TF activity tests. Data are shown as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001. NS, not significant.