Neutrophil extracellular traps released by neutrophils impair revascularization and vascular remodeling after stroke methods
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Stroke induces NET formation
To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. The results revealed a significantly higher number of neutrophils and H3Cit + neutrophils in ischemic mice compared with sham-operated mice (Fig. ). In line with this, elevated levels of circulating DNA was found in the plasma from these mice (Fig. ). We then isolated neutrophils from the peripheral blood of these mice and incubated them with or without lipopolysaccharide (LPS) stimulation. We found that either unstimulated or LPS-stimulated neutrophils from ischemic mice showed a significant increase in H3Cit + neutrophils and NET formation (Fig. ), indicating that neutrophils from mice subjected to stroke are primed to undergo NETosis. Fig. 3 Neutrophils form NETs presenting in the brain after...
Stroke induces NET formation
As NETs can injure host tissue, we next asked whether NETs were produced in the ischemic brain and affect stroke outcomes. Western blot analysis of the ischemic cortex showed an increased amount of H3Cit that was most robust from 3-5 days (Fig. ), suggesting that NETs may play an important role during the delayed phases after stroke. Immunostaining revealed that the peri-infarct cortex was extensively labeled with H3Cit + cells at 3 days (Fig. ). To identify which type of cells expressed H3cit after stroke, double immunofluorescence with confocal microscopy was performed on brain sections. This analysis revealed that H3Cit was colocalized with Ly6G-positive neutrophils, F4/80-positive macrophages/microglial cells, Iba1-positive microglial cells, NeuN-positive neurons, and glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. ). Importantly, 78.7% of...
Disruption of NETs enhances vascular remodeling after stroke
We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrophils in the peri-infarct cortical areas (Supplementary Fig. ) but reduced H3Cit levels (Fig. ). Compared with the vehicle controls, administration of DNase 1 significantly reduced BBB permeability (Fig. ) and extravascular IgG deposits (Fig. ), and enhanced both Pdgfr-β + and CD13 + pericyte coverage on brain microvessels (Fig. and Supplementary Fig. ). Vascular branches (Supplementary Fig. ), microvascular length (Fig. ), perfused capillary length (Fig. ), and tomato-lectin perfused vessels (Fig. ) were also increased in the brains of mice treated with DNase 1. Next, we investigated the importance of neutrophil NETs in vascular remodeling. Our re...
Disruption of NETs enhances vascular remodeling after stroke
Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extravascular IgG deposits at 14 days (Fig. ). Furthermore, we observed significant increases in microvascular length (Fig. ) and perfused cortical vessels (Fig. ) in anti-Ly6G antibody-treated mice compared with control IgG-treated mice. Treatment with DNase 1 starting at 7 days after stroke also attenuated BBB disruption (Fig. ), increased microvessels (Fig. ), and improved capillary perfusion (Fig. ) at 14 days. Fig. 5 Increased vascular remodeling by delayed inhibition of NET formation. a - d Representative confocal images ( a, c ) and quantitative analysis of IgG extravascular deposits ( b, d ) in the peri-infarct cortex at 14 days. Mice were subjected to stroke and...
PAD4 regulates BBB permeability and neovascularization
Peptidylarginine deiminase 4 (PAD4) is a histone-modifying enzyme that is critical for NET formation,. Quantitative PCR revealed a sustained 29.3-fold increase in PAD4 mRNA expression in the ischemic cortex at 3 days after stroke compared with sham-operated brains (Fig. ). Immunoblotting found a marked 3.8-fold upregulation of PAD4 protein expression in these areas (Fig. ). To test the hypothesis that increased release of NETs, orchestrated by PAD4, participates in stroke recovery, we first studied the role of overexpression of PAD4 on vascular remodeling. Immunohistochemical analysis indicated extensive expression of recombinant adeno-PAD4-flag-infected cells in the cortex at 4 days after injection (Fig. ). Overexpression of PAD4 in the cortex was also confirmed by immunoblotting (Supplementary Fig. ). PAD4-flag was present in the neurons, neutrophils, macro...
PAD4 regulates BBB permeability and neovascularization
To further establish the role of NETs in stroke recovery, we compared wild type (WT) with PAD4-deficient (PAD4 -/- ) mice or treated mice with the PAD inhibitor Cl-amidine. There was no significant difference in ischemic lesion between WT and PAD4 -/- mice at 14 days (Supplementary Fig. ). Thus, this approach allowed us to provide evidence that effects of PAD4 deficiency on long-term outcomes are not secondary to the reduced lesion size. PAD4 deficiency did not alter the number of infiltrating neutrophils in the ischemic brain tissue (Supplementary Fig. ), whereas the levels of H3Cit in the lysates from the ischemic cortex at 3 days were greatly reduced in PAD4 -/- mice and mice receiving Cl-amidine (Fig. and Supplementary Fig. ). Similarly, examination of the brain tissues revealed that PAD4 deficiency or Cl-amidine reduced...
NETs are responsible for STING-mediated vascular remodeling
The free DNA binds with cyclic GMP-AMP synthase to promote STING-dependent type I interferon (IFN) synthesis,. We found a marked tenfold increase in the levels of IFN-β in the cortical areas at 3 days after stroke (Fig. ), suggesting activation of the STING pathway. We then detected significantly increased levels of STING (Fig. and Supplementary Fig. ) and robust induction of phosphorylated TANK-binding kinase 1 (pTBK1) and TBK1-dependent IFN regulatory factor 3 (IRF3) activation (Fig. and Supplementary Fig. ). Treatment with the PAD inhibitor Cl-amidine reduced the levels of stroke-induced STING-meditated signaling in the cortical areas compared with vehicle-treated mice (Fig. and Supplementary Fig. ). Neutrophil depletion also resulted in a significant reduction in the levels of STING and the STING downstream signaling molecules...
Methods
Animal protocols were reviewed and approved by the Animal Care and Use Committee of the Institutes of Brain Science of Fudan University and were conducted in accordance with the ethical regulations. PAD4 -/- mice on a C57BL/6J background were purchased from The Jackson Laboratory. Age-matched WT C57BL/6 mice (SLAC Laboratory Animal Co. Ltd, Shanghai, China) were used as controls. All mice were housed in a temperature-controlled environment (22 ± 2 °C) on a 12 h light-dark cycle with food and water available ad libitum. Room humidity was controlled at 55 ± 5%. Focal cortical cerebral ischemia was induced by electrocoagulation of the distal portion of the right middle cerebral artery (MCA),. Male mice weighing 23-26 g were anesthetized with 1-1.5% isoflurane in 30% oxygen and 70% nitrous oxide. Afte...
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To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. The results revealed a significantly higher number of neutrophils and H3Cit + neutrophils in ischemic mice compared with sham-operated mice (Fig. ). In line with this, elevated levels of circulating DNA was found in the plasma from these mice (Fig. ). We then isolated neutrophils from the peripheral blood of these mice and incubated them with or without lipopolysaccharide (LPS) stimulation. We found that either unstimulated or LPS-stimulated neutrophils from ischemic mice showed a significant increase in H3Cit + neutrophils and NET formation (Fig. ), indicating that neutrophils from mice subjected to stroke are primed to undergo NETosis. Fig. 3 Neutrophils form NETs presenting in the brain after stroke. a Representative images of H3Cit (green) and Ly6G (red) double-positive cells in cytospins from sham-operated mice and ischemic mice at 3 days. DNA was visualized with Hoechst 33342 (blue). Bar = 30 µm. b, c Quantification of Ly6G-positive neutrophils in the total le...
As NETs can injure host tissue, we next asked whether NETs were produced in the ischemic brain and affect stroke outcomes. Western blot analysis of the ischemic cortex showed an increased amount of H3Cit that was most robust from 3-5 days (Fig. ), suggesting that NETs may play an important role during the delayed phases after stroke. Immunostaining revealed that the peri-infarct cortex was extensively labeled with H3Cit + cells at 3 days (Fig. ). To identify which type of cells expressed H3cit after stroke, double immunofluorescence with confocal microscopy was performed on brain sections. This analysis revealed that H3Cit was colocalized with Ly6G-positive neutrophils, F4/80-positive macrophages/microglial cells, Iba1-positive microglial cells, NeuN-positive neurons, and glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. ). Importantly, 78.7% of the H3Cit-positive cells were Ly6G-positive neutrophils. H3Cit + neutrophils were observed inside the blood vessels and cerebral parenchyma (Fig. ). Hematoxylin and eosin staining clearly indicated that DNA fibers were present in these areas (Supplementary Fig. ). To confirm these obser...
We next investigated whether degradation of NETs with DNase 1 could improve vascular remodeling after stroke in mice. Treatment with DNase 1 did not affect the amount of neutrophils in the peri-infarct cortical areas (Supplementary Fig. ) but reduced H3Cit levels (Fig. ). Compared with the vehicle controls, administration of DNase 1 significantly reduced BBB permeability (Fig. ) and extravascular IgG deposits (Fig. ), and enhanced both Pdgfr-β + and CD13 + pericyte coverage on brain microvessels (Fig. and Supplementary Fig. ). Vascular branches (Supplementary Fig. ), microvascular length (Fig. ), perfused capillary length (Fig. ), and tomato-lectin perfused vessels (Fig. ) were also increased in the brains of mice treated with DNase 1. Next, we investigated the importance of neutrophil NETs in vascular remodeling. Our results showed that treatment with DNase 1 in combination with anti-Ly6G antibody did not further improve neovascularization and BBB leakage compared with mice treated with anti-Ly6G antibody alone (Fig. ). These data indicate that DNase 1 primarily digests NETs generated by neutrophils, and tha...
Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extravascular IgG deposits at 14 days (Fig. ). Furthermore, we observed significant increases in microvascular length (Fig. ) and perfused cortical vessels (Fig. ) in anti-Ly6G antibody-treated mice compared with control IgG-treated mice. Treatment with DNase 1 starting at 7 days after stroke also attenuated BBB disruption (Fig. ), increased microvessels (Fig. ), and improved capillary perfusion (Fig. ) at 14 days. Fig. 5 Increased vascular remodeling by delayed inhibition of NET formation. a - d Representative confocal images ( a, c ) and quantitative analysis of IgG extravascular deposits ( b, d ) in the peri-infarct cortex at 14 days. Mice were subjected to stroke and treated with either anti-Ly6G antibody, control antibody, DNase 1, or vehicle starting at 7 days ( n = 6), unpaired two-tailed Student's t -test was applied with * P = 0.0392 ( b ), * P = 0.0384 ( d ). Bar = 10 µm. e - l Repre...
Peptidylarginine deiminase 4 (PAD4) is a histone-modifying enzyme that is critical for NET formation,. Quantitative PCR revealed a sustained 29.3-fold increase in PAD4 mRNA expression in the ischemic cortex at 3 days after stroke compared with sham-operated brains (Fig. ). Immunoblotting found a marked 3.8-fold upregulation of PAD4 protein expression in these areas (Fig. ). To test the hypothesis that increased release of NETs, orchestrated by PAD4, participates in stroke recovery, we first studied the role of overexpression of PAD4 on vascular remodeling. Immunohistochemical analysis indicated extensive expression of recombinant adeno-PAD4-flag-infected cells in the cortex at 4 days after injection (Fig. ). Overexpression of PAD4 in the cortex was also confirmed by immunoblotting (Supplementary Fig. ). PAD4-flag was present in the neurons, neutrophils, macrophages/microglial cells, and microglial cells, but was rarely detected in astrocytes (Supplementary Fig. ). Injection of PAD4 adenovirus in ischemic mice produced ~2.6-fold more NETs than control virus (Fig. and Supplementary Fig. ). We then observed a significant increase in the n...
To further establish the role of NETs in stroke recovery, we compared wild type (WT) with PAD4-deficient (PAD4 -/- ) mice or treated mice with the PAD inhibitor Cl-amidine. There was no significant difference in ischemic lesion between WT and PAD4 -/- mice at 14 days (Supplementary Fig. ). Thus, this approach allowed us to provide evidence that effects of PAD4 deficiency on long-term outcomes are not secondary to the reduced lesion size. PAD4 deficiency did not alter the number of infiltrating neutrophils in the ischemic brain tissue (Supplementary Fig. ), whereas the levels of H3Cit in the lysates from the ischemic cortex at 3 days were greatly reduced in PAD4 -/- mice and mice receiving Cl-amidine (Fig. and Supplementary Fig. ). Similarly, examination of the brain tissues revealed that PAD4 deficiency or Cl-amidine reduced Sytox green-positive extracellular DNA fibers (Fig. ) in the ischemic cortex. Previous studies have shown that PAD4 may also function in other cells,. We found that PAD4 deficiency or Cl-amidine substantially reduced neutrophil NETs, as seen by the decrease in H3Cit + neutrophils (Fig....
The free DNA binds with cyclic GMP-AMP synthase to promote STING-dependent type I interferon (IFN) synthesis,. We found a marked tenfold increase in the levels of IFN-β in the cortical areas at 3 days after stroke (Fig. ), suggesting activation of the STING pathway. We then detected significantly increased levels of STING (Fig. and Supplementary Fig. ) and robust induction of phosphorylated TANK-binding kinase 1 (pTBK1) and TBK1-dependent IFN regulatory factor 3 (IRF3) activation (Fig. and Supplementary Fig. ). Treatment with the PAD inhibitor Cl-amidine reduced the levels of stroke-induced STING-meditated signaling in the cortical areas compared with vehicle-treated mice (Fig. and Supplementary Fig. ). Neutrophil depletion also resulted in a significant reduction in the levels of STING and the STING downstream signaling molecules including pTBK1, pIRF3, and IFN-β in the ischemic brain relative to IgG-treated controls (Fig. and Supplementary Fig. ). To test whether the signaling part mediated by STING is related to neutrophils, we isolated neutrophils from bone marrow of ischemic mice and stimulated them with...
Animal protocols were reviewed and approved by the Animal Care and Use Committee of the Institutes of Brain Science of Fudan University and were conducted in accordance with the ethical regulations. PAD4 -/- mice on a C57BL/6J background were purchased from The Jackson Laboratory. Age-matched WT C57BL/6 mice (SLAC Laboratory Animal Co. Ltd, Shanghai, China) were used as controls. All mice were housed in a temperature-controlled environment (22 ± 2 °C) on a 12 h light-dark cycle with food and water available ad libitum. Room humidity was controlled at 55 ± 5%. Focal cortical cerebral ischemia was induced by electrocoagulation of the distal portion of the right middle cerebral artery (MCA),. Male mice weighing 23-26 g were anesthetized with 1-1.5% isoflurane in 30% oxygen and 70% nitrous oxide. After making a 2 cm curved skin incision between the right eye and the right ear using surgical scissors, the temporal muscle was retracted laterally. Under an operating microscope, a 1.5 mm-diameter window was opened using a high-speed micro drill (Stoelting, CellPoint Scientific, Maryland)...
Machine-readable layer
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"text": "To test whether NETs are present in the circulation of ischemic mice, blood cells from mice at 3 days after stroke were stained for citrullinated histone H3 (H3Cit) and Ly6G. The results revealed a significantly higher number of neutrophils and H3Cit + neutrophils in ischemic mice compared with sham-operated mice (Fig. ). In line with this, elevated levels of circulating DNA was found in the plasma from these mice (Fig. ). We then isolated neutrophils from the peripheral blood of these mice and incubated them with or without lipopolysaccharide (LPS) stimulation. We found that either unstimulated or LPS-stimulated neutrophils from ischemic mice showed a significant increase in H3Cit + neutrophils and NET formation (Fig. ), indicating that neutrophils from mice subjected to stroke are primed to undergo NETosis. Fig. 3 Neutrophils form NETs presenting in the brain after..."
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"text": "As NETs can injure host tissue, we next asked whether NETs were produced in the ischemic brain and affect stroke outcomes. Western blot analysis of the ischemic cortex showed an increased amount of H3Cit that was most robust from 3-5 days (Fig. ), suggesting that NETs may play an important role during the delayed phases after stroke. Immunostaining revealed that the peri-infarct cortex was extensively labeled with H3Cit + cells at 3 days (Fig. ). To identify which type of cells expressed H3cit after stroke, double immunofluorescence with confocal microscopy was performed on brain sections. This analysis revealed that H3Cit was colocalized with Ly6G-positive neutrophils, F4/80-positive macrophages/microglial cells, Iba1-positive microglial cells, NeuN-positive neurons, and glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. ). Importantly, 78.7% of..."
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"text": "Next, we detected whether NETosis regulates vascular remodeling during repair processes. We found that injection of anti-Ly6G antibody beginning 7 days after stroke reduced extravascular IgG deposits at 14 days (Fig. ). Furthermore, we observed significant increases in microvascular length (Fig. ) and perfused cortical vessels (Fig. ) in anti-Ly6G antibody-treated mice compared with control IgG-treated mice. Treatment with DNase 1 starting at 7 days after stroke also attenuated BBB disruption (Fig. ), increased microvessels (Fig. ), and improved capillary perfusion (Fig. ) at 14 days. Fig. 5 Increased vascular remodeling by delayed inhibition of NET formation. a - d Representative confocal images ( a, c ) and quantitative analysis of IgG extravascular deposits ( b, d ) in the peri-infarct cortex at 14 days. Mice were subjected to stroke and..."
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"text": "Peptidylarginine deiminase 4 (PAD4) is a histone-modifying enzyme that is critical for NET formation,. Quantitative PCR revealed a sustained 29.3-fold increase in PAD4 mRNA expression in the ischemic cortex at 3 days after stroke compared with sham-operated brains (Fig. ). Immunoblotting found a marked 3.8-fold upregulation of PAD4 protein expression in these areas (Fig. ). To test the hypothesis that increased release of NETs, orchestrated by PAD4, participates in stroke recovery, we first studied the role of overexpression of PAD4 on vascular remodeling. Immunohistochemical analysis indicated extensive expression of recombinant adeno-PAD4-flag-infected cells in the cortex at 4 days after injection (Fig. ). Overexpression of PAD4 in the cortex was also confirmed by immunoblotting (Supplementary Fig. ). PAD4-flag was present in the neurons, neutrophils, macro..."
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"text": "The free DNA binds with cyclic GMP-AMP synthase to promote STING-dependent type I interferon (IFN) synthesis,. We found a marked tenfold increase in the levels of IFN-β in the cortical areas at 3 days after stroke (Fig. ), suggesting activation of the STING pathway. We then detected significantly increased levels of STING (Fig. and Supplementary Fig. ) and robust induction of phosphorylated TANK-binding kinase 1 (pTBK1) and TBK1-dependent IFN regulatory factor 3 (IRF3) activation (Fig. and Supplementary Fig. ). Treatment with the PAD inhibitor Cl-amidine reduced the levels of stroke-induced STING-meditated signaling in the cortical areas compared with vehicle-treated mice (Fig. and Supplementary Fig. ). Neutrophil depletion also resulted in a significant reduction in the levels of STING and the STING downstream signaling molecules..."
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"text": "Animal protocols were reviewed and approved by the Animal Care and Use Committee of the Institutes of Brain Science of Fudan University and were conducted in accordance with the ethical regulations. PAD4 -/- mice on a C57BL/6J background were purchased from The Jackson Laboratory. Age-matched WT C57BL/6 mice (SLAC Laboratory Animal Co. Ltd, Shanghai, China) were used as controls. All mice were housed in a temperature-controlled environment (22 ± 2 °C) on a 12 h light-dark cycle with food and water available ad libitum. Room humidity was controlled at 55 ± 5%. Focal cortical cerebral ischemia was induced by electrocoagulation of the distal portion of the right middle cerebral artery (MCA),. Male mice weighing 23-26 g were anesthetized with 1-1.5% isoflurane in 30% oxygen and 70% nitrous oxide. Afte..."
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