Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps methods
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NE degrades histones to promote nuclear decondensation
Because histones pack DNA, we examined whether histones H3 and H4 are degraded in nuclei treated with LSS. To distinguish between histone degradation and histone release, we separated the soluble unbound proteins (SF) from the nuclear fraction (NF) by filtration. Interestingly, H4, but not H3, was degraded in an NE-dependent manner ( ). We also monitored the kinetics of nuclear localization of NE and MPO in relation to histone degradation and nuclear decondensation. Both NE and MPO were detected in the nuclear fraction within the first 30 min. Notably, H1 was degraded early but decondensation coincided with H4 degradation at 150 min ( ), which suggests that nuclear decondensation is driven primarily by the degradation of core histones ( ). However, H1 may have to be degraded first to allow for the subsequent degradation of core histones.
NE degrades histones to promote nuclear decondensation
Moreover, purified NE and PR3, but not CG, promoted nuclear decondensation in vitro (, Fig. S1 F, and not depicted). NE degraded H4 processively, whereas the other histones were only partially degraded ( ). Histone degradation was detectable by 30 min and coincided with nuclear decondensation ( ). As a control, we showed that NE completely degraded soluble histones purified from neutrophils ( ), which indicates that the pattern of histone degradation depends on chromatin structure and not on the intrinsic susceptibility of histones to this protease. Similarly, purified PR3 cleaved nuclear histones in vitro (Fig. S1 E). In contrast, and consistent with our previous observations, nuclear and purified histones were poor substrates for CG (unpublished data).
NE and MPO synergize to promote nuclear decondensation
Because MPO localizes to NETs and binds to nuclei in vitro ( ), we tested whether MPO promotes nuclear decondensation ( ). At 1 µM, MPO alone had little effect, but it dramatically enhanced chromatin decondensation in the presence of NE ( ). Interestingly, MPO did not affect histone degradation, which suggests that it does not up-regulate NE activity ( ). MPO promoted nuclear decondensation in a dose-dependent manner that did not require its substrate H 2 O 2 and was not inhibited by ABAH ( ). Furthermore, horseradish peroxidase, used as a control for enzymatic activity, did not promote chromatin decondensation (unpublished data). We concluded that MPO enhances NET formation independent of its enzymatic activity.
NE and MPO synergize to promote nuclear decondensation
NE and MPO synergize. (A) Effect of MPO titration on nuclear decondensation in vitro. Nuclei were incubated with the indicated concentrations of MPO for 30 (open circles), 60 (gray circles), or 120 min (closed circles). Decondensation was concentration- but not time-dependent. (B) MPO promotes decondensation independent of its enzymatic activity. Nuclei were treated with 5 µM MPO alone or MPO in the presence of 100 µM ABAH (an MPO inhibitor), 100 µM H 2 O 2 (MPO substrate), or both. (C) Nuclear decondensation was driven by titration of NE at different concentrations of MPO. MPO increases NE-mediated decondensation. (D) The effect of NE titration on nuclear decondensation. Nuclei were incubated with the indicated concentrations of NE in the absence (black line) or presence (red line) of 10 µM NEi for 120 min before measuring nuclear decondensation. (E) NE-mediated d...
NE translocates to the nucleus during NET formation
During NET formation, NE localized to the nucleus 60 min after stimulation, accompanied by reduced granular staining (, ii). In contrast, only low levels of PR3 were detected in the heterochromatin areas of the nucleus, arguing for a selective translocation of NE. After 120 min of stimulation, NE, but not PR3, was found predominantly in the decondensing nucleus in a diffuse gradient-like pattern (, iii). Interestingly, when neutrophils were treated with NEi, NE remained granular (, v), which suggests that NE activity is required for NE translocation. Because NE bound efficiently to isolated nuclei in the presence of NEi, NE activity may be required for granular release ( ).
NE translocates to the nucleus during NET formation
NE, PR3, and MPO localization during NET formation. (A-C) Naive and PMA-activated neutrophils in the presence or absence of NEi, fixed at the indicated time points and immunolabeled for NE (red) and PR3 (green; A), or NE (red) and MPO (green; B and C). DNA was stained with DRAQ5 (blue). (A) NE, and to a lesser extent PR3, translocate to the nucleus within 60 min after stimulation. (B) MPO associates with DNA before cell lysis but later than NE. (C) NEi prevents NE and MPO translocation to the nucleus, and chromatin decondensation. Bar, 5 µm. (D) NE is released from the granules during activation. Lysates from naive and activated neutrophils were prepared after 60 min of incubation and separated into cytoplasmic (HSS) and granule (HSP) fractions. The enzymatic activity (initial rate of change in absorbance) was normalized to the total amount of MPO (MPOt), which remains unch...
Mouse peritoneal cells
Mouse peritoneal cells were collected 5 h after injection of 1 ml of thioglycolate into the peritoneal cavity of 10-wk-old mice. The peritoneum was lavaged with 10 ml of PBS to collect the neutrophils. Neutrophils were washed in PBS and plated as described in the procedure for human neutrophils. NET formation was monitored by microscopy for 24 h after stimulation. Quantitation of NET formation was performed by measuring the area of DNA for each cell by staining with Sytox, as described in the procedures for human neutrophils.
NE is required for NET formation in vivo
We examined NET formation in the lungs of wild type (WT) and NE knockout mice infected intranasally with a sublethal dose of Klebsiella pneumoniae, a Gram-negative bacterium that causes pneumonia. Neutrophils were massively recruited to the lungs 48 h after infection in both groups of animals, confirming that the loss of NE does not interfere with neutrophil recruitment ( ). The lungs of WT mice contained decondensed web-like structures that stain with antibodies against MPO and an H2A-H2B-DNA complex (, i; ). NETs have also been observed in a mouse pneumococcal pneumonia model ( ). In contrast, in the lungs of infected NE knockout animals, neutrophils exhibited condensed nuclei, and MPO did not colocalize with the DNA marker but remained granular (, ii).
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Because histones pack DNA, we examined whether histones H3 and H4 are degraded in nuclei treated with LSS. To distinguish between histone degradation and histone release, we separated the soluble unbound proteins (SF) from the nuclear fraction (NF) by filtration. Interestingly, H4, but not H3, was degraded in an NE-dependent manner ( ). We also monitored the kinetics of nuclear localization of NE and MPO in relation to histone degradation and nuclear decondensation. Both NE and MPO were detected in the nuclear fraction within the first 30 min. Notably, H1 was degraded early but decondensation coincided with H4 degradation at 150 min ( ), which suggests that nuclear decondensation is driven primarily by the degradation of core histones ( ). However, H1 may have to be degraded first to allow for the subsequent degradation of core histones.
Moreover, purified NE and PR3, but not CG, promoted nuclear decondensation in vitro (, Fig. S1 F, and not depicted). NE degraded H4 processively, whereas the other histones were only partially degraded ( ). Histone degradation was detectable by 30 min and coincided with nuclear decondensation ( ). As a control, we showed that NE completely degraded soluble histones purified from neutrophils ( ), which indicates that the pattern of histone degradation depends on chromatin structure and not on the intrinsic susceptibility of histones to this protease. Similarly, purified PR3 cleaved nuclear histones in vitro (Fig. S1 E). In contrast, and consistent with our previous observations, nuclear and purified histones were poor substrates for CG (unpublished data).
Because MPO localizes to NETs and binds to nuclei in vitro ( ), we tested whether MPO promotes nuclear decondensation ( ). At 1 µM, MPO alone had little effect, but it dramatically enhanced chromatin decondensation in the presence of NE ( ). Interestingly, MPO did not affect histone degradation, which suggests that it does not up-regulate NE activity ( ). MPO promoted nuclear decondensation in a dose-dependent manner that did not require its substrate H 2 O 2 and was not inhibited by ABAH ( ). Furthermore, horseradish peroxidase, used as a control for enzymatic activity, did not promote chromatin decondensation (unpublished data). We concluded that MPO enhances NET formation independent of its enzymatic activity.
NE and MPO synergize. (A) Effect of MPO titration on nuclear decondensation in vitro. Nuclei were incubated with the indicated concentrations of MPO for 30 (open circles), 60 (gray circles), or 120 min (closed circles). Decondensation was concentration- but not time-dependent. (B) MPO promotes decondensation independent of its enzymatic activity. Nuclei were treated with 5 µM MPO alone or MPO in the presence of 100 µM ABAH (an MPO inhibitor), 100 µM H 2 O 2 (MPO substrate), or both. (C) Nuclear decondensation was driven by titration of NE at different concentrations of MPO. MPO increases NE-mediated decondensation. (D) The effect of NE titration on nuclear decondensation. Nuclei were incubated with the indicated concentrations of NE in the absence (black line) or presence (red line) of 10 µM NEi for 120 min before measuring nuclear decondensation. (E) NE-mediated degradation of histones is dose-dependent during nuclear decondensation. Western immunoblot analysis of MPO, H4, and NE was performed. Nuclei mixed with increasing concentrations of NE and 0.3 µM MPO for 120 min were separated into nuclear (NF, left) and soluble (SF, right) fractions. Reactions...
During NET formation, NE localized to the nucleus 60 min after stimulation, accompanied by reduced granular staining (, ii). In contrast, only low levels of PR3 were detected in the heterochromatin areas of the nucleus, arguing for a selective translocation of NE. After 120 min of stimulation, NE, but not PR3, was found predominantly in the decondensing nucleus in a diffuse gradient-like pattern (, iii). Interestingly, when neutrophils were treated with NEi, NE remained granular (, v), which suggests that NE activity is required for NE translocation. Because NE bound efficiently to isolated nuclei in the presence of NEi, NE activity may be required for granular release ( ).
NE, PR3, and MPO localization during NET formation. (A-C) Naive and PMA-activated neutrophils in the presence or absence of NEi, fixed at the indicated time points and immunolabeled for NE (red) and PR3 (green; A), or NE (red) and MPO (green; B and C). DNA was stained with DRAQ5 (blue). (A) NE, and to a lesser extent PR3, translocate to the nucleus within 60 min after stimulation. (B) MPO associates with DNA before cell lysis but later than NE. (C) NEi prevents NE and MPO translocation to the nucleus, and chromatin decondensation. Bar, 5 µm. (D) NE is released from the granules during activation. Lysates from naive and activated neutrophils were prepared after 60 min of incubation and separated into cytoplasmic (HSS) and granule (HSP) fractions. The enzymatic activity (initial rate of change in absorbance) was normalized to the total amount of MPO (MPOt), which remains unchanged, and plotted as the fraction of NE activity over total MPO activity (open bars). The distribution of MPO activity in each sample over total MPO activity is also shown (shaded bars). Samples were also resolved by SDS-PAGE and analyzed by immunoblotting against MPO, NE, and histone H2B.
Mouse peritoneal cells were collected 5 h after injection of 1 ml of thioglycolate into the peritoneal cavity of 10-wk-old mice. The peritoneum was lavaged with 10 ml of PBS to collect the neutrophils. Neutrophils were washed in PBS and plated as described in the procedure for human neutrophils. NET formation was monitored by microscopy for 24 h after stimulation. Quantitation of NET formation was performed by measuring the area of DNA for each cell by staining with Sytox, as described in the procedures for human neutrophils.
We examined NET formation in the lungs of wild type (WT) and NE knockout mice infected intranasally with a sublethal dose of Klebsiella pneumoniae, a Gram-negative bacterium that causes pneumonia. Neutrophils were massively recruited to the lungs 48 h after infection in both groups of animals, confirming that the loss of NE does not interfere with neutrophil recruitment ( ). The lungs of WT mice contained decondensed web-like structures that stain with antibodies against MPO and an H2A-H2B-DNA complex (, i; ). NETs have also been observed in a mouse pneumococcal pneumonia model ( ). In contrast, in the lungs of infected NE knockout animals, neutrophils exhibited condensed nuclei, and MPO did not colocalize with the DNA marker but remained granular (, ii).
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