1. In Vivo Imaging of Inflamed Glomeruli Reveals Dynamics of Neutrophil Extracellular Trap Formation in Glomerular Capillaries 
Clare L.V. Westhorpe, James E. Bayard, Kim M. O'Sullivan, Pam Hall, Qiang Cheng, A. Richard Kitching, Michael J. Hickey 
The American Journal of Pathology 
Volume 187, Issue 2, Pages (February 2017)
DOI: /j.ajpath
Copyright © 2017 American Society for Investigative Pathology Terms and Conditions

2. Figure 1
NETs are generated in glomeruli after anti-GBM Ab administration. Kidneys of mice were examined for NET formation via immunohistochemistry 2 hours after induction of anti-GBM Ab-induced glomerulonephritis. A: Staining is shown for citrullinated histone H3 (H3Cit; green), myeloperoxidase (MPO; red), and peptidyl arginine deiminase 4 (PAD4; white) in kidneys of mice treated with either normal sheep globulin (NSG) or anti-GBM Ab (glomeruli defined by dotted line). Left column shows merged images, which also include DNA staining via DAPI (blue). Boxed areas are shown at higher magnification in the insets. In NSG example, insets display magnified image of a non-NETing neutrophil (ie, lacking extracellular DNA). In anti-GBM Ab example, insets display magnified image of a NETing neutrophil (ie, positive for all three markers and extracellular DNA). B: Staining for H2A-H2B (green), MPO (red), and DNA (DAPI; blue) in kidney of mouse treated with either NSG or anti-GBM Ab (glomeruli defined by dotted line). Left column shows merged image. In NSG example, insets display magnified image of a non-NETing neutrophil. In anti-GBM Ab example, insets display magnified image of a NETing neutrophil (ie, positive for H2A-H2B, MPO, and extracellular DNA). Scale bars: 20 μm (A and B, main images); 5 μm (A and B, insets).
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2017 American Society for Investigative Pathology Terms and Conditions

3. Figure 2
Extracellular DNA is detectable in inflamed glomeruli via multiphoton microscopy. A–C: Examples of the morphology of Sytox Orange–stained structures (red; arrowheads) in glomerular capillaries 2 hours after administration of anti-GBM Ab. Neutrophils are stained via anti–Gr-1 (cyan), and the vasculature is labeled using fluorescein isothiocyanate dextran (green). D: Quantitation of Sytox Orange–stained structures in glomeruli of mice treated with either normal sheep globulin (NSG) or anti-GBM Ab (15 or 20 mg). E: Three-dimensional surface rendering of a neutrophil (anti–Gr-1; cyan) with an adjacent extracellular Sytox Orange–stained structure (red), consistent with NET generation. See also Supplemental Video S1. Data are shown as means ± SEM (D). n = 6 per group (D). ∗P < 0.05, ∗∗P < 0.01 versus NSG. Scale bars: 20 μm (A–C); 5 μm (E).
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2017 American Society for Investigative Pathology Terms and Conditions

4. Figure 3
Extracellular myeloperoxidase (MPO) is detectable in inflamed glomeruli via multiphoton microscopy and is colocated with DNA. A: Multiphoton images of glomeruli from mice treated with either normal sheep globulin (NSG; top row) or anti-GBM Ab (bottom row), showing individual staining for neutrophils (anti–Gr-1; cyan), extracellular DNA (Sytox Orange; red), extracellular MPO (green), and the merged image with the vasculature shown in gray (glomerulus defined by dotted lines). An area of colocalization of DNA and MPO (arrows) is present in the anti-GBM Ab-treated mouse, but not the NSG-treated mouse. B: Percentage of glomeruli showing either Sytox-stained structures or structures positive for both Sytox and MPO staining, in NSG-treated mice, anti-GBM Ab-treated mice, and anti-GBM Ab-treated mice after neutrophil depletion. C: Histogram of timing of occurrence of MPO+ structures during the second hour after anti-GBM Ab treatment, segregated into 10-minute windows. D: Percentage of glomeruli showing either cell-associated (cell-assoc.) or cell-free MPO in the second hour after either NSG or anti-GBM Ab treatment. Data are shown as means ± SEM (B–D). n = 6 to 9 mice per group (B–D). ∗P < 0.05, ∗∗∗P < 0.001, and ∗∗∗∗P <  versus NSG. Scale bar = 10 μm (A). PMN Depl, neutrophil depletion.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2017 American Society for Investigative Pathology Terms and Conditions

5. Figure 4
Extracellular H2Ax and neutrophil elastase are less frequent in inflamed glomeruli. A and B: Percentage of glomeruli showing either anti–H2Ax-stained structures (A) or anti–neutrophil elastase (NE)–positive structures (B) in mice treated with either normal sheep globulin (NSG) or anti-GBM Ab. C: Multiphoton images of a glomerulus from an anti-GBM Ab-treated mouse, showing individual staining for neutrophils (anti–Gr-1; cyan), neutrophil elastase (green), and the merged image with the vasculature shown in gray (glomerulus defined by dotted line). Arrows denote position of positive elastase staining. Data are shown as means ± SEM (A and B). n = 6 mice per group (A and B). ∗∗P < 0.01 versus NSG. Scale bar = 10 μm (C).
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2017 American Society for Investigative Pathology Terms and Conditions

6. Figure 5
Myeloperoxidase (MPO)+ structures present in glomerular capillaries are transient. A: Multiphoton images of the same glomerulus taken over a 60-second interval, showing neutrophils (anti–Gr-1; blue), MPO (green), and vasculature (gray). An MPO+ structure present in the first image (arrows) is absent in the second. B and C: High-power image sequence (B) and three-dimensional surface rendering (C) of neutrophils and MPO from A, taken at 30-second intervals, illustrating disappearance of MPO from the neutrophil surface. Dotted lines define perimeter of glomerulus (A and B). D: Histogram showing the duration of MPO+ structures in the second hour of the anti-GBM Ab model, with data derived from either post-hydronephrotic (hydro) kidneys or intact (intact) kidneys from young mice. See also Supplemental Video S2. n = 7 mice per group (D, hydro kidneys); n = 6 mice per group (D, intact kidneys). Scale bars = 10 μm (A and C). Original magnification, ×60 (B).
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2017 American Society for Investigative Pathology Terms and Conditions

7. Figure 6
NETs are unstable under glomerular shear conditions. NETs were generated in a microfluidic flow chamber under static conditions, stained with Sytox Orange and Alexa 488–anti–Gr-1 (green), and then imaged (A and B) while being exposed to either constant low shear equivalent to that in the hepatic sinusoids (1 dyn/cm2; A) or increasing to shear equivalent to that in glomerular capillaries (10 and 20 dyn/cm2) at 2-minute intervals (B). (Experimental timelines indicated at the top of the graph in C). A: NET (arrows) in constant shear shown after 2-, 2.5-, and 5-minute exposure to 1 dyn/cm2. B: NET (arrows) under increasing shear shown after exposure to 1 dyn/cm2 (2 minutes), 10 dyn/cm2 (30 seconds), and 20 dyn/cm2 (1 minute). C: DNA staining intensity normalized to that at the start of the experiment. Data are shown as means ± SEM (C). n = 4 (C, constant shear); n = 6 (C, increasing shear). ∗P < 0.05 versus constant shear group. Original magnification, ×20 (A and B). AU, arbitrary unit.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2017 American Society for Investigative Pathology Terms and Conditions

8. Figure 7
Effects of inhibition of NET generation or DNase I treatment on NET generation, albuminuria, and hematuria in anti-GBM Ab model. A and B: Effect of peptidyl arginine deiminase-4 (PAD4) inhibition via Cl-amidine and DNase I treatment on intraglomerular NET formation, as determined via immunohistochemistry 2 hours after induction of anti-GBM Ab-induced glomerulonephritis. Staining is shown for citrullinated histone H3 (H3Cit; green), myeloperoxidase (MPO; red), and PAD4 (white) in kidneys of mice treated with anti-GBM Ab alone or with anti-GBM Ab plus either Cl-amidine or DNase I (glomeruli defined by dotted line). Left column shows merged images, which also include DNA staining via DAPI (blue). Boxed areas are shown at higher magnification in the insets. In anti-GBM Ab example, inset displays a magnified image of a NETing neutrophil, defined as for Figure 1. In Cl-amidine and DNase I examples, insets display magnified images of non-NETing neutrophils (ie, lacking extracellular DNA). B: Quantitative analysis of NET frequency in corresponding mice. Data are shown for mice treated with normal sheep globulin (NSG), anti-GBM Ab alone, and anti-GBM Ab plus either Cl-amidine or DNase I. C and D: Overnight urinary albumin/creatinine ratio (Alb/Cr; C) and hematuria score (D) were measured in untreated mice (albumin/creatinine only), anti-GBM Ab-treated mice, and anti-GBM Ab-treated mice pretreated with either Cl-amidine or DNase I. In D, data for individual animals are shown, and Kruskal-Wallis analysis was used. Data are means ± SEM (B–D). n = 4 mice per group (B); n = 8 mice per group (C and D). ∗P < 0.05 versus NSG; †P < 0.05 versus anti-GBM Ab; ‡P < 0.05 versus baseline. Scale bars: 20 μm (A, main images); 5 μm (A, insets). gcs, glomerular cross section.
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2017 American Society for Investigative Pathology Terms and Conditions

9. Supplemental Figure S1
NET formation in hepatic microcirculation assessed via intravital microscopy and immunohistochemistry. Livers of untreated mice, or mice that underwent a model of systemic endotoxemia, were examined for NET formation via either intravital spinning disk confocal microscopy (A) or immunohistochemistry (B and C). A: Images from intravital spinning disk confocal microscopy experiments showing in vivo staining for H2Ax (blue) in hepatic microcirculation of livers of untreated [no lipopolysaccharide (LPS); left panel] or LPS-treated (LPS; middle panel) mice. H2Ax+ structures are denoted by arrows. Right panel shows in vivo staining for H2A-H2B (blue) in liver of LPS-treated mouse, demonstrating the inability to detect this protein via this approach. In these experiments, hepatocytes are detectable in the green channel via autofluorescence. B and C: Immunohistochemical assessment of NET formation in livers of endotoxemic mice. In B, staining is shown for citrullinated histone H3 (H3Cit; green), myeloperoxidase (MPO; red), and peptidyl arginine deiminase 4 (PAD4; white). In C, staining is shown for H2A-H2B (green), MPO (red), and DNA (blue). In both sets of images, the left hand column shows merged images, which include DNA staining via DAPI (blue). Boxed areas are shown at higher magnification in the insets. Insets: Magnified images of NETing neutrophils (ie, positive for all markers and extracellular DNA). Scale bars: 20 μm (main images, B and C); 5 μm (insets, B and C). Original magnification, ×20 (A–C).
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2017 American Society for Investigative Pathology Terms and Conditions

10. Supplemental Figure S2
NET generation by neutrophils adherent to endothelial cell monolayers in vitro. A–D: Neutrophils were allowed to adhere to endothelial cell (MS1) monolayers and stimulated with lipopolysaccharide for at least 1 hour. NETs were detected by Sytox Orange staining. A–D: Images show two examples, demonstrating adhesion of neutrophils (green, anti–Gr-1) to endothelial monolayers (visible via phase contrast) (A and C) with corresponding images showing the Sytox Orange staining alone (B and D), with NETs indicated by arrows. Scale bar = 50 μm (B and D). Original magnification, ×20 (A–D).
The American Journal of Pathology  , DOI: ( /j.ajpath )
Copyright © 2017 American Society for Investigative Pathology Terms and Conditions