1. Volume 149, Issue 7, Pages 1920-1931.e8 (December 2015)
Neutrophil Extracellular Traps Induce Trypsin Activation, Inflammation, and Tissue Damage in Mice With Severe Acute Pancreatitis 
Mohammed Merza, Hannes Hartman, Milladur Rahman, Rundk Hwaiz, Enming Zhang, Erik Renström, Lingtao Luo, Matthias Mörgelin, Sara Regner, Henrik Thorlacius 
Gastroenterology 
Volume 149, Issue 7, Pages e8 (December 2015)
DOI: /j.gastro
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2. Figure 1
NET formation in AP. (A) Scanning electron microscopy showing extracellular web-like structures in the pancreas from a mouse exposed to taurocholate. Scale bar: 5 μm. (B) A higher magnification of the indicated area of interest from Figure 1A showing fibrillar structures compatible with NETs in the inflamed pancreas. Scale bar: 2 μm. (C) Transmission electron microscopy of the indicated area of interest from Figure 1B incubated with gold-labeled anti–histone 2B (large gold particles) and anti-elastase (small gold particles) antibodies. Scale bar: 0.25 μm. (D) Quantification of extracellular DNA in the pancreas after IV administration of Sytox Green in vivo by measuring the relative area of fluorescence per high-power field. Plasma levels of cf-DNA were determined as described in the Supplementary Materials and Methods section. (E) Pancreatic levels of histone 3 and histone 4 were determined by enzyme-linked immunosorbent assay. Pancreatitis (black bars) was induced by infusion of sodium taurocholate (5%) into the pancreatic duct. Control mice (grey bars) were infused with saline alone. Animals were treated with IP injections of DNase I, an antibody directed against Ly6G (anti-Ly6G) or vehicle (saline) as described in the Materials and Methods section. Samples were collected 24 hours after induction of pancreatitis. Data represent means ± SEM and n = 4–7. #P < .05 vs control mice and ¤P < .05 vs taurocholate without DNase I or anti-Ly6G.
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3. Figure 2
(A) Representative H&E sections of the head of the pancreas from indicated groups. Scale bars: 100 μm. Histologic quantification of (B) acinar cell necrosis, (C) edema, (D) hemorrhage, and (E) leukocyte infiltration is shown from samples collected 24 hours after induction of pancreatitis. Pancreatitis (black bars) was induced by infusion of sodium taurocholate (5%) into the pancreatic duct. Control mice (grey bars) were infused with saline alone. Animals were treated with IP injections of the DNase I or vehicle (saline) as described in the Materials and Methods section. Data represent means ± SEM and n = 4–10. #P < .05 vs control mice and ¤P < .05 vs taurocholate without DNase I.
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4. Figure 3
Quantitative measurements of (A) blood amylase levels, (B) pancreatic MPO, lung MPO, and the number of neutrophils in the bronchoalveolar lavage fluid. (C) Pancreatic levels of CXCL2 and Mac-1 expression on circulating neutrophils. Pancreatitis (black bars) was induced by infusion of sodium taurocholate (5%) into the pancreatic duct. Control mice (grey bars) were infused with saline alone. Animals were treated with IP injections of the DNase I or vehicle (saline) as described in the Materials and Methods section. Samples were collected 24 hours after induction of pancreatitis. Data represent means ± SEM and n = 4–6. #P < .05 vs control mice and ¤P < .05 vs taurocholate without DNase I.
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5. Figure 4
Plasma levels of (A) HMGB1, (B) IL6, (C) CXCL2, and (D) MMP-9. Pancreatitis (black bars) was induced by infusion of sodium taurocholate (5%) into the pancreatic duct. Control mice (grey bars) were infused with saline alone. Animals were treated with IP injections of the DNase I or vehicle (saline) as described in the Materials and Methods section. Samples were collected 24 hours after induction of pancreatitis. Data represent means ± SEM and n = 4–5. #P < .05 vs control mice and ¤P < .05 vs taurocholate without DNase I.
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6. Figure 5
Trypsin activation and STAT3 phosphorylation. Trypsinogen activation was determined by measuring enzymatic activity of trypsin fluorometrically, using Boc-Glu-Ala-Arg-MCA as the substrate as described in detail in the Supplementary Materials and Methods section. Trypsin levels were calculated using a standard curve generated by assaying purified trypsin and normalized to protein concentrations. Total and phosphorylated STAT3 were determined by Western blot and the ratio of phosphorylated STAT3 divided by total STAT3 was quantified. (A) Acinar cells were co-incubated with caerulein (grey bars) or neutrophil-derived NETs (black bars) with or without DNase I. STAT3 phosphorylation was analyzed after incubation with neutrophil-derived NETs (black bars) with or without DNase I or saline (white bars) with or without DNase I. (B) Acinar cells were co-incubated with caerulein (grey bars) or histone 3 and histone 4 (black bars). STAT3 phosphorylation was analyzed after incubation with histone 3 or histone 4 (black bars) or saline (white bars). (C) Acinar cells were co-incubated with caerulein (grey bars) or neutrophil-derived NETs (black bars) with or without polysialic acid (PSA). Data represent means ± SEM and n = 5. #P < .05 control cells and ¤P < .05 vs NET alone.
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7. Figure 6
NET formation in L-arginine–induced AP. (A) Intravascular administration of Sytox Green was used to visualize extracellular DNA in the inflamed pancreas. (B) Quantification of extracellular DNA in the pancreas by measuring the relative area of fluorescence per high-power field. (C) Plasma levels of cf-DNA, (D) blood amylase levels, and (E) pancreatic and lung MPO activity. (F) Histologic quantification of acinar cell necrosis, leukocyte infiltration, hemorrhage, and edema were determined as described in the Materials and Methods section. Pancreatitis (black bars) was induced by IP challenge with L-arginine. Control mice (grey bars) were treated IP with saline alone. Animals were treated with IP injections of the DNase I or vehicle (saline) as described in the Materials and Methods section. Samples were collected 72 hours after induction of pancreatitis. Data represent means ± SEM and n = 6. #P < .05 vs control mice and ¤P < .05 vs L-arginine without DNase I.
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8. Figure 7
NET components in patients with AP. Plasma was drawn at admission and 24 hours after admission from patients with severe AP. Plasma levels of (A) cf-DNA and (B) DNA–histone complexes. Healthy individuals served as controls. Data represent means ± SEM and n = 10. #P < .05 vs healthy controls.
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9. Supplementary Figure 1
Plasma levels of DNase I were determined by use of enzyme-linked immunosorbent assay. Pancreatitis (black bars) was induced by infusion of sodium taurocholate (5%) into the pancreatic duct. Animals were treated with IP injections of the DNase I as described in the Materials and Methods section. Control mice (grey bars) were infused with saline alone. Samples were collected 6 and 24 hours after induction of pancreatitis. Data represent means ± SEM and n = 5. #P < .05 vs control mice.
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10. Supplementary Figure 2
Transmission electron microscopy of the indicated area of interest from Figure 1A incubated with gold-labeled anti-elastase antibodies (small gold particles, arrowheads) showing polymorphonuclear neutrophils containing granules (G) and elastase with the plasma membrane (PM) indicated. Scale bar: 0.5 μm. Pancreatitis was induced by infusion of sodium taurocholate (5%) into the pancreatic duct. (A) Control mice were infused with saline alone. Animals were treated with IP injections of (B) DNase I or (C) vehicle as described in the Materials and Methods section. Samples were collected 24 hours after induction of pancreatitis.
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11. Supplementary Figure 3
NET formation in AP. Quantification of extracellular DNA in the pancreas by measuring the relative area of fluorescence per high-power field. Pancreatitis (black bars) was induced by infusion of sodium taurocholate (5%) into the pancreatic duct. Control mice (grey bars) were infused with saline alone. Animals were treated with IP injections of DNase I or vehicle (saline) as described in the Materials and Methods section. Samples were collected 6 hours after induction of pancreatitis. Data represent means ± SEM and n = 5. #P < .05 vs control mice and *P < .05 vs taurocholate without DNase I.
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12. Supplementary Figure 4
Neutrophil depletion. An anti-Ly6G antibody or a control antibody (Control) was administered IP. The percentage of neutrophils (Ly6G+/Ly6C+ cells) was quantified by flow cytometry. Data represent means ± SEM and n = 4. #P < .05 vs control mice.
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13. Supplementary Figure 5
(A) Representative H&E sections of the lung from indicated groups. Pancreatitis (black bars) was induced by infusion of sodium taurocholate (5%) into the pancreatic duct. Control mice (grey bars) were infused with saline alone. Animals were treated with IP injections of the DNase I or vehicle (saline) as described in the Materials and Methods section. Histologic quantification of (B) alveolar collapse, (C) alveolar septa thickness, (D) neutrophil infiltration, and (E) alveolar fibrin deposition is shown from samples collected 24 hours after induction of pancreatitis. Scale bars: 100 μm. Data represent means ± SEM and n = 4–5. #P < .05 vs control mice and ¤P < .05 vs taurocholate without DNase I.
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14. Supplementary Figure 6
Mac-1 expression and ROS production in isolated neutrophils. (A) Aggregate data and representative histogram of Mac1-expression on neutrophils. (B) Quantification of ROS production in neutrophils by use of a fluorescence microplate reader. Murine neutrophils were isolated from bone marrow and incubated with neutrophil-derived NETs (black bars) or PMA (grey bars). Unstimulated cells (white bars) served as controls. Neutrophils were co-incubated with DNase I as indicated. Data represent means ± SEM and n = 5. #P < .05 vs control and ¤P < .05 vs NET without DNase I.
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15. Supplementary Figure 7
(A) Trypsin and (B) chymotrypsin activation were determined as described in detail in the Supplementary Materials and Methods section. Trypsin and chymotrypsin levels were calculated using a standard curve generated by assaying purified trypsin and chymotrypsin normalized to protein concentrations. Acinar cells were co-incubated with caerulein (grey bars) or histone 2A and histone 2B (black bars). Data represent means ± SEM and n = 5. #P < .05 control cells. RCU, relative chymotrypsin units; RTU, relative trypsin units.
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16. Supplementary Figure 8
Trypsinogen activation was determined by measuring enzymatic activity of trypsin fluorometrically using Boc-Glu-Ala-Arg-MCA as the substrate as described in detail in the Supplementary Materials and Methods section. Trypsin levels were calculated using a standard curve generated by assaying purified trypsin and normalized to protein concentration. Acinar cells were co-incubated with caerulein (black bars) with or without polysialic acid. Data represent means ± SEM and n = 5. RTU, relative trypsin units.
Gastroenterology  , e8DOI: ( /j.gastro )
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17. Supplementary Figure 9
Histone cytotoxicity. Viability of acinar cells was determined by the Trypan blue exclusion test as described in detail in the Supplementary Materials and Methods section. Acinar cells were co-incubated with 50 μg/mL of histone 2A, histone 2B, histone 3, or histone 4 for 60 minutes. Data represent means ± SEM and n = 10.
Gastroenterology  , e8DOI: ( /j.gastro )
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