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. 2016 Mar 11:7:10973.
doi: 10.1038/ncomms10973.

Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis

Moritz Leppkes  1 , Christian Maueröder  2 , Sebastian Hirth  3 , Stefanie Nowecki  1 , Claudia Günther  1 , Ulrike Billmeier  1 , Susanne Paulus  1 , Mona Biermann  2 , Luis E Munoz  2 , Markus Hoffmann  2 , Dane Wildner  1 , Andrew L Croxford  3 , Ari Waisman  3 , Kerri Mowen  4   5 , Dieter E Jenne  6 , Veit Krenn  7 , Julia Mayerle  8 , Markus M Lerch  8 , Georg Schett  2 , Stefan Wirtz  1 , Markus F Neurath  1 , Martin Herrmann  2 , Christoph Becker  1
Affiliations

Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis (V体育2025版)

Moritz Leppkes et al. Nat Commun. .

Abstract

Ductal occlusion has been postulated to precipitate focal pancreatic inflammation, while the nature of the primary occluding agents has remained elusive. Neutrophils make use of histone citrullination by peptidyl arginine deiminase-4 (PADI4) in contact to particulate agents to extrude decondensed chromatin as neutrophil extracellular traps (NETs). In high cellular density, NETs form macroscopically visible aggregates. Here we show that such aggregates form inside pancreatic ducts in humans and mice occluding pancreatic ducts and thereby driving pancreatic inflammation VSports手机版. Experimental models indicate that PADI4 is critical for intraductal aggregate formation and that PADI4-deficiency abrogates disease progression. Mechanistically, we identify the pancreatic juice as a strong instigator of neutrophil chromatin extrusion. Characteristic single components of pancreatic juice, such as bicarbonate ions and calcium carbonate crystals, induce aggregated NET formation. Ductal occlusion by aggregated NETs emerges as a pathomechanism with relevance in a plethora of inflammatory conditions involving secretory ducts. .

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Figures

Figure 1
Figure 1. Delivery of IL-17A induces pancreatitis with intraductal neutrophils.
(a,b) Haematoxylin and eosin staining of tissue sections obtained from human chronic pancreatitis (a), malignancy-related pancreatic inflammation (b) and murine pancreatitis induced by IL-17A delivery. (specific features are marked in the lower picture: +, fatty degeneration; §, fibrotic stroma; *, pseudotubular complex; dotted line and arrow, healthy area). (a) Note the focal nature of the disease with remodelled adjacent to intact glandular tissue (representative of n=13 (human) and n⩾15 (mouse); bar, 500 μm); (b) note cell-containing aggregates (coloured) inside pancreatic ducts (grey scale) in murine (15/15) and human samples (0/5 benign chronic pancreatitis, 3/8 malignancy associated). (c) Top: cells with segmented nuclei inside intraductal aggregates in human pancreatitis showed dual labelling for MPO and IL-17A (3/3); bottom: a close-up shows IL-17A immunoreactivity in a human granulocyte with a typical segmented nucleus (bar, 5 μm). (d) In IL-17A-induced pancreatitis, IL-17A+MPO+ aggregates were observed in the lumen of acini and ducts. No MPO- or IL-17A-positive cells were observed in control tissue (n=4 per group). (e) Note wasting in IL-17A-treated mice (n=8 per group, mean+s.e.m.). (f) Analysis by flow cytometry of bone marrow and spleen cells demonstrated IL-17A-enforced granulopoesis and neutrophil mobilization (n⩾15 per group). (g) Luminol bioluminescence imaging showed MPO activity in the upper abdomen of IL-17A-treated mice only, projected on the mesenteric part of the pancreas (n=4 per group; bars, 1 cm). (h) Tryptic activity of pancreas homogenates was fluorometrically assessed and showed a significant increase after IL-17A delivery as compared with control (two independent experiments, n=6 per group). (i,j) Analyses at day 10 and day 28 after IL-17A delivery demonstrated leukocyte infiltration at day 10, diminishing over time and a progressive remodelling of the pancreas. The control vector does not induce morphologic changes (n⩾8 per time point and group). (i) Immunohistochemistry reveals infiltration of neutrophils and macrophages into the pancreas 10 days after IL-17A delivery (n⩾10 per group). MPO+ cells displays patchy aggregation. After 28 days, MPO+ aggregates were found inside a pancreatic duct and in the periductal area. The aggregates are negative for F4/80, which is confined to myeloid cells throughout the fibroinflammatory stroma. Black scale bars, 200 μm; white scale bars, 50 μm, unless stated otherwise. (*P<0.05, ***P<0.001, Student's t-test).
Figure 2
Figure 2. IL-17A-induced pancreatitis is driven by PADI4-dependent neutrophil aggregates.
(a) Ly6G immunohistochemistry detected neutrophil granulocyte aggregates in the pancreas after IL-17A challenge only. (b,c) Mice treated with IL-17A delivery or control were injected with a neutrophil-depleting anti-Ly6G antibody (1A8) or isotype control (2A3) (three independent experiments with n=12 per group, ***P<0.001 (analysis of variance, Tukey honest significant difference). (b) Haematoxylin and eosin (H&E) staining (top) and MPO immunofluorescence (bottom) showed that IL-17A-induced pancreatitis depends on Ly6G+ granulocytes. (c) The area subject to fibroinflammatory remodelling was strongly reduced by anti-Ly6G treatment. The isotype antibody was without protective effect. (mean+s.e.m.). (d) A close-up of an aggregate after IL-17A delivery displayed neutrophils bound together by amorphous haematoxylin-stained fibres (day 28; top left). 4,6-Diamidino-2-phenylindole (DAPI) staining revealed DNA configured in non-nuclear morphology inside these ducts (bottom left). Immunohistochemistry of citrullinated histone H3 (H3cit) was detected on extracellular DNA (centre, right: staining control). (e) EpCAM/H3cit co-staining revealed aggregates formed in pancreatic ducts and metaplastic acini (day 10 after IL-17A delivery). Co-staining of neutrophil elastase (ELANE), Cramp and H3cit showed ELANE and Cramp on intraductal aggregates with PADI4 activity, even in areas, in which extracellular DNA was below detection level. The cellular density of intact neutrophils inside aggregates was increased at day 10 as compared with day 28, precluding a closer examination of non-nuclear DNA. H3cit was also detectable inside granulocyte nuclei at this time point. (f) Thioglycolate-elicited neutrophils from wild-type and PADI4−/− mice were stimulated with lipopolysaccharide (100 ng ml−1), and chromatin morphology was determined by Sytox Green fluorescence. Wild-type cells showed an increased size of the area covered by DNA and bizarrely configured DNA tangles, reflecting chromatin decondensation. Chromatin appeared more condensed in PADI4−/− neutrophils (>3 independent experiments). (g–i) Wild-type and PADI4−/− mice were treated with IL-17A or a mock control vector. (g) H&E-staining revealed no pancreatitis development in PADI4−/− mice in response to IL-17A. (h) MPO and ELANE immunohistochemistry showed only few MPO+ or ELANE+ cells in the pancreata of PADI4−/− IL-17A-challenged mice. In wild-type controls, patchy neutrophil accumulation was noted all-over the section (two independent experiments with a total of n⩾12 per group. (i) The area affected by fibroinflammatory remodelling was strongly diminished in PADI4−/− mice (mean+s.e.m.; black scale bars, 200 μm; white scale bars, 50 μm, unless stated otherwise. ***P<0.001, Student's t-test).
Figure 3
Figure 3. Components of pancreatic juice facilitate externalization of decondensed neutrophil chromatin.
(a) Immunofluorescence of myeloperoxidase (MPO) and citrullinated histone H3 (H3cit) revealed intraductal neutrophil aggregates in human pancreatic inflammation (immunopositivity rates of samples: malignancy-related pancreatitis (3/8) and benign chronic (0/5) pancreatitis, respectively); single channels are provided to appreciate low-intensity extracellular DNA between intact neutrophils. (b) Intraductal aggregates display CD66b+ cells and co-labelling of H3cit and extracellular DNA (3/3). (c) Cytospins from fresh patient-derived pancreatic fluid punctates of patients with benign pancreatitis revealed DNA webs positive for (left) neutrophil elastase (ELANE) and (right) H3cit with the respective staining control (right column; n=3/3). (d,e) Freshly isolated human blood neutrophils were cultured in human pancreatic juice or isotonic HBSS-based buffers containing varying amounts of NaHCO3, as indicated. DNA in the cell culture was quantified with a Sytox Green fluorimetric assay detecting extracellular DNA and chromatin of permeabilized cells only (DNASYTOX). (d) Quantitative assessment indicated strong increases in DNASYTOX of human neutrophil cultures in response to human pancreatic juice (n=20; 3 h of stimulation). (e) NaHCO3 dose dependently raises DNASYTOX as compared with NaHCO3-free conditions. This effect was facilitated by ambient pCO2 levels. Five percent of CO2 was able to inhibit DNA detection induced by 10 mM NaHCO3, yet failed to inhibit DNA detection at higher concentrations of NaHCO3 (50 mM; n=3 independent experiments, mean+s.e.m.). (f) Freshly isolated human blood neutrophils grown on coverslips developed decondensed chromatin and aggregates positive for (left) neutrophil elastase and citrullinated histones (right) in response to calcium carbonate crystals (5 mg ml−1, ⩾4 independent experiments). (All white scale bars, 50 μm. *P<0.05, **P<0.01, ***P<0.001, Student's t-test.)
Figure 4
Figure 4. NaHCO3-induced cellular changes support PADI4 activity.
(a) Freshly isolated human neutrophils from the peripheral blood of healthy donors were cultured on glass coverslips for 120 min at 37 °C/5% CO2 and subjected to isotonic HBSS media containing different concentrations of HCO3−. The calculated pH equilibrium of the bicarbonate–CO2 buffer system of these media at 37 °C/5% CO2 is specified. Immunocytochemistry of H3cit (top) and neutrophil elastase (bottom), and the respective DNASYTOX counterstain in both overlay as well as single-channel analyses are provided. Please note the absence of H3cit+ ELANE+ extracellular chromatin in the absence of NaHCO3. The presence of NaHCO3 in the media leads to marked increases in H3cit+ ELANE+ extracellular chromatin reminiscent of NETs (representative pictures of one of three independent experiments are shown). (b) Ratiometric determination of the cytosolic Ca2+ concentration revealed the elevation of [Ca2+]I induced by 75 mM NaHCO3 (n=4 independent experiments). (c) The intracellular pH of human neutrophils was measured by flow cytometry employing SNARF as pH-sensitive dye under ambient pCO2. Note the time- and bicarbonate-concentration-dependent increase of the cytoplasmic pH (n=3 independent experiments, mean+s.e.m.). (d) Chromatin externalization by 50 mM bicarbonate was induced in human granulocytes from healthy donors in the presence or absence of the PADI inhibitor Cl-amidine (1 mM) and images of propidium iodide fluorescence as in Supplementary Fig. 8H were morphometrically analysed. The nuclear decondensation is reflected by an increased ratio of chromatin area to mean fluorescence intensity (MFI) in flow cytometry. The data are normalized to 100 for an average healthy nucleus. Note the nuclear decondensation induced by bicarbonate, which was reduced in the presence of Cl-amidine (⩾3 independent experiments). (All white scale bars, 50 μm. *P<0.05, **P<0.01, ***P<0.001, Student's t-test).
Figure 5
Figure 5. Intraductal formation of carbonate-induced aggNETs causes segmental pancreatic atrophy.
(a–c) Neutrophil-rich peritonitis was induced in mice by means of intraperitoneal thioglycolate injection. After 18 h, 2 ml of 150 mM sodium bicarbonate, calcium carbonate crystals (20 mg) or 2 ml of saline control were injected into the peritoneal cavity. Thirty minutes later, a peritoneal HBSS lavage was aspirated, aggregates were collected and processed for consecutive immunocytochemistry; (a) note that aggNETs visible to the naked eye could only be aspirated after injection of NaHCO3 but not in saline control (b). Carbonate-induced aggNETs formed in vivo were immunopositive for citrullinated histone H3 and neutrophil elastase (ELANE) (three independent experiments of a total of n=6 per group). (c) Model of the aggNET transfer technique. We carefully transferred calcium carbonate crystals (0.5 mg) and thioglycolate-induced neutrophils (106 cells) in 10 μl each to the biliopancreatic duct to form aggNETs in situ, as well as saline or single-component controls. The biliary duct was ligated with a suture close to the liver hilus. (d) Intraoperative situs post aggNET transfer. The biliopancreatic duct filled with aggNETs and the stomach are marked by arrows and asterisks, respectively (bar, 10 mm). (e) Pancreatic duct filled with an aggregate after in situ aggNET formation (bar, 100 μm). (f) Immunohistochemical detection of citrullinated histone and myeloperoxidase in intraductal aggNETs 4 h after aggNET transfer. (g) Haematoxylin and eosin staining and (i) vimentin immunohistochemistry revealed the marked segmental fibroinflammatory area and mesenchymal cell expansion 6 days after aggNET transfer. (h) The sectional area of the pancreas affected by fibroinflammatory remodelling 6 days after aggNET transfer was calculated for each experimental group (two independent experiments, n=6 per group, mean+s.e.m., **P<0.01 one-way analysis of variance/post hoc Tukey honest significant difference analysis). (j–l) Crystals and neutrophil preparations of PADI4-deficient and wild-type mice were placed in biliopancreatic ducts of the respective recipients of the same genotype (two independent experiments, n=9 per group, **P<0.01 Student's t-test). In both experimental groups, fibroinflammatory remodelling was evident (j), yet the affected fibroinflammatory area was markedly reduced in PADI4-deficient mice (mean+s.e.m.) (k). Mesenchymal expansion as assessed by vimentin immunohistochemistry was attenuated as compared with wild-type controls (l). Black scale bars, 200 μm; white scale bars, 50 μm, unless stated otherwise.
Figure 6
Figure 6. Model of neutrophil-mediated ductal occlusion.
Model of intrapancreatic intraductal neutrophil accumulation in response to IL-17A and its downstream targets followed by increased intraductal chromatin extrusion and aggregation in response to HCO3− leading to microocclusion on the acinar level (left) or macroocclusion on the lobular ductal level (right).
Figure 7
Figure 7. Hypothesis chart.
Danger signals, exemplified by IL-17A challenge, instigate enforced granulopoesis, neutrophil mobilization and increased chemoattraction to the pancreas. When neutrophils encounter stimuli in the pancreatic juice such as elevated bicarbonate levels or CaCO3 precipitations, they form aggNETs. The large chromatin tangles of the latter reduce the fluidity of the pancreatic juice and consequently hamper secretory flow and lead to focal occlusion of the ductal tree. In occluded areas, digestive zymogens undergo premature activation. AggNET-borne serine proteases in static fluid may amplify this process. Dependent acini are destroyed, inflammation is perpetuated and finally tissue remodelling ensues.
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Comment in

  • Pancreatitis: NETs clog pancreatic ducts.
    Dickson I. Dickson I. Nat Rev Gastroenterol Hepatol. 2016 May;13(5):252. doi: 10.1038/nrgastro.2016.57. Epub 2016 Apr 1. Nat Rev Gastroenterol Hepatol. 2016. PMID: 27033127 No abstract available.

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