Extracellular cold-inducible RNA-binding protein regulates neutrophil extracellular trap formation and tissue damage in acute pancreatitis


Neutrophil extracellular traps (NETs) play a key role in the development of acute pancreatitis (AP). In the present study, we studied the role of extracellular cold-inducible RNA-binding protein (eCIRP), a novel damage-associated-molecular-pattern molecule, in severe AP. C57BL/6 mice underwent retrograde infusion of taurocholate into the pancreatic duct. C23, an eCIRP inhibitor, was given 1 h prior to induction of AP. Pancreatic, lung, and blood samples were collected and levels of citrullinated histone 3, DNA-histone complexes, eCIRP, myeloperoxidase (MPO), amylase, cytokines, matrix metalloproteinase-9 (MMP-9), and CXC chemokines were quantified after 24 h. NETs were detected by electron microscopy in the pancreas and bone marrow-derived neutrophils. Amylase secretion was analyzed in isolated acinar cells. Plasma was obtained from healthy individuals and patients with mild and moderate severe or severe AP. Taurocholate infusion induced NET formation, inflammation, and tissue injury in the pancreas. Pretreatment with C23 decreased taurocholate-induced pancreatic and plasma levels of eCIRP and tissue damage in the pancreas. Blocking eCIRP reduced levels of citrullinated histone 3 and NET formation in the pancreas as well as DNA-histone complexes in the plasma. In addition, administration of C23 attenuated MPO levels in the pancreas and lung of mice exposed to taurocholate. Inhibition of eCIRP reduced pancreatic levels of CXC chemokines and plasma levels of IL-6, HMGB-1, and MMP-9 in mice with severe AP. Moreover, eCIRP was found to be bound to NETs. Coincubation with C23 reduced NET-induced amylase secretion in isolated acinar cells. Patients with severe AP had elevated plasma levels of eCIRP compared with controls. Our novel findings suggest that eCIRP is a potent regulator of NET formation in the inflamed pancreas. Moreover, these results show that targeting eCIRP with C23 inhibits inflammation and tissue damage in AP. Thus, eCIRP could serve as an effective target to attenuate pancreatic damage in patients with AP.

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Fig. 1: eCIRP and NETs formation in AP.
Fig. 2: NETs formation in AP.
Fig. 3: Quantitative measurements of blood amylase levels.
Fig. 4: Pancreatic tissue damage in AP.
Fig. 5: Neutrophil recruitment and chemokines in the inflamed pancreas.
Fig. 6: Neutrophil recruitment in the lung and systemic inflammation during AP.
Fig. 7: In vitro experiment showing eCIRP in NETs and amylase secretion by acinar cells.
Fig. 8: NF-κB signalling in AP.
Fig. 9: eCIRP in patients with AP.


  1. 1.

    Isenmann R, Beger HG. Natural history of acute pancreatitis and the role of infection. Baillieres Best Pract Res Clin Gastroenterol. 1999;13:291–301.

    CAS  PubMed  Google Scholar 

  2. 2.

    Bhatia M, Wong FL, Cao Y, Lau HY, Huang J, Puneet P, et al. Pathophysiology of acute pancreatitis. Pancreatology. 2005;5:132–44.

    PubMed  Google Scholar 

  3. 3.

    Abdulla A, Awla D, Thorlacius H, Regner S. Role of neutrophils in the activation of trypsinogen in severe acute pancreatitis. J Leukoc Biol. 2011;90:975–82.

    CAS  PubMed  Google Scholar 

  4. 4.

    Regner S, Manjer J, Appelros S, Hjalmarsson C, Sadic J, Borgstrom A. Protease activation, pancreatic leakage, and inflammation in acute pancreatitis: differences between mild and severe cases and changes over the first three days. Pancreatology. 2008;8:600–7.

    CAS  PubMed  Google Scholar 

  5. 5.

    Sandoval D, Gukovskaya A, Reavey P, Gukovsky S, Sisk A, Braquet P, et al. The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis. Gastroenterology. 1996;111:1081–91.

    CAS  PubMed  Google Scholar 

  6. 6.

    Frossard JL, Saluja A, Bhagat L, Lee HS, Bhatia M, Hofbauer B, et al. The role of intercellular adhesion molecule 1 and neutrophils in acute pancreatitis and pancreatitis-associated lung injury. Gastroenterology. 1999;116:694–701.

    CAS  PubMed  Google Scholar 

  7. 7.

    Bhatia M, Hegde A. Treatment with antileukinate, a CXCR2 chemokine receptor antagonist, protects mice against acute pancreatitis and associated lung injury. Regul Pept. 2007;138:40–8.

    CAS  PubMed  Google Scholar 

  8. 8.

    Awla D, Abdulla A, Syk I, Jeppsson B, Regner S, Thorlacius H. Neutrophil-derived matrix metalloproteinase-9 is a potent activator of trypsinogen in acinar cells in acute pancreatitis. J Leukoc Biol. 2012;91:711–9.

    CAS  PubMed  Google Scholar 

  9. 9.

    Castanheira FVS, Kubes P. Neutrophils and NETs in modulating acute and chronic inflammation. Blood. 2019;133:2178–85.

    CAS  PubMed  Google Scholar 

  10. 10.

    Merza M, Hartman H, Rahman M, Hwaiz R, Zhang E, Renstrom E, et al. Neutrophil extracellular traps induce trypsin activation, inflammation, and tissue damage in mice with severe acute pancreatitis. Gastroenterology. 2015;149:1920–31 e8.

    CAS  PubMed  Google Scholar 

  11. 11.

    Madhi R, Rahman M, Taha D, Morgelin M, Thorlacius H. Targeting peptidylarginine deiminase reduces neutrophil extracellular trap formation and tissue injury in severe acute pancreatitis. J Cell Physiol. 2019;234:11850–60.

    CAS  PubMed  Google Scholar 

  12. 12.

    De Leeuw F, Zhang T, Wauquier C, Huez G, Kruys V, Gueydan C. The cold-inducible RNA-binding protein migrates from the nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as a translational repressor. Exp Cell Res. 2007;313:4130–44.

    PubMed  Google Scholar 

  13. 13.

    Qiang X, Yang WL, Wu R, Zhou M, Jacob A, Dong W, et al. Cold-inducible RNA-binding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis. Nat Med. 2013;19:1489–95.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Zhou Y, Dong H, Zhong Y, Huang J, Lv J, Li J. The cold-inducible RNA-binding protein (CIRP) level in peripheral blood predicts sepsis outcome. PLoS One. 2015;10:e0137721.

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Yoo IS, Lee SY, Park CK, Lee JC, Kim Y, Yoo SJ, et al. Serum and synovial fluid concentrations of cold-inducible RNA-binding protein in patients with rheumatoid arthritis. Int J Rheum Dis. 2018;21:148–54.

    CAS  PubMed  Google Scholar 

  16. 16.

    Gong JD, Qi XF, Zhang Y, Li HL. Increased admission serum cold-inducible RNA-binding protein concentration is associated with prognosis of severe acute pancreatitis. Clin Chim Acta. 2017;471:135–42.

    CAS  PubMed  Google Scholar 

  17. 17.

    Zhang F, Brenner M, Yang WL, Wang P. A cold-inducible RNA-binding protein (CIRP)-derived peptide attenuates inflammation and organ injury in septic mice. Sci Rep. 2018;8:3052.

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Yang WL, Sharma A, Wang Z, Li Z, Fan J, Wang P. Cold-inducible RNA-binding protein causes endothelial dysfunction via activation of Nlrp3 inflammasome. Sci Rep. 2016;6:26571.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Ode Y, Aziz M, Jin H, Arif A, Nicastro JG, Wang P. Cold-inducible RNA-binding protein induces neutrophil extracellular traps in the lungs during sepsis. Sci Rep. 2019;9:6252.

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    McGinn J, Zhang F, Aziz M, Yang WL, Nicastro J, Coppa GF, et al. The protective effect of a short peptide derived from cold-inducible RNA-binding protein in renal ischemia-reperfusion injury. Shock. 2018;49:269–76.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Laukkarinen JM, Van Acker GJ, Weiss ER, Steer ML, Perides G. A mouse model of acute biliary pancreatitis induced by retrograde pancreatic duct infusion of Na-taurocholate. Gut. 2007;56:1590–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Luo L, Zhang S, Wang Y, Rahman M, Syk I, Zhang E, et al. Proinflammatory role of neutrophil extracellular traps in abdominal sepsis. Am J Physiol Lung Cell Mol Physiol. 2014;307:L586–96.

    CAS  PubMed  Google Scholar 

  23. 23.

    Schmidt J, Rattner DW, Lewandrowski K, Compton CC, Mandavilli U, Knoefel WT, et al. A better model of acute pancreatitis for evaluating therapy. Ann Surg. 1992;215:44–56.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Saluja AK, Bhagat L, Lee HS, Bhatia M, Frossard JL, Steer ML. Secretagogue-induced digestive enzyme activation and cell injury in rat pancreatic acini. Am J Physiol. 1999;276:G835–42.

    CAS  PubMed  Google Scholar 

  25. 25.

    Banks PA, Bollen TL, Dervenis C, Gooszen HG, Johnson CD, Sarr MG, et al. Classification of acute pancreatitis-2012: revision of the Atlanta classification and definitions by international consensus. Gut. 2013;62:102–11.

    PubMed  Google Scholar 

  26. 26.

    Aziz M, Brenner M, Wang P. Extracellular CIRP (eCIRP) and inflammation. J Leukoc Biol. 2019;106:133–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Zhong P, Huang H. Recent progress in the research of cold-inducible RNA-binding protein. Future Sci OA. 2017;3:FSO246.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    McGinn JT, Aziz M, Zhang F, Yang WL, Nicastro JM, Coppa GF, et al. Cold-inducible RNA-binding protein-derived peptide C23 attenuates inflammation and tissue injury in a murine model of intestinal ischemia-reperfusion. Surgery. 2018;164:1191–7.

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303:1532–5.

    CAS  PubMed  Google Scholar 

  30. 30.

    Byrd AS, O’Brien XM, Johnson CM, Lavigne LM, Reichner JS. An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans. J Immunol. 2013;190:4136–48.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Pilsczek FH, Salina D, Poon KK, Fahey C, Yipp BG, Sibley CD, et al. A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol. 2010;185:7413–25.

    CAS  PubMed  Google Scholar 

  32. 32.

    Wang J, Hossain M, Thanabalasuriar A, Gunzer M, Meininger C, Kubes P. Visualizing the function and fate of neutrophils in sterile injury and repair. Science. 2017;358:111–6.

    CAS  PubMed  Google Scholar 

  33. 33.

    Leppkes M, Maueroder C, Hirth S, Nowecki S, Gunther C, Billmeier U, et al. Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis. Nat Commun. 2016;7:10973.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Ode Y, Aziz M, Wang P. CIRP increases ICAM-1(+) phenotype of neutrophils exhibiting elevated iNOS and NETs in sepsis. J Leukoc Biol. 2018;103:693–707.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Zhang F, Yang WL, Brenner M, Wang P. Attenuation of hemorrhage-associated lung injury by adjuvant treatment with C23, an oligopeptide derived from cold-inducible RNA-binding protein. J Trauma Acute Care Surg. 2017;83:690–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Godwin A, Yang WL, Sharma A, Khader A, Wang Z, Zhang F, et al. Blocking cold-inducible RNA-binding protein protects liver from ischemia-reperfusion injury. Shock. 2015;43:24–30.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Arita K, Hashimoto H, Shimizu T, Nakashima K, Yamada M, Sato M. Structural basis for Ca(2+)-induced activation of human PAD4. Nat Struct Mol Biol. 2004;11:777–83.

    CAS  PubMed  Google Scholar 

  38. 38.

    Awla D, Abdulla A, Zhang S, Roller J, Menger MD, Regner S, et al. Lymphocyte function antigen-1 regulates neutrophil recruitment and tissue damage in acute pancreatitis. Br J Pharmacol. 2011;163:413–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Hartman H, Abdulla A, Awla D, Lindkvist B, Jeppsson B, Thorlacius H, et al. P-selectin mediates neutrophil rolling and recruitment in acute pancreatitis. Br J Surg. 2012;99:246–55.

    CAS  PubMed  Google Scholar 

  40. 40.

    Bacon KB, Oppenheim JJ. Chemokines in disease models and pathogenesis. Cytokine Growth Factor Rev. 1998;9:167–73.

    CAS  PubMed  Google Scholar 

  41. 41.

    Li X, Klintman D, Liu Q, Sato T, Jeppsson B, Thorlacius H. Critical role of CXC chemokines in endotoxemic liver injury in mice. J Leukoc Biol. 2004;75:443–52.

    CAS  PubMed  Google Scholar 

  42. 42.

    Zhang H, Neuhofer P, Song L, Rabe B, Lesina M, Kurkowski MU, et al. IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality. J Clin Invest. 2013;123:1019–31.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Urban CF, Ermert D, Schmid M, Abu-Abed U, Goosmann C, Nacken W, et al. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathog. 2009;5:e1000639.

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Wang Y, Du F, Hawez A, Morgelin M, Thorlacius H. Neutrophil extracellular trap-microparticle complexes trigger neutrophil recruitment via high-mobility group protein 1 (HMGB1)-toll-like receptors(TLR2)/TLR4 signalling. Br J Pharmacol. 2019;176:3350–63.

    CAS  PubMed  Google Scholar 

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Swedish Medical Research Council (2017–01621) and Einar and Inga Nilsson foundation. Raed Madhi is supported by a doctoral fellowship from Misan University, College of Science, Iraq.

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Correspondence to Henrik Thorlacius.

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Linders, J., Madhi, R., Rahman, M. et al. Extracellular cold-inducible RNA-binding protein regulates neutrophil extracellular trap formation and tissue damage in acute pancreatitis. Lab Invest (2020). https://doi.org/10.1038/s41374-020-0469-5

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