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Inhibition of PAD2 Improves Survival in a Mouse Model of Lethal LPS-Induced Endotoxic Shock

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Abstract

Endotoxemia induced by lipopolysaccharide (LPS) is an extremely severe syndrome identified by global activation of inflammatory responses. Neutrophil extracellular traps (NETs) play an important role in the development of endotoxemia. Histone hypercitrullination catalyzed by peptidylarginine deiminases (PADs) is a key step of NET formation. We have previously demonstrated that simultaneous inhibition of PAD2 and PAD4 with pan-PAD inhibitors can decrease NETosis and improve survival in a mouse model of LPS-induced endotoxic shock. However, the effects of PAD2 specific inhibition during NETosis and endotoxic shock are poorly understood. Therefore, in the present study, we aimed to investigate the effect of the specific PAD2 or PAD4 inhibitor on LPS-induced endotoxic shock in mice. We found that PAD2 inhibition but not PAD4 inhibition improves survival. Also, the levels of proinflammatory cytokines and NETosis were significantly reduced by PAD2 inhibitor. To our knowledge, this study demonstrates for the first time that PAD2 inhibition can reduce NETosis, decrease inflammatory cytokine production, and protect against endotoxin-induced lethality. Our findings provided a novel therapeutic strategy for the treatment of endotoxic shock.

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References

  1. Kulp, A., and M.J. Kuehn. 2010. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annual Review of Microbiology 64: 163–184. https://doi.org/10.1146/annurev.micro.091208.073413.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Opal, S.M. 2010. Endotoxins and other sepsis triggers. Contributions to Nephrology 167: 14–24. https://doi.org/10.1159/000315915.

    Article  CAS  PubMed  Google Scholar 

  3. Ramnath, R.D., S.W. Ng, A. Guglielmotti, and M. Bhatia. 2008. Role of MCP-1 in endotoxemia and sepsis. International Immunopharmacology 8 (6): 810–818. https://doi.org/10.1016/j.intimp.2008.01.033.

    Article  CAS  PubMed  Google Scholar 

  4. Grigoleit, J.S., J.R. Oberbeck, P. Lichte, P. Kobbe, O.T. Wolf, T. Montag, A. del Rey, E.R. Gizewski, H. Engler, and M. Schedlowski. 2010. Lipopolysaccharide-induced experimental immune activation does not impair memory functions in humans. Neurobiology of Learning and Memory 94 (4): 561–567. https://doi.org/10.1016/j.nlm.2010.09.011.

    Article  CAS  PubMed  Google Scholar 

  5. Hirz, T., and C. Dumontet. 2016. Neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents. Journal of Visualized Experiments 109: e53846. https://doi.org/10.3791/53846.

    Article  CAS  Google Scholar 

  6. Yipp, B.G., and P. Kubes. 2013. NETosis: How vital is it? Blood 122 (16): 2784–2794. https://doi.org/10.1182/blood-2013-04-457671.

    Article  CAS  PubMed  Google Scholar 

  7. Brinkmann, V., U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, D.S. Weiss, Y. Weinrauch, and A. Zychlinsky. 2004. Neutrophil extracellular traps kill bacteria. Science 303 (5663): 1532–1535. https://doi.org/10.1126/science.1092385.

    Article  CAS  PubMed  Google Scholar 

  8. Camicia, G., R. Pozner, and G. de Larranaga. 2014. Neutrophil extracellular traps in sepsis. Shock 42 (4): 286–294. https://doi.org/10.1097/shk.0000000000000221.

    Article  CAS  PubMed  Google Scholar 

  9. Wong, S.L., M. Demers, K. Martinod, M. Gallant, Y. Wang, A.B. Goldfine, C.R. Kahn, and D.D. Wagner. 2015. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. Nature Medicine 21 (7): 815–819. https://doi.org/10.1038/nm.3887.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Li, R.H.L., G. Ng, and F. Tablin. 2017. Lipopolysaccharide-induced neutrophil extracellular trap formation in canine neutrophils is dependent on histone H3 citrullination by peptidylarginine deiminase. Veterinary Immunology and Immunopathology 193-194: 29–37. https://doi.org/10.1016/j.vetimm.2017.10.002.

    Article  CAS  PubMed  Google Scholar 

  11. Wang, S., and Y. Wang. 2013. Peptidylarginine deiminases in citrullination, gene regulation, health and pathogenesis. Biochimica et Biophysica Acta 1829 (10): 1126–1135. https://doi.org/10.1016/j.bbagrm.2013.07.003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wang, Y., M. Li, S. Stadler, S. Correll, P. Li, D. Wang, R. Hayama, L. Leonelli, H. Han, S.A. Grigoryev, C.D. Allis, and S.A. Coonrod. 2009. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. The Journal of Cell Biology 184 (2): 205–213. https://doi.org/10.1083/jcb.200806072.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang, X., M. Bolt, M.J. Guertin, W. Chen, S. Zhang, B.D. Cherrington, D.J. Slade, C.J. Dreyton, V. Subramanian, K.L. Bicker, P.R. Thompson, M.A. Mancini, J.T. Lis, and S.A. Coonrod. 2012. Peptidylarginine deiminase 2-catalyzed histone H3 arginine 26 citrullination facilitates estrogen receptor alpha target gene activation. Proceedings of the National Academy of Sciences of the United States of America 109 (33): 13331–13336. https://doi.org/10.1073/pnas.1203280109.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Liang, Y., B. Pan, H.B. Alam, Q. Deng, Y. Wang, E. Chen, B. Liu, Y. Tian, A.M. Williams, X. Duan, Y. Wang, J. Zhang, and Y. Li. 2018. Inhibition of peptidylarginine deiminase alleviates LPS-induced pulmonary dysfunction and improves survival in a mouse model of lethal endotoxemia. European Journal of Pharmacology 833: 432–440. https://doi.org/10.1016/j.ejphar.2018.07.005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Martinod, K., T.A. Fuchs, N.L. Zitomersky, S.L. Wong, M. Demers, M. Gallant, Y. Wang, and D.D. Wagner. 2015. PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock. Blood 125 (12): 1948–1956. https://doi.org/10.1182/blood-2014-07-587709.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Muth, A., V. Subramanian, E. Beaumont, M. Nagar, P. Kerry, P. McEwan, H. Srinath, K. Clancy, S. Parelkar, and P.R. Thompson. 2017. Development of a selective inhibitor of protein arginine deiminase 2. Journal of Medicinal Chemistry 60 (7): 3198–3211. https://doi.org/10.1021/acs.jmedchem.7b00274.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Park, S.Y., S. Shrestha, Y.J. Youn, J.K. Kim, S.Y. Kim, H.J. Kim, S.H. Park, W.G. Ahn, S. Kim, M.G. Lee, K.S. Jung, Y.B. Park, E.K. Mo, Y. Ko, S.Y. Lee, Y. Koh, M.J. Park, D.K. Song, and C.W. Hong. 2017. Autophagy primes neutrophils for neutrophil extracellular trap formation during sepsis. American Journal of Respiratory and Critical Care Medicine 196 (5): 577–589. https://doi.org/10.1164/rccm.201603-0596OC.

    Article  CAS  PubMed  Google Scholar 

  18. Deng, Q., T. Zhao, B. Pan, I.S. Dennahy, X. Duan, A.M. Williams, B. Liu, N. Lin, U.F. Bhatti, E. Chen, H.B. Alam, and Y. Li. 2018. Protective effect of tubastatin A in CLP-induced lethal sepsis. Inflammation 41 (6): 2101–2109. https://doi.org/10.1007/s10753-018-0853-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gotts, J.E., and M.A. Matthay. 2016. Sepsis: Pathophysiology and clinical management. Bmj 353: i1585. https://doi.org/10.1136/bmj.i1585.

    Article  PubMed  Google Scholar 

  20. Pan, B., H.B. Alam, W. Chong, J. Mobley, B. Liu, Q. Deng, Y. Liang, Y. Wang, E. Chen, T. Wang, M. Tewari, and Y. Li. 2017. CitH3: A reliable blood biomarker for diagnosis and treatment of endotoxic shock. Scientific Reports 7 (1): 8972. https://doi.org/10.1038/s41598-017-09337-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gloude, N.J., P. Khandelwal, N. Luebbering, D.T. Lounder, S. Jodele, M.N. Alder, A. Lane, A. Wilkey, K.E. Lake, B. Litts, and S.M. Davies. 2017. Circulating dsDNA, endothelial injury, and complement activation in thrombotic microangiopathy and GVHD. Blood 130 (10): 1259–1266. https://doi.org/10.1182/blood-2017-05-782870.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. van den Boogaard, M., B.P. Ramakers, N. van Alfen, S.P. van der Werf, W.F. Fick, C.W. Hoedemaekers, M.M. Verbeek, L. Schoonhoven, J.G. van der Hoeven, and P. Pickkers. 2010. Endotoxemia-induced inflammation and the effect on the human brain. Critical Care 14 (3): R81. https://doi.org/10.1186/cc9001.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Okeke, E.B., Z. Mou, N. Onyilagha, P. Jia, A.S. Gounni, and J.E. Uzonna. 2017. Deficiency of phosphatidylinositol 3-kinase delta signaling leads to diminished numbers of regulatory T cells and increased neutrophil activity resulting in mortality due to endotoxic shock. Journal of Immunology 199 (3): 1086–1095. https://doi.org/10.4049/jimmunol.1600954.

    Article  CAS  Google Scholar 

  24. Stiel, L., F. Meziani, and J. Helms. 2018. Neutrophil activation during septic shock. Shock 49 (4): 371–384. https://doi.org/10.1097/SHK.0000000000000980.

    Article  CAS  PubMed  Google Scholar 

  25. Delabranche, X., L. Stiel, F. Severac, A.C. Galoisy, L. Mauvieux, F. Zobairi, T. Lavigne, F. Toti, E. Anglès-Cano, F. Meziani, and J. Boisramé-Helms. 2017. Evidence of netosis in septic shock-induced disseminated intravascular coagulation. Shock 47 (3): 313–317. https://doi.org/10.1097/SHK.0000000000000719.

    Article  CAS  PubMed  Google Scholar 

  26. Czaikoski, P.G., J.M. Mota, D.C. Nascimento, F. Sonego, F.V. Castanheira, P.H. Melo, G.T. Scortegagna, et al. 2016. Neutrophil extracellular traps induce organ damage during experimental and clinical sepsis. PLoS One 11 (2): e0148142. https://doi.org/10.1371/journal.pone.0148142.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pieterse, E., N. Rother, C. Yanginlar, L.B. Hilbrands, and J. van der Vlag. 2016. Neutrophils discriminate between lipopolysaccharides of different bacterial sources and selectively release neutrophil extracellular traps. Frontiers in Immunology 7: 484. https://doi.org/10.3389/fimmu.2016.00484.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gupta, S., and M.J. Kaplan. 2016. The role of neutrophils and NETosis in autoimmune and renal diseases. Nature Reviews. Nephrology 12 (7): 402–413. https://doi.org/10.1038/nrneph.2016.71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Porto, B.N., and R.T. Stein. 2016. Neutrophil extracellular traps in pulmonary diseases: Too much of a good thing? Frontiers in Immunology 7: 311. https://doi.org/10.3389/fimmu.2016.00311.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. McDonald, B., R.P. Davis, S.J. Kim, M. Tse, C.T. Esmon, E. Kolaczkowska, and C.N. Jenne. 2017. Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. Blood 129 (10): 1357–1367. https://doi.org/10.1182/blood-2016-09-741298.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Shen, X.F., K. Cao, J.P. Jiang, W.X. Guan, and J.F. Du. 2017. Neutrophil dysregulation during sepsis: An overview and update. Journal of Cellular and Molecular Medicine 21 (9): 1687–1697. https://doi.org/10.1111/jcmm.13112.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kaufman, T., D. Magosevich, M.C. Moreno, M.A. Guzman, L.P. D'Atri, A. Carestia, M.E. Fandino, C. Fondevila, and M. Schattner. 2017. Nucleosomes and neutrophil extracellular traps in septic and burn patients. Clinical Immunology 183: 254–262. https://doi.org/10.1016/j.clim.2017.08.014.

    Article  CAS  PubMed  Google Scholar 

  33. Sakurai, K., T. Miyashita, M. Okazaki, T. Yamaguchi, Y. Ohbatake, S. Nakanuma, K. Okamoto, et al. 2017. Role for neutrophil extracellular traps (NETs) and platelet aggregation in early sepsis-induced hepatic dysfunction. In Vivo 31 (6): 1051–1058. https://doi.org/10.21873/invivo.11169.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Jimenez-Alcazar, M., C. Rangaswamy, R. Panda, J. Bitterling, Y.J. Simsek, A.T. Long, R. Bilyy, et al. 2017. Host DNases prevent vascular occlusion by neutrophil extracellular traps. Science 358 (6367): 1202–1206. https://doi.org/10.1126/science.aam8897.

    Article  CAS  PubMed  Google Scholar 

  35. Biron, B.M., C.S. Chung, X.M. O'Brien, Y. Chen, J.S. Reichner, and A. Ayala. 2017. Cl-amidine prevents histone 3 citrullination and neutrophil extracellular trap formation, and improves survival in a murine sepsis model. Journal of Innate Immunity 9 (1): 22–32. https://doi.org/10.1159/000448808.

    Article  CAS  PubMed  Google Scholar 

  36. Leshner, M., S. Wang, C. Lewis, H. Zheng, X.A. Chen, L. Santy, and Y. Wang. 2012. PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures. Frontiers in Immunology 3: 307. https://doi.org/10.3389/fimmu.2012.00307.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Kolaczkowska, E., C.N. Jenne, B.G. Surewaard, A. Thanabalasuriar, W.Y. Lee, M.J. Sanz, K. Mowen, G. Opdenakker, and P. Kubes. 2015. Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature. Nature Communications 6: 6673. https://doi.org/10.1038/ncomms7673.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Li, P., M. Li, M.R. Lindberg, M.J. Kennett, N. Xiong, and Y. Wang. 2010. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. The Journal of Experimental Medicine 207 (9): 1853–1862. https://doi.org/10.1084/jem.20100239.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Rohrbach, A.S., D.J. Slade, P.R. Thompson, and K.A. Mowen. 2012. Activation of PAD4 in NET formation. Frontiers in Immunology 3: 360. https://doi.org/10.3389/fimmu.2012.00360.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Asaga, H., K. Nakashima, T. Senshu, A. Ishigami, and M. Yamada. 2001. Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils. Journal of Leukocyte Biology 70 (1): 46–51.

    CAS  PubMed  Google Scholar 

  41. Takahara, H., M. Tsuchida, M. Kusubata, K. Akutsu, S. Tagami, and K. Sugawara. 1989. Peptidylarginine deiminase of the mouse. Distribution, properties, and immunocytochemical localization. The Journal of Biological Chemistry 264 (22): 13361–13368.

    CAS  PubMed  Google Scholar 

  42. Horibata, S., K.E. Rogers, D. Sadegh, L.J. Anguish, J.L. McElwee, P. Shah, P.R. Thompson, and S.A. Coonrod. 2017. Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration. BMC Cancer 17 (1): 378. https://doi.org/10.1186/s12885-017-3354-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Knight, J.S., V. Subramanian, A.A. O'Dell, S. Yalavarthi, W. Zhao, C.K. Smith, J.B. Hodgin, P.R. Thompson, and M.J. Kaplan. 2015. Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice. Annals of the Rheumatic Diseases 74 (12): 2199–2206. https://doi.org/10.1136/annrheumdis-2014-205365.

    Article  CAS  PubMed  Google Scholar 

  44. Muller, S., and M. Radic. 2015. Citrullinated autoantigens: From diagnostic markers to pathogenetic mechanisms. Clinical Reviews in Allergy and Immunology 49 (2): 232–239. https://doi.org/10.1007/s12016-014-8459-2.

    Article  CAS  PubMed  Google Scholar 

  45. Mishra, N., L. Schwerdtner, K. Sams, S. Mondal, F. Ahmad, R.E. Schmidt, S.A. Coonrod, P.R. Thompson, M.M. Lerch, and L. Bossaller. 2019. Cutting edge: Protein arginine deiminase 2 and 4 regulate NLRP3 inflammasome-dependent IL-1beta maturation and ASC speck formation in macrophages. Journal of Immunology 203 (4): 795–800. https://doi.org/10.4049/jimmunol.1800720.

    Article  CAS  Google Scholar 

  46. Ott, L.W., K.A. Resing, A.W. Sizemore, J.W. Heyen, R.R. Cocklin, N.M. Pedrick, H.C. Woods, J.Y. Chen, M.G. Goebl, F.A. Witzmann, and M.A. Harrington. 2007. Tumor necrosis factor-alpha- and interleukin-1-induced cellular responses: Coupling proteomic and genomic information. Journal of Proteome Research 6 (6): 2176–2185. https://doi.org/10.1021/pr060665l.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was funded by grants from Kickstart N022142 to Dr. Yongqing Li, UMHS-PUHSC Joint Institute U050150 and the National Institutes of Health Grant 5 R01 GM084127 to Dr. Hasan B. Alam and by the National Institutes of Health Grant R35 GM118112 to Paul R. Thompson.

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  1. Zhenyu Wu and Qiufang Deng contributed equally to this work.

Authors and Affiliations

  1. Department of Surgery, University of Michigan Medical School, Ann Arbor, MI, USA

    Zhenyu Wu, Qiufang Deng, Baihong Pan, Hasan B. Alam, Yuzi Tian, Umar F. Bhatti, Baoling Liu & Yongqing Li

  2. Department of Infectious Diseases, Xiangya Second Hospital, Changsha, Hunan, China

    Zhenyu Wu

  3. Department of Endocrinology, Xiangya Third Hospital, Changsha, Hunan, China

    Qiufang Deng

  4. Xiangya Hospital, Changsha, Hunan, China

    Baihong Pan & Yuzi Tian

  5. Department of Surgery, University of Michigan Hospital, Ann Arbor, MI, USA

    Hasan B. Alam & Yongqing Li

  6. University of Massachusetts School of Medicine, Worcester, MA, USA

    Santanu Mondal & Paul R. Thompson

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  1. Zhenyu Wu
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Contributions

YL and HA designed the study. QD and BP carried out the experiments. YL and ZW wrote the manuscript, and YL and HA made a critical revision. QD, BP, YT, and UFB reviewed manuscript, and XD, BL, YT, and ZW provided experimental support. All authors read and approved the final manuscript.

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Correspondence to Hasan B. Alam or Yongqing Li.

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The protocol for the animal experiments was approved by the University of Michigan Institutional Animal Care and Use Committee (PRO00008861). All experiments complied with animal welfare and research regulations.

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Wu, Z., Deng, Q., Pan, B. et al. Inhibition of PAD2 Improves Survival in a Mouse Model of Lethal LPS-Induced Endotoxic Shock. Inflammation 43, 1436–1445 (2020). https://doi.org/10.1007/s10753-020-01221-0

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