Inhibition of Netosis with PAD Inhibitor Attenuates Endotoxin Shock Induced Systemic Inflammation
Abstract
:1. Introduction
2. Results
2.1. Chloroacetamide Based PAD Inhibitor YW3-56 Inhibits Histone Citrullination and NET Formation
2.2. YW3-56 Decreased Endotoxin LPS-Induced Mouse Death and Serum NETs, cfDNA, TNFα, IL-6, IL-1β Levels
2.3. YW3-56 Inhibited Netosis, Decreased TNFα, IL-6 and cfDNA Levels and Increased the Amounts of Neutrophils in Peritoneal Cavity
2.4. YW3-56 Inhibits Endotoxin Shock Induced Lung Inflammation
2.5. YW3-56 Reversed the Expression of Inflammatory Genes Elevated in Lung Tissues by LPS
3. Discussion
4. Materials and Methods
4.1. Healthy Blood Donors
4.2. Animals and LPS-Induced Lethal Endotoxic Shock
4.3. Reagents
4.4. PAD4 Functional Assay
4.5. Human Blood Neutrophils Isolation
4.6. Mouse Bone Marrow Neutrophils Isolation
4.7. Immunocytochemistry Staining
4.8. Immunohistochemistry and H & E Staining
4.9. Peritoneal Lavage Fluid (PLF) and Bronchoalveolar Lavage Fluid (BALF) Collection
4.10. Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
4.11. Western Blot Analysis
4.12. NETs, cfDNA and Cytokines Analysis in Serum, PLF and BALF
4.13. Flow Cytometry Detection
4.14. RNA Sequencing Analyses
4.15. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Uhl, B.; Vadlau, Y.; Zuchtriegel, G.; Nekolla, K.; Sharaf, K.; Gaertner, F.; Massberg, S.; Krombach, F.; Reichel, C.A. Aged neutrophils contribute to the first line of defense in the acute inflammatory response. Blood 2016, 128, 2327–2337. [Google Scholar] [CrossRef] [Green Version]
- Fukata, M.; Vamadevan, A.S.; Abreu, M.T. Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in inflammatory disorders. Semin. Immunol. 2009, 21, 242–253. [Google Scholar] [CrossRef] [PubMed]
- Beutler, B.; Rietschel, E.T. Innate immune sensing and its roots: The story of endotoxin. Nat. Rev. Immunol. 2003, 3, 169–176. [Google Scholar] [CrossRef]
- Brinkmann, V.; Reichard, U.; Goosmann, C.; Fauler, B.; Uhlemann, Y.; Weiss, D.S.; Weinrauch, Y.; Zychlinsky, A. Neutrophil extracellular traps kill bacteria. Science 2004, 303, 1532–1535. [Google Scholar] [CrossRef] [PubMed]
- Urban, C.F.; Ermert, D.; Schmid, M.; Abu-Abed, U.; Goosmann, C.; Nacken, W.; Brinkmann, V.; Jungblut, P.R.; Zychlinsky, A. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS. Pathog. 2009, 5, e1000639. [Google Scholar] [CrossRef] [Green Version]
- Vossenaar, E.R.; Zendman, A.J.; van Venrooij, W.J.; Pruijn, G.J. PAD, a growing family of citrullinating enzymes: Genes, features and involvement in disease. Bioessays 2003, 25, 1106–1118. [Google Scholar] [CrossRef]
- Darrah, E.; Rosen, A.; Giles, J.T.; Andrade, F. Peptidylarginine deiminase 2, 3 and 4 have distinct specificities against cellular substrates: Novel insights into autoantigen selection in rheumatoid arthritis. Ann. Rheum. Dis. 2012, 71, 92–98. [Google Scholar] [CrossRef] [Green Version]
- Ying, S.; Dong, S.; Kawada, A.; Kojima, T.; Chavanas, S.; Mechin, M.C.; Adoue, V.; Serre, G.; Simon, M.; Takahara, H. Transcriptional regulation of peptidylarginine deiminase expression in human keratinocytes. J. Dermatol. Sci. 2009, 53, 2–9. [Google Scholar] [CrossRef]
- Zhang, X.; Gamble, M.J.; Stadler, S.; Cherrington, B.D.; Causey, C.P.; Thompson, P.R.; Roberson, M.S.; Kraus, W.L.; Coonrod, S.A. Genome-wide analysis reveals PADI4 cooperates with Elk-1 to activate c-Fos expression in breast cancer cells. PLoS Genet. 2011, 7, e1002112. [Google Scholar] [CrossRef] [Green Version]
- Lewis, H.D.; Liddle, J.; Coote, J.E.; Atkinson, S.J.; Barker, M.D.; Bax, B.D.; Bicker, K.L.; Bingham, R.P.; Campbell, M.; Chen, Y.H.; et al. Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation. Nat. Chem. Biol. 2015, 11, 189–191. [Google Scholar] [CrossRef]
- Li, P.; Li, M.; Lindberg, M.R.; Kennett, M.J.; Xiong, N.; Wang, Y. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J. Exp. Med. 2010, 207, 1853–1862. [Google Scholar] [CrossRef]
- Chang, X.; Han, J.; Pang, L.; Zhao, Y.; Yang, Y.; Shen, Z. Increased PADI4 expression in blood and tissues of patients with malignant tumors. BMC Cancer 2009, 9, 40. [Google Scholar] [CrossRef] [Green Version]
- Wang, S.; Wang, Y. Peptidylarginine deiminases in citrullination, gene regulation, health and pathogenesis. Biochim. Biophys. Acta 2013, 1829, 1126–1135. [Google Scholar] [CrossRef] [Green Version]
- Sonego, F.; Castanheira, F.V.; Ferreira, R.G.; Kanashiro, A.; Leite, C.A.; Nascimento, D.C.; Colon, D.F.; Borges, V.F.; Alves-Filho, J.C.; Cunha, F.Q. Paradoxical Roles of the Neutrophil in Sepsis: Protective and Deleterious. Front. Immunol. 2016, 7, 155. [Google Scholar] [CrossRef] [Green Version]
- McDonald, B.; Urrutia, R.; Yipp, B.G.; Jenne, C.N.; Kubes, P. Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis. Cell Hos. Microbe 2012, 12, 324–333. [Google Scholar] [CrossRef] [Green Version]
- Meng, W.; Paunel-Görgülü, A.; Flohé, S.; Hoffmann, A.; Witte, I.; MacKenzie, C.; Baldus, S.E.; Windolf, J.; Lögters, T.T. Depletion of neutrophil extracellular traps in vivo results in hypersusceptibility to polymicrobial sepsis in mice. Crit. Care 2012, 16, R137. [Google Scholar] [CrossRef] [Green Version]
- McDonald, B.; Davis, R.P.; Kim, S.J.; Tse, M.; Esmon, C.T.; Kolaczkowska, E.; Jenne, C.N. Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. Blood 2017, 129, 1357–1367. [Google Scholar] [CrossRef] [Green Version]
- Luo, Y.; Arita, K.; Bhatia, M.; Knuckley, B.; Lee, Y.H.; Stallcup, M.R.; Sato, M.; Thompson, P.R. Inhibitors and inactivators of protein arginine deiminase 4: Functional and structural characterization. Biochemistry 2006, 45, 11727–11736. [Google Scholar] [CrossRef] [Green Version]
- Wang, Y.; Li, P.; Wang, S.; Hu, J.; Chen, X.A.; Wu, J.; Fisher, M.; Oshaben, K.; Zhao, N.; Gu, Y.; et al. Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity. J. Biol. Chem. 2012, 287, 25941–25953. [Google Scholar] [CrossRef] [Green Version]
- Grommes, J.; Soehnlein, O. Contribution of neutrophils to acute lung injury. Mol. Med. 2011, 17, 293–307. [Google Scholar] [CrossRef]
- Cheng, Z.; Abrams, S.T.; Toh, J.; Wang, S.S.; Wang, Z.; Yu, Q.; Yu, W.; Toh, C.H.; Wang, G. The Critical Roles and Mechanisms of Immune Cell Death in Sepsis. Front. Immunol. 2020, 11, 1918. [Google Scholar] [CrossRef] [PubMed]
- Denning, N.L.; Aziz, M.; Gurien, S.D.; Wang, P. DAMPs and NETs in Sepsis. Front. Immunol. 2019, 10, 2536. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wang, H.; Ward, M.F.; Sama, A.E. Targeting HMGB1 in the treatment of sepsis. Expert Opin. Ther. Targets. 2014, 18, 257–268. [Google Scholar] [CrossRef] [Green Version]
- Abraham, E.; Arcaroli, J.; Carmody, A.; Wang, H.; Tracey, K.J. HMG-1 as a mediator of acute lung inflammation. J. Immunol. 2000, 165, 2950–2954. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Angus, D.C.; Yang, L.; Kong, L.; Kellum, J.A.; Delude, R.L.; Tracey, K.J.; Weissfeld, L. Circulating high-mobility group box 1 (HMGB1) concentrations are elevated in both uncomplicated pneumonia and pneumonia with severe sepsis. Crit. Care Med. 2007, 35, 1061–1067. [Google Scholar] [CrossRef]
- Sunden-Cullberg, J.; Norrby-Teglund, A.; Rouhiainen, A.; Rauvala, H.; Herman, G.; Tracey, K.J.; Lee, M.L.; Andersson, J.; Tokics, L.; Treutiger, C.J. Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock. Crit. Care Med. 2005, 33, 564–573. [Google Scholar] [CrossRef]
- Liang, Y.; Pan, B.; Alam, H.B.; Deng, Q.; Wang, Y.; Chen, E.; Liu, B.; Tian, Y.; Williams, A.M.; Duan, X.; et al. Inhibition of peptidylarginine deiminase alleviates LPS-induced pulmonary dysfunction and improves survival in a mouse model of lethal endotoxemia. Eur. J. Pharmacol. 2018, 833, 432–440. [Google Scholar] [CrossRef]
- Wang, Y.; Li, M.; Stadler, S.; Correll, S.; Li, P.; Wang, D.; Hayama, R.; Leonelli, L.; Han, H.; Grigoryev, S.A.; et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J. Cell Biol. 2009, 184, 205–213. [Google Scholar] [CrossRef] [Green Version]
- Zhan, Y.; Ling, Y.; Deng, Q.; Qiu, Y.; Shen, J.; Lai, H.; Chen, Z.; Huang, C.; Liang, L.; Li, X.; et al. HMGB1-Mediated Neutrophil Extracellular Trap Formation Exacerbates Intestinal Ischemia/Reperfusion-Induced Acute Lung Injury. J. Immunol. 2022, 208, 968–978. [Google Scholar] [CrossRef]
- Huang, H.; Tohme, S.; Al-Khafaji, A.B.; Tai, S.; Loughran, P.; Chen, L.; Wang, S.; Kim, J.; Billiar, T.; Wang, Y.; et al. Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury. Hepatology 2015, 62, 600–614. [Google Scholar] [CrossRef]
- Wong, S.L.; Demers, M.; Martinod, K.; Gallant, M.; Wang, Y.; Goldfine, A.B.; Kahn, C.R.; Wagner, D.D. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. Nat. Med. 2015, 21, 815–819. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Du, M.; Yang, W.; Schmull, S.; Gu, J.; Xue, S. Inhibition of peptidyl arginine deiminase-4 protects against myocardial infarction induced cardiac dysfunction. Int. Immunopharmacol. 2020, 78, 106055. [Google Scholar] [CrossRef] [PubMed]
- Rabadi, M.; Kim, M.; D’Agati, V.; Lee, H.T. Peptidyl arginine deiminase-4-deficient mice are protected against kidney and liver injury after renal ischemia and reperfusion. Am. J. Physiol. Renal. Physiol. 2016, 311, F437–F449. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Martinod, K.; Witsch, T.; Erpenbeck, L.; Savchenko, A.; Hayashi, H.; Cherpokova, D.; Gallant, M.; Mauler, M.; Cifuni, S.M.; Wagner, D.D. Peptidylarginine deiminase 4 promotes age-related organ fibrosis. J. Exp. Med. 2017, 214, 439–458. [Google Scholar] [CrossRef] [PubMed]
- Fuchs, T.A.; Abed, U.; Goosmann, C.; Hurwitz, R.; Schulze, I.; Wahn, V.; Weinrauch, Y.; Brinkmann, V.; Zychlinsky, A. Novel cell death program leads to neutrophil extracellular traps. J. Cell Biol. 2007, 176, 231–241. [Google Scholar] [CrossRef]
- Bawadekar, M.; Shim, D.; Johnson, C.J.; Warner, T.F.; Rebernick, R.; Damgaard, D.; Nielsen, C.H.; Pruijn, G.J.M.; Nett, J.E.; Shelef, M.A. Peptidylarginine deiminase 2 is required for tumor necrosis factor alpha-induced citrullination and arthritis, but not neutrophil extracellular trap formation. J. Autoimmun. 2017, 80, 39–47. [Google Scholar] [CrossRef]
- Buchanan, J.T.; Simpson, A.J.; Aziz, R.K.; Liu, G.Y.; Kristian, S.A.; Kotb, M.; Feramisco, J.; Nizet, V. DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr. Biol. 2006, 16, 396–400. [Google Scholar] [CrossRef] [Green Version]
- Yu, H.H.; Yang, Y.H.; Chiang, B.L. Chronic Granulomatous Disease: A Comprehensive Review. Clin. Rev. Allergy. Immunol. 2021, 61, 101–113. [Google Scholar] [CrossRef]
- Dhawan, U.K.; Bhattacharya, P.; Narayanan, S.; Manickam, V.; Aggarwal, A.; Subramanian, M. Hypercholesterolemia Impairs Clearance of Neutrophil Extracellular Traps and Promotes Inflammation and Atherosclerotic Plaque Progression. Arterioscler. Thromb. Vasc. Biol. 2021, 41, 2598–2615. [Google Scholar] [CrossRef]
- Lu, Y.C.; Yeh, W.C.; Ohashi, P.S. LPS/TLR4 signal transduction pathway. Cytokine 2008, 42, 145–151. [Google Scholar] [CrossRef]
- Gomes, N.E.; Brunialti, M.K.; Mendes, M.E.; Freudenberg, M.; Galanos, C.; Salomão, R. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood. Braz. J. Med. Biol. Res. 2010, 43, 853–858. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Brinkmann, V.; Laube, B.; Abu, A.U.; Goosmann, C.; Zychlinsky, A. Neutrophil extracellular traps: How to generate and visualize them. J. Vis. Exp. 2010, 1724–1726. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wang, Y.; Wysocka, J.; Sayegh, J.; Lee, Y.H.; Perlin, J.R.; Leonelli, L.; Sonbuchner, L.S.; McDonald, C.H.; Cook, R.G.; Dou, Y.; et al. Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 2004, 306, 279–283. [Google Scholar] [CrossRef] [PubMed]
- Yao, H.; Wang, T.; Deng, J.; Liu, D.; Li, X.; Deng, J. The development of blood-retinal barrier during the interaction of astrocytes with vascular wall cells. Neural. Regen. Res. 2014, 9, 1047–1054. [Google Scholar]
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Yao, H.; Cao, G.; Liu, Z.; Zhao, Y.; Yan, Z.; Wang, S.; Wang, Y.; Guo, Z.; Wang, Y. Inhibition of Netosis with PAD Inhibitor Attenuates Endotoxin Shock Induced Systemic Inflammation. Int. J. Mol. Sci. 2022, 23, 13264. https://doi.org/10.3390/ijms232113264
Yao H, Cao G, Liu Z, Zhao Y, Yan Z, Wang S, Wang Y, Guo Z, Wang Y. Inhibition of Netosis with PAD Inhibitor Attenuates Endotoxin Shock Induced Systemic Inflammation. International Journal of Molecular Sciences. 2022; 23(21):13264. https://doi.org/10.3390/ijms232113264
Chicago/Turabian StyleYao, Huanling, Guojie Cao, Zheng Liu, Yue Zhao, Zhanchao Yan, Senzhen Wang, Yuehua Wang, Zhengwei Guo, and Yanming Wang. 2022. "Inhibition of Netosis with PAD Inhibitor Attenuates Endotoxin Shock Induced Systemic Inflammation" International Journal of Molecular Sciences 23, no. 21: 13264. https://doi.org/10.3390/ijms232113264