Stroke-Homing Peptide-DNase1 Alleviates Intestinal Ischemia Reperfusion Injury by Selectively Degrading Neutrophil Extracellular Traps

Abstract

Neutrophil extracellular traps (NETs) act as a vital first line of defence against tissue damage and pathogens, playing a significant role in improving diseases such as intestinal ischemia reperfusion injury (IRI). However, we observed that after intestinal injury, intestinal bacteria and lipopolysaccharides (LPS) can enter the circulatory system, leading to a significant secondary increase in NETs production and the subsequent activation of a coagulation cascade. This phenomenon contributes to a pathological process known as the ‘second strike’ of NETs, which exaggerates intestinal damage and microcirculation disturbance. Selectively mitigating the detrimental effects associated with this second strike presents a promising therapeutic strategy. We developed an innovative conjugate of stroke-homing peptide (SHp) and DNase1 (SHp-DNase1) to enhance the stability of DNase in the bloodstream while selectively targeting NETs in thromboembolic events. The effects of SHp-DNase1 on blood flow, ischemia, and vascular leakage were evaluated in a mouse model using laser Doppler flowmetry and an in vivo imaging system. Levels of LPS and NETs were elevated in patients with IRI. Similarly, the expression of NETs and LPS was upregulated in mice with intestinal IRI. In vivo imaging revealed disturbances in intestinal microcirculation, accompanied by intestinal leakage, which were effectively reversed by the administration of SHp-DNase1. Almost all of the SHp-DNase1 localised to the gastrointestinal tract, demonstrating the effective targeting of DNase1 to the site of intestinal injury via SHp guidance. Furthermore, the combination of SHp-DNase1 and CRO significantly reduced the expression of ischemia-inducible factors, leading to a marked decrease in mortality in the mouse model. These findings suggest that intestinal LPS leakage correlated with NETs exacerbation plays a critical role in IRI. The combination of SHp-DNase1 and CRO is an effective treatment strategy by simultaneously controlling inflammation and addressing microcirculatory disorders induced by NETs in the therapy of IRI.

Keywords

ceftriaxone sodium / intestinal ischemia reperfusion injury / microcirculatory disturbance / neutrophil extracellular traps / stroke-homing peptide–DNase1

References

[1] M. B. Sayhan, S. Oguz, Ö. Salt, N. Can, T. Ozgurtas, and T. D. Yalta, “Sesamin Ameliorates Mucosal Tissue Injury of Mesenteric Ischemia and Reperfusion in an Experimental Rat Model,” Archives of Medical Science 15, no. 6 (2019): 1582-1588, https://doi.org/10.5114/aoms.2017.68535.

[2] F. Deng, Z. B. Lin, Q. S. Sun, et al., “The Role of Intestinal Microbiota and Its Metabolites in Intestinal and Extraintestinal Organ Injury Induced by Intestinal Ischemia Reperfusion Injury,” International Journal of Biological Sciences 18, no. 10 (2022): 3981-3992, https://doi.org/10.7150/ijbs.71491.

[3] L. M. Gonzalez, A. J. Moeser, and A. T. Blikslager, “Animal Models of Ischemia-Reperfusion-Induced Intestinal Injury: Progress and Promise for Translational Research,” American Journal of Physiology-Gastrointestinal and Liver Physiology 308, no. 2 (2015): G63-G75, https://doi.org/10.1152/ajpgi.00112.2013.

[4] T. Zuo, F. Zhang, G. C. Lui, et al., “Alterations in Gut Microbiota of Patients With COVID-19 During Time of Hospitalization,” Gastroenterology 159, no. 3 (2020): 944-955, https://doi.org/10.1053/j.gastro.2020.05.048.

[5] P. Chen and T. Billiar, “Gut Microbiota and Multiple Organ Dysfunction Syndrome (MODS),” Advances in Experimental Medicine and Biology 1238 (2020): 195-202, https://doi.org/10.1007/978-981-15-2385-4_11.

[6] B. Freitas, Y. Bausback, J. Schuster, et al., “Thrombectomy Devices in the Treatment of Acute Mesenteric Ischemia: Initial Single-Center Experience,” Annals of Vascular Surgery 51 (2018): 124-131, https://doi.org/10.1016/j.avsg.2017.11.041.

[7] R. W. Byard, “Acute Mesenteric Ischaemia and Unexpected Death,” Journal of Forensic and Legal Medicine 19, no. 4 (2012): 185-190, https://doi.org/10.1016/j.jflm.2011.12.023.

[8] P. M. Potey, A. G. Rossi, C. D. Lucas, and D. A. Dorward, “Neutrophils in the Initiation and Resolution of Acute Pulmonary Inflammation: Understanding Biological Function and Therapeutic Potential,” Journal of Pathology 247, no. 5 (2019): 672-685, https://doi.org/10.1002/path.5221.

[9] V. Brinkmann, U. Reichard, C. Goosmann, et al., “Neutrophil Extracellular Traps Kill Bacteria,” Science 303, no. 5663 (2004): 1532-1535, https://doi.org/10.1126/science.1092385.

[10] Z. Tian, Y. Zhang, Z. Zheng, et al., “Gut Microbiome Dysbiosis Contributes to Abdominal Aortic Aneurysm by Promoting Neutrophil Extracellular Trap Formation,” Cell Host and Microbe 30, no. 10 (2022): 1450-1463, https://doi.org/10.1016/j.chom.2022.09.004.

[11] Y. Zuo, M. Zuo, S. Yalavarthi, et al., “Neutrophil Extracellular Traps and Thrombosis in COVID-19,” Journal of Thrombosis and Thrombolysis 51, no. 2 (2021): 446-453, https://doi.org/10.1007/s11239-020-02324-z.

[12] C. Chu, X. Wang, C. Yang, et al., “Neutrophil Extracellular Traps Drive Intestinal Microvascular Endothelial Ferroptosis by Impairing Fundc1-Dependent Mitophagy,” Redox Biology 67 (2023): 102906, https://doi.org/10.1016/j.redox.2023.102906.

[13] Y. N. Zhang, Z. N. Chang, Z. M. Liu, et al., “Dexmedetomidine Alleviates Gut-Vascular Barrier Damage and Distant Hepatic Injury Following Intestinal Ischemia/Reperfusion Injury in Mice,” Anesthesia and Analgesia 134, no. 2 (2022): 419-431, https://doi.org/10.1213/ane.0000000000005810.

[14] A. Hosseinnejad, N. Ludwig, A. K. Wienkamp, et al., “DNase I Functional Microgels for Neutrophil Extracellular Trap Disruption,” Biomaterials Science 10, no. 1 (2021): 85-99, https://doi.org/10.1039/d1bm01591e.

[15] M. Boettcher, G. Eschenburg, S. Mietzsch, et al., “Therapeutic Targeting of Extracellular DNA Improves the Outcome of Intestinal Ischemic Reperfusion Injury in Neonatal Rats,” Scientific Reports 7, no. 1 (2017): 15377, https://doi.org/10.1038/s41598-017-15807-6.

[16] S. Wang, T. Xie, S. Sun, et al., “DNase-1 Treatment Exerts Protective Effects in a Rat Model of Intestinal Ischemia-Reperfusion Injury,” Scientific Reports 8, no. 1 (2018): 17788, https://doi.org/10.1038/s41598-018-36198-2.

[17] H. Y. Hong, J. S. Choi, Y. J. Kim, et al., “Detection of Apoptosis in a Rat Model of Focal Cerebral Ischemi Using a Homing Peptide Selected From In Vivo Phage Display,” Journal of Controlled Release: Official Journal of the Controlled Release Society 131, no. 3 (2008): 167-172, https://doi.org/10.1016/j.jconrel.2008.07.020.

[18] C. Y. Wang, T. T. Lin, L. Hu, et al., “Neutrophil Extracellular Traps as a Unique Target in the Treatment of Chemotherapy-Induced Peripheral Neuropathy,” eBioMedicine 90 (2023): 104499, https://doi.org/10.1016/j.ebiom.2023.104499.

[19] R. Struck, M. Wittmann, S. Müller, P. Meybohm, A. Müller, and S. Bagci, “Effect of Remote Ischemic Preconditioning on Intestinal Ischemia-Reperfusion Injury in Adults Undergoing On-Pump CABG Surgery: A Randomized Controlled Pilot Trial,” Journal of Cardiothoracic and Vascular Anesthesia 32, no. 3 (2018): 1243-1247, https://doi.org/10.1053/j.jvca.2017.07.027.

[20] B. Adamik, A. Kübler, A. Gozdzik, and W. Gozdzik, “Prolonged Cardiopulmonary Bypass Is a Risk Factor for Intestinal Ischaemic Damage and Endotoxaemia,” Heart, Lung and Circulation 26, no. 7 (2017): 717-723, https://doi.org/10.1016/j.hlc.2016.10.012.

[21] W. Zhang, B. Zhou, X. Yang, et al., “Exosomal circEZH2_005, an Intestinal Injury Biomarker, Alleviates Intestinal Ischemia/Reperfusion Injury by Mediating Gprc5a Signaling,” Nature Communications 14, no. 1 (2023): 5437, https://doi.org/10.1038/s41467-023-41147-3.

[22] M. Nguyen, A. Tavernier, T. Gautier, et al., “Glucagon-Like Peptide-1 Is Associated With Poor Clinical Outcome, Lipopolysaccharide Translocation and Inflammation in Patients Undergoing Cardiac Surgery With Cardiopulmonary Bypass,” Cytokine 133 (2020): 155182, https://doi.org/10.1016/j.cyto.2020.155182.

[23] X. Liu, T. Li, H. Chen, L. Yuan, and H. Ao, “Role and Intervention of PAD4 in NETs in Acute Respiratory Distress Syndrome,” Respiratory Research 25, no. 1 (2024): 63, https://doi.org/10.1186/s12931-024-02676-7.

[24] D. Lin, Y. Zhang, S. Wang, et al., “Ganoderma Lucidum Polysaccharide Peptides GL-PPSQ(2) Alleviate Intestinal Ischemia-Reperfusion Injury via Inhibiting Cytotoxic Neutrophil Extracellular Traps,” International Journal of Biological Macromolecules 244 (2023): 125370, https://doi.org/10.1016/j.ijbiomac.2023.125370.

[25] N. Hayase, K. Doi, T. Hiruma, et al., “Recombinant Thrombomodulin on Neutrophil Extracellular Traps in Murine Intestinal Ischemia-Reperfusion,” Anesthesiology 131, no. 4 (2019): 866-882, https://doi.org/10.1097/ALN.0000000000002898.

[26] M. L. Lähdeaho, M. Scheinin, P. Vuotikka, et al., “Safety and Efficacy of AMG 714 in Adults With Coeliac Disease Exposed to Gluten Challenge: A Phase 2a, Randomised, Double-Blind, Placebo-Controlled Study,” Lancet Gastroenterology and Hepatology 4, no. 12 (2019): 948-959, https://doi.org/10.1016/s2468-1253(19)30264-x.

[27] K. Dickson, H. Malitan, and C. Lehmann, “Imaging of the Intestinal Microcirculation During Acute and Chronic Inflammation,” Biology 9, no. 12 (2020): 418, https://doi.org/10.3390/biology9120418.

[28] T. Hua, Z. Lu, M. Wang, et al., “Shenfu Injection Alleviate Gut Ischemia/Reperfusion Injury After Severe Hemorrhagic Shock Through Improving Intestinal Microcirculation in Rats,” Heliyon 10, no. 11 (2024): e31377, https://doi.org/10.1016/j.heliyon.2024.e31377.

[29] L. Zou, X. Song, L. Hong, et al., “Intestinal Fatty Acid-Binding Protein as a Predictor of Prognosis in Postoperative Cardiac Surgery Patients,” Medicine 97, no. 33 (2018): e11782, https://doi.org/10.1097/md.0000000000011782.

[30] A. Kiwit, Y. Lu, M. Lenz, et al., “The Dual Role of Neutrophil Extracellular Traps (NETs) in Sepsis and Ischemia-Reperfusion Injury: Comparative Analysis Across Murine Models,” International Journal of Molecular Sciences 25, no. 7 (2024): 3787, https://doi.org/10.3390/ijms25073787.

[31] T. A. Fuchs, A. Brill, D. Duerschmied, et al., “Extracellular DNA Traps Promote Thrombosis,” Proceedings of the National Academy of Sciences of the United States of America 107, no. 36 (2010): 15880-15885, https://doi.org/10.1073/pnas.1005743107.

[32] C. Chu, X. Wang, F. Chen, et al., “Neutrophil Extracellular Traps Aggravate Intestinal Epithelial Necroptosis in Ischaemia-Reperfusion by Regulating TLR4/RIPK3/FUNDC1-Required Mitophagy,” Cell Proliferation 57, no. 1 (2024): e13538, https://doi.org/10.1111/cpr.13538.

[33] E. Kolaczkowska, C. N. Jenne, B. G. Surewaard, et al., “Molecular Mechanisms of NET Formation and Degradation Revealed by Intravital Imaging in the Liver Vasculature,” Nature Communications 6 (2015): 6673, https://doi.org/10.1038/ncomms7673.

[34] R. Reiner, U. Weiss, U. Brombacher, et al., “Ro 13-9904/001, a Novel Potent and Long-Acting Parenteral Cephalosporin,” Journal of Antibiotics 33, no. 7 (1980): 783-786, https://doi.org/10.7164/antibiotics.33.783.

[35] A. E. Yellin, J. M. Hassett, A. Fernandez, et al., “Ertapenem Monotherapy Versus Combination Therapy With Ceftriaxone Plus Metronidazole for Treatment of Complicated Intra-Abdominal Infections in Adults,” International Journal of Antimicrobial Agents 20, no. 3 (2002): 165-173, https://doi.org/10.1016/s0924-8579(02)00160-7.

[36] D. Tsai, P. Stewart, R. Goud, et al., “Total and Unbound Ceftriaxone Pharmacokinetics in Critically Ill Australian Indigenous Patients With Severe Sepsis,” International Journal of Antimicrobial Agents 48, no. 6 (2016): 748-752, https://doi.org/10.1016/j.ijantimicag.2016.09.021.

[37] S. Bárcena-Varela, G. Martínez-de-Tejada, L. Martin, et al., “Coupling Killing to Neutralization: Combined Therapy With Ceftriaxone/Pep19-2.5 Counteracts Sepsis in Rabbits,” Experimental and Molecular Medicine 49, no. 6 (2017): e345, https://doi.org/10.1038/emm.2017.75.

[38] Z. Gong, J. Guo, Q. Li, and H. Xie, “Molecular Mechanism of Lipopolysaccharide (LPS) in Promoting Biomineralization on Bacterial Surface,” Biochimica et Biophysica Acta, General Subjects 1867, no. 3 (2023): 130305, https://doi.org/10.1016/j.bbagen.2023.130305.

[39] J. S. Simões, R. F. Rodrigues, B. Zavan, R. M. P. Emídio, R. Soncini, and V. B. Boralli, “Endotoxin-Induced Sepsis on Ceftriaxone-Treated Rats' Ventilatory Mechanics and Pharmacokinetics,” Antibiotics 13, no. 1 (2024): 83, https://doi.org/10.3390/antibiotics13010083.

[40] E. Arslan, M. O. Biyik, M. Kosucu, A. R. Guvercin, A. Bodur, and A. Alver, “Is Ceftriaxone Effective in Experimental Brain Ischemia/Reperfusion Injury?,” Bratislavské Lekárske Listy 121, no. 12 (2020): 858-863, https://doi.org/10.4149/bll_2020_141.

[41] Z. Duan, T. Xie, C. Chu, et al., “De-Escalation Antibiotic Therapy Alleviates Organ Injury Through Modulation of NETs Formation During Sepsis,” Cell Death Discovery 7, no. 1 (2021): 345, https://doi.org/10.1038/s41420-021-00745-0.

[42] H. Englert, J. Göbel, D. Khong, et al., “Targeting NETs Using Dual-Active DNase1 Variants,” Frontiers in Immunology 14 (2023): 1181761, https://doi.org/10.3389/fimmu.2023.1181761.

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