Treatment, Prevention, and Mouse Models of Neutrophil-Derived NET Toxicity in Thrombosis, Cancer and Diabetes
Inventors: Denisa Wagner
Invention Types: Therapeutics, Diagnostic/Prognostic
Research Areas: Allergy/Respiratory/Pulmonary Disease, Immunology, Oncology/Hematology, Surgery/Wound Healing
Keywords: Animal Model (mouse)For More Information Contact: Dietz, Ryan
Neutrophils are one cell type involved in the innate immune response and when activated, they die releasing fibers of DNA associated with histones and antimicrobial proteins, called neutrophil extracellular traps (NETs). These NETs entangle and may kill pathogens, possibly impeding bacterial infection.
While NETs formation can serve a healthy purpose, Denisa Wagner shows that they also promote the development of blood clots in deep vein thrombosis, cancer, lung injury, and potentially impair wound healing in diabetes. Additional studies are underway to determine how NET formation might be pharmacologically impaired to prevent these dangerous medical conditions.
Targeting NETs to Prevent Deep Vein Thrombosis (DVT):
Dr. Wagner shows that plasma DNA levels are increased in patients with DVT indicating the presence of DVT in a patient, as it reflects the degradation of NETs within a thrombus. To demonstrate that blocking NET formation reduces DVT, Dr. Wagner’s lab pioneered the use of PAD4 knockout mice for DVT studies, since PAD4 is required for NETs formation. Her data show that wild type mice produce thrombi 90% of the time, in contrast to PAD4 knockout mice which produced thrombi less than 10% of the time. This suggests PAD4 inhibition may be an effective approach in preventing or treating DVT.
Studies of NETs and Blood Clots in Cancer:
Dr. Wagner’s lab shows that NETs play a critical role in blood clot formation in cancer. In a mouse model of chronic myeloid leukemia (CML), both malignant and healthy neutrophils were found to be more prone to generating extracellular DNA traps. Increased NETs formation is also observed in other animal models of solid tumors including breast and lung cancer xenografts. Further, Dr. Wagner’s lab shows that G-CSF boosts neutrophil abundance and promotes NET production supporting previous reports that cancer patients with high G-CSF levels are predisposed to a pro-thrombotic state.
Studies of NETs in Transfusion-Related Acute Lung Injury (TRALI):
Dr. Wagner’s lab showed that NET biomarkers are present in the blood of patients with lung injury due to TRALI. In vitro studies show that neutrophils produce NETs following exposure to anti-HNA-3a antibodies, which are implicated in TRALI. In addition, her lab demonstrated NETs prevention in TRALI mouse models when treated with DNase I, suggesting that NET formation in the lungs can be targeted to treat TRALI or other inflammatory lung conditions.
Studies of NETs and Impaired Wound Healing in Diabetes:
Dr. Wagner showed that NETs are induced by diabetes and can impair wound healing in diabetes patients. Diabetic subjected to excisional skin wounds displayed abundant citrullinated histone H3, a marker of NETs, and the presence of this protein was associated with delayed healing. DNase1 treatment cleared NETs and accelerated healing these diabetic mice suggesting that inhibition or cleavage of NETs may provide an effective means of improving wound healing in diabetic patients.
• Treatment with DNase or anti-histone antibodies to reduce thrombosis related to DVT, cancer or other indications, or defective wound healing in diabetes.
• Drug discovery target: Inhibition of PAD4 to reduce thrombosis related to DVT, cancer or other indications, or defective wound healing in diabetes.
• Methods (such as DNase 1 treatment) and devices for treating stored blood products to reduce the accumulation of NETs.
• DNase 1 inhalation as a prophylactic agent or treatment for TRALI
• A method for studying NET formation in healthy or cancer-bearing mice; Neutrophils are isolated, treated with PAF, and NET formation is monitored
• Using biomarkers, such as plasma DNA or histone markers, that indicate NET presence to determine a patient’s risk for blood clots
• The success rate of pharmacological catheter-directed thrombolysis ranges from 59% to 100%.
• Morbidity from venous thromboembolism (300,000/year in the US) has not substantially changed within the last 2 decades, and contemporary prophylaxis is not always efficient.
• This technology could lead to a targeted therapy for patients with DVT or other clotting/thrombotic or wound healing conditions.
• All blood products can cause TRALI, and no specific treatment or biomarkers are available.
• G-CSF and LPS inducible system to model a prothrombotic state induced by NETosis in research animals is simpler and more rapid than surgical methods currently used to induce thrombi
Exclusive License or Sponsored Research
Martinod K, Demers M, Fuchs TA, Wong SL, Brill A, Gallant M, Hu J, Wang Y, Wagner DD. Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8674-9. doi: 10.1073/pnas.1301059110. Epub 2013 May 6. PubMed PMID: 23650392
Martinod K, Fuchs TA, Zitomersky NL, Wong SL, Demers M, Gallant M, Wang Y, Wagner DD. PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock. Blood. 2015 Mar 19;125(12):1948-56. doi: 10.1182/blood-2014-07-587709. Epub 2015 Jan 26. PubMed PMID: 25624317
Demers M, Krause DS, Schatzberg D, Martinod K, Voorhees JR, Fuchs TA, Scadden DT, Wagner DD. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci U S A. 2012 Aug 7;109(32):13076-81. doi: 10.1073/pnas.1200419109. Epub 2012 Jul 23. PubMed PMID: 22826226
Wong SL, Demers M, Martinod K, Gallant M, Wang Y, Goldfine AB, Kahn CR, Wagner DD. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. Nat Med. 2015 Jul;21(7):815-9. doi: 10.1038/nm.3887. Epub 2015 Jun 15. PubMed PMID: 26076037
Fuchs TA, Alvarez JJ, Martinod K, Bhandari AA, Kaufman RM, Wagner DD. Neutrophils release extracellular DNA traps during storage of red blood cell units. Transfusion. 2013 Apr 8. doi: 10.1111/trf.12203. [Epub ahead of print] PubMed PMID: 23560771
Borissoff JI, Joosen IA, Versteylen MO, Brill A, Fuchs TA, Savchenko AS, Gallant M, Martinod K, Ten Cate H, Hofstra L, Crijns HJ, Wagner DD, Kietselaer BL. Elevated levels of circulating DNA and chromatin are independently associated with severe coronary atherosclerosis and a prothrombotic state. Arterioscler Thromb Vasc Biol. 2013 Aug;33(8):2032-40. doi: 10.1161/ATVBAHA.113.301627. Epub 2013 Jul 1. PubMed PMID: 23818485.
Thomas GM, Carbo C, Curtis BR, Martinod K, Mazo IB, Schatzberg D, Cifuni SM, Fuchs TA, von Andrian UH, Hartwig JH, Aster RH, Wagner DD. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice. Blood. 2012 Jun 28;119(26):6335-43. doi: 10.1182/blood-2012-01-405183. Epub 2012 May 17. PubMed PMID: 22596262