The prevalence of chronic inflammatory and thrombotic diseases is rising with the general increase in population longevity. Our application addresses the role of neutrophil extracellular traps (NETs), chromatin released by activated neutrophils, in such age-related diseases. We hypothesize that aging promotes NET formation and NETs in turn escalate chronic inflammatory and thrombotic diseases. We propose that genes regulating NETosis, such as peptidylarginine deiminase 4 (PAD4), impact aging. Our preliminary results indicate that neutrophils from old mice make more NETs and PAD4, which orchestrates NETosis, promotes cardiac fibrosis in older mice. We observed that the mitochondrial deacetylase sirtuin 3 (SIRT3), whose regulation of reactive oxygen species (ROS) is implicated in aging, is expressed by neutrophils and platelets and is a potent down-regulator of neutrophil ROS production and NETosis. We will address our hypothesis with in vitro studies and disease models in wild type (WT) and genetically engineered mutant mice. We propose three Aims:
Aim 1. To study the relationship of NETosis and aging in WT mice, the effect of calorie restriction (CR) on NETosis and resulting inflammatory, thrombotic, and fibrotic responses. We will examine NETosis and the susceptibility to cardiac and lung fibrosis as a function of mouse age. We will evaluate whether DNase1, that helps clear NETs, will reduce experimental lung fibrosis. We will study the effect of CR, known to slow down aging, on NET formation and susceptibility to thrombosis and organ fibrosis.
Aim 2. To investigate whether mutations in PAD4, an enzyme required for NET formation, affect aging and age-related inflammatory and thrombotic diseases. We will study the effect of PAD4- deficiency on age-related heart fibrosis and susceptibility to experimental lung fibrosis. We will follow aging healt and longevity of PAD4-/- and WT littermates. We will genetically engineer transgenic mice overproducing PAD4 in neutrophils. We anticipate that they will produce NETs more readily and have an increased propensity towards thrombosis and interstitial lung fibrosis as well as delayed wound healing.
Aim 3. To evaluate the role of SIRT3 in NETosis and in disease models involving NETs. Study the function of platelet SIRT3 in platelet activation and in their priming of neutrophils towards NETosis. We are keen to evaluate the role of SIRT3 in neutrophils, platelets and in the interaction between platelets and neutrophils that may enhance NETosis. SIRT3-/- and already generated SIRT3- overexpressing mice will be evaluated in inflammation and thrombosis. Bone marrow transplant chimeric animals will be prepared to distinguish the role of blood cell versus tissue SIRT3. We hope to uncover new pharmacological targets to reduce NETs' contribution to chronic inflammatory and thrombotic processes and that this may eventually improve the health of our aging population.

Public Health Relevance

The prevalence of chronic inflammatory and thrombotic diseases is rising with the increase in longevity. We have found that as mice age they activate more and more of their white blood cells to release highly toxic material from their nuclei, called NETs, that causes both inflammation and thrombosis. The goal of this application is to figure out how the release of NETs could be prevented to improve the health of our aging population.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Kindzelski, Andrei L
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Children's Hospital Boston
United States
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Hayashi, Hideki; Cherpokova, Deya; Martinod, Kimberly et al. (2017) Sirt3 deficiency does not affect venous thrombosis or NETosis despite mild elevation of intracellular ROS in platelets and neutrophils in mice. PLoS One 12:e0188341
Martinod, Kimberly; Witsch, Thilo; Erpenbeck, Luise et al. (2017) Peptidylarginine deiminase 4 promotes age-related organ fibrosis. J Exp Med 214:439-458
Demers, Mélanie; Wong, Siu Ling; Martinod, Kimberly et al. (2016) Priming of neutrophils toward NETosis promotes tumor growth. Oncoimmunology 5:e1134073
Pillai, Padmini S; Molony, Ryan D; Martinod, Kimberly et al. (2016) Mx1 reveals innate pathways to antiviral resistance and lethal influenza disease. Science 352:463-6
Martinod, K; Witsch, T; Farley, K et al. (2016) Neutrophil elastase-deficient mice form neutrophil extracellular traps in an experimental model of deep vein thrombosis. J Thromb Haemost 14:551-8
Gavillet, Mathilde; Martinod, Kimberly; Renella, Raffaele et al. (2015) Flow cytometric assay for direct quantification of neutrophil extracellular traps in blood samples. Am J Hematol 90:1155-8