The Role of Peptidyl Arginine Deiminase, Type IV (PAD4) in the Pathology of Shock/Sepsis

The Role of Peptidyl arginine deiminase, type IV (PAD4) in the Pathology of Shock/Sepsis

Bethany Marie Biron

B.A., Worcester State College

M.S., Johns Hopkins University

Dissertation Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Division of Biology and Medicine at Brown University

Providence RI

May 2017

This dissertation by Bethany Marie Biron is accepted in its present form by the Department of Pathobiology as satisfying the dissertation requirement for the degree of Doctor of Philosophy.

Date______Alfred Ayala, Co-Advisor

Date______Jonathan Reichner, Co-Advisor

Advisor Recommended to the Graduate Council

Date______Craig Lefort, Reader

Date______Daithi Heffernan, Reader

Date______Bruce Levy, Outside Reader

Approved by the Graduate Council

Date______Andrew Campbell, Dean of the Graduate School

Bethany M. Biron Girard, BA, MS

Pathobiology Graduate Program-Pre-doctoral Student Co-Mentored by Alfred Ayala/Jonathan Reichner Division of Surgical Research Rhode Island Hospital Brown Medical School Aldrich 239, 593 Eddy Street Providence, RI 02903 Email: [email protected]

Primary Research Field My current research involves studying the citrullination of histones via Protein Arginine Deiminase 4 (PAD4) and its involvement in Neutrophil Extracellular trap formation in disease states. Acute lung injury (ALI) is a common sequela of shock/trauma and is associated with significant mortality. Trauma patients exposed to a secondary infectious challenge are considered at significant risk for inflammatory lung injury. Neutrophil extracellular traps (NETs) are capable of microbial killing essential for combating systemic infection, but are also associated with detrimental inflammation in the host. My research entails defining the role of NET formation in the protection of the host from indirect acute lung injury as a result of hemorrhage and subsequent septic challenge, as well as elucidates the importance of NETs to host defense and whether the host is benefited or harmed by NET release by using an array of basic science laboratory protocols and methods..

Education 2013-present Ph.D. Pending. Pathobiology, Brown University, Providence, RI

2011 M.S. in Biotechnology concentration Biodefense, Johns Hopkins University, Rockville MD.

2007 B.A. in Spanish and Natural Sciences, Worcester State College, Worcester, MA

Professional Experience

Aug 2009- Aug2012 Research Microbiologist, Center for Aerobiological Sciences, USAMRIID, Frederick, MD

I participated in GLP and non-GLP infectious disease vaccine research and aerosol model development research experiments in BSL-3 laboratory environments. Under the supervision of the division chief for Aerobiological Sciences I developed, wrote, and carried out animal protocols in accordance to IACUC standards. More specifically I was in charge of developing and executing adjunct therapy studies against inhalational anthrax infection. In addition, I performed aerosolization of bacterial and viral agents in a class III biosafety cabinet, and then employed various biological techniques to determine aerosol concentrations of biological agents as well as

ii for protein quantization, antibody assays, and tissue culture assays. Upon completion of studies I analyzed data and wrote scientific reports and papers for publication. Completed all background checks and obtained top secret clearance to perform duties in BSL 3-4 laboratories, as well as enrolled in Personal Reliability Program (PRP) for work with select agents.

Oct 2007- Aug 2009 Research Laboratory Technician II, Charles River Labs, Shrewsbury, MA I served as lead/primary technician for multiple basic and complex in-life studies simultaneously. This included generating data with minimal supervision in the performance of studies as well as being responsible for handling and restraining animals, clinical observations, sample collections, monitoring food consumption, animal husbandry, and performing accurate data collection and reporting. Specialized in small animal procedures and worked in compliance with Good Laboratory Practice Regulations (GLP’s), study protocols, and Standard Operation Procedures (SOP’s)

Awards, Honors, Fellowships, Grants:

Phi Eta Sigma National Honor Society, 2004 Johns Hopkins University Research Fellowship, USAMRIID, 2009-2011 Shock Society-Travel Award to support presentation of a Poster/Abstract as a part of the 38th Annual Conference on Shock in Denver, CO, June 6-9, 2015 Society for Leukocyte Biology – Travel award to support presentation of Poster/Abstract as part of the 49th Annual SLB Meeting in Verona Italy, Sep 15-18, 2016

Societies: SHOCK Society: Student member Society of Leukocyte Biology: Student member American Society for Investigative Pathology: Graduate student member

Publications:  Biron BM, Beck K, Dyer D, Mattix M, Twenhafel N, Nalca A. Efficacy of ETI-204 monoclonal antibody as an adjunct therapy in a New Zealand white rabbit partial survival model for inhalational anthrax. Antimicrob. Agents Chemother. 59(4):2206-14, 2015.

 Biron BM, Ayala A, Lomas-Neira JL. Biomarkers for Sepsis: What Is and What Might Be? Biomark. Insights 15;10(Suppl 4):7-17, 2015 (review).

 Cheng T, Bai J, Chung CS, Chen, Y, Biron BM, Ayala, A. Enhanced innate inflammation induced by anti-BTLA antibody in dual insult model of hemorrhagic shock/sepsis. Shock 45:40-49, 2016

 Biron BM, Chen Y, Pulido S, Chung CS, Reichner J, Ayala A. Cl-amidine Prevents Histone 3 Citrullination/NET Formation and Improves Survival in a Murine Sepsis Model. J. Innate Immun. Sep 14, 2016. (Epub ahead of print)

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 O’Brian X, Biron BM, Reichner J. Consequences of Extracellular Trap Formation in Sepsis. Current Opinions in Hematology. (in press), 2016 (review) Abstracts (published):

 Biron BM, Chen Y, Pulido S, Chung CS, Reichner J, Ayala A. The role of PAD4 histone 3 citrullination and net formation in a murine polymicrobial sepsis model. Shock 43:49 (supplt), 2015.

 Biron BM, Chung C-S, Heffernan DS, Ayala A. 2016. Citrullinated histone modification and NET production is increased the septic patients. Shock 45: supplement 1, 2016

 Bethany Biron Girard, Chun-Shiang Chung, Yaping Chen , Jonathan Reichner, Alfred Ayala. Do NETs Matter? Establishing a Role for Neutrophil Extracellular Traps in Patho- Biology of Indirect Acute Lung Injury. Inflammation, Immunity and Cancer: Neutrophils and Other Leukocytes, Verona Italy, 2016

 Bethany Biron Girard, Yaping Chen, Chun-Shiang Chung, Jonathan Reichner, Alfred Ayala. PAD4 Deficiency Limits Kidney Dysregulation in a Murine Model of Shock/Sepsis. Experimental Biology, Chicago Il, 2017

Presentations (Oral & Poster):

 Bethany Biron Girard, Chun-Shiang Chung, Yaping Chen, Jonathan Reichner, Alfred Ayala. Do NETs Matter? Establishing a Role for Neutrophil Extracellular Traps in Patho- Biology of Sepsis and Indirect Acute Lung Injury. Brown University-Graduate Pathobiology Retreat, East Providence, RI (2016)

 Biron BM, Chen Y, Chung CS, Reichner J, Ayala A. Cl-amidine Prevents Histone 3 Citrullination and Improves Survival in a Murine Sepsis Model. 23rd Annual Hospital Research Celebration, Providence, RI (2015).

 Biron BM, Chen Y, Chung CS, Reichner J, Ayala A. Cl-amidine Prevents Histone 3 Citrullination and Improves Survival in a Murine Sepsis Model. Brown University- Graduate Pathobiology Retreat, West Greenwich, RI (2015).

 Biron BM, Chen Y, Pulido S, Chung CS, Reichner J, Ayala A. The Role of Peptidylarginine Deiminase 4 Catalyzed Histone 3 Citrullination and NET formation in Murine Polymicrobial Sepsis Model. 22nd Annual Hospital Research Celebration, Providence, RI (2014).

 Biron BM, Chen Y, Pulido S, Chung CS, Reichner J, Ayala A. The Role of Peptidylarginine Deiminase 4 Catalyzed Histone 3 Citrullination and NET formation in Murine Polymicrobial Sepsis Model. Brown University-Graduate Pathobiology Retreat, Bristol, RI (2014).

 Biron BM, Chung CS, Ayala A. Expression of Soluble PD-L1 in a Mouse Model of Polymicrobial Sepsis. Brown University-Graduate Pathobiology Retreat, Bristol, RI (2013).

 Biron BM, Dyer D, Pitt ML. Development of a New Zealand White Rabbit Partial Survival Model for Inhalational Anthrax to Demonstrate Added Value of Adjunct Therapies, Aerobiology in Biodefense IV, Richmond VA (2011)

 Biron BM, Dyer D, Pitt, ML. Indication Markers for the Treatment of Pneumonic Plague in African Green Monkeys, JHU research symposium, Rockville MD (2011); Ft Detrick Spring Research Festival, Frederick MD (2011)

 Biron BM, Dyer D, Pitt ML. Comparison of Inhalational Anthrax in African Green Monkeys, Rhesus Macaques, and Cynomologous Macaques. JHU research symposium , Rockville MD (2010); Ft. Detrick Spring Research festival, Frederick MD (2010)

Teaching Experience Fall 2013: Teaching Assistant for Dr. Peter Shank PhD. Biology of AIDS, Brown University, Providence RI

ACKNOWLEDGEMENTS

The journey to this point has been long and winding with many ups as well as downs. I could not have made it this far without the support system I have.

To my mom: I feel like this dissertation is equally yours as it is mine. You have always been there to push me when I didn’t feel like I could keep going. Whether it was school, work, or life in general, you have always listened and gave me the advice I needed (even if it wasn’t the advice I wanted). As you always say “I’ve worked too hard for this PhD to give up now”, so this is for you!

To my dad: I attribute my love of science to you and your own quest to always learn something new. From performing experiments from our children’s science books, building bridges out of coffee stirrers, building catapults, or just naming different plants and trees as we walked along the many trails we have hiked as a family, you taught me to be curious about the world and that if there is a will there is a way to figure it out . I will be forever grateful to you for being my first science teacher and for my ability to name plant life found in the forests of New England

To Adam: What can I say other than thank you for always being there. You have been there for this whole process, and have never been anything but supportive of my endeavors, even if they take us up and down the east coast. When I have questioned myself you were there to tell me how awesome and smart I am, and always gave me the boost I needed no matter what. Love you always!

To my Kids: You guys are my everything. You mean so much to me and couldn’t imagine my life without you. I hope that momma can always make you proud and teaches you that you can do anything you want as long as you put your mind to it.

Dr. Alfred Ayala: Thank you for taking a chance on me and letting me be a part of your lab. You have been one of the best people I have ever worked for. Through trying to find a project and many failed experiments, to juggling the balance of work and being a first time mom, you have been nothing but supportive of my scientific career. I have learned so much in my short time in your lab, and for that I will be forever grateful.

Yaping and Shiang: You are the best Chinese mommas ever. Thank you for always being willing to help me out on my experiments, or offering advice on how to make assays and procedures work for me. I couldn’t have completed this dissertation without you guys.

The Ayala Lab: To everyone who has come and gone and who is still around, you guys make coming to work fun. Thank you for everything.

Pathobiology Program: Thank you to the Pathobiology program and all the people in it for making this PhD experience as smooth as possible. I will never forget the people I have met and the support given to me to complete this degree.

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Table of Contents

Chapter 1 INTRODUCTION ...... 1 Sepsis and Severe Shock in the Critically Ill and/or Injured Patient/Animal ...... 2 Sepsis: ...... 2 Organ Injury and the Critically Ill Patient/Animal: ...... 3 Acute Respiratory Distress Syndrome (ARDS): ...... 3 Neutrophils ...... 5 Migration: ...... 6 Effector Functions: ...... 7 Neutrophil Extracellular Traps (NETs) ...... 8 ROS Dependent pathway for NET formation: ...... 9 ROS independent pathway for NET formation: ...... 9 Protein Arginine Deiminases (PADs) ...... 11 Peptidyl arginine deiminase, type IV (PAD4): ...... 11 NETs: Good or Bad? ...... 13 The Good ...... 13 The Bad ...... 14 Autoimmunity: ...... 14 Vascular Disease: ...... 15 The role of Neutrophils in Sepsis and Indirect Acute Respiratory Distress Syndrome Resultant from the Sequential Insults of Shock/Sepsis...... 17 Sepsis: ...... 17 Alterations in cell migration: ...... 17 Alterations in effector functions: ...... 19 Indirect ARDS: ...... 19 Mechanisms for neutrophil recruitment: ...... 20 Endothelial dysfunction: ...... 22 The Role of NETs in Sepsis and indirect ARDS ...... 23

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Sepsis: ...... 23 Cf/DNA and DNAse: ...... 23 Citrullinated Histone Inhibitors ...... 26 Cl-amidine ...... 26 PAD4 gene deletion ...... 27 NET Activators: ...... 27 Indirect ARDS: ...... 28 ARDS and AKI overlap ...... 30 Animal Models to study the Pathobiology of Sepsis and the Sequential Insults of Shock/Sepsis...... 31 Cecal Ligation and puncture a model of polymicrobial sepsis: ...... 31 Hemorrhage/cecal ligation and puncture (Hem/CLP): an experimental model of the sequential insults of shock/sepsis (indirect ARDS model) ...... 32 Hemorrhage ...... 32 CLP ...... 33 Significance of Our Work ...... 33 Central Hypothesis ...... 34 Aim 1: Establish the role of peptidylarginine deiminase IV (PAD4) on histone 3 citrullination and NET formation during sepsis (Chapter 2 and 3) ...... 34 Aim2: Determine the role of PAD4 in lung injury (iARDS) produced as a result of the sequential insults of shock (Hemorrhage)/sepsis (CLP) (Chapter 4) ...... 35 REFERENCES ...... 40 Chapter 2 Cl-amidine Prevents Histone 3 Citrullination, NET formation, and Improves Survival in a Murine Sepsis model ...... 73 ABSTRACT ...... 75 INTRODUCTION ...... 76 MATERIALS AND METHODS ...... 78 RESULTS ...... 82 DISCUSSION...... 86 REFERENCES ...... 100 Chapter 3 PATIENTS WITH SEPSIS EXHIBIT EVIDENCE OF CITRULLINATED HISTONE MODIFICATION AND NET PRODUCTION IN THEIR BLOOD: a small observational study ...... 108 ABSTRACT ...... 110

INTRODUCTION ...... 112 MATERIALS AND METHODS ...... 113 RESULTS ...... 115 DISCUSSION...... 117 REFERENCES ...... 125 Chapter 4 Innate immune mechanisms in organ injury: PAD4 deficiency leads to decreased organ dysfunction and improved survival in a murine model of indirect-acute lung injury .... 130 ABSTRACT ...... 132 INTRODUCTION ...... 134 MATERIAL AND METHODS ...... 136 RESULTS ...... 142 DISCUSSION...... 146 REFERENCES ...... 161 Chapter 5 DISCUSSIONS AND CONCLUSIONS ...... 171 OVERVIEW ...... 172 Chapter 2 and 3 Conclusions ...... 173 Implications and potential applications of research ...... 174 Questions and Future Directions ...... 176 Chapter 4 Conclusions ...... 180 Implications and potential applications of research ...... 182 Questions and Future Directions ...... 183 The Big Picture and Final Thoughts ...... 186 REFERENCES ...... 188

List of Figures and Tables

Figure 1-1 ...... 36 Figure 1-2 ...... 37 Figure 1-3 ...... 38 Figure 1-4 ...... 39 Figure 2-1 ...... 91 Figure 2-2 ...... 92 Figure 2-3 ...... 93 Figure 2-4 ...... 94

Figure 2-5 ...... 95 Figure 2-6 ...... 96 Figure 2-7 ...... 97 Figure 2-8 ...... 98 Supplemental Figure 2-9 ...... 99 Figure 3-1 ...... 121 Figure 3-2 ...... 122 Figure 3-3 ...... 123 Figure 3-4 ...... 123 Figure 4-1 ...... 152 Figure 4-2 ...... 153 Figure 4-3 ...... 154 Figure 4-4 ...... 155 Figure 4-5 ...... 156 Figure 4-6 ...... 157 Figure 4-7 ...... 158 Figure 4-8 ...... 159 Figure 4-9 ...... 160

Table 1-1 ...... 39 Table 3-1 ...... 121

Chapter 1 INTRODUCTION

Sepsis and Severe Shock in the Critically Ill and/or Injured Patient/Animal

Sepsis: The concept of Sepsis has been around since the beginning of ancient civilization.

The term Sepsis is derived from the ancient Greek word “sipsi” which means “to make rotten”(1). It has been described in the writings of Homer as well as Hippocrates who is known as the “father of founding medicine” (1, 2). As history has progressed, the definition of sepsis has changed and evolved with our understanding of the disease. As recently as 2016, the definition for sepsis has been updated and redefined. Today sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection (3).

Patients die as a result of organ failure as the disease elicits an exacerbated immune response with approximately 250,000 fatal cases in the USA annually (4, 5). As of 2009 the CDC listed sepsis as the eleventh leading cause of death in the United States (6, 7).

Sepsis is also costly. Total hospital costs for patients with severe sepsis increased from

$15.4 billion in 2003 to $24.3 billion in 2007.

Traditionally, the host immune response to sepsis was thought to be characterized by two sequential stages: (1) an initial hyper-inflammatory response, sometimes referred to as a cytokine storm. In this initial phase the innate immune system releases pro- inflammatory cytokines to combat infection, while also recruiting members of the adaptive system to mount an intense immune response. This initial response then thought to be followed by (2) a compensatory anti-inflammatory response syndrome

(CARS). In this second phase, there is systemic deactivation of the immune system

2 tasked with restoring homeostasis from an inflammatory state (8). This complex process can become dysregulated leading to persistent immune suppression and high risk for reoccurring infections (9, 10). Recent data suggests that both aspects of the pro- inflammatory and anti-inflammatory stages of the host immune response to severe injury and/or sepsis often occur concurrently (figure 1-1) (9). This in turn leads to tissue damage and Multiple Organ Dysfunction (MODs) (9, 11).

In critically ill patients that suffer from trauma, complex surgery, gastrointestinal bleeding, obstetrical bleeding, etc., there can be a severe deterioration of the pro- inflammatory cytokine response leading to a state of immunosuppression. This can be associated with an increased susceptibility to bacterial infections (12). These patients can then go on to develop Acute Respiratory Distress Syndrome (ARDS) with approximately 25% of ARDS cases stemming from severe sepsis (13, 14).

Organ Injury and the Critically Ill Patient/Animal:

Acute Respiratory Distress Syndrome (ARDS): ARDS is a critical condition associated with significant mortality (15). 40% of all ARDS cases result in death, accounting for around

75,000 deaths per year in the USA (16). ARDS is characterized by bilateral alveolar infiltrates, decreased lung function, pulmonary edema, increased lung micro-vascular permeability, and an influx/sequestration of activated neutrophils into lung interstitium and alveolar space (13, 17). In 1994 ARDS was formally defined by the American-

European Consensus Conference (AECC) (18). The AECC defined ARDS as the acute onset of hypoxemia (arterial partial pressure of oxygen to fraction of inspired oxygen

[PaO2/FIO2]200 mm Hg) with bilateral infiltrates on frontal chest radiograph, with no evidence of left atrial hypertension (18). Acute lung injury (ALI) was also defined using similar criteria, but with less severe hypoxemia (PaO2/FIO2300 mm Hg ARDS was also categorized into direct injury and indirect injury with direct pulmonary insults, such as pneumonia, aspiration, contusion, and reperfusion causing direct injury to the lung, and extra (indirect) pulmonary insults (iARDS) like extra pulmonary sepsis, burn injury, massive transfusion, and trauma causing indirect injury (17).

In 2011 an initiative of the European Society of Intensive Care Medicine endorsed by the

American Thoracic Society and the Society of Critical Care Medicine developed the

Berlin Definition for ARDS, which focused on feasibility, reliability, validity, and objective evaluation of its performance (table 1-1). The updated definition classifies ARDS as mild, moderate, or severe according to the value of PaO2/FiO2 ratio with the

PaO2/FiO2 ratio value being considered only with a Continuous positive airway pressure

(CPAP) or Positive end-expiratory pressure (PEEP) value of at least 5 cmH2O (17, 19).

One of the most notable changes to the definition was the removal of the term ALI; as it was felt that the term was not being used correctly, with many clinicians and researchers viewing ALI as a category of patients (i.e., PaO2/FiO2 201–300) that was distinct from ARDS rather than an umbrella term for all patients, thus leading to the frequent use of the term ALI/ARDS (20).

The pathophysiology of ARDS is complex and multifactorial. However, in comparison to direct ARDS, iARDS is not as well understood. This may be due to the

4 possible heterogeneity of the factors involved (13). Proposed mechanisms of iARDS involve a priming event of immune cell populations as a result of the initial insult that acts upon lung cells, leading to a breakdown of epithelial and endothelial barrier function, leukocyte infiltration, and a dysregulated immune response (21–23). In experimental mouse models of iARDS, hemorrhage has been shown to prime immune cell populations, including neutrophils, macrophage, and T cells, such that exposure to a secondary challenge (such as CLP) elicits a dysregulated and detrimental inflammatory response, as in ARDS (figure 1-2) (23–25). Our lab has demonstrated a murine model of hemorrhage (Hem)/ cecal ligation and puncture (CLP) where the hemorrhagic shock serves as the priming event followed by subsequent CLP challenge leads to the pathology characteristics of iARDS seen in patients (23, 26, 27).

Neutrophils

Neutrophils are short-lived terminally differentiated leukocytes that are generated in the bone marrow from myeloid precursors (28, 29). Neutrophils are continuously released into circulation. This process is regulated by the cytokine granulocyte colony- stimulating factor (G-CSF)(30, 31). Under basal conditions, neutrophils circulate for a brief period (approximately 6 h) before being cleared by the liver, spleen, or bone marrow (30, 32). In addition to the neutrophil population in circulation, there is a large storage pool of mature neutrophils in the bone marrow. This reservoir is called the bone marrow reserve. This reserve of neutrophils is maintained in the bone marrow by stromal cell-derived factor (SDF-1α) and chemokine receptor 4 (CXCR4) (33). During

5 inflammation or in a response to infection, this reserve of neutrophils is released from the bone marrow, leading to a rise in circulating neutrophil numbers (30, 34).

Migration: During periods of inflammation or infection, neutrophils are recruited from the bloodstream to the site of inflammation and/or infection through a process termed chemotaxis. This process is regulated by a number of chemoattractants such as

Complement component 5a (C5a), small lipid mediators, as well as secreted pro- inflammatory chemokines (31, 35). These chemoattractants act as immediate mediators of inflammatory responses by regulating the neutrophil recruitment cascade.

In most tissues, the leukocyte recruitment cascade involves the following steps: tethering, rolling, adhesion, crawling and, transmigration (36). Neutrophil recruitment from the bloodstream is initiated by changes on the surface of the endothelium. This is a result of stimulation by pro-inflammatory mediators, such as IL-1 β, TNF- α, CXCR2.

These mediators are released from tissue resident sentinel leukocytes, which are activated by the ligation of various pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) molecules (37, 38).

Within minutes, the endothelium up-regulates the release of pre-stored P- selectin from Weibel–Palade bodies. E-selectin is also up regulated within 90 minutes

(39) . P-selectin and E-selectin then bind to their ligand, P-selectin glycoprotein ligand 1

(PSGL-1). This allows for the neutrophil to adhere to the endothelium. Additionally, chemokines released by macrophages activate leukocytes allowing for β2 integrins to switch from the default low-affinity state to a high-affinity state. In the activated/high

6 affinity state, integrins bind tightly to complementary receptors on endothelial cells such as ICAM-1 (36). This causes the arrest of the neutrophil movement on the endothelium. Before crossing the walls of post capillary venules, neutrophils crawl inside blood vessels in a MAC1- and ICAM1-dependent manner (40), in search of preferred sites of endothelial cell transmigration. This transmigration through the endothelial cell layer occurs paracellularly (between endothelial cells) or transcellularly

(through an endothelial cell) (41, 42), with neutrophils predominately choosing the paracellular route of transmigration under inflammatory conditions (43).

Effector Functions: Once at the site of infection neutrophils are capable of eliminating pathogens through different mechanisms (29, 44). These killing mechanisms include phagocytosis of microbes in which the microbe is encapsulated into a phagolysosome and reactive oxygen species and/or antibacterial proteins are released to kill the microbe (45). The second mechanism termed degranulation utilizes azurophilic granules, specific granules, and tertiary granules found within the neutrophil

(46), all of which contain antimicrobial proteins that aid in the killing of pathogens.

During this process, these antimicrobial proteins are released from neutrophil granules either intracellularly or extracellularly to combat pathogens (47). The third killing mechanism used by neutrophils to combat pathogens is the release of neutrophil extracellular traps (NETs) (44). NETs are comprised of histones, granule proteins, neutrophil elastase, myeloperoxidase, and bactericidal permeability increasing protein

(BPI) that line the DNA backbone and then are released by the neutrophil into the extracellular space as a way to entrap microbes (44, 48). To date, our understanding of

7 the role of this last form of microbial clearance is the least well-understood. Hence, we have chosen to discuss what is known about the basic biology of NET formation in greater detail below.

Neutrophil Extracellular Traps (NETs)

NET formation, a process also known as NETosis, is a neutrophil effector function in which cells release a web like structure of chromatin fibers complexed with granule- derived antimicrobial peptides and enzymes (44). Microbes are then ensnared within the chromatin network and are killed via the extracellular microbicidal activity associated with granular enzymes within the NET (49–51). NETs have the ability to trap and kill a broad range of microorganisms, including gram-negative and gram-positive bacteria, fungi, viruses, and protozoa (35–41).

NETosis is a slow process that occurs over hours post-stimulation. For NET formation to occur the following is required: (1) the production of reactive oxygen species (ROS), (2) the migration of the protease neutrophil elastase (NE) and myeloperoxidase (MPO) from granules to the nucleus, (3) the decondensation of histones, and (4) the rupture of the cell which leads to cell death (figure 1-3) (51–53).

To release NETs, activated neutrophils undergo dramatic morphological changes.

Minutes after activation, they flatten and firmly attach to a surface that stimulates MAC-

1, as this is needed for efficient NET formation (54). The nucleus then loses its lobules, chromatin decondenses, and the inner and outer nuclear membranes progressively detach from each other. Concurrently, intracellular granules disintegrate. After 1 hour,

8 the nuclear envelope disaggregates into vesicles and the nucleoplasm and cytoplasm form a homogenous mass. Finally, the cell rounds up and seems to contract until the cell membrane ruptures and the interior of the cell is ejected into the extracellular space forming NETs (51, 53).

ROS Dependent pathway for NET formation: The classical pathway of NET formation as described above is dependent on the production of ROS, where nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived ROS acts at the intracellular level and is required to initiate the formation of NETs (53, 55). Inhibition of

NADPH oxidase in human and mouse neutrophils have been shown to prevent NET formation (56, 57). Additionally, chronic granulomatous disease (CGD) patients that have impaired NADPH oxidase function are unable to generate NETs (58). The mechanism of how NADPH oxidase-derived ROS influence NET formation inside the cell is not fully understood particularly how ROS influences the main features of NET formation. Studies exploring NADPH oxidase role in NET formation showed that singlet oxygen produced during ROS production is essential for NET formation in human neutrophils upon stimulation with PMA (59). Additionally, ROS production allows for the release of NE and MPO from the azurophilic granules in the neutrophil cytoplasm, and is needed for the translocation of these enzymes to the nucleus, where NE aids in histone degradation and MPO promotes chromatin decondensation (60, 61).

ROS independent pathway for NET formation: An alternative form of NET formation, termed ‘Vital’ or early/rapid NETosis, differs in that it occurs within minutes

9 of stimulation. This process is activated by complement-opsonized targets, and it occurs in mostly in extravasated neutrophils. These neutrophils then lose their nuclei and can be observed as nuclear cytoplasts. Vital NETosis is thought to be independent of ROS as well as PAD4 activation (62, 63). However, it does rely on signaling through TLRs, as neutrophils deficient in either MyD88 or TLR2 do not initiate the response after microbial challenge. During vital NETosis the neutrophil remains enucleated and capable of additional functions such as migration and phagocytosis (64). Studies with s. auerus and Leishmania suggest that rapid, ROS-independent, NET formation has the capacity to ensnare and kill pathogens (62, 63).

While both pathways are able to generate NETs that are capable of microbial killing, the specific mechanisms whereby oxidants participate in NET formation remain to be clarified as requirements for ROS generation seeming to depend on the stimulus.

Parker et al. (65) demonstrated that NET formation requires ROS when induced with

PMA or by bacterial stimulation; however NET formation is independent of ROS production when they are induced via calcium influx mediated by the bacterial calcium ionophore ionomycin. Other studies have also shown that PMA-induced NET release from human neutrophils is dependent on ROS activation through the MAPK/ERK pathway (56). The requirement for MPO for NET formation also seems to depend on the stimulus. MPO was shown to be essential for NET formation in a model of Candida albicans infection with the same study also demonstrating that neutrophils from donors who are completely deficient in MPO fail to form NETs after stimulation with PMA (66).

Alternatively, other studies have demonstrated that MPO-deficient neutrophils release

NETs when stimulated with P. aeruginosa, S. aureus or E. coli, but fail to respond to PMA

(65). Likewise, a model of Leishmania-induced NETosis demonstrated that NET production is not dependent on MPO activity (62).

Protein Arginine Deiminases (PADs)

Protein Arginine Deiminases (PADs) are enzymes that catalyze the hydrolysis of peptidyl-arginine to form peptidyl-citrulline on histones, fibrinogen, and other biologically relevant proteins (67). The PAD family is composed of five calcium dependent isozymes. PADs 1–4 and PAD6 and are found in numerous cell and tissue types (67, 68). These include the epidermis and uterus (PAD1), skeletal muscle, brain, inflammatory cells, several cancer cell lines, and secretory glands (PAD2), hair follicles and keratinocytes (PAD3), granulocytes and various cancer cell lines (PAD4), and oocytes and embryos (PAD6) (69, 70) . PADs are Ca2+ dependent and are activated during a high influx of Ca2+ concentrations due to the influx of calcium ions from the extracellular environment as well as the release from intracellular calcium stores (71). Over- expression and/or increased PAD activity has been linked to disease. Citrullination of proteins by PAD4 in particular has been implicated in several disease states including rheumatoid arthritis (RA), multiple sclerosis (MS), ulcerative colitis (UC), systemic lupus erythematosus (SLE), sepsis and thrombosis (68). A key point is that PAD4 also plays an integral role in the formation of NETs (50, 72).

Peptidyl arginine deiminase, type IV (PAD4): PAD4 is a 74kDa protein that exists as a head to tail dimer and is found mainly in granulocytes and monocytes. It contains a

11 nuclear localization signal at its N terminus (73–77). PAD4 binds five calcium molecules,

Ca1–Ca5 (73, 77, 78). Ca1 and Ca2 bind in the C-terminal catalytic domain to induce major conformational changes that move several active site residues into positions important for catalytic activity. Calcium binding also induces large structural changes in the N-terminus of the protein. These conformational changes may provide, or remove, docking sites for other proteins. These changes may in turn serve to further regulate

PAD activity (73). There are four key catalytic residues, Asp350, His471, Asp473, and

Cys645 in the deimination of arginine to citrulline (70). Cys645 and His471 are situated on opposite sides of the guanidinium group of the arginine and are positioned to promote catalysis via nucleophilic attack on the guanidinium carbon (Cys645) and protonation of the developing tetrahedral intermediate. The collapse of the intermediate leads to the loss of ammonia and the formation of the stable S-alkyl thiouronium intermediate, which is subsequently hydrolyzed via a second tetrahedral intermediate to form citrulline (79). His471 likely activates the water molecule for nucleophilic substitution, and Asp350 and Asp473 having a role in substrate binding

(79).

PAD4 was first discovered in the nucleus during study of the terminal differentiation of HL-60 cells into granulocytes and monocytes (75). This research initially suggested a role for PAD4 in cellular differentiation, though the authors were unable to define a state in this process where it was directly involved. Using cancer cell lines, PAD4 has been shown to play a regulatory role in gene expression function as a transcription co-repressor that regulates the expression of multiple p53 target genes

(80–82). PAD4 also acts on Estrogen receptor (ER) target genes in a breast cancer MCF-

7 cell line, in which PAD4 is recruited to the promoters of ER regulated genes, and citrullinates histones H3 and H4 at R17 and R3 respectively. This citrullination correlates with the decreased expression of ER regulated genes (83, 84). Immunologically, PAD4 has a regulatory role in NET formation through mediating chromatin decondensation via citrullination of target histones H3, H4, and H2A (50, 54, 85, 86). PAD4 activity is essential for histone citrullination and subsequent chromatin decondensation, which are critical steps needed for NET formation. Histone citrullination is thought to promote

NET formation by inducing chromatin decondensation and aiding in the release of chromosomal DNA coated with antimicrobial molecules into the extracellular space (50,

54, 85). Without the catalyzing activity of PAD4, NETs cannot be released from the neutrophil (50, 87, 88). Various stimuli and signaling pathways have been implicated in

PAD4 activation and NET formation. These include direct contact with pathogen, pathogen associated molecular patterns (PAMPS), pro-inflammatory signaling, and calcium influx (50, 54, 70, 89). ROS generation is required for NET formation, and therefore has been speculated to be required for PAD4 activation (53, 85, 90, 91).

NETs: Good or Bad?

The Good - NETS have been shown to have a beneficial role in the immune response to infection. NETs are comprised of several proteins that kill or functionally inhibit microbes via enzymes (lysozyme, proteases), antimicrobial peptides (BPI, defensins), ion chelators (calgranulin), and histones. The antimicrobial activity of NETs is

13 the result of the combination of these components in a concentrated space within the

NET (44). NET release has been demonstrated to capture a wide array of pathogens included Gram-positive and Gram-negative bacteria, fungi, protozoa, and viruses (92–

98). They also act as a physical barrier preventing the spread of microorganisms (44,

99). NETs also have the ability to inactivate virulence factors, further limiting the pathogen. Neutrophil elastase on the NETs specifically cleaves virulence factors of Shigella flexneri, Salmonella typhimurium, and Yersinia enterocolitica (44, 51, 100)

Various pathogens have adapted different methods to evade NET capture. This includes

Streptococcus pneumonia which masks itself within a capsule to prevent binding to NETs

(101). Group A Streptococcus attach nucleases to their surfaces to disengage themselves from NETs (102). Additionally, Group A S. pyogenes pneumococcus (103), and Staphylococcus aureus (93) encode endonucleases that free them from NETs, allowing for further spreading into deeper tissues (92).

The Bad - Despite to the role they play in pathogen containment and killing, NETs have been implicated in a number of deleterious effects. At times they have even been described as pathogenic. Increasing evidence has linked NET formation to disease states suggesting that they contribute to excessive inflammation and tissue damage

(104–106).

Autoimmunity: In Small Vessel Vasculitis (SVV), patients can develop anti- neutrophil cytoplasmic antigens (ANCAs) which induce NETosis, with autoantigens recognized by ANCAs being found in the NET structure. Additionally, NET fragments can

14 be detected in blood and in kidney biopsies from a large proportion of SVV patients

(107). In patients who suffer from systemic lupus erythematosus (SLE), there is an imbalance between NET formation and NET clearance (108–110), leaving patients prone to NET-associated tissue damage. An abnormal neutrophil subset, termed “low density granulocytes” has also been identified in lupus patients. This cell subset has been shown to have an increased tendency to form NETs leading to endothelial cell damage and increased levels of pro-inflammatory cytokines and type I interferons (109).

NETs have been studied extensively in Rheumatoid arthritis as the source of citrullination of extracellular proteins. RA patients make antibodies to deiminated proteins, collectively identified as anti-citrullinated proteins/peptides antibodies (ACPA).

The targets of ACPA include auto-antigens as well as exogenous antigens. These proteins become the target of ACPA after citrullination (111). A major contributor to the generation of Citrullinated proteins in RA comes from the enhanced ability to form

NETs, by RA neutrophils from the periphery or synovial fluid (111).

In synovial fluid, NETs release active PAD2 and PAD4, which are in turn able to citrullinate extracellular proteins in the inflamed joint (112). Together, these data suggests that neutrophils have an active role in the inflammatory process of RA in part by being the source of and post-translationally modified by auto antigens through NET formation (113, 114).

Vascular Disease: NETs have been demonstrated to induce vascular damage in both murine models as well as in experimental human studies (115, 116), giving NET

15 formation a potentially important role in diseases such as atherosclerosis, thrombosis and vasculitis (99, 117). NETs have been shown to have a significant role in pathogenic thrombosis through platelet and PMN recruitment to the endothelial wall, subsequent activation and NETosis. This leads to the activation of intrinsic and extrinsic coagulation cascades (106, 115, 118). NET related proteins such as PAD4 have been shown to be required for thrombus formation in the inferior vena cava (IVC) as well as in a stenosis model of murine deep vein thrombosis (DVT) (119). In clinical studies of thrombosis, biomarkers of NET formation are used to correlate disease severity with NET formation.

For example, circulating nucleosomes and markers of neutrophil activation (elastase-α1- antitrypsin and MPO) have been shown to be significantly increased in patients with

DVT, compared to symptomatic patients with unconfirmed DVT diagnosis (120, 121).

NETs are indeed a double-edged sword with both positive and negative attributes. NETs function as a valuable antimicrobial defense mechanism by containing and eliminating pathogens; however, NETs can lead to a dysregulation/accumulation of pro-inflammatory mediators within tissues and vasculature, leading to tissue damage and organ failure. It is this double-edged nature of NETs that has made it difficult to determine the role of NETs in the immune response to insults like trauma and/or sepsis, with studies demonstrating mixed results as to whether NETs are beneficial or detrimental to the host. Currently, determining if the role of PAD4 citrullination and

NET release in sepsis and subsequent indirect Acute Respiratory Distress Syndrome is a critical component of the host response has not been fully investigated. It is the goal of this dissertation to elucidate the role of NET formation in the protection of the host

16 from septic insult and subsequent indirect acute lung injury as a result of hemorrhage/septic insult.

The role of Neutrophils in Sepsis and Indirect Acute Respiratory Distress Syndrome

Resultant from the Sequential Insults of Shock/Sepsis

Sepsis: During septic infection, innate immune function becomes dysregulated.

This includes neutrophil response to infection. There is an underwhelming antimicrobial response, and impairment in neutrophil recruitment during severe sepsis (122, 123).

Additionally, there is also a detrimental accumulation of neutrophils within vital organs typically remote to the nidus of peritoneal sepsis, such as lungs, liver, kidneys, and heart. This is thought to lead to bystander tissue damage and organ dysfunction/failure

(28, 124–126). This Impairment in neutrophil chemotactic responses and changes in neutrophil function are directly correlated with increases in sepsis-mediated morbidity and mortality (123). Multiple mechanisms have been shown to contribute to the sepsis- induced impairment of neutrophil migration /function, however, our full understanding remains incomplete as to how these processes take place and what their significance is to the developing pathology of severe shock and/or sepsis (127).

Alterations in cell migration: Processes that are involved in neutrophil impairment as a result of sepsis include marked increase in the rigidity of neutrophil cell membranes thought to contribute to their accumulation in capillary beds, especially those of the lung. These sequestered neutrophils appear to neither migrate through endothelium nor complete their passage through the capillary (128, 129). This capillary

17 bed sequestration of neutrophils leads to microvascular occlusion in some cases, contributing to tissue ischemia and subsequent multiple organ failure (128).

Nitric Oxide production, which is increased during sepsis, contributes to impairment in neutrophil migration as well. Nitric oxide (NO) has been identified as an important modulator of neutrophil migration (130). Nitric oxide directly inhibits leukocyte–endothelial cell interactions, primarily by inhibiting ICAM-1 and VCAM-1 (131,

132). This role for NO was further confirmed by Mestriner et al. (133) who demonstrated that acute-phase protein α-1 inhibited neutrophil migration into the rat peritoneal cavity following carrageenan injection or CLP in a dose-dependent manner, and that this effect could be eliminated either by pharmacological inhibition of iNOS or by genetic deficiency of this enzyme.

Another characteristic of a septic response is the increase in the release of leukocyte chemoattractants that function through activation of G protein-coupled receptors (GPCR). The sustained elevated levels can result in functional ‘desensitization’ of receptor responsiveness, which can occur as a result of down regulation of GPCR cell surface expression (127, 134). This increase in GPCR ligand expression can also diminish chemotactic responses through phosphorylation of agonist-occupied GPCR by GPCR kinases (GRKs) (135). Neutrophils isolated from septic patients display this increase of

GRK2 and GRK5 suggesting that increased GRK activation in septic patients may induce neutrophil desensitization to chemoattractants (136).

Alterations in effector functions: As sepsis evolves, neutrophil gene expression is altered, leading to the suppression of pro-inflammatory and immunomodulatory genes and decreased production of ROS (126). Microarray analysis of neutrophils collected from patients with sepsis within 24h from the time of admission exhibited suppression of several gene clusters including inflammatory response genes, immune modulation genes, and genes required for oxidant production (137). The effect of sepsis on the phagocytic capacity is less clear. During the immune-suppressive stage of sepsis, phagocytosis is conserved or even increases in patients (138). Alternatively, another study concluded that severe sepsis is associated with a significant increase in circulating immature neutrophils. These immature neutrophils have decreased phagocytosis and aberrant calcium signaling, which suggests an undermining of overall phagocytic efficacy (139).

Indirect ARDS: One of the key features of ARDS is the dysregulation and recruitment of activated neutrophils to the lung microvasculature, interstitium, and alveolar space (13, 23, 140). This excessive neutrophil activation and accumulation leads to increased ROS and pro-inflammatory mediator production and a decrease in

PMN apoptosis. This results in epithelial and endothelial cell damage (13, 25). In bronchoalveolar lavage fluid (BALF) of ARDS patients, neutrophil concentrations have

19 been correlated to disease severity and poor outcome (141, 142). In cases of iARDS, both non immune cells (epithelial/endothelial) and immune cells (neutrophils and macrophages) are primed, which leads to the recruitment of neutrophils to the lung and subsequent pathogenesis (13).

Mechanisms for neutrophil recruitment: Neutrophil recruitment into the lung takes place in the small capillaries in a sequence of activation, sequestration from blood to interstitium and transepithelial migration (143). In this regard, neutrophils need to alter their shape to pass through the pulmonary capillaries. Inflammatory mediators such as C5a, leukotrienes, platelet activating factor, IL-8 or endotoxin can induce neutrophils to become stiffer, increase in size and prolong transit through lung capillaries, thereby facilitating endothelial–neutrophil interactions (144). Activated

PMNs from ARDS patients appeared even more rigid than those from septic patients

(128).

In animal models of ARDS, numerous factors have been associated with neutrophil recruitment into the lung and alveolar spaces. Examples include cell adhesion molecules, cytokines, and chemokines (145). Adhesion molecules regulate the emigration of activated PMNs and passage through the endothelium (144). These include the up-regulation of L-selectin and CD11b/CD18 on neutrophils to initiate contact with the endothelium, and CD31 (aka PECAM-1) for vascular diapedesis (13,

146, 147). The role of adhesion molecules in the recruitment of neutrophils in ARDS is complex. Trauma patients have been shown to have an acute increase of PMN L-

20 selectin and CD11b expression (148). On the other hand, decreases in L-selectin expression in response to trauma have also been described (149). Doerschuk et al (150) described both a CD11b/CD18-dependent and CD11b/CD18 independent pathway for neutrophil emigration into the lung. In this study, PMN emigration in response to Escherichia coli, LPS, Pseudomonas aeruginosa, IgG immune complexes and IL-1 was mediated by CD11b/CD18. However, in response to Streptococcus pneumoniae, Group

B Streptococcus, Staphylococcus aureus, hyperoxia or C5a, neutrophil emigration is

CD11b/CD18 independent (150). Taken together, these results would suggest that neutrophils do not seem to require adhesion molecules, but they allow neutrophils to stay adhered for a longer period of time (144, 150).

Chemokine IL-8 (CXCL8) and the rodent homologues CXCL1 (KC) and CXCL2 (MIP-

2) are considered to be central to neutrophil recruitment into the lung during ARDS.

High concentrations of IL-8 in BAL fluid from ARDS patients were associated with increased neutrophil influx into the airspace (151, 152) and have been correlated to disease severity (152, 153). IL-8 is able to bind to receptors, CXCR1 or CXCR2. Mouse homologs CXCL1 and CXCL2 both signal through CXCR2. While a functional CXCR1 homologue has now been discovered in mice, its importance in neutrophil migration is not clear (154). Using a mouse model of Hem/CLP as a model for iARDS, blockade of

CXCR2 significantly reduced neutrophil influx, lung protein leak, and lung-tissue content of interleukin IL-6, CXCL1, and CXCL2 and increased tissue IL-10 levels. Suggesting that blockade of CXCR2 signaling attenuates shock-induced priming and ARDS (155). In the same model of Hem/CLP CXCL2 siRNA significantly reduced IL-6 levels in the lung tissue

21 as well as systemically. It also reduced neutrophil influx into the lung (156). CXCL1 siRNA treatment reduced local and systemic levels of CXCL1, but there was no significant reduction of systemic IL-6 or neutrophil infiltration in the lung. Together these data support the role of these chemokines in neutrophil recruitment to the lung and the pathogenesis of ARDS.

After an acute insult such as trauma or septic infection, there is systemic release of cytokines and mediators including LPS, TNF, IL-1 and IL-6. These have a wide range of effects on endothelium, epithelium and on circulating and resident immune cells. Pro- inflammatory cytokines IL-1β and TNF-α have been identified in BAL fluids from patients with ARDS along with their specific antagonists IL-1RA and soluble TNF receptors (157).

This secretion of pro-inflammatory cytokines and chemokines by lung epithelial cells, has been shown in mouse models to activate Fas/Fas ligand (FasL) system leading to increased expression of TNF-α, MIP-1α, MIP-2, MCP-1 and IL-6. The alveolar–capillary barrier becomes compromised (158, 159) leading to increased inflammation in the lung tissue.

Endothelial dysfunction: Endothelial cells (ECs) are important mediators of the critical balance between fluid homeostasis, immunity and inflammation (160). They form a tight barrier between the pulmonary circulation and lung parenchyma and maintain normal gas exchange and tissue oxygenation. With the initiation of the inflammatory response, endothelial cells promote neutrophil transepithelial migration and favor platelet aggregation and coagulation that result in microvascular obstruction

22 of pulmonary capillaries (161). Endothelial activation and subsequent damage has been described in sepsis related ARDS cases (162, 163), and severe hemorrhage/hemorrhagic shock have shown to be causative for the development of indirect ARDS and are associated with severe endothelial cell injury (164, 165). In addition, circulating endothelial cells have been documented as being significantly higher in patients with moderate or severe sepsis-related ARDS compared to patients with sepsis but no

ARDS, suggesting circulating ECs could be used as a potential biomarker of EC activation in ARDS (166).

The Role of NETs in Sepsis and indirect ARDS

Sepsis: NETs have been shown to play a role in sepsis. Components of NETs have been shown to be elevated in septic and septic shock patients (167, 168) suggesting that

NETs may play an important part in the innate immune response to infection (50).

However, increasing evidence has linked NET formation to various disease states, such as autoimmune diseases, as well as sepsis, suggesting that they contribute to excessive inflammation and tissue damage (104–106). Various methods have been utilized to study the role of NETs in sepsis including characterizing different components of NETs as a marker for NET formation.

Cf/DNA and DNAse: Recently, there has been interest in the use of circulating – free DNA (cf-DNA) as a potential biomarker in septic patients. The measurement of cf-

DNA has been shown to be useful for early risk stratification and prediction of in

23 hospital and overall morbidity and mortality in a range of conditions including stroke, myocardial infarction, cancer, and trauma (169). Circulating free DNA (cf-DNA) levels in the blood are increased in various infectious diseases, including sepsis (167, 170).

Recent studies have reported that cf-DNA is associated with NETs, (44, 167). To this end, a wide array of studies have used DNase as a way to degrade cf-DNA/NETs as an indirect way to study the impact of NETs in sepsis (171–175)

DNase has been utilized in mouse models of sepsis to study the role of NETs in terms of inflammatory response, and antimicrobial activity. Degradation of NETs, using

DNase to test differences in survival has also been undertaken (171–175). Using CLP and LPS models it has been determined that NETs are involved in pro-inflammatory and pro-coagulant responses, and they potentially contribute to immune dysregulation and organ damage (171, 176). However, taken together, results are mixed as to whether

DNase treatment is beneficial or detrimental under septic conditions. While overall

NETs appear to be associated with the pro-inflammatory response in sepsis (173), one of the big questions is whether NETs are actually required to kill pathogens and reduce microbial burden in the host. Using cf-DNA as a marker of NETs and DNase as a NET inhibitor, microbial burden has been evaluated in both the bloodstream, the peritoneal cavity (source of infection), and within tissues of CLP mice. These results have of microbial burden documented with DNase treatment varied. Some have determined that DNase treated mice at 6hr and 24hr post CLP have no significant effect on microbial burden in blood, peritoneal cavity, lungs, spleen, and liver (175, 177), while Mai et al determined that delayed DNase treatment decreased colony forming units (CFU) in the

24 lungs, blood, and peritoneal cavity (172). Others have showed that DNase treatment immediately before CLP and then after CLP led to an increased microbial burden as compared to CLP saline control mice (171). This same study also demonstrated that, when DNase was used in conjunction with antibiotics, there was a decrease in bacterial dissemination in the blood that was comparable to the “antibiotics alone” group. This suggests that NETs play a role in containing the microbial burden.

To support the idea that NETs do have a bacteriocidal activity, a recent study showed that the DNA backbone of NETs have direct anti-microbial properties by disrupting the bacterial outer membrane via cation chelation-mediated disruption (49).

With all of these studies using DNase to study NETs have varying results, it is no surprise that the overall survival data and NET inhibition is mixed as well. In the CLP model

RNase treatment appears to have no real survival benefit. However, a modified treatment regimen such as late/delayed DNase administration or DNase+antibiotic treatment has some protective effect. Using a LPS model, mice treated with DNase have a higher survival rate as compared to saline treated mice (171). This difference between CLP and LPS survival studies adds to the debate as to whether NETs play a role in the inflammatory process, while having no direct role in microbial host interactions or is involved in direct microbial killing. That being said, it has been questioned whether cf-

DNA is really an accurate biomarker of NET formation. Recent studies have shown that the majority of cf-DNA found after CLP is non-immune cell derived and not associated with neutrophils (178), therefore eliminating plasma cf-DNA as a reliable marker of NET

25 formation. However, utilization of cf-DNA in conjunction with other NET components, such as MPO, could improve this method of describing NET formation in vivo. (171).

Citrullinated Histone Inhibitors: Extracellular histones are considered biomarkers of disease progression during septic infection and have been established as potential therapeutic targets (105). Citrullinated histones have been identified as components of

NETs, which are released into the extracellular space as part of the neutrophil response to infection. Citrullinated histone 3 (H3Cit) is released in both the CLP and LPS models of sepsis (168, 179). Histone inhibitors that have been utilized include suberoylanilide hydoxamic acid (SAHA), Cl-amidine, and antibodies against H3Cit (168, 179, 180). All of these inhibitor treatments have improved survival in both the LPS and CLP models.

Cl-amidine: Nα-(2-carboxyl) benzoyl-N5 - (2- chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine) is a PAD inhibitor that irreversibly inactivates PAD1, PAD3, and PAD4 by covalently modifying an active site cysteine that is important for its catalytic activity

(181). There are two mechanisms that have been proposed for this inactivation of PAD4

(182). Mechanism 1 involves direct substitution of the halide, whereas mechanism 2 involves the formation of a tetrahedral intermediate, which first evolves into a three- membered sulfonium ring and subsequently rearranges to a thioether with the collapse of the tetrahedral intermediate. The latter mechanism is invoked to account for the poor leaving group potential of fluoride. Cl-amidine treatment has been used extensively to study the role of NET formation in colitis (183, 184) and arthritis (185).

However, its effects on experimental polymicrobial sepsis/ CLP are not known.

PAD4 gene deletion: In 2010 a PAD4-/- mouse was created to study a purpose for

NETs in the innate immune response (50). This lack of NETs in an immune response to both Gram-negative and Gram-positive microbial infection leads to increased susceptibility to disease, supporting the concept mentioned earlier that NETs may be beneficial against infection. To take this finding further and more specific to septic conditions PAD4 deficient mice were subjected to CLP to study the affect NETs have in survival in a septic model (186). No significant benefit or detriment was found in a severe model of CLP where mortality rates were high in the control group and the PAD4-

/- group. When mice were subjected to low grade CLP model, no difference was noted between groups. However, the high survival noted in the control group may have given too narrow a window to detect any difference.

The same study utilized the PAD4 deficient mice in a lethal LPS model and determined that PAD4 deficiency leads to an increased time to death. It is worthy to note that in both the CLP model and the LPS model PAD4-/- appears to have an acute protective effect. Overall, these data contradict each other on whether NETs have any role in the innate immune response to microbial infection, and more specifically, the immune dysregulation that occurs with septic infection.

NET Activators: Along with using inhibitor strategies above that directly block

NET components and/or known aspects of the process of their release; others have observed NET production under septic conditions appears to be affected by otherwise non-NET specific inhibitors/pathways. In a CLP model of sepsis, peroxisome

27 proliferator-activated receptor gamma (PPARγ) agonist (rosiglitazone) has been shown to increase NET production as well as modulate the inflammatory response, thereby increasing NET dependent elimination of bacteria (187). Another approach was genetic or pharmaceutical targeting of PLD2. After blocking PLD2 there was enhanced neutrophil recruitment, increased NET formation, and increased microbial killing (188).

Both these models argue that NET production is needed for enhanced bacterial killing and improved survival.

However, establishing pathological significance of NETs in a clinical setting of sepsis in the critically ill/injured patient not only has been a challenge, but is vital if we are to determine whether NETs are a potentially viable therapeutic target. Since a majority of septic patients in the ICU also have underlying morbid issues of trauma, e.g., tissue injury/shock, it is important to understand the role of NETs in model that better reflects the contribution of significant tissue injury/shock in the development organ dysfunction among critically ill/injured patients who develops sepsis. Inasmuch, we have chosen to look not only at a model of simple polymicrobial sepsis (e.g., a ruptured appendicitis), but a model that reflects the impact of shock (a common component of significant traumatic injury) and its impact on the neutrophil/NET response to subsequent septic insult, much as is encountered in the severely injured critically ill patient (23).

Indirect ARDS: Components of NETs including neutrophil derived circulating free

DNA (CF-DNA/NETS) as well as circulating histones have been implicated in acute lung

28 injury (167, 189, 190). Infused circulating histones have been shown to lead to alveolar capillary obstruction, histone toxicity in lung tissue and coagulation activation all leading to lung injury as well as multiple organ failure (MOF) (190). Histones have been detected in the BALF and plasma of patients who developed ARDS after trauma and gastric acid aspiration (190, 191). Extracellular histones have been implicated in lung damage in various animal models of lung injury and they directly cause alveolar damage

(105). It is suggested that these extracellular histones are products of NET formation, as depletion or neutralization of neutrophils in mouse models leads to decreased in circulating histones and reduced lung injury (192, 193). However, as stated earlier, the source of CF-DNA and circulating histones remains a source of controversy (178).

NET related damage in lungs has also been reported in LPS injury models where

NET formation was detected in both lung tissue and BALF (189). Following infection with influenza virus H1N1, NETs were found to be associated with damaged alveoli in the lungs of challenged mice (194). While NETs and NET components have been observed in various models of direct ALI as mentioned above, it is not understood how they function in a model of trauma/shock (hemorrhage) followed by septic insult, which is thought to more closely resemble the process of extra (indirect)-pulmonary ALI. Thus, it would be beneficial to determine whether NET formation in an iALI model is overall beneficial or detrimental to the host.

ARDS and AKI overlap

Sepsis leads not only to ARDS (a very common organ injury noted in the critically ill patient) (13, 195), but also to multi-organ injury. Clinically, 35% of patients with ARDS develop acute kidney injury (AKI), and the development of this secondary incident of organ dysfunction dramatically increases patient mortality (196, 197). In this respect, many of the biomarkers of ARDS, such as inflammatory cytokines, endothelial and epithelial cell injury, neutrophil–endothelial interactions, and alterations in adhesion molecules are thought to contribute to the development of acute kidney injury

(197). It is this close association that makes the kidney an organ of interest when studying the MOF associated with sepsis induced iARDS.

In addition to the ARDS AKI overlap, PAD4 and NET formation have both been implicated in renal damage that has been reported in experimental models of acute ischemic kidney injury. In mice, after renal ischemia reperfusion (I/R) injury, activation of platelets and to increased platelet–granulocyte interactions led to the formation of

NETS, which increased renal inflammation and further increase in tissue injury (198).

PAD4 specifically has been implicated in the damage seen in renal I/R injury by increasing renal tubular inflammatory responses and neutrophil infiltration into the kidney. In mice, renal I/R injury leads to an up regulation of renal tubular PAD4 expression, an increase in histone H3 citrullination, and an increase in indices of inflammation. Furthermore, inhibition of PAD4 either chemically (199) or genetically with a PAD4 deficient animal (200) reduced renal tubular necrosis, inflammation, and

30 apoptosis. PAD4-/- also demonstrates decreases in neutrophil infiltration after I/R in the kidneys and liver via immunohistochemistry. These data together suggest that renal tubular PAD4 plays a critical role in renal I/R injury by increasing the renal tubular inflammatory response and neutrophil infiltration after renal I/R.

Animal Models to study the Pathobiology of Sepsis and the Sequential Insults of Shock/Sepsis.

Cecal Ligation and puncture a model of polymicrobial sepsis: To better understand the pathology and mechanisms of sepsis, animal models have been developed to attempt to delineate reliable diagnostic biomarkers specific for sepsis, and to develop therapeutics. There are various mouse models that have been developed to study sepsis, all of which come with their advantages and disadvantages. The two most prominent models currently utilized to study sepsis are LPS injection (simulating endotoxemia) and cecal ligation and puncture (CLP) (replicating an abdominal source of polymicrobial sepsis) (201–203). Polymicrobial sepsis induced by CLP is the most frequently used model because it closely resembles the progression and characteristics of human sepsis (204, 205). CLP creates a bowel perforation with leakage of fecal contents into the peritoneum. This establishes an infection with mixed bacterial flora as well as an inflammatory source of necrotic tissue. CLP generates both the early hypermetabolic and late hypometabolic phases of sepsis (204, 205), and produces a more chronic condition as mortality rate develops over days. This is thought to make it more comparable to the clinical conditions associated with the development of sepsis in patients. Additionally, CLP seems to create a similar immune response seen in human

31 disease (206, 207), with systemic cytokine profiles in CLP resembling the delayed elevation of cytokine levels observed in septic patients (208).

Because of its reproducibility and similarities to clinical sepsis, our lab uses this

CLP model in our study of sepsis. Briefly, an incision is made into the murine abdomen, whereupon the cecum is exposed, ligated and then punctured twice with a 22- gauge needle. A small amount of fecal material is then extruded from the punctured cecum, and the cecum is then returned to the abdomen where the laparotomy incision is sutured closed and the mouse is resuscitated with lactate ringers solution. (202, 209–

211).

Hemorrhage/cecal ligation and puncture (Hem/CLP): an experimental model of the sequential insults of shock/sepsis (indirect ARDS model): As sepsis is often preceded by trauma/shock, our lab uses a “double hit” model that incorporates this clinical component (23, 27, 212). Our mouse model of sequential hemorrhagic shock induces

‘‘priming’’, which predisposes the neutrophil to inflammatory stimuli. This leads to an altered response after a subsequent secondary challenge, namely the induction of peritoneal sepsis. This “double-hit” approach creates an environment that leads to

ARDS, a phenomenon not typically seen in sepsis alone. Of note the development of lung injury/ARDS is not a consistent result peritoneal septic/CLP challenge alone typically (27, 213).

Hemorrhage: mice are anesthetized using an isofluorane vaporizer setup. They are secured in supine position and have catheters inserted into bilateral femoral

32 arteries. When fully conscious, as determined by a mean blood pressure of 95 mm Hg, the mice are then bled (0.8–1.0 mL) over a 5- to 10- min period to a mean blood pressure of 35 mm Hg (5 mm Hg) and kept stable for 90 min. Immediately following hemorrhage mice are resuscitated (IV) with Ringers lactate at four times the drawn blood volume.

CLP: Twenty-four hours post-hemorrhage, sepsis was induced as a secondary challenge via cecal ligation and puncture as described above.

Significance of Our Work

While Neutrophils have been studied extensively in both sepsis and iARDS, the role of NETs and whether they are beneficial or detrimental to the host in these conditions is poorly understood. NETs are capable of ensnaring and killing a wide array of pathogens. On the other hand, they have been implicated in damage to the host by acting as pro-inflammatory mediators. The enzyme PAD4 regulates the formation of

NETs by controlling the citrullination of histones needed for chromatin decondensation and subsequent NET release in the classic pathway of NETosis. This makes PAD4 a potential target for studying NET formation. The results in this dissertation demonstrate a role for NET formation by utilizing both chemical and genetic inhibition of PAD4 in sepsis and as well iARDS. These data taken together suggest that PAD4-mediated NET formation in both disease states contributes to the morbidity and mortality associated with each, and that NET inhibition should be further explored as a possible therapeutic

33 maneuver against the damaging pro-inflammatory response seen in polymicrobial sepsis and iARDS.

Central Hypothesis

The PAD4 catalyzed Histone 3 citrullination and resultant formation of NETs contributes systemic inflammation and bystander tissue injury, which in turn contributes to increased morbidity/mortality seen in response to septic insult or the combined insults of hemorrhagic shock and sepsis.

Aim 1: Establish the role of peptidylarginine deiminase IV (PAD4) on histone 3 citrullination and NET formation during sepsis (Chapter 2 and 3)

Sub-Hypothesis tested: Cl-amidine treatment (NET inhibitor) will eliminate or significantly decrease histone 3 citrullination expression in mice subjected to CLP model.

Ia: Compare and measure histone 3 citrullination (H3cit) systemically as well as in the peritoneal cavity in mice after septic insult (CLP)

Ib: Characterize inflammation, and organ damage in PAD inhibitor treated mice vs. non treated mice

Ic: To determine if Cl-amidine treatment increases survival after CLP

Id: Characterize citrullination of histones in circulating neutrophils from septic patient as well as in plasma and subsequently evaluate NET formation from in septic ICU patients

Aim2: Determine the role of PAD4 in lung injury (iARDS) produced as a result of the sequential insults of shock (Hemorrhage)/sepsis (CLP) (Chapter 4)

Sub-Hypothesis tested: Inhibition of PAD4 will decrease organ damage associated with iARDS by decreasing the pro-inflammatory response well as neutrophil influx to the lung.

IIa: To determine if PAD4-/- increases survival after shock/septic insult (Hem/CLP model)

IIb: Evaluate the effect PAD4 gene deletion has on the pro and anti-inflammatory response as well as neutrophil influx to the lung

IIc: To assess kidney dysfunction after double hit of Hem/CLP

Figure 1-1

Sequential stages of Sepsis: activation the pro-inflammatory and anti-inflammatory stages of the host immune response to severe injury and/or sepsis often occur concurrently. Cells of the innate immune system including monocytes and neutrophils release large amounts of pro- inflammatory cytokines leading to a “Cytokine storm”. Early death in sepsis is usually due to this hyper-inflammatory response. Over the course of there is a systemic deactivation of the immune system (CARs) which hypothetically is tasked with restoring immune balance, but often leads to immune dysregulation and persistent immune suppression along with a high risk for reoccurring infections. Death is generally caused by this inability to clear either initial infection or secondary infections

Figure 1-2

Neutrophil priming and iARDS: hemorrhagic shock induced ‘‘priming’’ predisposes the neutrophil to inflammatory stimuli that leads to an altered response after a subsequent secondary challenge , such as the induction of sepsis. This altered neutrophil response leads to a dysregulation and recruitment of activated neutrophils to the lung microvasculature, interstitium, and alveolar space. Excessive neutrophil activation and accumulation leads to increased ROS production, as well as pro-inflammatory mediators and a decrease in PMN apoptosis leading to epithelial and endothelial cell damage leading to the development of iARDS.

Figure 1-3

Neutrophil Extracellular Trap Formation: Neutrophils are activated either through DAMP/PAMP signaling or direct interaction with microbe. Reactive Oxygen Species (ROS) and Ca2+increases within the neutrophil and NE and MPO migrate from the granules to the nucleus. This leads to PAD4 activation and histone citrullination and subsequent chromatin decondensation. The nuclear envelope disaggregates into vesicles and the nucleoplasm and cytoplasm form a homogenous mass. Finally, the cell membrane ruptures and the interior of the cell is ejected into the extracellular space, forming NETs . Microbes are then ensnared within the chromatin meshwork and are killed via the extracellular microbiocidal activity by associated granular enzymes within the NET

Figure 1-4

Acute Respiratory Distress Syndrome Definition

Timing Within 1 week of known clinical insult or new or worsening respiratory symptoms Chest imaging (chest x-ray or CT scan) Bilaterial opacites-not fully explained by effusions lobar/lung collapse, or nodules

Origin of Edema Respiratory failure not explained by cardiac failure or fluid overload

Oxygenation

Mild 200 < PaO2/FiO2 ≤ 300 with PEEP or CPAP ≥5 cmH2O

Moderate 100 < PaO2/FiO2 ≤ 200 with PEEP ≥5 cmH2O

Severe PaO2/FiO2 ≤100 with PEEP ≥5 cmH2O

Adapted from: Force, A. D. T. (2012). Acute respiratory distress syndrome. Jama, 307(23), 2526- 2533.

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Chapter 2 Cl-amidine Prevents Histone 3 Citrullination, NET formation, and Improves Survival in a Murine Sepsis model

(Accepted, September 2016, Journal of Innate Immunology)

Cl-amidine Prevents Histone 3 Citrullination, NET Formation, and Improves Survival in a Murine Sepsis Model

Bethany M. Biron, Chun-Shiang Chung, Xian M. O’Brien, Yaping Chen, Jonathan S.

Reichner, Alfred Ayala

Brown University, Division of Surgical Research/Department of Surgery, Rhode Island

Hospital, Providence RI 02903

Short Title: Role of NETs in sepsis

Key Words: Neutrophils, CLP, inflammation, post translational modification

ACKNOWLEDGMENTS

This work was funded by NIH R01-GM46354 & R35 GM118097 (A.A.), GM066194 (J.S.R).

Authors would also like to thank Ms. Lauren Watts for her assistance with flow cytometry.

ABSTRACT

Sepsis refers to the presence of a serious infection that correlates with systemic and uncontrolled immune activation. Posttranslational histone modification plays an important role in chromatin decondensation, which is regulated by citrullination.

Citrullinated histone H3 (H3cit) has been identified as a component of Neutrophil

Extracellular Traps (NET), which are released into the extracellular space as part of the neutrophil response to infection. The conversion of arginine to citrulline residues on histones is catalyzed by PAD4. This study’s goals were to characterize the presence of

PAD4 catalyzed H3cit and NET formation during the onset of sepsis and elucidate the effects on the immune response when this mechanism of action is blocked.

Adult C57BL/6 male mice were treated with Cl-amidine, an inhibitor of PAD4, 1 h prior to sepsis induced by cecal ligation and puncture (CLP). 24hrs post CLP, cytokine levels, H3cit protein expression, neutrophil counts, and NET production were evaluated in the peritoneal cavity. Survival studies were also performed. Here we demonstrate that Cl-amidine treatment prior to CLP improves overall survival in sepsis and the abrogation of PAD4 has minimal effects to the pro-inflammatory immune response to sepsis, while has no effect on overall neutrophil migration to the peritoneum.

INTRODUCTION

Sepsis is a life threatening condition that elicits an exacerbated and damaging immune response with approximately 250,000 cases leading to death in the United

States annually (1–3). As of 2009, the CDC listed sepsis as the 11th leading cause of death in the United States making sepsis a significant health care problem (4, 5).

Neutrophils are considered one of the most crucial components of the innate immune system during septic infection (6). Neutrophil extracellular traps (NETs) are complex structures made of nuclear chromatin, histones, granular antimicrobial proteins, and some cytoplasmic proteins(7). NETs are capable of physically ensnaring bacteria and facilitating the interactions between bacteria and antimicrobial effectors, ultimately leading to enhanced bacterial killing(7). Components of NETs have been shown to be elevated in septic and septic shock patients(8, 9) suggesting that NETs may play an important part in the innate immune response to infection(10). However, increasing evidence has linked NET formation to various disease states, such as autoimmune diseases, as well as sepsis, suggesting that they contribute to excessive inflammation and tissue damage (6, 11, 12).

Posttranslational histone modification plays an important role in chromatin decondensation, which is regulated by citrullination (13). Citrullinated H3 (H3cit) has been identified as a component of NETs, which is released into the extracellular space as part of the neutrophil response to infection (10, 14–16). Recently, it has been revealed that Peptidylarginine deiminase 4 (PAD4) has a regulatory role in NET formation

76 through mediating chromatin decondensation through hypercitrullination of target histones H3, H4, and H2A (16–18). This histone citrullination and NET formation are essential elements of host defense and has been shown to be necessary in innate immunity during bacterial infection (10).

In order to determine a putative role of NETs in sepsis, it is necessary to characterize the presence of PAD4 catalyzed H3cit and NET formation during the onset of sepsis as well as the effects on the immune response when this mechanism of action is blocked. While genetic PAD4 knockout mice have been used to study the role of NETs in various disease states, including sepsis (10, 19–22), to date there is no inhibitor available that specifically targets NET formation. N-α-benzoyl-N5-(chloro-iminoethyl)-L- ornithine amide, or Cl-amidine, is a pharmaceutical inhibitor of Peptidylarginine deiminases (PADs) including PAD4 (23, 24). It irreversibly inactivates PADs by covalently modifying an active site cysteine that is important for its catalytic activity (23). It was previously found to repress the formation of NET-like structures in HL-60 cells (17, 25), and had also been used in various studies to examine the mechanism of PAD4 and NET inhibition (10, 21, 24).

Here we first assessed the level of H3cit protein modification in a mouse cecal ligation and puncture (CLP) model of sepsis. We then suppressed H3cit in vivo using a dose of Cl-amidine prior to CLP and studied what effect this had on H3cit modification as well as its effect on the immune response and overall survival. We found that Cl- amidine treatment prior to surgery significantly improves overall survival in a CLP model

77 of sepsis, but it seems to have little effect on the pro-inflammatory or anti-inflammatory cytokine response and no effects on overall neutrophil migration to the source of infection.

MATERIALS AND METHODS

Mice: C57BL/6 male mice, ages 8-12 weeks, were obtained from the Jackson Laboratory

(Bar Harbor, ME, USA) and used in all experiments. All protocols carried out with animals were done according to NIH Guide for Animal Use and Care, and were approved by the Rhode Island Hospital Institutional animal care and use committee (AWC 0110-

13).

Sepsis model induced by cecal ligation and puncture (CLP): Mice were anesthetized with isoflurane and a midline incision was made in the abdomen. The cecum was isolated and ligated at a point approximately 1 cm from the cecal tip, punctured twice with a 22- gauge needle, then gently squeezed to extrude a small amount of feces from the perforation sites. In the sham/CLP mice, the cecum was exposed but neither ligated nor punctured. Then the cecum was placed back into the peritoneal cavity and the incision was sutured closed in two layers. Mice were resuscitated with 1 ml Ringers lactate by subcutaneous injection (26).

Cl-Amidine calculations and dosing: Cl-amidine (Cayman Chemical, Ann Arbor, MI) was reconstituted in EtOH for a stock solution of 20 mg/mL and kept at -20°C. Working solutions were created by diluting Cl-amidine stock solution with PBS to a concentration of 2.0 mg/mL. Mice in Cl-amidine treatment groups were given a 50 mg/kg(27, 28) subcutaneously 30-60 min prior to CLP (control animals received PBS).

Survival study: Both Cl-amidine treated and control mice (n = 12/group) were subjected to CLP, and received additional doses once a day for seven days. Survival was observed.

Log-Rank statistical analysis was used to determine if a statistical significant difference in septic mortality was evident between the two groups at P < 0.05.

Sample collection: 24 or 48hrs post sham/CLP procedures, mice were euthanized with a

CO2 overdose. Blood was collected in a heparinized tube via cardiac puncture and centrifuged to obtain plasma. To collect peritoneal fluid for western blot and cytokine analysis, 1mL of 1x PBS was injected IP, recollected, and centrifuged at 10,000xg for 10 min. Supernatant was collected. For the collection of peritoneal cells, 5mL of 1X PBS was injected and recollected via IP, and centrifuged at 10,000xg for 10 min. Cell pellet was collected for NET analysis, and morphological analysis. For western blot analysis cell pellets were lysed and spun down again at 10,000xg for 10 min and lysate was collected. The lung, liver, spleen, and kidney were also harvested for cytokine analysis.

Protein concentration in lavage fluid and cell/tissue lysate was assessed by Bradford assay. All samples collected were stored at -80°C until needed.

H3 citrullination (H3cit) protein modification: Samples were probed for H3cit via

Western blot. Proteins (25 μg per lane) were separated by 16% SDS-page gels and transferred on to Polyvinylidene fluoride membranes (Novex, San Diego, CA).

Membranes were blocked with 5% milk in PBST and probed with anti-Histone H3

(citrulline R2+R8+R17)(ab5103) or anti-Histone H3 (ab8898)(Abcam, Cambridge, MA) at

1 μg/ml overnight at 4°C and subsequently incubated with a HRP-conjugated anti-rabbit

IgG (1:10,000) at room temperature for 1 h. Chemiluminescence detection was performed using ECL reagent (GE healthcare, Pittsburgh, PA ), and films were developed using standard procedure. Abundance of modified histone H3 protein was densitometrically assessed on an Alpha-Innotech image analyzer (San Leandro, CA) (29).

H3cit protein was then normalized to either β-actin for the cell lysates or to the Ponceau stained membrane for secretory proteins in peritoneal fluid (30).

Measurement of cytokines: Concentrations of interleukin(IL)-6 and IL-10 from the peritoneal fluid, plasma, and tissue samples were assessed via ELISA according to manufacturer’s protocols (BD Bioscience) (31).

NET visualization/ Flow cytometry: Cells collected from peritoneal lavage were re suspended (1:20) in 1X PBS and placed on tissue culture plates coated with 1% BSA and incubated at 37°C for 1-2 hrs. 5μM of Sytox green (Thermo Fischer Sci, Waltham, MA,) was added 10 min prior to imaging. Images were captured using a Nikon TE-2000U inverted microscope (Nikon, Melville, NY) coupled to an iXonEM + 897E back illuminated

EMCCD camera (Andor, Belfast, UK). Bright field images were captured using NIS-

Elements software (Nikon). A xenon lamp illuminated cells through a 33 mm ND4 filter and 20× Nikon Plan Apochromat objective using a Nikon B2-A long-pass emission filter set cube.

For flow cytometry, all the antibodies used for analysis were purchased from eBioscience (San Diego, CA). LY6G+ cell populations in the peritoneal lavage were determined with FACS Array flow cytometer (BD Bioscience, San Jose, CA ) and analyzed with FlowJo software (Tree Star, Ashland, OR) was used for analysis (31).

NET quantification: Neutrophils were prepared as described above for NET visualization/ Flow cytometry. NETs were visualized with Sytox green and multiple images obtained per well. Images were thresholded using the default thresholding algorithm in ImageJ (NIH, Bethesda, MD) and gated to include extruded NETs and exclude stained nuclei. NET formation quantified as the percent area of the totaled imaged field. Well averages were then ensemble averaged. Data represents 4-6 wells per condition.

Statistics: Results are expressed as Mean ± SEM. Statistical significance of the results presented were determined by one-way ANOVA (for multiple comparison) with All

Pairwise Multiple Comparison Procedures (Holm-Sidak or Dunn’s method), unpaired two-tailed Student’s t-test (for normal distribution data), or log-rank test (for survival study) where appropriate. Statistical software used was Sigmaplot 11.0 (Systat Software,

Inc., San Jose, CA). P ≤ 0.05 was used as a cutoff for significance. For NET quantification

ANOVA analysis with Newman-Keuls posthoc analysis or unpaired-sample two-tailed

Student’s t-test as appropriate performed using MATLAB (Mathworks, Natick, MA) or

Excel (Microsoft, Redmond, WA) running the statistiXL data package (statistiXL,

Nedlands, Australia). The null hypothesis rejected if P < 0.05.

RESULTS

Citrullination of histone 3 is increased 24 h post CLP

Histone H3 citrullination has been used as a marker for PAD4 activity and NET formation(10, 17). First, we wanted to determine if H3Cit protein modification was evident in our CLP model of sepsis. The abundance of H3cit protein modification was measured in plasma, peritoneal fluid, and peritoneal cells 24 and 48 h post CLP via

Western blot assay. Histone H3 was also measured in whole cell lysates and peritoneal fluid. To further ensure that H3cit modification was independent of Histone H3, we blotted for Histone H3 as well as H3cit in nuclear protein isolated from peritoneal cells and determined that H3cit levels were separate from Histone H3 levels. (Supplemental fig. 2-1) At 24 h post CLP, H3cit protein abundance was significantly elevated in the peritoneal cells (Fig. 1A-B) as well as in peritoneal fluid (Fig. 2-1C-D) when compared to sham mice; however, H3Cit was not detected in the plasma of sham or CLP mice (Fig. 2-

1E-F). At 48 h post CLP, a significant increase of H3Cit was still detected in peritoneal cell lysates, however, H3cit was no longer present in the peritoneal fluid. Therefore, a

24 h time point was chosen for later experiments.

Cl-amidine reduces the abundance of the H3cit protein modification in peritoneal fluid and peritoneal cells after CLP

We wanted to determine whether inhibition of PAD4 activity would eliminate or significantly decrease H3cit protein modification in mice subjected to CLP. Cl-amidine was administered at a dose of 50 mg/kg given subcutaneously 30-60 min prior to CLP.

Overall, in vivo Cl-amidine treatment significantly reduced H3cit protein modification at

24 h post CLP in peritoneal cells (Fig. 2-2A-B) as well as in peritoneal fluid (Fig. 2-2C-D).

While elimination of an H3cit protein band was not seen in every treated animal, there was still a consistent reduction in comparison to the vehicle treated mice.

Cl-amidine treatment reduces NET formation in peritoneal cells after CLP

To support the observation that Cl-amidine treatment reduces the H3cit protein modification implicated in NET formation after CLP, the capacity for NET formation by peritoneal neutrophils was assessed ex vivo. Peritoneal content was collected via lavage

(which is a compilation of mostly CLP-activated neutrophils, and macrophages) from mice 24 h post CLP. Peritoneal cells were cultured on BSA-coated plates with no ex vivo stimulation, and stained with Sytox green for extracellular nucleic acid visualization, which has been used to identify NET formation in stimulated cells(32). After 1-2 h incubation, there was a marked reduction of NET formation in mouse cells taken from the Cl-amidine treated as compared to the vehicle treated CLP mice (Fig. 2-3A-B).

Cl-amidine treatment does not alter neutrophil recruitment to the peritoneum after

CLP

To determine whether the reduction of H3cit protein modification in the peritoneal cavity was due to PAD4 inhibition, as opposed to a possible off target effect of Cl-amidine that may hamper neutrophil migration to the site of infection, we compared the number of neutrophils in the peritoneal cavity in the Cl-amidine treated and vehicle treated groups after CLP. Cell numbers were significantly increased in both treated groups subjected to CLP as compared to sham animals (Fig. 2-4A). However, there was no difference in cell numbers between the Cl-amidine and vehicle treated CLP groups. As the peritoneum after CLP is inundated with not just neutrophils, but also other innate immune cells (33–36), we further examined the cell heterogeneity in the peritoneum by performing a cytospin and Wright’s stain on cells collected from the peritoneum after CLP. Once again there were comparable cell numbers as well as cell sub-types in both groups (Fig 2-4B-C). Cells from the peritoneum were also stained for

CD11b and Ly6G to compare the percentages of macrophages (CD11bhigh/Ly6Glow) and neutrophils (CD11bhigh/Ly6Ghigh) present at the site of infection (Fig 4D). There was no difference in the percentage of CD11bhigh/Ly6Glow or CD11bhigh/Ly6Ghigh cells in the peritoneum in the treated CLP mice as compared to the vehicle treated CLP mice.

Cl-amidine treatment improves survival in CLP sepsis model

Extracellular histones as well as circulating free DNA (cf-DNA) have been implicated in increased organ injury and mortality in the CLP model (9, 11). To better understand PAD4’s role in sepsis mortality, we performed CLP on two groups: the first group was treated with Cl-amidine 30-60 min prior to the procedure, and the second with vehicle control. Mice were then given subsequent doses of Cl-amidine or vehicle, respectively, every day for seven days. We observed that the Cl-amidine treated animals were significantly protected from CLP-induced mortality, with a 100% survival rate at seven days, as compared to the vehicle treated group who had a 45% survival rate (Fig. 2-5). Animals undergoing sham surgery had a 100% survival rate.

Cl-amidine treatment leads to a minimally decreased pro-inflammatory response

To further investigate how Cl-amidine increases survival, we measured alterations in the pro-inflammatory cytokine IL-6 as well as anti- inflammatory cytokine

IL-10 in tissues known to be affected by sepsis (37–39), in the plasma, and in peritoneal fluid (40). Extracellular histones and NETs have been attributed to causing excessive inflammation (12, 41, 42). Here we found that there was a significant, marked reduction of IL-6 in the spleen. However, there was no significant reduction of IL-6 in the kidney, lung, and liver tissues (Fig. 2-6). This may be attributed to the increase of other cell types, such as macrophages, which also secrete IL-6 after CLP as part of the initial innate immune response to infection (36). In addition, there was also no difference in IL-10 levels found in tissues (Fig. 2-7). When looking systemically (plasma) and at the site of

85 infection (peritoneal fluid) there was no reduction of IL-6, but there was a significant increase of IL-10 levels in the bloodstream in the Cl-amidine treated mice as compared to both the sham and vehicle control CLP mice (Fig. 2-8).

DISCUSSION

NETs are complex structures comprised of nuclear chromatin, histones, granular antimicrobial proteins, and cytoplasmic proteins (7). While NET formation physically ensnares bacteria and facilitates the interactions between bacteria and antimicrobial effectors, ultimately leading to enhanced bacterial killing, they also contribute to detrimental effects such as excessive inflammation and tissue damage in the host (6, 11,

12).

PAD4 activity is an important component of neutrophil activation, NET formation, and the innate immune response (10, 13). Alternatively, PAD4 activity and histone citrullination has also been shown to be a contributor to various autoimmune diseases such as rheumatoid arthritis, lupus, and IBD, and serves as a convergence point for many inflammatory signals that prompt the neutrophils’ response to infections (27,

41–43). In the case of sepsis, these extracellular histones have been linked to tissue damage as well as increased mortality (11). PAD4 gene deletion studies have been variable. PAD4-/- mice have been shown to be susceptible to bacterial infections and their neutrophils display a significant reduction in bacterial killing in vitro (10). However, when additional sepsis in vivo studies utilizing these PAD4-/- mice using the CLP model were performed, PAD4 gene deletion appears to have no real affect on survival (19).

This discrepancy in survival results as compared to our findings may be attributed to the differences in the model procedure as well as differences in control mortality rates.

Moreover, PAD inhibition as compared to PAD4 gene deletion may also account for the differences as the use of Cl-amidine or neutralization of circulating citrullinated histones using anti-Cit H3 antibody as a therapeutic strategy has been made known to increase survival in septic mice (44). Our results confirm this increase in survival in the CLP model using a different dosing strategy, which included a smaller subcutaneous dose of Cl- amidine given prior to CLP. Thus, demonstrating that chemical inhibition of PADs, and subsequently histone citrullination and NET formation, leads to increased survival against septic insult. Protein expression analysis demonstrated that PAD inhibitor, Cl- amidine, did not fully negate all histone citrullination, but greatly suppresses it as compared to vehicle treated CLP mice. Furthermore, the reduction of H3cit protein modification after Cl-amidine treatment is likely due to PAD4 inhibition and not a reduction of neutrophils emigrating into the site of infection.

With extracellular cf-DNA and citrullinated histones having been reported to be detected within the bloodstream during a septic infection(19, 45) and circulating citrullinated histone H3 has been reported as a potential biomarker for the early diagnosis of septic shock (8), H3cit has been proposed as a target considered for improving survival in the CLP model (44). In our model we were unable to detect H3cit within the bloodstream using protein expression by Western immunoblot as our method of detection at 24 or 48 h post CLP. Furthermore, others have suggested that the elevated cf-DNA levels reported during early-phase are not derived from neutrophils

87 or NETs, but from other factors such as necrotic tissue or apoptotic cells (46). The H3cit protein modification was detected in the peritoneal cavity after CLP. As neutrophils are one of the first immune cells to respond to infection (47), it is possible that the increase in citrullination is due to PAD4 activity. Collectively, our data suggests that NETosis is occurring within the peritoneum 24 h after CLP (11, 16, 17) and that the H3cit protein modification, and by extension NET formation, is reduced in the peritoneal cavity with

Cl-amidine treatment.

Cl-amidine treatment has been demonstrated to improve various inflammatory diseases phenotypes in autoimmune disorders (27, 28, 43). Interestingly, Cl-amidine treatment has also been shown to have an effect on dendritic cell (DC) maturation induced by TLR agonists to reduce pro-inflammatory cytokine levels as well as impair the proliferation of naïve CD4+ and CD8+ T cells both in vitro and in vivo (48). A reduction in pro-inflammatory markers was also seen in our study where there was a reduction in IL-

6 levels in the spleen after CLP. These data taken together suggests that Cl-amidine treatment may have a hand in reducing inflammation that is caused by extracellular histones and NETs. Surprisingly, IL-10 levels were significantly increased in the bloodstream in the Cl-amidine treated mice compared to vehicle treated mice after CLP indicating that Cl-amidine treatment may be having an effect on the systemic anti- inflammatory response after CLP. However, IL-10 levels were not significantly affected by Cl-amidine treatment in the peritoneal cavity, or in the various organ tissues analyzed. Therefore, it is unclear if PAD4 inhibition has any type of effect on the compensatory anti-inflammatory response that occurs concomitantly with the pro-

88 inflammatory response (49). In our model we cannot say definitively that these alterations in pro- and anti-inflammatory signaling are a direct result of NET inhibition, as Cl-amidine is a non-selective PAD inhibitor (not specific to PAD4 and NET formation alone). It would be interesting to further explore the effect of a selective inhibitor of

PAD4 on the inflammatory response as it becomes available.

Overall, the results here demonstrate that histone H3 citrullination is present in the peritoneal cavity as well as within cells collected from the site of infection 24 h after being subjected to CLP procedure. This citrullination is modulated when mice are treated with the PAD inhibitor Cl-amidine. Daily treatment significantly improved survival after CLP, suggesting that chemical inhibition of PAD4 is beneficial against polymicrobial septic mortality. Treatment did not affect overall neutrophil emigration into the site of infection. Nonetheless, Cl-amidine did diminish activated neutrophils’ ability to release NETs. While Cl-amidine has been implicated in improving inflammation in various autoimmune disease states, its direct mechanism of action is not clear (24, 27, 43). Cl-amidine does not seem to have a significant impact on pro- inflammatory markers of infection, but does alter systemic IL-10 levels. Thus having a possible an effect on the immunosuppressive response in our model. It is possible that the increase in survival is due to secondary effects caused by Cl-amidine, such as an increase in apoptosis of other inflammatory cells, like that seen in a murine colitis model

(43). While PAD4 inhibitors are under development (50), there is still no specific inhibitor available to study the direct correlation between NET inhibition and subsequent host improvement during a septic infection. Our observations suggest that

NET inhibition using Cl-amidine should be further explored as a possible therapeutic maneuver against the damaging pro-inflammatory response seen in polymicrobial sepsis

Figure 2-1

Fig. 2-1: H3Cit protein modification is present after CLP (A-B) The H3cit protein modification is highly abundant in cells collected from the peritoneal cell lysates collected 24 h post CLP. 48 h after CLP there is still a increase in H3cit protein modification. (C-D) Fluid collected from the peritoneal cavity is also increased H3cit protein modification at 24 h post CLP while levels decrease after 48 h. (E-F) At both time points, the H3cit protein modification was undetectable in the bloodstream. H3cit protein was then normalized to either β-actin for the cell lysates or to the Ponceau stained membrane that demonstrated equal loading in the peritoneal fluid. Sham n = 4, CLP n = 5-6 per group. *P < 0.05, One way ANOVA.

Figure 2-2

Figure 2-2: H3cit protein modification is significantly diminished in the peritoneal cavity in mice treated with Cl-amidine 24 h post CLP 30-60 min prior to CLP mice were either treated with Cl-amidine given at 50 mg/kg by subcutaneous injection or vehicle control (1x PBS). Peritoneal cell lysates and peritoneal fluid were then collected from both groups of mice 24 h post CLP. (A-B) The abundance of H3cit protein modification was significantly diminished in the peritoneal cell lysates collected from treated mice as compared to vehicle control mice. (C-D) The levels of H3cit protein was also significantly diminished in peritoneal fluid after treatment. Overall, treatment with Cl-amidine did not fully eliminate the H3cit protein modification in every treated animal; however, there was still a consistent reduction in comparison to the vehicle treated mice. H3cit protein was then normalized to either β- actin for the cell lysates or a non- specific 64Kd band located on the Ponceau stained membrane that demonstrated equal loading in the peritoneal fluid gels. Sham n = 4, CLP n = 6-8 per group. *P < 0.05 sham vs. CLP, #P < 0.05 CLP vehicle vs. CLP Cl-amidine One-way ANOVA.

Figure 2-3

Figure 2-3: Cl-amidine treatment in vivo reduces NET formation in murine neutrophils ex vivo: Septic mouse lavage contents were placed on tissue culture plates coated with 1% BSA, (with no additional stimulation) incubated at 37°C for 1-2 h, and stained with Sytox green for extracellular nucleic acid visualization. (A) Peritoneal cells from Cl- amidine treated mice display a decrease in extracellular nuclear material (NET formation) compared to control cells at 1-2 h. (B) This reduction in NET formation is statistically significant when quantified as the percent area of the totaled imaged field. (Data represents 4-6 wells per condition.) *P < 0.05 ANOVA analysis with Newman-Keuls post-hoc analysis or unpaired-sample two-tailed Student’s t-test as appropriate. All images were obtained at 20X original magnification.

Figure 2-4

Figure 2-4: Cl-amidine treatment has no effect on neutrophil migration to the peritoneum after CLP: Cells were collected from the peritoneum by injecting 5 mL of 1x PBS into the abdomen and then harvesting an equal volume. (A) The total number of cells as determined by hemocytometer was not different between the Cl-amidine- and vehicle-treated groups. (B-C) Cytospins of peritoneal cells were Wright stained and analyzed for neutrophil counts at 20x using RGB, DIC N1 filter (0.33µM/pixel). The number of neutrophils did not significantly differ between the vehicle control and the Cl-amidine treated animals. (D) The percentage of Ly6G+ cells in the peritoneum in both groups 24h post CLP demonstrated no significant difference between vehicle and CL- amidine treated animals consistent with neutrophil counts. Sham n = 4, CLP n = 12/group. *P < 0.05, One way ANOVA.

Figure 2-5

Figure 2-5: Cl-amidine treatment improves survival in CLP sepsis model: Mice were divided into randomized groups and treated with a 50 mg/kg dose of Cl-amidine or 1x PBS (vehicle control) by subcutaneous injection 30-60 min prior to CLP and then once a day for seven days post procedure. The Cl-amidine treated group had a 100% survival rate compared to the control group which had a 45% survival rate. Sham animals had a 100% survival rate (data not shown) Sham n = 3, CLP n = 12/group. *P < 0.05, log-rank test.

Figure 2-6

Figure 2-6: Cl-amidine treatment significantly alters pro-inflammatory IL-6 cytokine levels in spleen after CLP, but not in other tissues: Tissue homogenate lysates were normalized using a Bradford protein assay and then were assessed for IL-6 levels by ELISA. Tissues assessed were (A) kidney, (B) lung, (C) spleen, and (D) liver 24 h post CLP. IL-6 levels were not significantly altered between vehicle control and Cl-amidine treated mice in all tissues except in the spleen where Cl-amidine treatment significantly reduced IL-6 levels as compared to vehicle control CLP mice. Sham n = 6, CLP n = 12-16/group. *P < 0.05 sham vs. CLP, #P < 0.05 CLP vehicle vs. CLP Cl-amidine, One-way ANOVA.

Figure 2-7

Figure 2-7: Cl-amidine treatment does not significantly alter anti-inflammatory IL-10 cytokine levels in tissues: The same tissue homogenate lysates used for IL-6 level assessment were also assessed for IL-10 levels as a measurement of alteration in the anti-inflammatory response. Tissues assessed were (A) kidney, (B) lung, (C) spleen, and (D) liver 24 h post CLP. There was a significant increase in IL-10 levels in the kidneys in both the vehicle and Cl-amidine CLP groups compared to shams. However, IL-10 levels were not significantly different between vehicle control and Cl-amidine treated mice in all tissues. Sham n = 6, CLP n = 12-16/group. *P < 0.05 sham vs. CLP, One-way ANOVA.

Figure 2-8

Figure 2-8: Cl-amidine treatment increases systemic levels of IL-10 but has no impact on the systemic pro-inflammatory response after CLP: Plasma was collected from heparinized whole blood via cardiac puncture and peritoneal fluid was collected from the abdomen by injecting 1 mL 1x PBS IP and then recollected. Both samples were analyzed for IL-6 and IL-10 levels. (A-B) IL-6 levels were significantly increased in plasma and peritoneal fluid of both the vehicle and Cl-amidine treated groups after CLP. (C) Systemically IL-10 levels were significantly increased in the Cl-amidine treated mice as compared to the vehicle control mice after CLP. (D) IL-10 levels were comparable in both groups at the site of infection in the peritoneum. Sham n = 6, CLP n = 12-16/group. *P < 0.05 sham vs. CLP, #P < 0.05 CLP vehicle vs. CLP Cl-amidine, One-way ANOVA.

Supplemental Figure 2-9

Supplement Figure 2-9: Unmodified H3 protein levels are independent of Citrullinated H3 protein levels: Nuclear protein (NP) was isolated from peritoneal cells and probed for both H3 and H3cit protein. (A) H3 protein was detected in both the sham and CLP NP 24hr and 48hr post procedure, while H3cit was only detected in the CLP mice. (B) In the presence of Cl-amidine H3 protein was detected in all groups. H3cit protein was only found in all mice given the vehicle control and 2/3 mice treated with Cl-amidine after CLP.

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Chapter 3 PATIENTS WITH SEPSIS EXHIBIT EVIDENCE OF CITRULLINATED

HISTONE MODIFICATION AND NET PRODUCTION IN THEIR BLOOD: a small observational study

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PATIENTS WITH SEPSIS EXHIBIT EVIDENCE OF CITRULLINATED HISTONE MODIFICATION

AND NET PRODUCTION IN THEIR BLOOD: a small observational study

Bethany M. Biron, Chun-Shiang Chung, Catherine Dickinson, Daithi Heffernan, Jonathan

Reichner, Alfred Ayala

Brown University, Division of Surgical Research/Department of Surgery, Rhode Island

Hospital, Providence RI 02903

Short Title: Role of NETs in sepsis

Key Words: Neutrophils, inflammation, cf-DNA, PAD4

This work was funded by NIH R01-GM46354 & R35 GM118097 (A.A.)

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ABSTRACT

Sepsis remains a leading cause of death among ICU patients. Findings from the multiple failed clinical trials focusing on single receptors or cytokines have highlighted the need to better understand mechanisms of the septic response at a cellular level rather than a single receptor or cytokine antagonist. Neutrophil extracellular traps (NETs) have been identified as an integral aspect of neutrophil response to inflammatory stimuli and infection. This mechanism is regulated by the citrullination of histones by PAD4, allowing for chromatin decondensation. However, NETs and their components have been increasingly implicated in contributing to the pathogenesis of several diseases, including sepsis. The purpose of this study was to determine if citrullination of histones is increased in circulating neutrophils from septic patient as well as in plasma and subsequently evaluate NET formation from in septic ICU patients.

Critically ill septic ICU patients with either an abdominal or pulmonary source of sepsis were enrolled. Blood was collected and plasma and neutrophils were isolated.

Histone citrullination was measured in plasma as well as in circulating neutrophils via western blot. NET formations were visualized by immunoflourescence in blood smears.

These results were then correlated with clinical parameters. Both histone citrullination as well as NET visualization were compared to samples collected from healthy individuals.

We identified that histone citrullination modification was typically increased in circulating neutrophils collected from septic patients as compared to healthy

110 individuals, while the presence of citrullinated histone components in plasma were more variable between patients. NETs were also observed more frequently in septic patient blood smears.

In conclusion, the presence of citrullinated histone modification is increased in isolated neutrophils from septic patients and corresponds to increased NET visualization in blood smears suggesting that NETs may play a role in the host response to septic infection.

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INTRODUCTION

Sepsis is a severe clinical problem that elicits an exacerbated and damaging immune response and is associated with high mortality rates in ICU patients (1–3).

Neutrophils are considered one of the most crucial components of the innate immune response to septic infection (4). Neutrophil extracellular traps (NETs) are complex structures made of nuclear chromatin, histones, granular antimicrobial proteins, and some cytoplasmic proteins (5). NETs formation allows for the physical entrapment of bacteria and helps promote the interactions between bacteria and antimicrobial effectors, ultimately leading to enhanced bacterial killing (5).

Histone citrullination in neutrophils by the enzyme PAD4 has been shown to be a crucial step in the chromatin decondensation and NET release from the neutrophil. (6).

Citrullinated H3 (H3cit) has been identified as a component of NETs, which is released into the extracellular space as part of the neutrophil response to infection (7–10). In septic patients circulating Histone levels are significantly increased (11, 12) and may contribute to damaging inflammation and increased mortality. In addition to histones, components of NETs, as determined by cf-DNA, have been shown to be elevated in septic and septic shock patients (13, 14) suggesting that NETs may play an important part in the innate immune response to infection (9).

Using whole blood samples collected from septic patients and healthy controls we identified that histone citrullination modification was typically increased in circulating neutrophils collected from septic patients as compared to healthy

112 individuals, while the presence of citrullinated histone components in plasma were more variable between patients. NETs were also observed more frequently in septic patient blood smears.

MATERIALS AND METHODS

Patient enrollment: Blood was obtained from septic patients and healthy human volunteers. Critically ill septic patients requiring ICU level of care were prospectively enrolled (n=8) within 24 hours of the diagnosis of sepsis. Charts were reviewed for patient characteristics, including age, gender and white cell count at the time of the lab draw and source of the sepsis. All sources of sepsis were either acute intra-abdominal or active pneumonia in patients with respiratory failure requiring mechanical ventilation.

Abdominal sources of sepsis were clinically determined at the time of operation.

Clinical Pulmonary Infection Score (CPIS) was used as the trigger to consider the diagnosis of pneumonia. If CPIS was greater than 5, and the patient had clinical signs of pneumonia, then bronchoscopy was performed. Pneumonia was diagnosed by bronchoalveolar lavage (BAL) with >10,000 colony forming units (CFU)/mL. Charts and labs were also reviewed to calculate the APACHEII score for each patient at the time of the lab draw. Control samples were collected from healthy volunteers (n=4). Full Rhode

Island Hospital Institutional Review Board (IRB) approval was obtained prior to study initiation.

Neutrophil Isolation: Venous blood samples were collected in EDTA-containing

Vacutainer tubes (BD Biosciences, San Jose, CA) from either a peripheral line already in

113 place on the patient or by venipuncture of healthy volunteers. Histopaque - 1077 was used for initial leukocyte separation followed by sedimentation through 3% dextran

(400–500 kDa molecular weight). Contaminating erythrocytes were removed by hypotonic lysis, yielding a >95% pure neutrophil preparation of >90% viability by trypan dye exclusion. Neutrophils were than suspended in HBSS (without Ca+2/Mg+2) and placed on ice until use.

Western Blot: Neutrophils isolated from septic patient whole blood and then lysed. 75ug of protein per lane (as determine by Bradford protein assay, Bio-Rad, Hercules CA) was separated by SDS-page electrophoresis (Invitrogen, Carlsbad, CA) on 16% gels) and transferred on to Polyvinylidene fluoride membranes (Novex, San Diego, CA).

Membranes were blocked with 5% BSA in PBST and probed with primary antibody

(H3Cit rabbit polyclonal antibody (Abcam, Cambridge, MA) at 5μg/ml overnight at 4°C.

Primary antibody was detected by incubation with a HRP-conjugated anti-rabbit IgG

(1:10,000) (Abcam, Cambridge, MA). The chemiluminescence detection was performed using ECL reagent (GE healthcare, Pittsburgh, PA), and films was developed using standard procedure. H3 citrullination was normalized to β-actin and compared by densitometric assessment as previously described in our laboratory (15, 16).

Measurement of NET components in Sera: Measurement of MPO in soluble NET fragments from patient sera was performed using the Neutrophil Extracellular Trap

(MPO-Histone) ELISA kit developed by Cayman chemical®(Ann Arbor, MI)

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NET Visualization: Blood smears were created using 10-20ul of whole blood from healthy controls or septic patients. Cells were fixed to the slide and incubated with

Hoechst stain (5ug/ml in water) (Thermo Fisher Scientific, Waltham, MA) for 20 min.

Images were captured using a Nikon TE-2000U inverted microscope (Nikon, Melville, NY) coupled to an iXonEM + 897E back illuminated EMCCD camera (Andor, Belfast, UK) at

20x. A xenon lamp illuminated cells through a 33 mm ND4 filter and 20× Nikon Plan

Apochromat objective using a Nikon B2-A long-pass emission filter set cube.

Statistical analysis: Results are expressed as Mean ± SEM. Statistical significance of the results presented were determined unpaired two-tailed Student’s t-test. Statistical software used was Sigmaplot 11.0 (Systat Software, Inc., San Jose, CA). P ≤ 0.05 was used as a cutoff for significance.

RESULTS

Patient Data

8 critically ill septic patients were enrolled from the ICU within 24 hours of the diagnosis of sepsis. The average age was 61.7 +/-4.7 years, and 4 of the 8 (62.5%) were male. 5 of the 8 (62.5%) patients had an abdominal source of sepsis, all of whom required operative intervention. The other 3 patients had pneumonia as the source of sepsis, and all 3 patients with pneumonia had associated respiratory failure requiring mechanical ventilation. All patients were on antimicrobial therapy at the time of the blood draw. The average white cell count (WCC) at the time of the blood draw was 14.1

(+/-1.6) x109/L. The median APACHE II score was 19.5 (IQR 16.2 – 23.3). [Table 3-1] The

115 mortality within the septic patients was 25% (2/8 patients). There was no correlation between H3cit or NET formation and risk of death.

Citrullinated Histone 3 Modification is increased in the Isolated Neutrophils of Septic

Patients

Histone H3 citrullination has been used as a marker for PAD4 activity and NET formation(9, 17), and has been suggested as a potential biomarker for NET formation in murine models of sepsis (16, 18, 19). We next determined if an increase in H3 citrullination was evident in these critically ill septic patients. Neutrophils were isolated from whole blood samples and probed for H3cit protein. Densometric analysis of western blot images showed that there was statistically significant increase of H3cit in the neutrophil lysates collected from septic patients as compared to healthy controls

(Figure 3-1). This suggests that histone citrullination is present in the neutrophils

NET fragment associated MPO is increased in sera from septic patients

With Net components having been reported to be detected within the bloodstream during a septic infection(20, 21), we aimed to determine if the increase in citrullinated histone protein expression could also be correlated to an increase in NET formation in the circulation of septic patients. Sera was collected from the whole blood samples during the neutrophil isolation process, and was analyzed for MPO-histone conjugates as a marker of NET components within the bloodstream. While the amount of NET associated MPO-histone conjugates was slightly variable within the septic patient

116 population, there was a significant increase overall in the septic patient group as compared to the sera from the healthy controls (Figure 3-2).

NET visualization is increased in Sepsis

As both H3cit protein expression in septic neutrophils and NET associated fragments are elevated in the septic patient population enrolled in our study, we also analyzed the generation of NETs by neutrophils from septic and non-septic patients using DNA immunofluorescence staining. To visualize NETs in the bloodstream, blood smears were prepared and then stained with Hoechst stain for extracellular DNA.

Circulating neutrophils from septic patients displayed an increase in NET like structures with extracellular DNA protruding from the cell and a loss of cell membrane integrity

(Figure 3-3). These NET like structures were not found on the blood smear samples from healthy controls. These NET like structures correlate with both an increase in histone citrullination from circulating neutrophils and NET associated fragments in the bloodstream, suggesting that NET formation is occurring as a response to septic infection.

DISCUSSION

Components of NETs have been shown to be elevated in septic and septic shock patients(18, 13) suggesting that NETs may play an important part in the innate immune response to infection(9). However, NET formation has been associated with excessive inflammation as well as endothelial and tissue damage (4, 22, 23).

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Extracellular histones are released in response to inflammatory challenge and contribute to endothelial dysfunction, organ failure and death during sepsis (22, 24).

Clinical studies evaluating the impact of circulating free histones in septic patients demonstrated that histone levels were higher in septic patients compared to patients with multiple organ failure without infection (11), suggesting that the source of histones in circulation could be associated with the underlying infection. Additionally, circulating histones have also been shown to be mediators of cardiac injury, arrhythmias, and LV dysfunction in patients with sepsis (25). Overall, high circulating histones have been associated with SOFA scores, sepsis severity, organ dysfunction, and mortality (11, 12,

25). Since, circulating histones alone are not strictly indicative of NET formation, whereas citrullinated histones are a precursor of chromatin decondensation and NET formation (9, 10, 17), our method of measuring H3cit is more indicative of NET formation.

Our study demonstrated that citrullination of histone H3 is present in isolated neutrophils from the septic patient population, whereas no H3cit protein is detectable in neutrophils isolated from healthy populations.

Recent studies have reported that cf-DNA is associated with NETs, (5, 13). To this end, there has been interest in the use of cf-DNA as a potential biomarker of NET formation in critically ill patients. However, increased cf-DNA can also be associated with cellular necrosis, apoptosis, and NET formation in patients with cancer, trauma, stroke, or sepsis (13, 26, 27). In septic patients higher cf-DNA concentrations were

118 detected in comparison with healthy controls, and levels were correlated with sepsis severity and organ dysfunction (28). However, it is difficult to ascertain the origin of cf-

DNA and it has been questioned as to whether cf-DNA alone is a reliable biomarker of

NET formation. Recent studies have shown that the majority of cf-DNA found in a murine model of polymicrobial sepsis is non-immune cell derived in nature and not associated with neutrophils (29). To determine the amount of NET components in circulation, we also analyzed sera for MPO-histone conjugates. Both MPO and histones are molecules that are dotted along the backbone of DNA when it is extruded from the cell (5). Our data showed that while the amount of MPO-histone conjugates was variable amongst the patient population, there was an overall increase in our septic patient population as compared to healthy controls. This would suggest that there are most likely NETs within the circulation during a septic infection.

NET release has been demonstrated to capture a wide array of pathogens included gram positive and gram negative bacteria and also act as a physical barrier preventing the spread of microorganisms (9, 30). Using blood smears from patients, we able to detect NET like structures in the whole blood samples from septic patients. This observation is agreement with our previous mouse studies where peritoneal neutrophils from septic mice had increases in NET formation (16) as well as other murine studies that showed bone marrow derived neutrophils in mice with abdominal sepsis have the enhanced capability form NETs after ex vivo stimulation as compared with sham mice (31).

The observations in this study collaborate with our previous findings in a mouse model of polymicrobial sepsis that citrullianted histone protein expression is increased

119 in neutrophils in a septic setting, suggesting that citrullinated histones may serve as a potential biomarker for NET formation. Additionally, we demonstrated that NET formations as well as NET fragments are present in the circulation of septic patients, suggesting that NET formation is part of the host response to infection. While circulating histones have been associated with sepsis severity, cellular damage, and organ dysfunction further work is needed to determine if citrullinated histones and subsequent NET formation plays a critical role in the outcome in sepsis.

Limitations: this study was a single center study with a small sample size. Additionally our patient population was mostly older, while healthy controls were from a younger cohort. Although this is a relatively small study, our findings demonstrate a critical translation of previously demonstrated murine findings onto a relatively heterogenous population of critically ill surgical patients with sepsis.

120

Figure 3-1

Age 61.7 (+/-4.7) years

Male Gender 62.5%

Source of sepsis

Abdominal 62.5%

Pneumonia 37.5%

APACHE II 19.5 (IQR – 16.2 – 23.3)

White Cell Count 14.1 (+/- 1.6) x109/L

Table 3-1 Patient Data: 8 critically ill septic patients were enrolled from the ICU within 24 hours of the diagnosis of sepsis. 5 of the 8 (62.5%) patients had an abdominal source of sepsis, all of whom required operative intervention. The other 3 patients had pneumonia as the source of sepsis, and had associated respiratory failure requiring mechanical ventilation. All patients were on antimicrobial therapy at the time of the blood draw.

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Figure 3-2

Figure 3-1. Citrullinated Histone 3 Modification is Increased in the Isolated Neutrophils of Septic Patients: at time of blood collection neutrophils were isolated an probed for H3cit protein. H3cit protein is increased in septic neutrophils in comparison to healthy controls (a.) This increase is statistically significant via densitometric analysis *p<0.05(b.) healthy control n=3, septic n=7 (no data for patient 7)

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Figure 3-3

Figure 3-2. NET fragment associated MPO is increased in sera from septic patients: Serum was collected from enrolled septic patients as well as from healthy controls and assayed for NET fragment associated MPO using a sandwich ELISA kit developed by Cayman Chemical® (a.) H3cit-MPO fragments was significantly increased in the sera of septic patients as compared to the healthy controls *p<0.05 (b.)

Figure 3-4

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Figure 3-3. NET visualization is increased in Sepsis: Using a Hoechst stain to visualize extracellular DNA in whole blood it was found that there is an increase of NET structures () in the blood samples from septic patients vs. healthy controls n=4 healthy controls, n=5 septic

124

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SE, Windolf J, Lögters TT. 2012. Depletion of neutrophil extracellular traps in vivo

results in hypersusceptibility to polymicrobial sepsis in mice. Crit Care 16:R137.

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Chapter 4 Innate immune mechanisms in organ injury: PAD4 deficiency

leads to decreased organ dysfunction and improved survival in a

murine model of indirect-acute lung injury

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PAD4 deficiency leads to decreased organ dysfunction and improved survival in a Duel

Insult Model of Hemorrhagic Shock and Sepsis

Bethany M. Biron, Chun-Shiang, Yaping Chen, Zachary Wilson, Jonathan S. Reichner,

Alfred Ayala

Brown University, Division of Surgical Research/Department of Surgery, Rhode Island

Hospital, Providence RI 02903

Short Title: Role of PAD4 in the pathology of shock/sepsis

Key Words: Neutrophils, hemorrhage, sepsis, inflammation, post translational modification

FOOTNOTES:

This work was funded by NIH R01-GM46354 & R35 GM118097 (A.A.), GM066194 (J.S.R).

Acknowledgements: The authors of this manuscript would like to thank Dr. Kerri

Mowen (Scripps Research Institute, La Jolla, CA) for gifting the PAD4-/- mice used in these studies

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ABSTRACT

Acute Respiratory Distress Yyndrome (ARDS) represents a clinical syndrome of acute respiratory failure, with an incidence rate between 80 and 60 per 100,000 per year, respectively. Indirect ARDS (iARDS) is caused by a non-pulmonary inflammatory process as a result from insults such as non-pulmonary sepsis, hypotensive shock, etc.

Neutrophils are thought to have a significant role in mediating ARDS, with the development of iARDS being characterized by dysregulation and recruitment of activated neutrophils to the lung. Recently, a novel mechanism of microbial killing by neutrophils was identified through the formation of neutrophil extracellular traps

(NETs). NETs are comprised of large webs of decondensed chromatin coated with granule proteins that are released from activated neutrophils into the extracellular space and are regulated by the enzyme PAD4 through mediating chromatin decondensation via citrullination of target histones. Components of NETs including neutrophil derived circulating free DNA as well as circulating histones has been implicated in ARDS. However, it is unknown if there is any pathological significance of

NET formation in ARDS caused by non-pulmonary insult.

To determine if NET formation contributes to the mortality associated with shock/sepsis we subjected PAD4-/- mice and WT mice to a “2 hit” model of traumatic shock (fixed-pressure hemorrhage; Hem) followed by septic (CLP) insult (Hem/CLP).

Mice were hemorrhaged, resuscitated, and 24 hours post-Hem mice were then subjected to CLP. Overall, PAD4 deletion led to an improved survival as compared to WT

132 mice. PAD4-/- displayed a marked decrease in neutrophil influx to the lung, as well decreases in pro-inflammatory mediators. PAD4-/- mice were also able to maintain baseline kidney function after Hem/CLP.

These data taken together suggest PAD4-mediated NET formation contributes to the mortality associated with Shock/Sepsis and may play a role in the pathobiology of multiple organ injury in response to iARDS in the Hem/CLP model.

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INTRODUCTION

Acute respiratory distress syndrome (ARDS) is a type of respiratory failure characterized by bilateral alveolar infiltrates, decreased lung function, pulmonary edema, increased lung micro-vascular permeability, and influx/sequestration of activated neutrophils into lung interstitium and alveolar space (1–3). ARDS is associated with a mortality rate of about 40% and accounts for approximately 75,000 deaths per year in the United States (4, 5). ARDS can be caused by pulmonary (direct ARDS) or extra-pulmonary (indirect ARDS) insult. Direct ARDS (pneumonia, aspiration, and lung trauma) accounts for 57% of total cases, whereas indirect ARDS (iARDS), including extra- pulmonary sepsis and multisystem trauma, represents 43%, with approximately 25% of

ARDS cases stemming from severe sepsis (2, 6). However, compared with direct ARDS, the pathophysiology of iARDS is much less well understood, possibly due to heterogeneity of the disease and the involvement of multiple systemic factors.

One of the key features of ARDS is the dysregulation and recruitment of activated neutrophils to the lung microvasculature, interstitium, and alveolar space (2).

This excessive neutrophil activation and accumulation leads to increased Reactive oxygen species (ROS) production, as well as pro-inflammatory mediators and a decrease in neutrophil apoptosis leading to epithelial and endothelial cell damage (2, 7).

Neutrophil extracellular traps (NETs) are complex structures made of nuclear chromatin, histones, granular antimicrobial proteins, and some cytoplasmic proteins (8) that are capable of physically ensnaring bacteria and facilitating the interactions

134 between bacteria and antimicrobial effectors, ultimately leading to enhanced bacterial killing. However, there has been increasing evidence that NETs may have detrimental consequences for the host. Increased NET formation has been linked to various disease states, such as autoimmune diseases as well as sepsis, which suggests that they contribute to excessive inflammation and tissue damage (9–11). Components of NETs including neutrophil derived circulating free DNA (CF-DNA/NETs) as well as circulating histones have been implicated in acute lung injury (12–14). Histones have been detected in the bronchoalveolar lavage fluid (BALF) and plasma of patients who developed ARDS after trauma and acid aspiration (14, 15), and have been implicated in lung damage in various animal models of lung injury and directly cause alveolar damage

(10).

The process of NET formation is mediated by the enzyme Peptidylarginine deiminase 4 (PAD4), which is essential for histone citrullination and subsequent chromatin decondensation. This histone citrullination is thought to promote NET formation by inducing chromatin decondensation and aiding in the release of chromosomal DNA coated with antimicrobial molecules into the extracellular space (16–

18). Thus, making PAD4 a potential target for studying NET formation

While NETs and their components have been observed in various models of direct acute lung injury, it is not understood how they function in a model of traumatic shock (hemorrhage) followed by septic insult, which is thought to more closely resemble

135 the process of extra (indirect)-pulmonary ARDS encountered in many critically ill/injured patients.

We hypothesize that PAD4 catalyzed Histone 3 citrullination and the resultant formation of NETs contributes systemic inflammation and by-stander tissue injury that leads to increased morbidity/mortality seen in the combined insults of hemorrhagic shock and sepsis (which induce acute lung injury as well as range of other organ injuries) and, therefore, Inhibition of PAD4 will decrease organ damage associated with iARDS and increase overall survival in a “2 hit” model of traumatic shock (fixed-pressure hemorrhage; Hem) followed by septic cecal ligation and puncture (CLP) insult

(Hem/CLP).

To address this hypothesis, here we have subjected both PAD4-/- and WT mice to a dual hit model of Hem/CLP and assessed for survival, cytokine analysis, and neutrophil infiltration in the lung and kidney. Taken together, our data suggests that PAD4/NET formation contributes to the mortality associated with shock/sepsis and also plays a role in the multiple organ injury seen in response to pathological processes regulating iARDS in the Hem/CLP model.

MATERIAL AND METHODS

Mice: C57BL/6 male mice (WT), ages 8-12 weeks, were obtained from the Jackson

Laboratory (Bar Harbor, ME, USA) and used in all experiments. PAD4-deficient mice

(PAD4-/-) were gifted by Dr. Kerri Mowen (Department of Pharmacology and Chemical

136

Physiology, Scripps Research Institute, La Jolla, CA). LoxP sites were introduced into the introns flanking exons 9 and 10 of the PAD4 gene and PAD4 floxed mice were mated with CMV-Cre deleter mice to generate PAD4-deficient mice (19). PAD4-deficient mice are viable and have no gross anatomical abnormalities. All protocols carried out with animals were done according to NIH Guide for Animal Use and Care, and were approved by the Rhode Island Hospital Institutional animal care and use committee (AWC 0040-

16).

Analysis of neutrophil effector functions: Neutrophils were isolated from naïve WT and

PAD4-/- as previously described (20). Cells were then used for the following assays:

Respiratory burst capacity: Isolate blood neutrophils (3 × 105 cells/well) were placed on BSA coated 96-well plates with 2x Cytochrome C solution (Sigma Aldrich,

Natick MA) and stimulated with phorbol myristate acetate (PMA). The plate was placed in a Microplate Bio Kinetics reader (BIOTEK Instruments, Winooski, VT) at 37°C and optical density readings at dual wavelengths of 550/630 nm were made every 2 min for

60 min using DeltaSoft3 software (BIOTEK Instruments). Neutrophil burst = 13.258 (a predetermined absorbance constant) *(OD at Xmin-OD at 0min)

Phagocytosis: Phagocytosis capabilities were measured using a phagocytosis kit that utilizes fluorescently labeled bacterial particles (ThermoFisher Scientific, Waltham,

MA). 5x105 neutrophils/well were plated and incubated at 37°C for 1 hr. The plates were aspirated and then 100 µL of the prepared fluorescent BioParticle suspension were added to the wells and incubated at 37°C for 2 hrs. After aspiration, 100 µL of prepared

137 trypan blue suspension was added to all of the wells and incubated for 1 minute at room temperature. The trypan blue was removed and the microplate was placed in the fluorescence plate reader (FLx800, Bio-tek Instruments Inc., Winooski, VT) using ~480 nm excitation, ~520 nm emission and the appropriate sensitivity settings. Phagocytosis in response to the effector was then calculated.

NET formation: Thioglycolate elicited neutrophils were isolated and cultured on

Poly-L-lysine coated plates for 4 hours at 37°C post 20nM PMA stimulation to induce

NET production. 5μM of Sytox green (Thermo Fischer Sci, Waltham, MA,) was added

10 min prior to imaging. Images were captured using a Nikon TE-2000U inverted microscope (Nikon, Melville, NY) coupled to an iXonEM + 897E back illuminated EMCCD camera (Andor, Belfast, UK). Bright field images were captured using NIS-Elements software (Nikon). A xenon lamp illuminated cells through a 33 mm ND4 filter and 20×

Nikon Plan Apochromat objective using a Nikon B2-A long-pass emission filter set cube.

Hemorrhage (Hem)model: As previously described (21, 22) , mice were anesthetized using an isofluorane vaporizer setup, restrained in supine position and catheters were inserted into both femoral arteries. When awake, as determined by a mean blood pressure of ~85 mm Hg, the mice were bled (0.8–1.0 mL) over a 5- to 10-min period to a mean blood pressure of 35 mm Hg (±5 mm Hg) and kept stable for 90 min. Immediately following hemorrhage mice were resuscitated through the catheter with Ringers lactate at four times drawn blood volume. The incision was sutured closed and mice were then

138 returned to their cages. Sham mice were anesthetized and both femoral arteries were ligated. Incisions were sutured closed and mice were returned to their cages.

Sepsis induced by cecal ligation and puncture (CLP): Twenty-four hours after Hem, mice were anesthetized with isoflurane and a midline incision was made in the abdomen. The cecum was isolated and ligated at a point approximately 1 cm from the cecal tip, punctured twice with a 22-gauge needle, then gently squeezed to extrude a small amount of feces from the perforation sites. Then the cecum was placed back into the peritoneal cavity and the incision was sutured closed in two layers. Mice were resuscitated with 1 ml Ringers lactate by subcutaneous injection and then returned to their cages. In the sham mice, the cecum was exposed but neither ligated nor punctured

(23).

Survival study: Both WT and PAD4-/- mice (n = 16-17/group) were subjected to

Hem/CLP, and were then returned to their cages, given access to food and water and assessed for morbidity/mortality for 14 days post procedure.

Sample Collection: Twenty-four hours post Hem/CLP or sham/sham procedures; mice were euthanized with either a CO2 or isoflurane overdose. Blood was collected in a heparinized tube via cardiac puncture and centrifuged to obtain plasma. BALF was collected as previously described (22). Briefly, the trachea was exposed via a midline incision and cannulated with a sterile polypropylene catheter. The lungs were lavaged with 0.6 ml saline, two times, for an average of 1 ml lavage fluid collected/lung. Lavage

139 fluid was centrifuged at 1500 rpm for 10 min at 4°C, and the supernatant was tested for cytokine levels. The lung and kidney were also harvested for cytokine analysis as previously described (22). All samples were stored at -80°C until needed. Peritoneal fluid was collected by lavage with 1mL of PBS into the peritoneal cavity. 1xPBS was recollected for microbial burden assays. For lung and kidney histology, tissue was harvested and fixed in 10% formalin and paraffin embedded, and tissue sections were prepared as described previously (24, 25). Samples were then stained with H&E and examined by light microscopy.

Cytokine/chemokine analysis: Concentrations of interleukin(IL)-6, TNF-α, IL-10 (lung, kidney, BALF, and plasma), CXCL1 (KC) and CXCL2 ( MIP-2) (lung and kidney tissue) were assessed via ELISA according to manufacturer’s protocols (BD Bioscience, San Jose, CA)

(26). Mouse Myeloperoxidase (MPO) was measured using an enzyme-linked immunosorbent assay kit (ThermoFisher Scientific, Waltham, MA).

Flow cytometry: the left lobe of the lung underwent an enzymatic digestion, as described previously (24, 25). All the antibodies used for analysis were purchased from eBioscience (San Diego, CA). CD11b+ LY6G+ cell populations were determined using a

MACSQuant Analyzer (Miltenyi Biotec Inc, San Diego, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR) (26).

Kidney dysfunction: To quantify blood urea nitrogen (BUN) levels in sera, a Urea assay kit

(ab83362, Abcam, Cambridge, MA) was used. In brief, Urea reacts as substrate with compounds in the presence of enzymes to form a product that reacts with the probe to generate color (ODmax=570nm). For Creatinine quantification, Creatinine Assay Kit

140

(ab65340, Abcam, Cambridge, MA) was used to measure creatine concentration in serum (27). In the assay, creatinine is converted to creatine by creatininase, creatine is converted to sarcosine, which is specifically oxidized to produce a product which reacts with a probe to generate color (ODmax = 570 nm).

Evans Blue Dye (EBD) extravasation assay: Vascular permeability in the lungs and kidneys was assessed by measuring the EBD in the lung and kidney in ng/mg of tissue

(28). Briefly, EBD (2.5 mg in 0.5 mL 0.9N saline) was administered intravenously by tail vein injection 15 min before euthanasia. The Lungs and kidneys were harvested, weighed, and incubated in 2 mL of formamide for 24 h at 55°C. Following centrifugation,

EBD was measured in supernatant, dual wavelenths 620/740 nm, compared with EBD standard curve, and normalized with total EBD in collected plasma.

Statistics: Results are expressed as Mean ± SEM. Statistical significance of the results presented were determined by one-way ANOVA (for multiple comparison) with All

Inc., San Jose, CA). P ≤ 0.05 was used as a cutoff for significance.

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RESULTS

PAD4 deficiency does not affect other effector functions

Firstly, we compared overall neutrophil effector functions in neutrophils isolated from PAD4-/- and WT mice to ensure the only difference was the ability to produce NETs.

The ability of PAD4-/- neutrophils to phagocytize was equivalent to WT neutrophils (fig 4-

1A), and PAD4−/− neutrophils were also capable of producing ROS after PMA stimulation comparable to WT neutrophils (fig 4-1B). However, when isolated neutrophils were cultured on Poly-L-lysine for 4 hours post 20nM PMA stimulation, the

PAD4−/− neutrophils were unable to elicit NET formation; unlike WT neutrophils which produced NET structures after stimulation (fig 4-1C).

To compare overall circulating leukocyte percentages, we observed that they were similar in both PAD4-/- and WT mice (fig 4-2A-E). After Hem/CLP, PAD4−/− mice circulating lymphocyte (fig 2A), neutrophil (fig 4-2B), eosinophil (fig4- 2D) and basophil

(fig 4-2E) percentages were similar to WT mice indicating that PAD4-/- neutrophils were able to successfully transmigrate across the endothelium after Hem/CLP and were not trapped in circulation. However, PAD4-/- mice did display a reduction in circulating monocytes (fig 4-2C).

PAD4 deficiency reduces bacterial burden and mortality after Hem/CLP

Studies have shown that PAD4 plays an important role in neutrophil antibacterial function mediated by NETs to effectively contain various pathogens (8, 18), we

142 hypothesized that PAD4−/− mice would be more susceptible to an overwhelming bacterial burden, due to a reduced ability to kill bacteria via NETosis. Surprisingly, the bacterial load in the peritoneum 24 hours after CLP (48 hours post Hem), was significantly lower in the PAD4-/- mice than in the WT (fig 4-3). Both WT and PAD4-/- sham/sham groups) had no bacterial growth. This suggests that PAD4-/- mice had enhanced bacterial clearance capabilities compared to WT mice after Hem/CLP.

To better understand the role NETs play in shock/sepsis associated mortality, we performed Hem/CLP on WT and PAD4-/- mice, and then looked for changes in their mortality over 14 days. Overall, PAD4-/- mice displayed improved survival with a survival rate of 94% as compared to WT mice which had a 59% survival rate after 14 days post

Hem/CLP (fig 4-4). Animals undergoing sham/sham surgery in both the WT and PAD4-/- had a 100% survival rate (data not shown).

Effects of PAD4 on organ injury indices after Hem/CLP

One of the hallmarks of indirect ARDS is the sequestration and activation of neutrophils in the lung (1–3). MPO activity as an index of neutrophil influx was assessed in lung tissue lysates as well as histologically. Previous studies from our lab have shown that MPO activity is increased significantly in the lungs after Hem/CLP (21). PAD4-/- mice had attenuated levels of MPO in the lungs after Hem/CLP that was comparable to sham levels, whereas WT Hem/CLP mice had significant increases in MPO (fig 4-5A-B). To the extent that the reduction in MPO as an index of neutrophil influx was related to altered chemokine mediated chemoattraction, we looked at chemoattractant levels of CXCL-1

143 and CXCL-2 within the lung. However, there was no difference between Hem/CLP WT and PAD4-/- mice, with both groups displaying elevated chemokine levels (fig 4-5C-D).

While there was no reduction in chemoattractant levels, the PAD4-/- mouse exhibited significant decreases in CD11b+ Ly6G+ cells (neutrophils) as well as CD11b+ Ly6G-

(myeloid cell types) cell populations within the lung (fig 4-5E-F). Overall, this data demonstrates that PAD4-/- mice had a decrease in MPO activity in the lung as well as decreased neutrophil infiltration into the lung after Hem/CLP. Interestingly, when assessing MPO levels in the kidney, there was no difference between WT and PAD4-/- mice, with both groups displaying MPO levels comparable to sham levels after Hem/CLP

(fig 4-6A). Similarly, CXCL1 levels were unchanged in the kidney across all groups (fig 4-

6B). Baseline CXCL2 levels were significantly higher in the sham PAD4-/- kidneys as compared to WT (fig 6C). Subsequently, after Hem/CLP, CXCL2 levels in the PAD4-/- were decreased to comparable WT levels (fig 4-6C).

Loss of vascular integrity after a shock/sepsis insult leads to increased vascular permeability resulting in organ edema as well as tissue inflammatory cell infiltrates and an increase in tissue/vascular protein leak (1, 32, 33). In addition, NET formation has been shown to contribute to pathogenesis of numerous vascular disorders and recently has been implicated in the destabilization of intracellular cell junctions and subsequent increased endothelial cell permeability (34). In this respect, pulmonary and renal vascular permeability was assessed using Evan’s Blue Dye extravasation assay. In the lung, while there was a marked increase when compared to the sham animals, there was no difference in vascular leakage between WT and PAD4-/- Hem/CLP mice, with both

144 groups displaying a similar rise in lung permeability (fig 4-7A). This corresponded with the increase in protein concentrations seen in the BALF in WT and PAD4-/- Hem/CLP mice (fig 4-7B). While there was no difference seen in lung permeability, kidney permeability was significantly decreased in the PAD4-/- mice as measured by a significant decline of EBD recovered from the kidneys (fig 4-7C).

To further investigate the role of PAD4 gene deficiency on kidney function after

Hem/CLP, we measured BUN and creatinine levels in the plasma of WT and PAD4-/- mice. BUN levels were significantly increased in the WT mice, but were attenuated in the PAD4-/- mice (fig 4-8A). Creatinine levels in both groups were comparable to sham levels (fig 4-8B). These BUN and Creatine levels were used to determine the BUN:Crs.

WT mice displayed a significant increase in their BUN:Crs ratio suggesting that WT mice are in pre-renal failure, which can be caused by a decreased effective arterial blood volume, whereas PAD4-/- remained at sham levels (fig 4-8C). This reduced BUN:Cr in ratio compared to WT after Hem/CLP would suggest that the PAD4-/- mice were able to tolerate hemorrhage better than their WT counterparts, leading to a maintenance of normal kidney function.

Effects of PAD4 on inflammation in mice after Hem/CLP

Next we sought to determine if alterations in the inflammatory response after

Hem/CLP contributed to increased survival of PAD4-/- mice, since extracellular histones and NETs have been implicated in causing excessive inflammation and tissue damage

(11, 29, 30). We measured IL-6 and TNF-α levels as markers of systemic inflammation in

145 the blood and of lung tissue inflammation. We also assessed IL-10 levels, as IL-10 is a predominantly anti-inflammatory modulator that has been reported to be elevated and shown to play a role in the immune response to shock (1, 31). PAD4-/- lung homogenates displayed a significant reduction of pro-inflammatory cytokine IL-6 and

TNF-α levels (fig 4-9A-C) compared to WT mice after Hem/CLP. BALF IL-6 of PAD4-/- mice were also attenuated where in WT mice there was a significant increase detected after

Hem/CLP (fig 4-9D-F). In the kidney, there were no differences in IL-6 levels while PAD4-

/- kidney TNF-α levels were higher compared to WT after hem/CLP, with no difference in

TNF-α levels detected when PAD4-/- Hem/CLP is compared to PAD4-/- sham mice (fig 4-

9G-I). Anti-inflammatory cytokine IL-10 levels in WT mice were significantly increased in the lung (fig 4-9C) while it was decreased in the kidney (fig 4-9I). In PAD4-/- mice, there was the opposite effect with decreased levels measured in the lung and increased in the kidney compared to WT after Hem/CLP, albeit comparable to PAD4-/- kidney sham mouse levels. Systemically, there were no significant differences between the two groups with WT and the PAD4-/- displaying significant increases in systemic IL-6 levels

(data not shown). Overall, PAD4-/- mice showed evidence of a decreased pro- inflammatory cytokine response in the lung after Hem/CLP, with minimal differences detected within the kidney.

DISCUSSION

PAD4 activity is an essential component in histone citrullination and subsequent chromatin decondensation, which are critical steps needed for NET formation (18, 35).

Conversely, PAD4 activity and histone citrullination has also been shown to be a

146 contributor to various disease states, and serves as a mediator for many inflammatory signals that prompt the neutrophils’ response to infections (10, 29, 30, 36, 37), which have been linked to tissue damage as well as increased mortality.

Components of NETs, such as circulating free DNA (CF-DNA) as well as circulating histones have been implicated in acute lung injury and ARDS (12–14). Infused circulating histones have been shown to lead to alveolar capillary obstruction, histone toxicity in lung tissue and coagulation activation all leading to lung injury as well as multiple organ failure (MOF) (14). Extracellular histones have been implicated in lung damage in various animal models of lung injury and directly cause alveolar damage (10), whereas depletion or neutralization of neutrophils in mouse models leads to a decrease in circulating histones and reduced lung injury (38, 39).

PAD4-/- mice have been shown to be susceptible to bacterial infections and their neutrophils display a significant reduction in bacterial killing in vitro (18). However, this is in contrast to our results that found PAD4-/- mice had enhanced bacterial clearance in the peritoneum after shock/septic insult. Studies looking at the bactericidal ability of

NETs support the idea that bacterial entrapment rather than bacterial killing is the primary function of NETs in infection (40, 41). Therefore, a lack of NETs may not necessarily mean a reduction in bacterial killing, and the increased peritoneal bacterial clearance seen in PAD4-/- mice after Hem/CLP may be the result of other consequences of PAD4 gene deficiency, such as a reduction in immune dysfunction.

147

In a murine CLP model of sepsis, PAD4-/- mice seemingly had no real survival advantage over there PAD4+/+ counterparts (42). In our model of Hem/CLP we found that PAD4 gene deficiency provides an increase in survival over a 14 day period. While we cannot exclusively give credit to the improved outcome in the PAD4−/− mice to lack of NETs, however, our data is in line with survival data described in sepsis models that use chemical inhibition of PAD, which imparts an increase in survival after CLP (43–45).

One of the key features of ARDS is the dysregulation and recruitment of activated neutrophils to the lung microvasculature, interstitium, and alveolar space (1–

3). This excessive neutrophil activation and accumulation leads to increased ROS production, as well as pro-inflammatory mediators and a decrease in PMN apoptosis leading to epithelial and endothelial cell damage (2, 7). In case of iARDS there is a priming of both non immune (epithelial/endothelial cells) and immune cell (neutrophils and macrophages) that occurs and leads to the recruitment of neutrophils to the lung and subsequent pathogenesis (2). PAD4 deficiency does not seem to play a role in the increased lung micro-vascular permeability seen in iARDs, However, to our knowledge, our results show for the first time that PAD4-/- leads to a reduction of neutrophil sequestration in the lung after shock/septic insult resulting in reductions in MPO activity within the lung. In addition to decreased neutrophil numbers, PAD4-/- mice also display a decrease in CD11b+ Ly6G- myeloid cells in the lung. This could be of interest, as PAD4 is also found in granulocytes and monocytes (46–50), which also play a role in the innate immune response to infection. Nonetheless, it is unclear the exact mechanism in which

PAD4 alters neutrophil recruitment to the lung. There was significant increases in CXCL1

148 and CXCL2 in both PAD4-/- as well as WT mice, suggesting no alterations in chemotactic signaling. Our mouse model of hemorrhagic shock induces neutrophil ‘‘priming’’, which predisposes the neutrophil to inflammatory stimuli that leads to an altered response after a subsequent secondary challenge, CLP, (1, 51, 52). It is possible that this

“priming” event is altered by PAD4 deficiency, leading to a decrease of neutrophil sequestration into the lung, and should be further explored.

NETs can lead to a dysregulation/accumulation of pro-inflammatory mediators within tissues as well as the vasculature, leading to tissue damage and organ failure (9–

11). In various animal models of lung injury, extracellular histones have been found to be a major contributor of pro-inflammatory cytokines in ARDS leading to lung injury and directly cause alveolar damage (10). After Hem/CLP, PAD4-/- mice displayed significant decreases in TNF-α in lung tissue and IL-6 in both lung tissue and BALF. PAD4-/- also lead to a decrease in IL-10 production in the lung. The reduction in cytokine levels in the lung of Hem/CLP PAD4-/- mice may suggest that the lack of NET formation reduces the inflammatory mediators that are caused by extracellular histones and NETs.

Critically ill patients who suffer from sepsis are not only at risk of developing

ARDS (the most common organ injury noted in the critically ill patient), but also other organ system dysfunctions (53, 54). In this respect; 35% of patients with ARDS also develop acute kidney injury (AKI), and the development of this secondary insult dramatically increases patient mortality (55, 56). In this respect, many of the pathways that are implicated in the development of ARDS are also thought to play a role in the

149 development of acute kidney injury (56). It is this close association that makes the kidney an organ of interest when studying the MOF associated with sepsis induced iARDS. When analyzing kidney function after Hem/CLP, there were minimal changes in cytokine and chemokines production between groups, however, vascular leakage was significantly decreased in the PAD4-/- mice as compared to the WT mice. Additionally, while WT mice displayed significant increases in BUN levels after Hem/CLP, there was no statistically significant change in BUN levels between PAD4-/- sham and Hem/CLP mice.

All 4 mice groups maintained normal creatinine levels. However, when BUN:CR were calculated, PAD4-/- mice maintained normal ratios where WT Hem/CLP mice had a significantly increased ratios. This suggests that the PAD4-/- mice were able to tolerate hemorrhage better than WT mice and overall these mice were able to retained normal kidney function compared to WT mice. It is unclear if this maintenance of kidney function can be attributed to lack of NET formation, as indices of neutrophil influx were similar between groups. However, it is important to note that PAD4 specifically has been implicated in the damage seen in renal ischemia reperfusion (I/R) injury by increasing renal tubular inflammatory responses and neutrophil infiltration into the kidney (57). In addition, PAD4 deficient mice have been reported to display a reduction in renal tubular necrosis, inflammation, and apoptosis, as well as decreases in neutrophil infiltration after I/R in the kidneys and liver via immunohistochemistry (58).

Their data together suggests PAD4 plays a critical role in increasing the renal tubular inflammatory response and neutrophil infiltration after renal I/R injury. As MOF is progressive through the course of disease, it would be interesting to investigate

150 neutrophil recruitment and NET formation within the kidney to detect any differences that might occur further along in the course of the disease state caused by Hem/CLP.

In conclusion, our study demonstrated for the first time that inhibition of PAD4 through gene deletion and subsequent NET formation leads to a reduction of sequestered neutrophils to the lung and reductions in MPO as well as pro-inflammatory cytokine signaling within the lung in a mouse iARDS model induced by Hem/CLP.

Additionally, PAD4-/- mice maintain baseline kidney function, which decreases the chances of renal failure. Taken together, these alterations in the immune responses may be attributed to the overall increase in survival seen in PAD4-/- mice after Hem/CLP.

While these results seen in the PAD4-/- mice cannot be definitively attributed to inhibition of NET formation, it is a step in the right direction to study the effect of NETs on the immune response to shock/sepsis. While PAD4 specific inhibitors are under development (59), the use of Cl-amidine, which has been used extensively to study NET formation in various disease sates, including sepsis,(43, 60, 61) can be utilized to further study and understand the role of NET formation in the critically ill patient.

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Figure 4-1

Figure 1: PAD4 gene deficiency effects NET formation but does not inhibit other neutrophil effector functions: Neutrophils from C57BL/6 (WT) and PAD4 KO mice were analyzed for other anti-microbial functions. Phagocytosis capabilities were comparable in WT and PAD4-/- mice (A). ROS generation after stimulation with PMA was increased in WT and PAD4-/- mice (B). Neutrophils were isolated via thioglycolate injection and were cultured on Poly-L-lysine coated plates for 4 hours post 20nM PMA stimulation to elicit NET production. NETs were visualized via immunofluorescence with Sytox green (C). PAD4-/- neutrophils displayed a deficiency in producing NETs, while WT neutrophils were able to produce NET structures after PMA stimulation.

152

Figure 4-2

Figure 2: Circulating neutrophil percentages are equivalent between PAD4-/- and WT mice before and after Hem/CLP: Whole blood from WT and PAD4-/- mice was analyzed for circulating leukocyte percentages before and after Hem/CLP. Circulating neutrophils (A), lymphocytes (B), monocytes (C), eosinophils (D), and basophils (E), were similar in both PAD4-/- and WT mice. After

Hem/CLP, WT and PAD4-/- mice had increases in overall neutrophil percentages in circulation, while PAD4-/- did display a reduction in circulating monocytes compared to WT. One way ANOVA, #p≤0.05 sham vs. Hem/CLP; * p≤0.05 WT Hem/CLP vs PAD4-/- Hem/CLP. n=3-6/group.

153

Figure 4-3

Figure 3: PAD4-/- mice exhibit enhanced bacterial clearance at site of infection after Hem/CLP: twenty four hours after Hem/CLP, PBS was injected into the peritoneum, recollected, and plated on blood agar plates for 24hrs at 37°C. PAD4-/- mice had significantly reduced colony formations compared to WT mice. Sham animals from both groups had no colony formations. One way ANOVA, #p≤0.05 sham vs. Hem/CLP; * p≤0.05 WT Hem/CLP vs PAD4-/- Hem/CLP. n=4-6/group

154

Figure 4-4

Figure 4: PAD4-deficient mice display improved survival in Hem/CLP model of iARDS: Mice were subjected to hemorrhage and subsequent septic insult and then monitored daily for 14 days. PAD4-/- mice had 90% survival at 14 days compared to the 50% survival in WT mice. Sham animals from both groups had a 100% survival rate. log-rank test. P=0.013. (n=6 sham/sham (not shown); n=16-17 Hem/CLP per group)

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Figure 4-5

Figure 5: Neutrophil influx and MPO activity is decreased in PAD4-/- mouse lung tissue following Hem/CLP: Homogenized lung tissue was assessed for neutrophil infiltration and activation in the lung. MPO levels were significantly decreased in PAD4-/- compared to WT mice after Hem/CLP when analyzed via ELISA (A) as well as histologically (B). Chemoattractants CXCL1 (C) and CXCL2 (D) were up-regulated in WT and PAD4-/- lung tissue after Hem/CLP. Overall, Ly6G+ CD11B+ cells were significantly decreased in PAD4-/- lung homogenates after Hem/CLP via flow cytometry (E) and also displayed a significant decrease in Ly6G- CD11B+ cells (F). One way ANOVA, #p≤0.05 sham vs. Hem/CLP; * p≤0.05 WT Hem/CLP vs PAD4-/- Hem/CLP. n=6-9/group

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Figure 4-6

Figure 6: MPO levels are not altered in the kidneys of WT compared PAD4-/- mice following Hem/CLP: Homogenized kidney tissue was assessed for MPO activity after Hem/CLP. There were no significant changes detected in MPO levels between WT and PAD4-/- groups (A). CXCL1 levels were unchanged in the kidney across all groups (B). While CXCL2 levels were significantly higher in the sham PAD4-/- kidneys as compared to WT (C), after Hem/CLP, CXCL2 levels in the PAD4-/- were decreased to comparable WT levels. One way ANOVA, #p≤0.05 sham vs. Hem/CLP; * p≤0.05 WT Hem/CLP vs PAD4-/- Hem/CLP; **p≤0.05 WT sham vs PAD4-/- sham. n=6-9/group

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Figure 4-7

Figure 7: PAD4-/- while not altering the change Hem/CLP induced lung permeability, Decreased Vascular Leakage in the Kidneys after Hem/CLP: Pulmonary and renal vascular permeability was assessed using Evan’s Blue Dye extravasation assay 24hrs after Hem/CLP. No difference in vascular leakage between WT and PAD4-/- mice was detected after Hem/CLP, with both groups displaying a similar rise in lung permeability (A). This was consistent with an increase in protein concentrations seen in the BALF in WT and PAD4-/- mice (B). Kidney permeability was significantly decreased in the PAD4-/- after Hem/CLP as measured by a significant decline of Evan’s Blue Dye recovered from the kidneys (C). One Way ANOVA, #p≤0.05 sham vs. Hem/CLP; * p≤0.05 WT Hem/CLP vs PAD4-/- Hem/CLP. Lung n=3-8/group, BALF n=3-6/group, Kidney n=3-6/group.

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Figure 4-8

Figure 8: PAD4-/- mice maintain urea and creatinine levels more comparable sham protocol animals then WT after Hem/CLP: BUN and Creatinine levels were measured in plasma as an indicator of renal function. BUN levels were attenuated in the PAD4-/- mice whereas WT had significantly increased levels after Hem/CLP (A). Creatinine levels in both groups were comparable to their correspondent sham levels (B). Overall, WT mice displayed a significant increase in their BUN:Crs ratio, whereas PAD4-/- remained at sham levels (C). One Way ANOVA, #p≤0.05 sham vs. Hem/CLP; * p≤0.05 WT Hem/CLP vs PAD4-/- Hem/CLP. N=6-9/group

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Figure 4-9

-/- Figure 9: PAD4 results in decreased pro-inflammatory and anti-inflammatory cytokine levels in the lungs of Hem/CLP mice: Pro-inflammatory and anti-inflammatory cytokine levels were assessed in lung tissue, the bronchial-aveolar lavage fluid (BALF) and kidney tissue of WT and PAD4-/- mice. Within the lung tissue, there were significant decreases in IL-6 (A), TNF-α (B) and IL-10 levels (C) in PAD4-/- after Hem/CLP. In the BALF, PAD4-/- displayed no increase in IL-6 levels, unlike the WT mice (D), with no differences detected in TNF-α (E) and IL-10 levels (F) between groups. In the kidney, there were no differences in IL-6 levels (G). PAD4-/- kidney TNF-α levels were higher compared to WT -/- -/- after hem/CLP (H), however, TNF-α levels between PAD4 sham and PAD4 Hem/CLP was unchanged. IL-10 levels in WT mice were decreased in the kidney compared to WT sham levels after Hem/CLP (I), while In PAD4-/- , IL-10 levels in the kidney were unchang ed between sham and Hem/CLP animals. One Way ANOVA, #p≤0.05 sham vs. Hem/CLP; * p≤0.05 WT Hem/CLP vs PAD4-/- Hem/CLP; ** p≤0.05 WT Hem/CLP vs PAD4-/- sham. Lung tissue n=5-7/group, BALF n=6-9/group, Kidney tissue n=5-9/group

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Chapter 5 DISCUSSIONS AND CONCLUSIONS

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OVERVIEW

Neutrophils are a critical part of the innate immune response to infection, playing an important role in the elimination of pathogens through a variety of effector functions. While Neutrophils have been studied extensively in both sepsis and iARDS, the role of NETs in the immune response under these conditions is less well understood.

The studies in this thesis investigated the role of NET formation in the immune response to sepsis as well as in a model iARDS through the blockade or elimination of PAD4.

While many different mediators have been associated with NET formation, such as platelet activation, Platelet–neutrophil interactions, and NADPH oxidase-derived ROS

(1–6), these mediators can also be associated with numerous other pathways and functions.

PAD4 can be considered a more viable target to study the effects of NET formation, as it is upstream of NET formation, but has a limited role of citrullinating histones for chromatin decondensation and NET release. The results from these studies demonstrated that in a model of polymicrobial sepsis as well as in a more complex, yet more clinically relevant, model of shock/sepsis, PAD4 activation and subsequent NET formation contribute to the pro-inflammatory response and associated pathology and mortality. Additionally, this dissertation proposes that citrullination of histones and

NETosis play a part in the immune response of the septic patient.

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Chapter 2 and 3 Conclusions

Synopsis: The increase of histone citrullination is associated with the formation of NETs, which is catalyzed by peptidylarginine deiminase 4 (PAD4)(7, 8). Citrullinated

H3 has been identified as a component of NETs and is released into the extracellular space as part of the neutrophil response to infection or can be induced by LPS (9, 10).

While previous studies have used PAD4 deficient mice as a way to study the affect NETs have on survival in a septic model (11), no findings have been reported how chemical inhibition of NETs affects the innate immune response during sepsis in a mouse model.

As citrullinated histones and NETs are associated with damaging pro-inflammatory responses, we hypothesized that Cl-amidine treatment, a commercially available PAD inhibitor previously found to repress the formation of NETs (12, 13), would eliminate or significantly decrease histone 3 citrullination in mice subjected to CLP model.

In chapter 2 we established that citrullinated H3 (H3cit) protein modification was evident in our CLP model of sepsis. The abundance of H3cit protein modification was measured in plasma, peritoneal fluid, and peritoneal cells 24 and 48 h post CLP via

Western blot assay. We found that 24hr post CLP, H3cit protein modification was significantly increased in the peritoneum. We then treated mice with a dose of Cl- amidine prior to CLP and again measured H3cit protein modification in the same samples as mentioned above. Cl-amidine treatment reduced the abundance of the

H3cit protein modification in peritoneal fluid and peritoneal exudate cells after CLP. In addition to reductions in H3cit, we determined that Cl-amidine treatment also reduced

NET formation in the neutrophils collected from the peritoneum after CLP. Next, we

173 performed a survival study to determine if the inhibition of H3cit and NET formation altered outcome in our CLP model. To this end, we established that prophylactic treatment with Cl-amidine with subsequent doses after CLP significantly reduces overall morality induced by CLP.

In addition to studying H3cit and NET formation in a murine model of sepsis, in chapter 3 we looked at the presence of citrullinated histone modification and NET formation in septic patients. Our data showed that critically ill septic ICU patients with either an abdominal or pulmonary source of sepsis had increased H3cit protein modification in circulating neutrophil populations compared to healthy controls. We then looked for NET fragments (H3cit/MPO conjugates) within blood samples and found significant increases in septic sera samples. This correlated with an increase in NET visualization via immunofluorescence in whole blood smears in septic samples. This data taken together, suggests that NETs may play a role in the host response to septic infection.

Implications and potential applications of research

As more researchers delve into the field of NET biology, more is being understood about this novel killing pathway. In sepsis, neutrophils become systemically dysregulated and contribute to the morbidity and mortality associated with the disease (14). In a clinical setting NETs have been shown to be elevated in septic and septic shock patients(15, 10) suggesting that NETs may play an important part in the innate immune response to

174 infection (8). Hence, NETs and their role in the innate immune response to septic infection has become a significant area of interest; however the results are muddled at best making a definitive conclusion as to whether NETs play a critical role in the outcome in sepsis difficult.

while PAD4 inhibitors are under development (16), there is still no specific inhibitor available to study the direct correlation between NET inhibition and subsequent host improvement during a septic infection. Cl-amidine treatment has been used extensively to study the role of NET formation in colitis (17, 18) as well as arthritis

(19). Here, we presented data that demonstrated that Cl-amidine treatment in a murine sepsis model of CLP not only improves survival (20, 21), but inhibits NET formation in cells collected from septic mice while having no secondary effect on neutrophil recruitment to the site of infection (see chapter 2). Our observations suggest that NET inhibition using Cl-amidine should be further explored as a possible therapeutic maneuver against the damaging pro-inflammatory response seen in polymicrobial sepsis.

Many targets have been considered as potential biomarkers of NET formation.

Many of these have been measured in an ex vivo setting by culturing collected neutrophils, stimulating them with various stimuli, and probing for proteins that are associated with NET formation such as Neutrophil Elastase, DNA, MPO, and histones

(citation needed). In an in vivo setting, cf-DNA has been utilized as an indirect/surrogate marker of NET formation to study the role of NET formation in a sepsis model.

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Additionally, there has been interest in the use of cf-DNA as a potential biomarker of

NET formation in critically ill patients. However, increased cf-DNA can also be associated with cellular necrosis, apoptosis, in patients with cancer, trauma, stroke, or sepsis (15,

22, 23). Therefore, it is difficult to ascribe the origin of cf-DNA and it has been questioned as to whether cf-DNA alone is a reliable biomarker of NET formation (24). In this dissertation, we have shown that measuring citrullinated histones could be used as a more reliable indicator or NET formation in both a murine model of sepsis, as well as in a septic patient population (see results chapter 3). Additionally, we demonstrated that in septic patients the presence of H3cit in isolated neutrophils correlated to the presence of conjugated H3cit/MPO found in circulation further suggesting that H3cit could be used as a potential biomarker of NET formation.

Questions and Future Directions

What are the molecular Mechanisms involved with Cl-amidine inhibition?

Cl-amidine has been used in a wide array of studies to inhibit the citrullination of histones. It has been shown to suppress pro-inflammatory responses and in turn decrease the damaging effects associated with those responses (18, 20, 25, 26). It is important to remember that Cl-amidine is not PAD4 specific, but targets all PADs. While it has been demonstrated that inhibition of PADs by Cl-amidine suppresses the production of H3cit, and that administration of Cl-amidine improves survival in a CLP model of sepsis (21, 27), the underlying mechanisms which are affected by Cl-amidine treatment are less understood. In chapter 2 we found that Cl-amidine had no effect on

176 overall neutrophil numbers found at the site of infection compared to vehicle control animals, and that 24hr after CLP treatment there was a significant decrease in IL-6 levels in the spleen, with minimal effects on IL-6 levels systemically. However, a recent study using Cl-amidine in a CLP model found reductions of IL-1β and IL-6 in blood samples from Cl-amidine treated mice at 48hr post CLP (20). Interestingly, in our study, IL-10 levels were significantly increased in the bloodstream in the Cl-amidine treated mice compared to vehicle treated mice after CLP indicating that Cl-amidine treatment may be having an effect on the systemic anti-inflammatory response after CLP. Therefore, it cannot be definitively known that these alterations in pro- and anti inflammatory signaling are a direct result of NET inhibition, or an off target effect of Cl-amidine. To address this question, further studies should be performed comparing cytokine profiles from PAD4-/- mice, and Cl-amidine treated mice to look for correlations. Additionally, as selective PAD4 inhibitors become available, the ability to study the direct correlation between NET inhibition and subsequent host improvement during a septic infection will improve.

How does PAD4 inhibition play a role in bacterial burden and survival?

NETs have been demonstrated to capture a wide array of pathogens included gram positive and gram negative bacteria, fungi, protozoa, and viruses (28–34), and have been shown to create a physical barrier preventing the spread of microorganisms (35, 36). However, microbiocidal function of NETs does not seem to be a necessity for host protection during sepsis in vivo. Various studies have looked at the effect on bacterial burden in a

CLP model after NET inhibition. Data suggests that Physical entrapment within NETs

177 prior to and during sepsis is the predominant benefit of Net release, whereas the contribution of NETs to microbial killing may be not necessary with other effector pathways such as phagocytosis and oxidant production (37).

While we did not specifically look at bacterial burden in our study, it would be interesting to see the effect Cl-amidine has on the bacterial burden both within the peritoneum and within circulation to determine if it correlates to our survival data.

Based on previous data, I would hypothesize that Cl-amidine does not inhibit other neutrophil effector functions, and given that Cl-amidine does not affect neutrophil migration to the site of infection, bacterial burden would be comparable in vehicle and

Cl-amidine treated groups.

Does the source of Sepsis impact NET formation?

The study of NETs and their role in the immune response against infection has been an emerging interest in patient based studies. Net formation has been studied in various patient populations that met various criteria standards for study inclusion (15,

38–41). However, no one has looked at if the source of sepsis plays a role in the NET response. Our data (see Chapter 3) displayed a trend for patients with an abdominal source of sepsis to have more MPO/NET conjugates present in sera when compared to patients with pneumonia. While our patient enrollment was too small to detect any significant difference between patient populations, it would be of interest to determine if the source of septic infection plays any role in the activation of NETs.

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Can H3cit modification and subsequent NET formation be a predictor of mortality in a clinical setting?

H3cit modification has been shown to associate with the severity and outcome of sepsis using lethal and sub lethal doses of LPS in vivo (10). Based on our data from chapter 2, H3 citrullination protein expression increases in peritoneal cell populations in a CLP model of sepsis, which is considered an acceptable translational model to clinical sepsis (42, 43). When analyzing isolated neutrophils from septic patients from the ICU, there was indeed evidence of H3cit protein modification not seen in healthy controls

(see chapter 3). Additionally, we demonstrated that NET formations as well as NET fragments are present in the circulation of septic patients, suggesting that NET formation is part of the host response to infection. While circulating histones have been associated with sepsis severity, cellular damage, and organ dysfunction, further work is needed to determine if citrullinated histones and subsequent NET formation plays a critical role in the outcome in sepsis. Correlations between H3cit protein modification as well as increases in circulating H3cit/MPO conjugates and clinical scoring such as

APACHEII or SOFA scores could better determine if histone citrullination and subsequent

NET formation can be predictors of severity of disease and final outcome. Patient populations can be further divided into sub groups depending on the source of sepsis, as well as trauma vs. no trauma to further delineate the correlation between NET formation and shock/sepsis.

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Chapter 4 Conclusions

Synopsis: Critically ill patients that suffered from trauma, complex surgery, gastrointestinal bleeding, obstetrical bleeding, etc., can have an increased susceptibility to bacterial infections (44), and in turn go on to develop Acute Respiratory Distress

Syndrome (ARDS) (45, 46). Increasingly, NETs have been suggested to play a role in the pathogenesis of ALI/ARDS. Circulating free DNA (CF-DNA) as well as circulating histones, considered a major component of NETs, have been implicated in acute lung injury (15,

47, 48). In experimental models of LPS induced ALI, NET formation leads to an increased pro inflammatory response, lung tissue injury and contribute to host cell cytotoxicity leading to lung epithelial and endothelial cell injury and/or death (2, 49–51). Although

NETs have been studied in models of direct ALI, as mentioned above, it is less clear what role they play in a model of indirect ARDS where multiple systemic factors are involved.

Additionally, while critically ill patients who suffer from sepsis are at risk of developing indirect ARDS (the most common organ injury noted in the critically ill patient), they are also at risk to develop multiple organ dysfunction (52, 53). In this respect, many of the pathways that are implicated in the development of ARDS are also thought to play a role in the development of acute kidney injury (54). It is this close association that makes the kidney an organ of interest when studying Multiple Organ failure (MOF) associated with sepsis induced iARDS. Therefore, we aimed not only to study the role of NETs in lung pathology but also examine the effects on renal function.

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Due to the previously described pathogenicity of NETs in various models of disease, including ALI, we hypothesized that PAD4 catalyzed Histone 3 citrullination and the resultant formation of NETs contributed to the systemic inflammation and by-stander tissue injury which in turn contributes to increased morbidity/mortality seen in the combined insults of hemorrhagic shock and sepsis.

In chapter 4 we used PAD4-/- mice to study the effect NET inhibition in our double hit model of Hem/CLP. PAD4-/- displayed a survival advantage over WT mice when subjected to Hem/CLP. As one of the key features of ARDS is the dysregulation and recruitment of activated neutrophils to the lung microvasculature, interstitium, and alveolar space, we measured neutrophil recruitment and MPO activity in lung tissue

24hr post Hem/CLP. We found that PAD gene deletion leads to a decrease in CD11b+

Ly6G+ (neutrophils) and CD11b+ Ly6G- (myeloid cells other than neutrophils). PAD4-/- mice also displayed decreased MPO activity in the lung as shown by ELISA and histological analysis. In addition to decreased neutrophil recruitment/activity, we found that pro-inflammatory cytokines IL-6, TNF-α, and anti-inflammatory cytokine IL-10 were decreased in the lung tissue homogenates and/or Bronchial- alveolar lavage fluid

(BALF).

As indirect ARDS not only affects lung function, but causes organ failure in other systems as well, we assessed the effect PAD4 gene deficiency had on renal function after Hem/CLP. We found that there is reduced vascular leakage, as determined by

Evans Blue extravasation assay in PAD4-/- mice as compared to the WT mice.

Additionally, WT mice displayed significant increases in BUN levels after Hem/CLP but

181 maintained sham creatinine levels. However, there were no elevated BUN or creatinine levels detected in PAD4-/- mice. This lead to WT mice having increased BUN:CR ratio whereas PAD4-/- maintained normal ratios.

Implications and potential applications of research

The role of neutrophils in the pathogenesis of ARDS has been well documented.

Recently, accumulating evidence has suggested that the process of NETosis has a biological relevance in the inflammatory processes and pathogenesis seen in ARDS.

Histones, which have been used as a indirect marker of NET formation, have been detected in the BALF and plasma of patients who developed ARDS after trauma and acid aspiration (48, 55) and has been suggested to correlate with the severity or poor prognosis of these diseases (56).

By understanding the role and significance of NETs in the development of ARDS as well as other organ system dysfunctions that can occur in the critically ill septic patient (52, 53), NETs and/or their components may be targets for potential therapeutics for ARDS. Inflammatory protein involved in the NETosis pathway have become of increasing interest in potential pathological/ therapeutic targets, including

C5a, which has been shown to trigger the formation of NETs (57), extracellular histones, and TGFβ, which can be up regulated through NET-induced platelet activation (58).

While neutralizing antibodies, neutrophil depletion, or downstream signaling events has been utilized to study NETs indirectly , and their role in lung injury (59, 60), to date no one has inhibited NET formation through the inhibition of PAD4 to study NETs and their

182 implications in indirect ARDS and multiple organ dysfunction (MOD) that is encountered by the critically ill septic patient. Here we demonstrated that PAD4 deficiency leads to a reduction of sequestered neutrophils to the lung and reductions in MPO as well as pro- inflammatory cytokine signaling within the lung. Additionally, PAD4 inhibition also decreased kidney dysfunction leading to decreased chances of renal failure. The reduction in organ dysfunction in the lung and kidneys may account for the overall increase in survival seen in PAD4-/- mice after Hem/CLP. Taken together, our data implies that targeting PAD4 appears to be a more direct way of documenting that NETs specifically contribute to ARDS/MOD seen in critically ill patients

Questions and Future Directions

What effect does “priming” have on PAD4-/- neutrophils?

In our mouse model of iARDS, Hem/CLP, hemorrhage has been shown to prime immune cell populations, including neutrophils, such that exposure to a secondary challenge

(such as CLP) leads to an altered response after a subsequent secondary challenge, such as the potentiated induction of inflammation mediator release/ oxidant production/ migratory-phagocytic capacity/ etc. (see chapter 1, fig 2) (61–63). This altered responsiveness is thought to be responsible for the influx of leukocyte infiltration, and a dysregulated immune response that often culminates in by-stander tissue injury (62, 64,

65). In our study we report for the first time that PAD4-/- leads to a reduction of neutrophil sequestration in the lung after Hem/CLP resulting in reductions in MPO activity within the lung. To understand if the “priming” of neutrophils after hemorrhage

183 is altered by PAD4 deficiency, 24hrs after hemorrhage, PAD4-/- and WT neutrophils could be collected and isolated for ex-vivo analysis. The isolated neutrophils can then be assessed for chemotactic ability, as well as their neutrophil apoptotic response.

Additionally, while no differences in chemotactic signaling were seen between PAD4-/- and WT mice after Hem/CLP, we were unable to assess the receptor CXCR2. Differences in CXCR2 upregulation on neutrophils after Hem/CLP may serve as an alternative explanation for decreased neutrophil influx and should be further explored.

Are the decreases in organ dysfunction and improved survival due exclusively to NET inhibition?

We cannot exclusively solely credit the decreased organ dysfunction and improved outcome in the PAD4-/- mice to the lack of NETs, as there may be off target effects related to the global knock out of PAD4. However, our data is in line with survival data described in sepsis models by us and others that use Cl-amidine, a chemical inhibition of

PAD, which imparts an increase in survival after CLP (20, 21, 27). In addition to a PAD4-/- mouse, a PAD4fl/fl mouse was also created (66). The PAD4fl/fl can then be crossed with a lox-Cre mouse to delete PAD4 from neutrophil specific lineage. This strategy would help delineate if the alteration in the immune response to Hem/CLP is due to neutrophils inability to make NETs or the generated by off target effects of PAD4KO. However, as genetic deletion is not translatable to the clinical setting, the use of a PAD inhibitor, such as Cl-amidine, can also be utilized in the Hem/CLP model to determine if chemical

184 inhibition of NET formation corresponds to the results seen in the PAD4-/- mice. Finally, as selective PAD4 inhibitors become available the role of NET formation in indirect ARDS will be even easier to ascertain.

Does NET formation directly influence Kidney Function after Hem/CLP?

While the pathophysiology of ARDS is complex with multiple factors involved (45); many of the pathways that are implicated in the development of ARDS are also thought to play a role in the development of acute kidney injury (AKI) (54). Furthermore, in a renal ischemia reperfusion (I/R) injury model the formation of NETS has been implicated in the development of renal inflammation and increased tissue injury (67). More specifically, PAD4 has been implicated in renal I/R injury by increasing renal tubular inflammatory responses and neutrophil infiltration into the kidney. In this study, we did not see any direct evidence that linked neutrophils and/or NET formation to the improved kidney function seen in the PAD4-/- mice. However, as MOF is progressive through the course of the response to severe injury/shock/sepsis, it is possible that a difference may be detected further along in the course of the disease state. To address this question, I would propose a time course study that goes beyond the 24hr time point we use in the Hem/CLP model, to assess neutrophil infiltration/NET formation in the kidney. While a further out time point may show a temporal difference, it is also possible the neutrophil infiltration/NET formation does not play a important role in altering kidney function, but rather the differences seen between groups is an indirect effect to alterations in other pathways upstream of AKI or an indirect effect of PAD4

185 inhibition that is independent of NET formation. By performing a series of experiments that use neutrophil depletion in PAD4-/- vs. WT and re-examining kidney function after

Hem/CLP, we would be able to assess if the maintenance of kidney function seen in

PAD4 deficient animals is due to neutrophil activity or is an independent artifact of

PAD4 deficiency.

The Big Picture and Final Thoughts

Since Neutrophil Extracellular Traps were initially described as an additional effector function of neutrophils to kill pathogens in 2004 (36), they have studied extensively in the immune response to a wide ranging spectrum of disease states.

However, their overall impact on the immune response and the host, either beneficial or detrimental, is still not well understood. This dissertation set out to test the hypothesis that NETs contribute to the systemic inflammation and by-stander tissue injury that plays a part in the increased morbidity/mortality seen in response to septic insult, or the combined insults of hemorrhagic shock and sepsis (which induce acute lung injury as well as range of other organ injuries). Using pharmacological and genetic inhibition of

PAD4 to more directly target NET formation, we have shown that NET inhibition increases survival not only in a polymicrobial sepsis model (CLP), but a more complex model of Hemorrhagic shock and sepsis (Hem/CLP). This would suggest that NET formation does play a role in pathogenesis of sepsis as well as shock/sepsis and their formation is overall detrimental to the host.

186

While our data contributes to a better understanding of the role of NETosis in sepsis and shock/sepsis, limitations still exist. The nature of NETs, fine meshwork fibers extruded from the cell (36), make them difficult to clearly identify in vivo, thus, limiting ones ability to tie them to site of tissue damage. There has been successful visualization of NETs in vitro with the use of immunofluorescence microscopy, transmission electron microscopy, scanning electron microscopy etc. but limited available in vivo data (68).

While advances have been made using intra-vital imaging (1, 69, 70) to study NET formation in the vasculature and tissues, these techniques are not widely available, and difficult to utilize in different disease models. Therefore, to fully understand the mechanisms regulating NET formation and the role of NETs during health and disease, improved NET visualization and quantification techniques are needed. In the meantime, the use of more specific biomarkers for NET formation, such as PAD4 or Citrullinated histones as we used here, bring us closer to understanding the role of NET formation in the complex immune response to sepsis as well as the combined insults of hemorrhagic shock and sepsis.

187

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