Airway Nucleases and Surfactant Protein D Promote

Neutrophil Extracellular Traps Clearance

by

Lily Yip

A thesis submitted in conformity with the requirements for the degree of Master of Science Laboratory Medicine & Pathobiology

University of Toronto

© Copyright by Lily Yip 2014

ii

Airway Nucleases and Surfactant Protein D Promote

Neutrophil Extracellular Traps Clearance

Lily Yip

Master of Science

Laboratory Medicine & Pathobiology

University of Toronto

2014

Abstract

Neutrophils release neutrophil extracellular traps (NETs) to trap and kill invading pathogens.

However, NETs can damage host cells. A mechanism of NET clearance in the airways has not

been established. We investigated whether NETs are cleaved by airway nucleases. Our study

showed that nucleases are present in murine airways and that genomic DNA and NETs are

cleaved in a magnesium/calcium- and calcium-dependent manner, respectively. We also found

that these nucleases function optimally at specific pH ranges, such as near neutral pH and acidic

pH. We also assessed the role of SP-D in NET clearance. We found that SP-D-deficient mice

are defective in NET clearance compared to WT mice. SP-D supplemented to NET-alveolar

macrophage (AM) cultures had fewer remaining NETs. Thus, we conclude that SP-D enhances

the clearance of NETs by AMs. Overall, we conclude that nuclease digestion and SP-D are

involved in the clearance of NETs in the airways.

iii

Acknowledgements

First and foremost, I would like to thank my supervisor, Dr. Nades Palaniyar, for providing me

with this opportunity in research and for his guidance and support throughout. The passion that

you have for the work that you do resonates throughout the lab. I am very grateful to be

mentored by someone who believes in his students and motivates them to achieve their ultimate

goals.

I would also like to thank all the members of the Palaniyar lab for their friendship and support.

In particular, I would like to thank Dr. David Douda for his help in neutrophil and mouse

experiments, in teaching me the techniques of the lab and providing insightful knowledge and

advice. Pascal Djiadeu for his advice on macrophage isolation and experiments. Hayley Craig-

Barnes for her help on the manual quantification of NETs. Estelle Zhu-Yuan Chen for her help

on the DNA assays. Thanks to everybody who has lent a helpful hand. Also, to Stéphane

Gagnon for being so gracious in allowing me to use his lab equipment.

I would like to thank the advisory committee members, Drs. Martin Post, Christoph Licht,

Adam Gassas and David Bazett-Jones for their support and in giving critical suggestions

throughout my project.

I’d also like to thank the staff from the Imaging Facility and the University of Toronto LMP

Graduate Department for their technical assistance and guidance.

Lastly, I’d like to thank my friends, family and loved ones for their unconditional love and

support throughout these two years. I am very appreciative of your understanding and patience.

Work presented in this thesis would not have been possible without the support from our

funding agencies. This project was supported by operating grants from Canadian Institutes of

Health Research (MOP-111012) and Cystic Fibrosis Canada (grant 2619). My stipend was

partially supported by the University of Toronto Fellowship Award and Ontario Graduate

Scholarship.

iv

Table of Contents

Abstract .......................................................................................................................................... ii

Acknowledgements....................................................................................................................... iii

Table of Contents .......................................................................................................................... iv

List of Tables ................................................................................................................................ vi

List of Figures .............................................................................................................................. vii

Chapter 1 - Introduction................................................................................................................. 1

1.1 Neutrophils ......................................................................................................................... 2

1.1.1 Neutrophil Recruitment and Activation ...................................................................... 3

1.1.2 Neutrophil Extracellular Traps (NETs) ....................................................................... 4

1.1.2.1 Inducers of NETosis ............................................................................................ 6

1.1.2.2 NETosis vs. Apoptosis and Necrosis ................................................................... 7

1.1.2.3 Mechanism of NET Formation ............................................................................ 7

1.1.2.4 Viable Immune Cells Form Extracellular Traps ................................................ 11

1.1.3 NETs in Health and Disease ...................................................................................... 12

1.1.3.1 Cytotoxic Effects of NETs ................................................................................. 12

1.1.3.2 NETs in Blood Disorders ................................................................................... 12

1.1.3.3 NETs in Autoimmune Disorders ....................................................................... 14

1.1.3.4 NETs in Lung Disorders .................................................................................... 15

1.1.4 Resolution of Neutrophils and NETs ........................................................................ 17

1.2 Clearance in the Lung ....................................................................................................... 19

1.2.1 Mucociliary Clearance in the Airways ...................................................................... 19

1.2.1.1 Airway Surface Liquid ....................................................................................... 20

1.2.1.2 Antimicrobial Factors in the ASL ...................................................................... 21

1.2.1.3 Nucleases ........................................................................................................... 21

1.2.1.3.1 Nucleases in the ASL .................................................................................... 24

v

1.2.2 Clearance by Alveolar Macrophages ........................................................................ 25

1.2.2.1 Surfactant Protein D and Phagocytosis .............................................................. 26

1.3 Rationale and Hypothesis ................................................................................................. 28

Chapter 2 ...................................................................................................................................... 30

2.1 Abstract ......................................................................................................................... 31

2.2 Introduction ................................................................................................................... 31

2.3 Materials & Methods .................................................................................................... 32

2.4 Results ........................................................................................................................... 35

2.5 Discussion ..................................................................................................................... 53

Chapter 3 ...................................................................................................................................... 57

3.1 Abstract ......................................................................................................................... 58

3.2 Introduction ................................................................................................................... 58

3.3 Materials and Methods .................................................................................................. 59

3.4 Results ........................................................................................................................... 62

3.5 Discussion ..................................................................................................................... 70

Chapter 4 ...................................................................................................................................... 74

4.1 Overall Discussion ........................................................................................................ 75

4.1.1 Airway nucleases degrade genomic DNA and NET DNA .................................... 75

4.1.1.1 Maximal airway nuclease activity at neutral and acidic pH ............................. 76

4.1.2 SP-D enhances the clearance of NETs .................................................................. 77

4.1.3 Clearance of NET fragments by macrophages ...................................................... 78

4.2 Conclusions ................................................................................................................... 79

4.3 Future Directions .......................................................................................................... 80

References.................................................................................................................................... 82

Appendix.................................................................................................................................... 110

vi

List of Tables

Table 1.1 Summary of Identified NET proteins. ............................................................................. 5

Table 1.2 Pathological implications of NETs and co-localized effector molecules. ..................... 10

vii

List of Figures

Figure 1.1. NADPH-dependent pathway for NETosis regulation. .................................................. 9

Figure 2.1. Nucleases are present in non-flamed airways of naïve and PBS-instilled mice. ........ 38

Figure 2.2. Nucleases in the airways of naïve and PBS-instilled mice require cations for

activity. ........................................................................................................................................ 40

Figure 2.3. Neutrophils recruited to the airways of 4-week-old mice form NETs. ....................... 42

Figure 2.4. Nucleases are present in the BAL fluid of LPS-instilled mice. .................................. 43

Figure 2.5. Nucleases in the inflamed airways of LPS-instilled mice require cations for

activity… ....................................................................................................................................... 45

Figure 2.6. Degradation of NETs in the BAL fluid of LPS-instilled mice. ................................... 46

Figure 2.7. Nucleases are active with Mg2+

/Ca2+

and also without cations near acidic pH. ......... 48

Figure 2.8. Nucleases of inflamed airways have two pH optimums – PIPES buffer. ................... 49

Figure 2.9. Nucleases of inflamed airways have two pH optimums – MOPS buffer.................... 50

Supplementary Figure S2.1. Nucleases from naive airways of mice have two pH optimums –

MOPS buffer…………………………………………………………………………………..… 51

Supplementary Figure S2.2. Nucleases from PBS-instilled airways of mice have two pH

optimums – MOPS buffer……………………………………………………………………….. 52

Figure 3.1. Neutrophils recruited to the airways have NET-derived DNA-protein complexes in

SP-D KO mice. .............................................................................................................................. 64

Figure 3.2. SP-D-deficient mice are defective in NET clearance. ................................................. 65

Supplementary Figure S3.1. SP-A levels are variable in the BALF of both PBS- and LPS-

instilled WT and SP-D KO mice………………………………………………………………... 66

Figure 3.3. SP-D enhances the clearance of murine NETs by alveolar macrophages ex vivo. ..... 68

Figure 3.4. Digested NETs are cleared by macrophages by 2 h. ................................................... 69

viii

List of Abbreviations

ALI acute lung injury

AM alveolar macrophage

ANCA anti-neutrophil cytoplasmic autoantibody

ARDS acute respiratory distress syndrome

ASL airway surface liquid

ATA aurintricarboxylic acid

BALF bronchoalveolar lavage fluid

CAD caspase-activated DNase

CGD chronic granulomatous disease

CitH3 citrullinated histone H3

CRD carbohydrate recognition domain

CF cystic fibrosis

COPD chronic obstructive pulmonary disease

DAMP danger-associated molecular pattern

DC dendritic cell

DNA deoxyribonucleic acid

DNase deoxyribonuclease

DPI diphenylene iodonium

DVT deep vein thrombosis

EBC exhaled bronchial condensate

EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol tetraacetic acid

ERK extracellular signal-related kinase

ET extracellular trap

FBS fetal bovine serum

FCS fetal calf serum

fMLP N-formyl-methionine-leucine-phenylalanine

gDNA genomic deoxyribonucleic acid

GM-CSF granulocyte macrophage-colony stimulating factor

ix

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HMDM human monocyte-derived macrophages

HMGB1 high-mobility group box 1

IFN-ɣ interferon-gamma

IL interleukin

KO knockout

LPS lipopolysaccharide

MAPK mitogen-activated protein kinase

MEK mitogen-activated kinase/ERK kinase

MES 2-(N-morpholino)ethanesulfonic acid

MIP-2 macrophage inflammatory protein-2

MNase micrococcal nuclease

MOPS 3-(N-morpholino)propanesulfonic acid

MPO myeloperoxidase

MUC mucin

n/CRD neck CRD (recombinant molecule of SP-D made of trimeric neck domain and CRD)

NADPH nicotinamide adenine dinucleotide phosphate

NE neutrophil elastase

NET neutrophil extracellular trap

NETosis neutrophil extracellular trap formation

NOX NADPH oxidase

PAD4 peptidylarginine deiminase 4

PAP pulmonary alveolar proteinosis

PBS phosphate buffered saline

PCL periciliary liquid layer

PIPES piperazine-N,N′-bis(2-ethanesulfonic acid)

PK proteinase K

PKC protein kinase C

PMA phorbol 12-myristate 13-acetate

rhDNase recombinant human deoxyribonuclease

ROS reactive oxygen species

x

SCD sickle cell disease

SIRP-α signal regulatory protein-alpha

SLE systemic lupus erythematosus

SP-A surfactant protein A

SP-D surfactant protein D

SVV small vessel vasculitis

TFPI tissue factor pathway inhibitor

TGF-β tumor growth factor-beta

TLR toll like receptor

TNF-α tumor necrosis factor-alpha

TRALI transfusion related acute lung injury

VOC vaso-occlusive crisis

1

Chapter 1:

Introduction

2

Chapter 1 - Introduction

1.1 Neutrophils

Neutrophils are phagocytic leukocytes of the innate immune system and are one of the first

responders to microbial infections. Originating from the hematopoietic stem cells of bone

marrow, these cells undergo several maturation phases before being released into circulation as

neutrophils1,2

. Mature neutrophils constitute up to 60 % of circulating leukocytes in humans3.

The maturation sequence follows a continuous and consecutive series of granule expansions

elicited by several cytokines and growth factors in the bloodstream. These granules contain a

plethora of pro-inflammatory molecules and are categorized into the 3 distinct groups based on

its contents: peroxidase-positive primary (azurophilic) granules, peroxidase-negative secondary

(specific) granules, and gelatinase-positive tertiary (gelatinase) granules4. The formation of

these granules also coincides with the timing of neutrophil maturation. Hence, primary granules

are formed first, followed by secondary granules, and then tertiary granules. By the end of

maturation, neutrophils are fully equipped with an arsenal of proteins and peptides shown to

have antimicrobial effects. These terminally differentiated cells are characteristically recognized

by their multi-lobed nuclei, from which the synonymous name, “polymorphonuclear

neutrophils”, was derived.

Mature neutrophils are found to be 40 to 80 times more concentrated in the pulmonary

capillary bed than in the blood of larger vessels. These neutrophils emigrate from a concentrated

region known as the “marginated pool” into the distal lung during inflammatory conditions5. In

circulation, neutrophil longevity was always considered to be short-lived with a half-life about

1.5-3.3 and 8 hours in mice and humans, respectively6–8

. However, these ex vivo studies may

have underscored the lifespan of neutrophils in vivo. Recently, the half-life of circulatory

neutrophils has been reported to be as high as 12.5 hours in mice and 3.8 days in humans,

although some criticism has been received for the human data8. Nevertheless, activated

neutrophils during inflammation have lifespan increases by several folds which ensures

regulation of host resistance and inflammatory processes9.

3

1.1.1 Neutrophil Recruitment and Activation

Regarded as highly motile cells, neutrophils are constant patrollers of the bloodstream and play

a key role in inflammatory responses. Following a physical insult or invasion by a foreign

pathogen, danger-associated molecular patterns (DAMPs) released by dying cells are

subsequently sensed by sentinel cells such as macrophages, dendritic cells, mast cells, and

endothelial cells to produce signals that form a chemokine gradient10

. These signals include

host-derived interleukin (IL)-8 and C5a, and for rodents, MIP-2 and KC (an IL-8 analog)11

.

Bacteria-derived component lipopolysaccharide (LPS) and bacterial product N-formyl-

methionine-leucine-phenylalanine (fMLP) can also act as chemoattractants for activated

neutrophils. To assist with the recruitment cascade, adhesion molecules such as selectins (P-

selectin and E-selectin), CD44, and other glycosylated ligands, are mobilized to apical surfaces

of the endothelium. As such, transient interactions formed between ligands and incoming

neutrophils initiate cell tethering and rolling on the capillary wall. Further changes to the

neutrophil surface (expression of integrins) and cytoskeleton enables cell adhesion, crawling and

transmigration across the endothelium to the site of injury. Once extravasated, neutrophils

participate in elaborate signaling networks through the release of their own cytokines,

chemokines and growth factors to recruit additional immune cells. Activated neutrophils will

then function to defend against host infections by phagocytosis, degranulation, or reactive

oxygen species (ROS) production12

.

During phagocytosis, opsonised microbes are ingested into phagosomes and killed

intracellularly. Neutrophil oxidative bursts are induced following the fusion of the phagosome

with lysosomes or to its own granules (internal degranulation). This leads to the vast

consumption of molecular oxygen which becomes reduced by NADPH oxidase to generate

superoxide and hydrogen peroxide (H2O2). Myeloperoxidase from azurophilic granules further

convert H2O2 to hypochlorous acid (HOCl). The resulting combination of these reactive oxygen

species (ROS) and granular proteins destroys the phagocytosed microbe13

. Degranulation also

occurs extracellularly, of which primary and secondary granules fuse with the plasma membrane

to release its antimicrobial contents, creating an inhospitable environment for invading

pathogens. Neutrophil elastase, proteinase-3 and cathepsin G are serine proteases that cleave

peptide bonds to degrade microbial proteins, while defensins, lysozymes,

bactericidal/permeability-increasing protein and LL-37 (peptide of human cathelicidin) are

4

neutrophilic peptides that act to disrupt membrane integrity of the bacteria cell wall14,15

. Similar

to degranulation, ROS may also be released outside of the cell to perform its antimicrobial

duties.

1.1.2 Neutrophil Extracellular Traps (NETs)

Phagocytosis, degranulation and ROS production employed by neutrophils to fight infection

have been classically described for numerous decades16

. More recently, a new mechanism of

host defense involves the expulsion of neutrophilic DNA into the extracellular milieu. This

phenomenon was first documented as an unusual form of cell death following the activation of

neutrophils with phorbol-12-myristate-13-acetate (PMA)17

. Although described as being

morphologically distinct from typical apoptosis and necrosis, the mechanistic details and

functional relevance of this novel cell death program was not understood at the time17

. In 2004,

Brinkmann et al. defined these structures as “neutrophil extracellular traps (NETs)”. By using

electron microscopy and immunohistochemistry, the authors elegantly showed that NETs are a

meshwork of fibrous DNA decorated with granular proteins which served as platforms for

effectively trapping and killing both Gram-negative and Gram-positive bacteria18

. The authors

also showed that NETs were capable of trapping and killing fungi through the action of

calprotectin, a NET protein19,20

. Soon after, several other reports identified a number of

microorganisms that were susceptible to the antimicrobial properties of NETs.

NET-mediated killing is shown to be attributed to histones21

, elastases22

,

myeloperoxidases (MPOs)23

, LL-3724

, calprotectins20

and α-defensins23

. Table 1.1 outlines a list

of PMA-induced NET-associated proteins/peptides captured by nano-scale liquid

chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry (nano

LC-MALDI-MS)20

. The function of some of these molecules localized within NETs has not

fully been examined and remains to be elucidated. Despite the unknowns, NET formation

proves to have many beneficial outcomes and is not surprising that this course of action is

evolutionarily conserved across a number of species including humans18

, mice25

, cows26

, cats27

,

fishes28,29

, chickens (in heterophil cells)30

, and insects (in hemocytes)31

. Furthermore, similar

forms of DNA-protein structures identified in other immune cell types (macrophages32,33

, mast

cells34

, eosinophils35

) led to the redefinition of extracellular trap (ET) formation as “ETosis”36

.

5

Table 1.1 Summary of Identified NET proteins*.

*Proteins found on NETs from PMA-activated human neutrophils. Proteins were identified by

nano-scale liquid chromatography coupled to matrix-assisted laser desorption/ionization mass

spectrometry (nano LC-MALDI-MS). Accession numbers can be found in the NET Database

(http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/ls/index.cgi). Urban et al. (2009) PLoS Pathog,

5(10):e1000639. doi: 10.1371/journal.ppat.

6

1.1.2.1 Inducers of NETosis

NET formation, hereafter known as NETosis, is triggered by a variety of stimulants, many of

which are physiological activators of neutrophils. Infections by microorganisms and pathogens

such as bacteria18,37,38

, protozoa21,39

, fungi19,40

and viruses23,41

have all been shown to induce

NETosis. Several of these invaders are recognized by toll-like receptors (TLRs) on neutrophil

surfaces, and are presumably one of the common signalling pathways for NET formation.

Bacterial flagellin and viral nucleic acid of human immunodeficiency virus-1, for example,

signals through TLR5 and TLR7/8, repectively23,42

. Lipopolysaccharide (LPS), a constituent of

the outer membrane of Gram-negative bacteria, activates neutrophils by signalling through

TLR4. Despite numerous ex vivo studies showing direct stimulation of NETosis by LPS18,42–44

,

the effect of LPS remains to be highly controversial as other researchers have reported different

findings. In a septic model, the action of LPS required the intermediate activation of TLR4-

platelets to form platelet-neutrophil interactions before leading to NET production45

. Priming of

neutrophils with granulocyte/macrophage colony-stimulating factor (GM-CSF) was also

necessary for activation by LPS to generate mitochondrial NETs from viable cells46

.

Host-derived factors involved in immune cell activation and recruitment can also serve

as inducers of NETosis. NET formation was shown to occur in the presence of IL-8, a

chemokine for neutrophils released by activated cells during inflammation and infection47

.

High-mobility group box 1 (HMGB1), a nuclear nonhistone DNA-binding protein and a

released DAMP signal, was shown to signal through TLR4 to induce NET formation in wild

type mice compared to their TLR4-deficient counterparts48

. GM-CSF is a growth factor that

only when paired with complement anaphylatoxin C5a, acquires the ability to induce NET

formation. This has been described as a unique form of NETosis with DNA originating from the

mitochondria46

. Other activators of NETosis include platelet activating factor, HOCl and

oxidative species singlet oxygen49–51

.

Key discoveries to NET formation and its resulting properties have stemmed from the

use of synthetic molecules that act as potent inducers of NETosis. In vitro, PMA is commonly

used as an inducer of NET formation by signalling through the protein kinase C (PKC)

pathway52

. Calcium ionophores that increase intracellular calcium in the endoplasmic reticulum

are alternatively used for the rapid generation of NETs53

. Although these methods provide a

convenient way to study NETs, the use of non-biological molecules dampens the physiological

7

relevance of NET formation and limits the ability to recapitulate similar findings within a living

system.

1.1.2.2 NETosis vs. Apoptosis and Necrosis

NET release by dying cells is a novel cell death program that is, in many ways, morphologically

and molecularly distinct from apoptosis and necrosis54

. The word ‘death’ used to describe

NETosis can be easily mistaken as a process similar to apoptosis. It is, however, very different

from apoptosis as cells undergoing NETosis do not display surface phosphatidylserine (an ‘eat

me’ signal) before the rupture of the plasma membrane. This prevents immediate clearance by

macrophage which, in turn, provides optimal ‘working time’ for NETs to trap and kill microbes.

Morphological signs of apoptosis such as membrane blebbing, chromatin condensation and

DNA fragmentation by caspase-activated DNases (CADs) are all devoid in NETosing

neutrophils38,46

. Caspase activity is also absent in PMA-induced NETosis55,56

. Furthermore, we

have recently shown that PMA-mediated NETosis requires the upregulation of Akt, an apoptosis

inhibitor56

(see Appendix; “Akt is essential to induce NADPH dependent NETosis and to switch

the neutrophil death to apoptosis”).

Necrosis occurs as a result of tissue injury leading to premature cell death by autolysis.

Certain receptor-interacting serine-threonine (RIP) kinases, such as RIPK1 and RIPK3, have

been shown to drive necrosis via mitochondrial dysfunction57

. However, PMA-mediated

NETosis was not affected by RIP1 inhibitor necrostatin-1, indicating that NETosis and necrosis

are two separate cell death pathways55

.

1.1.2.3 Mechanism of NET Formation

The most extensively characterized pathway of NETosis is the nicotinamide adenine

dinucleotide phosphate (NADPH)-oxidase dependent pathway. Additionally, many of the

pathways for NETosis were mapped out from studies using PMA as the activator. NETosis is an

active form of cell death that requires both autophagy and ROS production by NADPH oxidase

(NOX) 255

. As such, inhibition of autophagy and NOX by Wortmannin and non-specific

diphenylene iodonium (DPI), respectively, prevents NET formation. In fact, individuals with

chronic granulomatous disease (CGD) have non-functional NOX2 and are unable to produce

8

NETs. Consequently, CGD patients continuously suffer from recurrent and severe infections58

.

Rac2 is also required for NET formation, as it is a key regulator of NOX and, therefore, ROS

production44

. Myeloperoxidase (MPO) found in azurophilic granules is vital for the conversion

of superoxide dismutase product H2O2 to HOCl, a superoxide previously mentioned to have

NET-inducing capabilities51

.

Neutrophils treated with PMA have increases in intracellular calcium which further

activates PKC to initiate several downstream signalling cascades (Fig. 1.1). One such cascade

involves the Raf-MEK-ERK kinase pathway which was discovered by a high-throughput

chemical genetic screen of small molecules incubated with PMA-activated neutrophils. The

same authors additionally showed that Raf-MEK-ERK is upstream of NOX-mediated ROS

production by using DPI and ERK inhibitors59

. The finding that activation of Raf-MEK-ERK

occurs prior to ROS production was widely accepted as the dogma of NET formation. However,

this was challenged by Keshari and colleagues, who later found that free radical generation

preceded Raf-MEK-ERK, as well as p38 MAPK60

. Despite these differences, it is known that

the requirement for ROS production, Raf-MEK-ERK phosphorylation and p38 MAPK

phosphorylation are all necessary for PMA-mediated NET formation. Recently, we have

discovered the involvement of Akt as a molecular switch for inhibiting apoptosis while

simultaneously promoting NET formation in PMA-activated neutrophils (see Appendix)56

.

Following the generation of ROS, neutrophil elastase (NE) translocates from azurophilic

granules to the nucleus where it partially degrades histones (H1, H2A, H2B, H4) to promote

chromatin decondensation. Shortly after, incoming MPO binds to chromatin and synergizes with

NE to enhance NE-mediated nuclear decondensation61

. This process is further mediated by

peptidylarginine deiminase 4 (PAD4) which hypercitrullinates positively charged arginine on

histones to uncharged citrulline (CitH3), a marker of NETs. Changes to histone residues relieves

electrostatic charges between chromatin coils and alters the density of nucleosome

structures62,63

. Eventually, the nucleus loses its multi-lobed form at the same time that nuclear

and granular membranes begin to break down. This allows for the mixing of nuclear DNA with

the contents released from granules. Final permeabilisation of the plasma membrane

accompanies NET release into the extracellular environment. NETs are now equipped with an

arsenal of antimicrobial proteins/peptides, ready to trap and kill pathogens. Table 1.2 outlines a

list of NET-associated antimicrobial molecules and their associated pathological implications38

.

9

Figure 1.1. NADPH-dependent pathway for NETosis regulation.

Following the binding of pro-inflammatory ligands, NETosis can be induced. Activation of PKC

leads to intracellular increases in calcium which stimulate downstream processes of autophagy,

NOX2 and PAD4. Activation of NETotic processes also leads to the inhibition of caspases to

prevent apoptosis. Both PAD4 and neutrophil elastase will translocate to the nuclease to assist

with deimination of histones as well as chromatin condensation. This is followed by the

disintegration of granular and nuclear membranes. Granular proteins mixes with decondensed

DNA and are together released as NETs. [Adapted with permission from Remijsen, Q.,

Kuijpers, T.W., Wirawan, E., Lippens, S., Vandenabeele, P., Vanden Berghe, T. (2011). Dying

for a cause: NETosis, mechanisms behind an antimicrobial cell death modality. Cell Death and

Differentiation, 18(4): 581-588. doi: 10.1038/cdd.2011.1.]

PMA

10

Table 1.2 Pathological implications of NETs and co-localized effector molecules*.

*Permission from Logters et al. (2009) Med. Microbiol. Immunology. 198:211-219. doi:

10.1007/s00430-009-0121-x.

11

Alternatively, NETosis may occur independently of the NADPH pathway and still

require ROS production64–66

. The mitochondrion is another source of ROS and can be activated

for production via calcium-mediated small conductance channels. However, in this pathway,

neutrophil death was documented to occur by apoptosis64

. The involvement of these channels in

the context of NETs has not been investigated. In other studies, the use of ROS inhibitors did

not prevent NET formation. This hinted at ROS-independent pathways as alternative routes for

NETosis. Indications of such a pathway was shown to be stimuli-specific65,66

. That is,

neutrophils stimulated with Staphylococcus aureus (S. aureus) or calcium ionophore produced

NETs much more rapidly (<1 h) than the conventional inducers and did not require ROS. The

authors also noted a unique difference in NET formation, whereby DNA-budding vesicles from

the nuclear membrane were extruded into the extracellular space without specific lysis of the

plasma membrane66

.

1.1.2.4 Viable Immune Cells Form Extracellular Traps

It has long been thought that NETosing neutrophils render themselves dead, presumably due to

the elaborate ejection of their nuclear (genomic) and cytoplasmic machinery. However, this was

based on initial observations of cells whose plasma membranes were ruptured and no longer

functioned as intact entities. Recent studies have suggested that the preceding findings may not

be necessarily true46,66

. In one study, priming of neutrophils with GM-CSF following a short-

term stimulation with C5a led to the rapid release of mitochondrial NETs rather than genomic

NETs. The authors showed that NET release were from viable cells based on the low percentage

of ethidium bromide-containing cells, and suggested that both GM-CSF/C5a prolonged

neutrophil survival46

. In another study, viable cells stimulated with S. aureus led to the rapid

release of NET-containing vesicles without rupturing the plasma membrane66

. Multitasking by

neutrophils was further demonstrated by Yipp and colleagues67

. Using an interdermal S. aureus

infectious mouse model, they showed that neutrophils post-NETosis existed as live, enucleated,

chemotactic and phagocytic cells. Viable neutrophils that have undergone NETosis but still

retain the ability to migrate and perform phagocytosis are synonymously referred to as

neutrophil “cytoplasts” or “cytokineplasts”67

. Of note, cytoplasts or cytokineplasts have long

been observed in human abscesses since the 1980s by Malawista and colleagues68,69

, but their

roles in vivo remained unknown until the recent work by Yipp and colleagues67

.

12

1.1.3 NETs in Health and Disease

NET formation is undeniably important to host immunity as the failure to produce NETs

weakens the organism’s ability to fend off pathogens55,58,62,70

. For instance, mice deficient in

PAD4 were more susceptible to bacterial infections than wild type mice due to the lack of

NETs62

. This is further highlighted in human neonate neutrophils whose unresponsiveness to

inflammatory agonists renders the conquest of bacterial invasion70

. It was later demonstrated

that these neutrophils exhibit a delayed response, rather than lack thereof, which might explain

how children are more prone to infections before reaching adulthood42

. The persistent challenge

of infections faced by patients with CGD is also due to the fact that NET production is inhibited

by inactive NOX. Recent advances indicate that infection control can be repaired by the

reactivation of NET formation by treating CGD neutrophils with NOX-complementing gene

therapy58

.

1.1.3.1 Cytotoxic Effects of NETs

Despite the beneficial properties of NETs, there is increasing evidence linking aberrant or

excessive NET formation and impaired NET clearance to the pathogenesis of diseases71

. Highly

concentrated antimicrobial molecules found on NETs have been shown to be key contributors to

tissue damage72

. This was highlighted by a study showing that the cytotoxicity of NETs on

alveolar cells was independent of DNA digestion. The authors found that NET proteins, such as

histones and MPOs, were directly responsible for lung epithelial and endothelial cell death73

. On

the other hand, activated endothelial cells can trigger NET formation and cause their own cells

to succumb to NET-mediated death74

. These cytotoxic effects can be eliminated by the use of

anti-histone antibodies or MPO inhibitors to promote cell survival73

. Table 1.2 compares the

functions and implications of other NET proteins in pathological diseases.

1.1.3.2 NETs in Blood Disorders

Before the discovery of NETs, increased levels of circulating free DNA was reported in several

diseases including sepsis75

, autoimmune disorders76

, and specific cancers77,78

. It was believed

that circulating free DNA originated from apoptotic and/or necrotic cells, but several research

groups later suggested that this DNA had similar properties to NETs75,79,80

. NETs are now

13

associated with various blood disorders including thrombosis81–84

, deep vein thrombosis

(DVT)85,86

and sickle cell disease87

.

Thrombosis is a disorder characterized by the formation of blood clots or thrombi within

the blood vessel. Numerous labs are now establishing the role of NETs in the pathogenesis of

thrombosis81–84

. NET formation triggers coagulation as NETs can serve as scaffolds for platelet

adhesion, activation and aggregation81

. Platelet activation may be achieved by the binding of

LPS to platelet TLR4 or by the presence of histones or cathepsin G found on NETs45,82,83,88

.

Also found on NETs are serine proteases such as neutrophil elastase which couples together

with cathepsin G to promote additional clotting. This occurs by the proteolytic cleavage of

tissue factor pathway inhibitor (TFPI)89,90

. A perfusion study also showed that NETs, together

with platelets and fibrin, bind to and accumulate red blood cells to enhance coagulation for

greater thrombus generation81

. Specifically, the combination of histones and DNA binding to

large fibrin yields a complex with higher mechanical stability and tensile strength that resists

clot lysis91

. It is noteworthy that during thrombosis, activation of platelets by LPS also leads to

additional activation of NETosis45

. Thereafter, the components on NETs will trigger more

platelet activation and contribute to a continuous loop of pro-thrombotic events. The interplay

between neutrophils and platelets suggests that the pathways of inflammation and thrombosis

are connected through these cell types.

Impairment of blood flow by NETs is further corroborated by the rising models of deep

vein thrombosis (DVT)86,92–94

. It was first noted in baboons with DVT that extracellular DNA

accumulated in the plasma, followed by additional detection of both DNA and histones in

venous thrombi81

. Presence of histones was usually correlated with increased thrombin

generation which implied that histones were involved in the suppression of anticoagulant

factors. This was validated by a DVT mouse model whose vein thrombi were found to contain

large amounts of CitH3 and, therefore, NETs. By administering intravenous deoxyribonuclease

1 (DNase 1), these mice were protected from DVT formation as a result of NET degradation93

.

Similarly, the depletion of NETs in neutropenia is associated with decreased thrombus weight86

.

Histone modifications are essential to DVT as mice deficient in PAD4 are protected against

developing DVT following stenosis of the inferior vena cava. Although these mice are unable to

form NETs, platelet plugging and coagulation remained normal94

. Having DVT exposes the risk

14

for fatal pulmonary embolisms which emphasizes the need for better treatment options aimed at

regulating neutrophils and its constituents.

Neutrophils also play an important role in sickle cell disease (SCD)-related

complications. SCD is characterized by the distortion of red blood cells as a result of atypical

hemoglobin molecules. Patients with SCD have elevated levels of neutrophil cell count and are

accompanied with a higher propensity for activation by inflammatory stimuli as evidenced by

the primed state of neutrophils95,96

. Mouse models of SCD also demonstrate that neutrophils

adhere to endothelial and sickle red blood cells which contribute to the development of vaso-

occlusions. Vaso-occlusions are characteristic of the painful state during the vaso-occlusive

crisis (VOC) and account for a vast majority of SCD-related hospitalizations97,98

. Previous work

on NETs and coagulation has hinted on the pathogenic effects of NETs on SCD. Indeed, a

cohort study comparing SCD conditions found significantly higher levels of NETs in the plasma

of patients during the painful crisis relative to their steady state controls. NETs were measured

by the presence of nucleosomes and neutrophil activation marker, elastase-α1-antitrypsin. The

authors also found a positive correlation between nucleosome levels to the length of hospital

stay87

. SCD is associated with chronic hemolysis and increased heme in the plasma99

. The direct

effect of heme on NET formation was demonstrated in vitro, as neutrophils activated with heme

released DNA containing CitH3 and elastase in a ROS- and heme-dependent manner100

. In vivo,

heme injection increased NET formation in control mice, whereas hemopexin administration

decreased plasma heme in SCD mice and reduced NET formation by 41 %100

. Interestingly,

patients with SCD also show deficiencies in vitamin C, which has recently been identified as a

suppressor of NETosis in a sepsis model101,102

. However, the mechanism of vitamin C inhibition

on SCD neutrophils has yet to be examined.

1.1.3.3 NETs in Autoimmune Disorders

The cytotoxic property of NETs causing host tissue damage is undoubtedly linked to the

growing field of autoimmune diseases. Systemic lupus erythematosus (SLE) is an autoimmune

disorder that is characterized by the generation of self-antibodies against DNA, histones, and in

many cases, against neutrophil proteins103

. Recent evidence points toward the imbalance of NET

formation and NET clearance in this disease. As such, patients with elevated levels of NETs

may be more susceptible to NET-mediated tissue damage. Impaired NET degradation is

15

observed in a subset of SLE patients whose sera contains elevated levels of anti-NET

antibodies103

. It was later found that the abundance of autoantibodies limited serum DNase I

from accessing and degrading NETs. This is in contrast to normal serum, where DNase I

degrades NETs in a calcium-dependent manner. C1q deposited on NETs have also been shown

to inhibit NET degradation by directly binding to DNase I104

. These authors further found that

impaired NET degradation led to additional C1q deposition and recruitment of neutrophils,

thereby, exacerbating the conditions of SLE104,105

. The consequent of impaired NET degradation

was further correlated with the development of lupus nephritis103

. One other study showed that

low density neutrophils isolated from the blood of individuals with SLE had a greater propensity

for NET formation106

.

Small-vessel vasculitis (SVV) is inflammation of the vessels which is strongly linked to

the generation of anti-neutrophil cytoplasm autoantibodies (ANCAs)107

. ANCAs developed

against proteinase-3 or MPO are inducers of NETosis. As such, SVV-related inflammation may

be associated with NET formation108,109

. Antimicrobial peptide LL-37 (cathelicidin) deposited

onto NET-DNA is also enhanced in SVV. LL-37 binding to NET-DNA can impair degradation

and has been shown to drive autoinflammatory conditions in SLE and psoriasis by activating

plasmacytoid dendritic cells to produce large amounts of interferon (IFN)-α110

.

Psoriasis is a T-cell mediated autoimmune inflammatory disease recognized for its skin

and joint manifestations. IL-17 and IL-23 are known to be absolutely essential to pathogenic

psoriasis as drugs targeting either cytokine has beneficial outcomes111,112

. Psoriatic lesions are

highly enriched with neutrophils and mast cells113,114

. Although the precise role of neutrophils

and mast cells in psoriatic lesions was not known then, we now know that these cells release IL-

17 by extracellular trap formation. The same authors further demonstrated that IL-17 and IL-1β

can, in turn, drive ETosis in mast cells to release additional proinflammatory cytokines115

.

It is without question that NETs contribute to the exacerbation of several autoimmune

diseases. Other autoimmune diseases affected by NETs not mentioned here include rheumatoid

arthritis116,117

and Felty’s syndrome118

.

1.1.3.4 NETs in Lung Disorders

Neutrophils are highly abundant in the marginated pools of lung capillary and are far more

concentrated in pulmonary blood (about 40 to 80 times) than in systemic blood as measured by

16

neutrophil to blood ratio5,119

. This allows neutrophils to readily emigrate into the lungs in

response to pro-inflammatory signals. As the main function of NETs is to trap and kill foreign

pathogens, it is also expected that pulmonary microbes induce airway NETosis. Pseudomonas

aeruginosa (P. aeruginosa) is the main cause of pulmonary infections in cystic fibrosis (CF)

lung disease and can actively stimulate NET production in healthy and CF neutrophils120,121

.

However, NET-mediated killing is only effective with early clinical isolates of P. aeruginosa, as

bacteria from the later stages of infection develop resistance121

. The result of P. aeruginosa

growth and NET production contributes to persistent infections and mucous thickening which

altogether impairs lung function121,122

. Tenacious lung secretions in CF patients can be

effectively reduced by inhaling DNase I which destroys necrotic DNA and presumably

NETs123,124

. Details on the effects of DNase I and NET clearance will be discussed in section

1.1.4 Resolution of Neutrophils and NETs. The precise mechanism of P. aeruginosa resistance

from NET-mediated killing is currently unknown. However, it is well documented in pneumonia

that the evasion of NETs by Streptococcus pneumonia occurs via endonuclease EndA secretions

to disintegrate NETs for further spreading of the disease-causing bacteria125

. In other bacteria,

such as S. aureus, nuclease and adenosine synthase secretion assists with NET DNA break down

and conversion of cleaved NET monophosphates (2′-deoxyadenosine-3′-phosphate) to

deoxyadenosines, respectively. These conversions trigger caspase-3-mediated death of immune

cells126,127

. Similar and novel evasion strategies are reported by NETosis inducers Vibrio

chloerae128

, group A Streptococci (including M1 and M1T1)24,129–131

and Neisseria

meningitidis132

. The ability of microbes to evade NETs is an evolutionary adaptation against

host defenses as the combined effect of liberated NET proteins, proliferating bacteria, and

continual NETosis leads to severe inflammation and perhaps fatal outcomes.

Excessive neutrophil recruitment and activation are known factors of acute lung injury

(ALI). Nowadays, the expanding field of NETosis identifies NETs at the center of ALI diseases.

Acute respiratory distress syndrome (ARDS) is a severe form of ALI with symptoms of

pulmonary edema, diffuse alveolar damage and respiratory failure133

. NETs were shown to be

associated with lung damage in mice developing ARDS from an influenza virus infection. This

was directly confirmed in vitro as neutrophils from infected lungs of mice with ARDS induced

cell death to human umbilical vein endothelial cells (HUVECs)41

. NETs have also been linked

to transfusion-related acute lung injury (TRALI) in humans and in mice. These studies

17

demonstrated that NETs were found in circulation and the lungs, while the treatment with

DNases or antibodies against histones protected against TRALI134,135

. Platelets involved with the

activation of neutrophils are also responsible for inducing NET formation in TRALI134

.

For as long as immune defenses are required to maintain homeostasis, the list of

pathologies associated with the negative effects of NETs seems limitless. Therefore, it is

important that we switch the focus from NET formation to identifying key processes involved in

NET clearance.

1.1.4 Resolution of Neutrophils and NETs

While neutrophils are granulocytes packed with an arsenal of cytotoxic proteins, these cells

must be cleared efficiently from the site of inflammation to prevent excessive tissue damage.

Indeed, neutrophil turnover is highly regulated as a result of neutrophils undergoing

spontaneous apoptosis following senescence or pathogen clearance. However, neutrophil

apoptosis is affected by most inflammatory conditions. That is, cells that have phagocytosed

bacteria will accelerate the cell death program to improve resolution of neutrophil-dependent

inflammation136,137

. Gram-negative bacteria-derived products such as LPS as well as other

proinflammatory factors including GM-CSF have been shown to delay neutrophil apoptosis by

prolonging survival138,139

. As with all apoptotic cells, dying neutrophils mobilize

phosphatidylserines (“eat me” signals) to membrane surfaces which become recognized by

scavenger macrophages for efferocytosis. Neutrophil clearance is not exclusive to apoptotic

populations, as activated and aging neutrophils can signal for removal before actually dying. For

instance, surface ligands similar to those on apoptotic neutrophils are generated upon activation

of neutrophil NADPH oxidase which enhances cell removal by macrophages in vitro and in

vivo140,141

. Senescent and pre-apoptotic murine neutrophils also re-express chemokine receptor 4

(CXCR4) for clearance by bone marrow macrophages from the circulation142,143

. Clearance of

neutrophils require the recruitment of macrophages which is accomplished by mobilizing

surface “find me” signals including lysophosphatidylserine140

, nucleotides ATP and UTP144

,

thrombospondin 1145

and soluble factor IL-6R146

. Several mediators are also involved in

potentiating the phagocytic activity of macrophages. Bridging molecules, including collectins

SP-A and SP-D, C1q, deposited complement after activation and IgM, assist with cell-to-cell

contact through ligand-receptor processes. Resolution mediators, such as lipoxins, resolvins,

18

protectins and annexin-1, stimulate neutrophil phagocytosis by macrophages at inflamed

sites147–154

. Macrophages themselves may contribute to clearance by secreting Fas-ligand and

TNF-α, which also triggers cell death of bystander leukocytes155,156

. Once ingested, neutrophils

are processed in macrophage phagosomes, followed by production of anti-inflammatory

cytokines IL-10 and/or tumor growth factor (TGF)-β to suppress proinflammatory conditions

and leukocyte recruitment157,158

.

Since the discovery of NETs, a vast array of literature has dissected the mechanisms and

signalling pathways for NETosis as well as the effects of NETs in health and disease. Even so,

many of the details relating to NETs remain to be examined, especially in regards to NET

clearance. To date, the molecular mechanism involved with NET clearance has only been

documented by one study159

. Farrera and colleagues demonstrated that NETs were only partially

degraded by DNase I at a physiological concentration (20 ng/ml) or by 10-20% healthy serum

(containing endogenous DNase I). The authors concluded that an additional mechanism, such as

phagocytosis, is required for the complete removal of NETs. Indeed, using human monocyte-

derived macrophages (HMDMs), they showed that NET removal is an active endocytic process

that is enhanced by DNase I digestion (non-physiological 5 µg/ml) or by NETs opsonised with

complement factor C1q. Although it is known that mammalian DNA stimulates type 1 IFN

production, macrophages that have phagocytosed NETs did not display such a response,

thereby, promoting an anti-inflammatory response similar to the clearance of apoptotic

cells147,159,160

.

While the above study showed that healthy serum DNase I partially degrades NETs, a

few labs have reported different findings using various and similar types of sera103,161,162

In

vitro, human-derived NETs isolated from PMA-activated neutrophils were fully degraded with

56 °C heat-inactivated fetal calf serum (FCS), whereas 70 °C heat-inactivated FCS had no

effect. This demonstrated that thermo-stable nucleases are present in FCS161

. In another study

looking at NET degradation, 10 % healthy serum completely degraded NETs after six hours,

whereas sera from a subset of SLE patients degraded less. They found that serum DNase I

required calcium ions for activity and was inhibitable by globular actin (G-actin) or blocked by

anti-NET antibodies103

. Furthermore, C1q deposition, as a result of complement activation by

non-degraded NETs, was shown to inhibit DNase I and restrict nuclease access to NETs (more

details on NETs in SLE found in 1.1.3.3 NETs in Autoimmune Disorders)104

. Contrary to the

19

findings outlined by Farrera and colleagues159

, these experiments show that serum DNases are

effective at degrading NETs and that C1q may not enhance NET clearance in individuals with

SLE.

Despite the limited knowledge on the clearance NETs, exogenous DNases are commonly

used in experiments to destroy NETs in vitro and in vivo. On the other hand, NET clearance

involving natural nucleases and phagocytic-promoting molecules have largely been unexplored.

Surfactant protein D (SP-D) is a collectin in the lung that enhances phagocytic processes in

alveolar macrophages. Our lab identified a novel interaction between SP-D and NETs in vitro

and in vivo25

. However, it has not been established whether SP-D enhances NET clearance in

the lung.

1.2 Clearance in the Lung

Inflammation in the airways occurs as a protective response to infectious pathogens and non-

infectious injury, representing the hallmark of most lung-associated diseases including the

common asthma, cystic fibrosis (CF), tuberculosis, and chronic obstructive pulmonary disease

(COPD). Adult humans inhale about 103 litres of air per day during resting conditions, with

values reaching as high as up to 203 during exercise

163,164. While oxygen and nitrogen make up

most of the air that we breathe in, we also inhale small amounts of other gases, bacteria, viruses,

oxidants, pollutants, and allergens. As such, the continuous flux of foreign particles into the

airways poses a challenge for the lungs to maintain sterility. Fortunately, lungs have evolved

innate defense mechanisms to constantly protect the airways by efficiently clearing foreign

matter via physical, chemical and biological means.

1.2.1 Mucociliary Clearance in the Airways

Mechanical clearance in the proximal airway is considered the primary mode of protection

which relies on the action of a mucociliary apparatus. The mucociliary apparatus is composed of

the ciliary escalator and mucus secreted from globlet cells and submucosal glands. There are

three main functions of the mucociliary apparatus: to serve as a physical barrier to trap

microparticles in the airway surface liquid covering the respiratory epithelium; to act as a

chemical shield by screening for inhaled pathogens and protecting the lung with epithelial-

20

secreted antioxidants, salt-sensitive defensins, and immunomodulatory proteins165–170

; and to

provide a biological barrier to prevent microorganisms and luminal inflammatory cells from

adhering to and migrating through the airway epithelium164

.

1.2.1.1 Airway Surface Liquid

The thin layer of watery solution (about 98 %) separating the airway epithelium from the gas of

the lumen is known as the airway surface liquid (ASL)171

. ASL assists with clearance of trapped

particles by working in concert with the beating activity of cilia to transport mucus from the

lungs to the esophagus where it gets expectorated or swallowed. The ASL consists of the “sol”

or periciliary liquid layer (PCL) that surrounds ciliated cells and the mucus gel layer which rests

on top of the PCL172

. This bifunctional property of the ASL enhances mucociliary clearance.

Abnormalities in the ASL are associated with lung diseases such as COPD and CF in which

both are characterized with dehydrated airway mucous and elevated levels of mucin that cause

mucous thickening173,174

.

The viscous-mucus gel layer represents about 1 % of the total mucin in ASL and is

comprised of high-molecular weight and heavily glycosylated macromolecules which behaves

as an entangled network of polymers to trap microparticles. Airway mucins (MUC2, MUC5AC

and MUC5B) contained in the gel layer are able to recognize and bind to a diverse group of

molecules through their carbohydrate side chains, thus, enhancing the clearance potential of

ASL175,176

. The mucus layer also serves as a liquid reservoir for ASL volume homeostasis by

accepting or donating liquid to or from the PCL. In doing so, the ionic composition and liquid

height of the PCL remains constant while the mucus layer shrinks or swells in response to

changes to airway osmolalities177

.

Unlike the viscous mucus gel layer, the PCL is more watery, stains weakly for mucins,

and is less electron-opaque178–180

. The low viscosity of PCL allows cilia to beat rapidly so that

ASL transport is constantly moving towards the mouth. Also, PCL height normally matches the

length of epithelial cilia to coordinate proper movement of the outer mucus gel layer. In the case

that the PCL thickness extends beyond the length of cilia, transport by ciliary beat might be

ineffective since the mucus gel layer would be floating too far from the actual motion.

Movement of the PCL-mucus gel layer is further aided by the presence of membrane-spanning

21

mucins in the PCL and large mucopolysaccharides that are tethered to cilia, microvilli and

epithelial surfaces172,181

. Additionally, mucopolysaccharides form a ‘brush’ between the PCL

and mucus layer to prevent compression of the PCL by the thick mucus172

. This gel-on-brush

model replaces the traditional view of the gel-on-liquid model which failed to explain how

mucus remained as its own distinct layer172,182,183

.

Clearance of mucus from the lungs can also be achieved by coughing. By definition,

cough clearance is independent of ciliary activity, but cough efficiency relies heavily on the

properties of ASL. The height and volume of ASL directly correlates to coughing efficiency,

whereas an inverse relationship is observed with ASL viscosity184,185

.

Surface liquids have been shown to extend all the way to the alveoli that separate the

surfactant layer from the alveolar epithelial cells169,186,187

. However, this ‘alveolar subphase’

does not contain a mucus sheet and its functions are much less studied. Contrary to the clearance

of mucus in the upper airways, microparticles (less than 5 µm) trapped in this distal region are

removed by resident phagocytes. The role of phagocytes in clearance will be discussed below in

section 1.2.2 Clearance by Alveolar Macrophages.

1.2.1.2 Antimicrobial Factors in the ASL

Aside from mucociliary transport, endogenous enzymes localized in the ASL protect the

airways from infections or impaired clearance. Lysozymes and lactoferrins are the two most

abundant ASL peptides with the ability to directly kill microorganisms188,189

. Among these are

other antimicrobial factors including LL-37, calgranulins, defensins and secretory leukocyte

protease inhibitor165,166,170,189–191

. These factors are secreted by airway epithelial cells,

submucosal glands, and in some cases, leukocytes. Molecules with immunomodulatory

functions, such as complements, immunoglobulins and fibronectins, are also found in the ASL

to opsonise microbes and mediate phagocytosis192–194

.

1.2.1.3 Nucleases

Of the many characterized nucleases, DNases are the most widely studied enzyme that is

categorized into three specific groups: Mg2+

/Ca2+

-dependent nucleases, Mg2+

-dependent

nucleases and cation-independent nucleases195

. DNase I is the best known Mg2+

/Ca2+

nuclease

22

generally found in the pancreas and parotid glands196,197

. This enzyme is strongly activated by

Mg2+

, whose function is maximized by the paring of Mg2+

with Ca2+ 198,199

. Titrating for

secondary phosphate groups showed that the activity of DNase I occurs in two phases198

. The

first phase, referred to as the ‘rapid phase’, is activated most by Mg2+

, while the second phase or

‘slow’ phase is activated by Ca2+ 198

. One other study showed that binding of bovine pancreatic

DNase I to Mg2+

and Ca2+

at sites III and IVa/b and sites I and II, respectively, modifies the

electrostatic fit of DNA to the hydrolytic pocket. Namely, Ca2+

at site II stabilizes the functional

structure of the nuclease while Mg2+

at site IVa/b coordinates two histidine residues involved

with DNA hydrolysis199

. These binding sites are remarkably conserved across diverse proteins

belonging to the DNase I family199

. The optimal pH for DNase I ranges between pH 7 to 8200,201

.

Other DNase I-like nucleases such as DNase ɣ and DNase X also require Mg2+

and Ca2+

for

activity and function optimally at neutral pH202–204

. Cu2+

, Ni2+

and Zn2+

inhibit activity of DNase

I, DNase ɣ and DNase X198,202,203

. NaCl, too, is known to inhibit DNase I205

. While

physiological concentrations of NaCl inhibit DNase I, it is likely that the balance of nuclease

activity is regulated by the interplay of NaCl and activating divalent cations during basal

conditions205,206

.

DNases may alternatively function in the presence of Mg2+

alone. Belonging to this group

are caspase-activated DNases (CADs). CADs are activated by caspase 3, DNA fragmentation

factor or other serine proteases during apoptosis and are responsible for generating multiples of

180 bp fragments (i.e. 180, 360, 540 bp and etc.) by cleaving between the nucleosomes of

DNA207–210

. As such, separation of these fragments by gel electrophoresis shows a specific DNA

laddering effect that is characteristic of CAD activity and apoptosis211

. CAD is able to bind to

DNA without Mg2+ 212,213

but requires Mg2+

for activity at a pH optimum of 7.5207

. The role of

CADs during apoptosis defines an intracellular function for the protein and is, therefore, less

likely to be found on extracellular surfaces such as the ASL. DNase I has recently been reported

to also mediate internucleosomal cleavage of chromosomal DNA in dying cells214–216

. However,

as DNase I is a secretory enzyme, extracellular DNase I likely takes part in other processes

involved with mediating tissue homeostasis and clearance of DNA.

DNase II, also known as DNase IIα, is a cation-independent nuclease found most in the

lysosome of engulfing cells217,218

. Lysosomes are intracellular acidic compartments which

normally fuse with cargo that has been phagocytosed by the cell. This acidic environment is

23

most favourable for DNase II activity, as the optimal pH for the enzyme ranges from pH 4.5 to

5197,219

. DNase II is also found in other secretory and body fluids, such as saliva, blood, urine

and testicular liquid, although at much lower levels compared to cells220,221

. Among 20 human

cell lines, DNase II activity was shown to be highest in epithelial cells and lowest in

hematopoietic cells222

. One other group also showed that DNase II is ubiquitously expressed in

human tissues including the lung223

. However, whether DNase II exists specifically in alveolar

epithelial cells has not been clearly established. Interestingly, a new gene of a DNase II-like

nuclease was detected in salivary glands as well as minimally in the trachea and pulmonary

epithelial cells223

. This protein, now denominated as DNase IIβ, shares 37 % of identity and 56

% of conservativeness with human DNase II223

. While the limited expression pattern suggests

that DNase IIβ functions primarily as a secretory enzyme, its existence or role in the ASL has

not been investigated.

While serum DNase I originates partly from the liver224

, the origin of nuclease secretion

in the airways is currently unknown. One potential source could be from AMs as their main

roles are to maintain lung tissue homeostasis by regulating immune responses and clearing up

debris. AMs contain lysosomal DNase II225

. As DNase II requires an acidic environment to

function, this protein would rarely be localized to the extracellular space where it could be

rendered inactive at normal airway pH values. On the other hand, DNase I contains a 22 amino

acid hydrophobic signal peptide designated for secretion. Although DNase I is found in the ER,

Golgi apparatus, and secretory granules of rat pituitary endocrine cells226

, direct evidence of

DNase I localized to the same compartments in alveolar macrophages has not been examined.

Rather, another DNase I-like enzyme, known as DNase ɣ, was reported to be found in

macrophage populations including Kupffer cells, alveolar macrophages and macrophages of the

brain and kidney227

. Whether DNase ɣ remains intracellular or extracellular has thus far been

controversial. Some authors have described DNase ɣ to be a non-secretory protein found near

the perinuclear space203

, whereas others have found active secretion of the protein into the

extracellular space227

. Very little is known about DNase ɣ. However, this protein may confer

physiological advantages to systems requiring active DNA hydrolysis in the presence of

accumulated actins known most for inhibiting DNase I. Thus far, it has not been established

whether DNase ɣ exists in the ASL. DNase IIβ has recently been discovered in secretory fluids,

lung tissues and in a pulmonary epithelial cell line223

. The role of non-activated or activated

24

pulmonary epithelial cells in secreting DNase IIβ to the ASL is another candidate source for

nuclease activity. During inflammation, serum leakage as well as recruited neutrophils might

also contribute to the pool of airway nucleases. While DNase II-like nucleases have been

identified in neutrophils228

, the release of such an enzyme to the ASL is not known. As NETs

have now been shown to be major contributors to pulmonary diseases, additional studies on

airway nucleases, such as on their properties, localization or effect on NETs, could improve our

current understanding of NET clearance in the lungs.

1.2.1.3.1 Nucleases in the ASL

In the case that an inflammatory response is elicited, dying cells and the release of DNA

contributes to mucous thickening and airway obstruction. This is a prevalent symptom of most

pulmonary ailments and persists to be a chronic problem in severe diseases such as asthma and

CF. Under normal conditions, endogenous nucleases are presumably responsible for the

clearance of DNA within the airways. The presence of nucleases in the airways was indirectly

verified by a transfection study showing that plasmid DNA was significantly degraded by

murine and macaque lung lavage fluids. The authors further demonstrated that plasmid

clearance by pulmonary nucleases could be delayed by co-administration of free plasmid with a

nuclease inhibitor, aurintricarboxylic acid (ATA)229,230

. One other study characterised that

DNases were present in the bronchoalveolar lavage fluid of healthy and CF patients and that its

activity was dependent on Mg2+

as the cofactor231

. This was further confirmed in sputum

samples of CF patients who did not respond to recombinant human DNase I (rhDNase I)

therapy. These “non-responder” were found to have lower levels of Mg2+

in the sputum

compared to those who benefited from rhDNase I. By supplementing Mg2+

ions in vitro,

rhDNase I was reactivated in DNA solutions as well as in the sputum of non-responders. While

oral intake of magnesium gluconate restored ion levels to physiological concentrations in non-

responders, the in vivo relationship between Mg2+

supplementation and rhDNase I activity was

not examined232

. These studies demonstrate the importance of nucleases in the airways as an

additional method of clearance for DNA released from dying cells. However, the action of

airway nucleases on other DNA structures, such as NETs, has not been investigated. Moreover,

the source of DNase secretion in the airways has not been clearly established.

25

1.2.2 Clearance by Alveolar Macrophages

Resident phagocytes of the alveoli rest beneath the surfactant layer and are primarily responsible

for the uptake and clearance of particles (< 5 µm) that bypass the barriers of the upper

respiratory tract. These surface phagocytes, also known as alveolar macrophages (AM), are

exposed to the alveolar lumen and are, therefore, the first line of innate immune defense. To

prevent damage to type I and II alveolar cells from harmful antigens, AMs are normally kept in

a quiescent state with limited production of proinflammatory cytokines. Cytokine production as

well as phagocytic activity is suppressed by TGF-β, an autocrine signal released from AMs

attached to alveolar epithelial cells233

. Surfactant protein A (SP-A) and D (SP-D) are also known

to tonically suppress macrophage function in the lungs via interaction with SIRPα or

calreticulin/CD91234,235

. AMs remain inactive and participate in high levels of spontaneous

(macro-) pinocytosis which allows the phagocyte to ‘sample’ the extracellular milieu for

immune-relevant factors and respond accordingly236,237

. In the case that infection occurs, TLRs

or other non-pattern recognition receptors on AMs are triggered to activate the cell for

phagocytosis.

Largely owing to the nature of the lung environment, AMs are a unique set of

macrophages that are, in many aspects, similar to the phenotype of dendritic cells (DCs).

Contrary to other macrophage populations which express high levels of CD11b, AMs express

high levels of CD11c, like DCs237

. This was further confirmed by a series of adoptive transfer

experiments in which bone marrow macrophages developed CD11blow

/CD11chigh

phenotypes in

the airways and CD11bhigh

/CD11clow

phenotypes in the peritoneal cavity. Expression of CD11c

was later shown by the same authors to be modulated by high concentrations of airway GM-

CSF and SP-D. AMs have also been described as poor phagocytes of early (large) apoptotic

cells compared to peritoneal macrophages238–240

. Our lab has shown that rather than ingesting

large particles, AMs are more efficient at phagocytosing late (small) apoptotic cells, especially

in the presence of apoptotic binding agents, IgM and SP-D153

. For the reason that AMs are

constantly burdened by microparticle invasion, it is likely that these cells prefer to clear small

particles over larger matter. It is also well documented that the phagocytic potential of AMs is

enhanced by surfactant proteins which act as opsonins to foster the clearance of cellular debris,

microbes, and extracellular DNA151,241–246

. The role of AMs or surfactant proteins in clearing

other DNA structures, such as NETs, has not been investigated to date. However, a recent

26

finding from our lab demonstrating that SP-D binds strongly to NETs enlists a possible role for

surfactant proteins in NET clearance in the airways25

.

1.2.2.1 Surfactant Protein D and Phagocytosis

Collectins, or collagen-containing C-type lectins, are innate immune proteins responsible for

tightly regulating inflammatory responses in the lung. SP-A and SP-D are collectins with pattern

recognition abilities and comprise >90 % (w/w) of the surfactant layer247

. Distinct from SP-B

and SP-C, which reduce surface tension and prevent collapse of the lung, SP-A and SP-D are

considered as antibodies of the immune system due to their resemblance in structure and

function to antibodies. Collectins are found most in mucosal surfaces such as in the airways,

saliva, tear fluid, and intestines248–251

. In the airways, collectins are secreted by the nasal

epithelium248

, upper airway non-ciliated or Clara cells252

and alveolar type II epithelial cells253

.

The basic monomer of a collectin consists of an N-terminal cysteine-rich region, a long fibrillar

collagen-like region, a trimerizing alpha-helical coiled ‘neck’ and a carbohydrate recognition

domain (CRD). Three of these monomers pre-assemble into trimeric subunits before forming

higher order oligomeric structures through disulfide-stabilized, non-covalent bonds. Native SP-

D is easily recognized as a structure resembling an “X” (4 trimers; tetramer) or asterisk (>10

trimers)254

.

The role of SP-D in regulating pulmonary innate immunity is rather complex as altered

levels and abnormal folding of the protein has been associated with many disorders. Mouse

models lacking SP-D develop symptoms that resemble features of pulmonary alveolar

proteinosis (PAP) and emphysema by 3 months after birth255–257

. On the contrary, humans with

PAP have SP-D expression levels increased to as high as 20-fold258

. Loss of AM inhibition in

SP-D-deficient mice results in elevated levels of superoxide production, increased NF-ĸB

activity and matrix metalloproteinase (MMP) production, all of which contribute to airspace

remodeling and destruction of the alveoli tissue256,257

. Furthermore, polymorphisms in the SP-D

allele such as Met11Met (homozygous for methionine at amino acid 11) or Thr11Thr

(homozygous for threonine at amino acid 11) are associated with respiratory syncytial virus

brochiolitis in infants and the reduced ability to bind to bacteria and viruses, respectively259–261

.

27

Whether SP-D levels increase or decrease depends on the specific disease and can, therefore, be

monitored as a potential biomarker for disease progression.

1.2.2.1.1 SP-D as an Opsonin

The ability for SP-D to opsonise several pathogens owes largely to its protruding CRD domains

which bind to sugar moieties of bacteria, viruses, yeast and fungi25,262–264

. SP-D reduces the

ability of pathogens to colonise by increasing bacterial cell wall permeability, exhibiting direct

killing effects and/or microagglutination. Additional interactions are mediated through the

collagen domain which forms ionic interactions with negatively charged molecules such as

phospholipids of the surfactant layer, LPS of Gram-negative bacteria and phosphates of

DNA265,266

. Furthermore, SP-D interactions with early and late apoptotic cells are dependent on

lectin and calcium-mediated binding, respectively267

. Multiple labs, including ours, have now

demonstrated that these SP-D interactions promote the clearance of pathogens, DNA and

apoptotic cells by alveolar macrophages151,241,243,246

.

AM receptors involved with SP-D-mediated clearance are not clearly established.

Currently, one existing model proposes that the phagocytosis of dying cells is mediated by a

bridging molecule, calreticulin, which complexes with endocytic receptor CD91 on AMs151

.

Calreticulin is usually present in the endoplasmic reticulum, but is also found on surfaces of

AMs268

. Although the mechanism as to how calreticulin mobilizes to the surface is unknown,

the authors claim that specific binding of calreticulin to SP-D enhances the uptake of cell debris

through CD91 signalling151

. This claim was later challenged by another study showing that no

such interaction between calreticulin and SP-D exists269

. SP-D also binds to glycoprotein-340, a

purified protein from bronchoalveolar lavages belonging to the scavenger receptor family on

AMs270

. However, the functional relevance of this interaction is not understood. We have

recently discovered a strong interaction between SP-D and alpha-2-macroglobulin (A2M)271

.

A2M is a serum protease inhibitor known to infiltrate the airways during inflammation272,273

.

A2M is also a ligand to the CD91 endocytic receptor (also known as A2M receptor or low

density lipoprotein receptor related protein 1 (LRP1)) present on AMs151,274–276

. A possible

mechanism for NET clearance could function through the SP-D-A2M-CD91 pathway on AMs.

However, this pathway has not been established. Therefore, additional studies are required to

28

confirm existing models or identify new receptors and/or adaptor molecules involved in SP-D-

mediated clearance by AMs.

1.2.2.1.2 SP-D and NETs

The timely removal of potentially harmful substances is essential in protecting the lungs from an

autoimmune attack. For example, our lab showed that the effect of SP-D in enhancing the

clearance of DNA by AMs reduces the generation of autoantibodies against DNA242

. The

removal of extracellular DNA by SP-D and AMs were originally thought of as a sweep up of

nucleic acids originating from necrotic cells. It is possible that the pool of extracellular DNA

also contains other DNA structures such as NETs. Indeed, NETs are formed by neutrophils in

inflamed lungs of mice following the intranasal administration of LPS25

. The nature of SP-D

binding to DNA also meant that SP-D could bind to NETs. Using a combination of pull-down

assays and measurements from the bronchoalveolar lavage, our lab was the first to determine a

novel interaction between SP-D and NETs. This dual complex also exhibited greater

microagglutination of P. aeruginosa than each of the components alone, thus, putting SP-D at

the forefront of innate immune defense25

. While it is known that SP-D enhances the clearance of

genomic DNA242,243

, it is possible that SP-D binding to NETs also promotes the uptake of NETs

by AMs. However, this model of NET clearance has not been examined.

1.3 Rationale and Hypothesis

Recently, the emerging field of NET formation within the lungs has been documented as a novel

mechanism of host defense by trapping and killing invading pathogens. Although beneficial at

first, persistent or aberrant NET formation as well as impaired NET clearance has been

attributed to a variety of lung disorders120,121,134,135

. Many efforts have focused on identifying

pathways involved in NETosis as well as elucidating the functions of proteins localized within

these DNA structures. With the present knowledge on NETs, numerous interventions have

targeted factors that block NET formation or used enzymes to degrade and neutralize the DNA

and cytotoxic proteins contained within it. Although NETs are commonly eliminated in vitro

and in vivo by the administration of exogenous DNases, very few studies showing direct

breakdown by endogenous DNases have been reported. Endogenous DNases present in the

29

blood serum are responsible for NET degradation, while excessive NETs are found in SLE

patients with reduced DNase activity104,162

. Endogenous nucleases are also found in the ASL to

interfere with gene therapy by hydrolyzing plasmids229,230

. The action of ASL nucleases on

NETs is virtually unknown and should be explored as NET degradation in the lungs has not

been documented. Furthermore, it is commonly assumed that lung phagocytes mediate NET

clearance, although this too has not been directly proven.

SP-D is an innate immune collectin of the lung and contains a unique structure that allows

the protein to bind to several molecules. The carbohydrate recognition domain is known to

opsonise dying cells and bacteria for the clearance by alveolar macrophages153,254,277,278,279,280

.

The collagen domain is also involved in binding to and enhancing the clearance of DNA by

alveolar macrophages which reduces the generation of anti-DNA antibodies242,243,266

. Recently,

our lab characterized a novel interaction between SP-D and NETs25

. However, the role of SP-D

in promoting NET clearance has not been examined.

Based on the lack of knowledge as to how NETs are removed from the lungs, I

hypothesize that NETs are degraded by nucleases present in the airway and are removed by

alveolar macrophages. This clearance is enhanced by the NET-interacting molecule, surfactant

protein D (SP-D). In order to test this hypothesis, I have two aims:

Aim 1: To determine whether NETs are fragmented by the presence of nucleases in the airways.

Aim 2: To determine whether SP-D enhances the clearance of NETs by alveolar macrophages.

30

Chapter 2

Airway nucleases fragment NETs in a calcium-dependent manner and work optimally at

narrow pH ranges

Lily Yip1,2

, Nades Palaniyar1,2,3

1Lung Innate Immunity Research Laboratory, Program in Physiology & Experimental Medicine

SickKids Research Institute

2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto,

Ontario, Canada

3Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada

Corresponding author: Nades Palaniyar, PhD, Lung Innate Immunity Research Laboratory, The

Hospital For Sick Children, 555 University Ave., Toronto, ON, Canada, M5G 1X8. Phone: 1-

416-813-7654 ext 302328. Fax: 1-416-813-5771. e-mail: nades.palaniyar@sickkids.ca

The work presented in this chapter is presented as a manuscript for future submission.

31

2.1 Abstract

A novel defense by neutrophils has been described as the formation of neutrophil extracellular

traps (NETs). These NETs are composed of nuclear DNA decorated with antimicrobial peptides

which assist with the trapping and localized killing of pathogens. Although seemingly beneficial

at first, excessive NETs or the inability to clear NETs has been shown to contribute to the

pathogenesis of several diseases. While treatment with DNases ex vivo can lead to sufficient

NET degradation, the characterization of endogenous nucleases in the airways is lacking. Also,

whether airway nucleases directly cleave NETs has not been shown. Our study shows that

nucleases are present in murine BALF of healthy and inflamed lungs which can degrade

genomic DNA (gDNA) in a dose-dependent and time-dependent manner. The degradation of

gDNA occurs in a Mg2+

/Ca2+

-dependent manner, which can be inhibited by divalent cation

chelators EGTA and EDTA. The presence of CitH3 and NET-derived DNA-protein complexes

confirms that BALF from LPS-instilled mice contain NETs. These NETs are fragmented by

BALF nucleases in a Ca2+

-dependent manner. This is different from the degradation of gDNA,

which occurs in a Mg2+

/Ca2+

-dependent manner. We further found that optimal activity of

BALF nucleases occurs at near neutral pH (6.8-7.0) and at acidic pH (6.6 or less). The presence

of both DNase I-like and DNase-II-like properties suggests that there are two types of nuclease

activities present in the airways that assist with DNA hydrolysis and eventual NET clearance.

2.2 Introduction

Neutrophils provide the first line of defense during inflammation and infection by typically

phagocytosing and killing pathogens intracellularly12

. Over the past decade however,

neutrophils have been described to control infection extracellularly by the formation of NETs18

.

These NETs are composed of nuclear DNA decorated with antimicrobial histones (CitH3) and

peptides (elastase and myeloperoxidase) which assist with the trapping and localized killing of

pathogens18,55,63

. Although NET formation is beneficial at first, excessive NETs or the inability

to clear NETs has been associated with the pathogenesis of several diseases including many

lung disorders93,121,133,134

. NETs exhibit direct cytotoxic effects on lung epithelial cells as well as

endothelial cells73

. In CF disease, inefficient clearance of necrotic DNA and NETs increases

mucus thickening and contributes to airway obstruction281

. Recombinant human DNase

32

(rhDNase) is commonly administered to CF patients to hydrolyze DNA and has been shown to

improve lung function123

. However, whether these nucleases specifically degrade NETs is

unknown. NETs have also been linked to transfusion-related acute lung injury (TRALI) as

demonstrated in both human and mouse models. These studies showed that NETs were found in

circulation and in the lungs, and that the treatment with DNases or antibodies against histones

protected the lungs from developing TRALI134,135

.

Although the use of exogenous DNases for degrading NETs is convenient in experimental

models, the role of pulmonary nucleases in NET clearance is unknown. Nucleases do exist in

the ASL and have been shown to cleave plasmid DNA in a magnesium-dependent manner.

Alternatively, intranasal administration of both plasmids and a DNase inhibitor, ATA, protected

degradation of plasmids by pulmonary nucleases229,230

. In this study, I sought to determine

whether NETs can be degraded by airway nucleases and whether cofactors and/or other

conditions would affect the activity of these nucleases.

2.3 Materials & Methods

Reagents

All reagents were purchased from Sigma-Aldrich unless otherwise stated.

Mice and bronchoalveolar lavage

Mouse experiments were approved by the Animal Care Committee at The Hospital for Sick

Children in accordance with the Canadian Council on Animal Care guidelines. For the analysis

of pulmonary NETs in vivo, BALB/c mice aged 4 - 6 weeks were sedated using oxygen-carried

isoflurane and 1 µg LPS (E. Coli O111:B4) in 50 µl PBS or a PBS control was intranasally

instilled for up to 1 day as described previously25

. Mice were sacrificed with 0.05 ml Euthanyl

(Bimeda-MTC, ON, CA) prior to the bronchoalveolar lavage (BAL). BAL was performed with

1 ml of chilled calcium- and magnesium-free Hanks Balanced Salt Solution (HBSS)

(Invitrogen) three times to a total of 3 ml. BAL fluid (BALF) was centrifuged at 400 g for 10

min to pellet the cells. The cell-free supernatant was analysed for DNA using a Quant-iT™

PicoGreen dsDNA reagent kit (Invitrogen). Red blood cells (RBCs) in the cell pellet were lysed

with a hypotonic saline solution (0.2 % (w/v) NaCl) for 30 seconds, followed by the addition of

an equal volume of buffered hypertonic saline solution (1.6 % (w/v) NaCl, 20 mM HEPES, pH

33

7.2) to achieve an isotonic equilibrium. The resulting RBC-free cell pellet was resuspended in

calcium- and magnesium-free HBSS for cell counting with a haemocytometer. Cytospin

preparations were made and further stained with Hemacolor™ histology staining kit (EMD

Chemicals, Gibbstown, NJ) for cell differential analysis. Histology images were taken with a

light microscope and images were randomly chosen for determining percent populations of each

cell type. A minimum of 100 cells were counted for each condition.

Western Blot

BALF isolated from naïve, PBS- and LPS-instilled mice will be denoted as “naïve BALF”,

“PBS-BALF” and “LPS-BALF”, respectively. Equal volume of BALF samples were prepared

with 5 loading dye (250 mM Tris-HCL, pH 6.8, 0.1 % (w/v) SDS, 0.5 % (v/v) glycerol, 0.05

% (w/v) bromophenol blue, 10 mM DTT) and incubated at 95 °C for 10 min. An equal volume

of each sample was separated in 12 % (w/v) polyacrylamide gels and transferred to

nitrocellulose membranes. The transferred membranes were blocked with 5 % (w/v) non-fat

powdered milk in PBS containing 0.05 % (v/v) Tween-20 (PBST) for 1 h, followed by three 10-

min washes with PBST. Primary antibodies were diluted in 1 % (w/v) non-fat powdered milk in

PBST and incubated for either 1 h shaking at room temperature or overnight shaking at 4 °C.

Primary antibodies used included rabbit polyclonal anti-histone H3 (citrulline 2 + 8 + 17)

(ab5107; Abcam, ON, CA) at 1:2000 dilution, rabbit polyclonal anti-SP-D (purified from

serum) at 1:10000 and rabbit polyclonal anti-SP-A (purified from serum) at 1:10000.

Membranes were washed five times, 5 min. each, and incubated with secondary goat anti-rabbit

conjugated to horseradish peroxidise (BD, ON, CA) at 1:10000 dilution in 1 % (w/v) non-fat

powdered milk in PBST for 1 h. After final washing for four times, 5 min. each, membranes

were coated with enhanced chemiluminescence substrate (PerkinElmer, ON, CA) and exposed

to radiographic films. Films were scanned with a Epson perfection 610 photo scanner.

Densitometry analysis was performed with ImageJ software (version 1.45s; NIH, Maryland,

US).

Gel electrophoresis of BAL fluid NETs

PBS-BALF and LPS-BALF (see notation in “Western Blot”) were incubated with Proteinase K

(PK; 200 µg/ml), MgCl2 (5 mM) and CaCl2 (5 mM) or without treatment in an equal volume of

34

DEPC-treated H2O at 37 °C for 30 min. PK-treated BALF was then supplemented with DNase I

(50 µg/ml) or RNase (250 µg/ml) and incubated at 37 °C for an additional hour. Twenty µl

samples were separated in a 1.5 % (w/v) agarose gel at 115 V for 25 min. and stained with

SYBR Gold (Invitrogen) for 35 min in TAE (40 mM Tris-Acetate, 1 mM EDTA). Gel images

were taken with AlphaImager HP.

Human polymorphonuclear neutrophils

Under the approval of the ethics board at The Hospital for Sick Children, a signed informed

consent was obtained from each healthy blood donor. Peripheral blood was drawn into K2

EDTA tubes (BD, Franklin Lakes, NJ), and neutrophils were isolated over a PolymorphPrep™

gradient (Axis-Shield, PoC, Oslo, NO) according to the manufacturer’s protocol with slight

modifications. Specifically, RBC) were lysed with a hypotonic saline solution (0.2 % (w/v)

NaCl) for 30 seconds, followed by the addition of an equal volume of buffered hypertonic saline

solution (1.6 % (w/v) NaCl, 20 mM HEPES, pH 7.2) to achieve an isotonic equilibrium.

Neutrophils were resuspended in RPMI 1640 (Invitrogen) supplemented with 10 mM HEPES

(pH 7.2) and total cell counts were determined by a haemocytometer. For gDNA isolation,

neutrophils were pelleted and kept at - 80 °C until needed for DNA extraction.

PMN genomic DNA isolation

Genomic DNA (gDNA) was isolated from neutrophils using the GenElute™ mammalian

genomic DNA miniprep kit (Sigma-Aldrich) according to the manufacturer’s protocol.

Quantification of gDNA was done by a Quant-iT™ PicoGreen dsDNA reagent kit (Invitrogen)

and the quality of the isolation was analyzed in a 1.5 % (w/v) agarose gel stained with SYBR

Gold as did before.

Characterization of nuclease activity in the fluid of lung lavages

To determine whether nucleases are present in the BALF of naive, PBS, and LPS-instilled mice,

gDNA (0.5 µg) isolated from human neutrophils were incubated with increasing BALF

concentrations (7.5 %, 15 %, and 30 % (v/v)) to a total reaction volume of 80 µl. gDNA-BALF

reactions were supplemented with MgCl2 (5 mM) and CaCl2 (5 mM) to activate any nucleases

requiring cations and incubated at 37 °C for 1 h and 3 h. BALF at 15 % (v/v) was efficient to

35

cause gDNA (0.5 µg) degradation and was used throughout the rest of the studies unless

otherwise stated.

The effect of individual cations (Mg2+

versus Ca2+

) on nuclease activity was also

assessed. Namely, gDNA-BALF was incubated at 37 °C for 3 h alone or with MgCl2 (2 mM),

CaCl2 (5 mM), EGTA (20 mM, pH 8.0) and/or EDTA (20 mM, pH 8.0). Naïve-, PBS, and LPS-

BALF conditions were all tested.

NETs are present in the BALF of LPS-instilled mice. To determine whether NETs become

degraded by endogenous nucleases contained within BALF, different combinations of MgCl2 (2

mM), CaCl2 (5 mM), EGTA (20 mM, pH 8.0), and/or EDTA (20 mM, pH 8.0) were added to

LPS-BALF and incubated for 3 h at 37 °C. LPS-BALF alone was included as a negative control.

To analyze the effect of pH on nuclease activity, buffers with different pH ranges were

added to gDNA-BALF reactions. BALF from both naïve, PBS- or LPS-instilled BALF were

tested. The pH of HEPES, PIPES, MOPS, or MES buffers were all adjusted with HCl or NaOH

in ddH2O. gDNA-BALF samples contained either 50 mM HEPES, PIPES, MOPS, or MES

alone or with cations (2 mM MgCl2 and/or 5 mM CaCl2) and/or chelators (20 mM EGTA and/or

20 mM EDTA). Samples were then incubated at 37 °C for 3 h.

Equally loaded samples (20 µl) from all of the above reactions were analyzed in a 1.5 %

(w/v) agarose gel, stained and imaged as did before (see “Gel electrophoresis of BAL fluid

NETs”).

Agarose gel signal intensity analysis

ImageJ software (version 1.45s) was used to assess the signal intensities of each condition in the

agarose gels. Measurements were taken from the bottom of the wells to the lowest DNA marker

(0.25 kb). Arbitrary values are plotted against molecular weight (kb) to show the distribution of

large, moderate and small DNA fragments.

2.4 Results

Nucleases are present in the airways of naïve and PBS-instilled mice

To determine whether nucleases are found in the airways of BALB/c mice under basal, non-

inflammatory conditions, the following experiment assesses gDNA degradation by naïve and

PBS-BALF DNases. Cations were added to the reactions to account for the presence of both

36

cation-dependent and cation-independent nucleases. Cell-free supernatant of lavage samples

from naïve or PBS-instilled mice were incubated in vitro with intact gDNA and cations (Mg2+

and Ca2+

) for 1 h and 3 h at 37 °C. Electrophoretic analysis of the reactions shows that gDNA is

degraded in a dose-dependent and time-dependent manner by BALF nucleases of naïve (Fig.

2.1A) and PBS-instilled mice (Fig. 2.1B). The controls, gDNA alone and gDNA plus Mg2+

and

Ca2+

, were not degraded and confirms that the source of nuclease activity originates from the

BALF. A plot of the agarose gel signal intensity (3 h samples) against molecular weight (kb)

illustrates the cleavage of high molecular weight gDNA (>10 kb) (Figs. 2.1C, D, top panels)

into a wide range of low molecular weight products (0.25 – 3 kb) (Figs. 2.1C, 1D, bottom

panels). Thus, a basal level of nucleases does exist in the airways of mice and can be activated

by supplementing both Mg2+

and Ca2+

to BALF.

Nucleases in the BAL fluid are active in the presence of cations

To assess the effect of specific cations on nuclease activity, individual or combinations of

magnesium, calcium and/or chelators were added to gDNA-BALF and incubated at 37 °C for 3

h. The agarose gel and density plots show that for both naïve (Fig. 2.2A, C) and PBS-instilled

mice (Fig. 2.2B, D), airway nucleases become active with the addition of Mg2+

alone. This

effect is not observed with Ca2+

alone. However, in the presence of both Mg2+

and Ca2+

,

maximal gDNA degradation is observed by BALF nucleases. A plot of the agarose gel signal

intensity against molecular weight (kb) confirms that high molecular weight gDNA (~10 kb)

(Figs. 2.2C, 2.2D, top panels) is cleaved into a wide range of low molecular weight products

(~0.75 – 6 kb) in the presence of both Mg2+

and Ca2+

(Fig. 2.2C, D, middle panels). By

chelating ions with EGTA or EDTA, gDNA was protected from nuclease activity and remained

as intact, high molecular weight structures (Fig. 2.2A, B agarose gel and Fig. 2.2C, D bottom

density plot). Therefore, naïve and PBS-BALF nucleases require Mg2+

for activation, whose

functions can be enhanced by the further addition of Ca2+

.

Murine neutrophils recruited by LPS form NETs in vivo

To study the formation of NETs in vivo, we used a lung injury mouse model that was

characterized to study NETs in our lab with some slight modifications25

. Specifically, we

intranasally instilled 1 µg LPS for 24 h into the airways of 4-6-week-old BALB/c mice. A

37

3

gDNA + + + + + - - -

Mg2+/Ca2+ - + + + + - - -

Naïve BALF (µl) - -

t = 1 hr

t = 3 hr

A

C

gDNA + + + + + - - -

Mg2+/Ca2+ - + + + + - - -

PBS BALF (µl) - -

t = 1 hr

t = 3 hr

D

B

10

0.25

0.75

2

3

10

0.25

0.75

2

3

10

0.25

0.75

2

3

10

0.25

0.75

2

kb kb

Naïve mouse BALF PBS-instilled mouse BALF

38

Figure 2.1. Nucleases are present in non-flamed airways of naïve and PBS-instilled mice.

A & B, Agarose gel of degraded gDNA from reactions containing gDNA, naïve BALF (Fig. 1A)

or PBS-BALF (Fig. 1B), Mg2+

and Ca2+

incubated at 37 °C. gNDA degradation is dose-

dependent and time-dependent, with slightly more degradation observed at higher BALF

concentrations and a longer incubation time (3 h). C & D, Density plots of the agarose gel signal

intensity (3 h) against the molecular weight (kb) showing that gDNA alone and gDNA with the

addition of Mg2+

(“M”) and Ca2+

(“C”) remain as high molecular weight (m.w.) structures.

gDNA incubated with naïve-(Fig. 1C, bottom panel) or PBS-BALF (Fig. 1D, bottom panel) in

the presence of Mg2+

and Ca2+

becomes degraded as shown by the appearance of low m.w.

bands. Images of agarose gels and signal intensity plots are representative of three independent

experiments. gDNA, genomic DNA; BALF, BAL fluid, MC, MgCl2 (5 mM) & CaCl2 (5 mM).

39

Naïve BALF

Ca2+

Mg2+

EGTA

- - + + + + + +

- + - - + + + +

- + - + - + + +- - - - - - + -

EDTA - - - - - - - +

gDNA + + + + + + + +PBS BALF

Ca2+

Mg2+

EGTA

- - + + + + + +

- + - - + + + +

- + - + - + + +- - - - - - + -

EDTA - - - - - - - +

gDNA + + + + + + + +A B

C D

3

10

0.25

0.75

2

3

10

0.25

0.75

2

kb kb

Naïve mouse BALF PBS-instilled mouse BALF

40

Figure 2.2. Nucleases in the airways of naïve and PBS-instilled mice require cations for

activity.

A & B, Agarose gel showing gDNA incubated with naïve BALF (Fig. 1A) or PBS-BALF (Fig.

1B) in the presence of cations and/or chelators at 37 °C for 3 h. gDNA controls remain as high

molecular bands. Addition of Mg2+

leads to slight gDNA degradation by BALF nucleases,

whereas Ca2+

does not. Both Mg2+

and Ca2+

enhance nuclease activity synergistically. EGTA

and EDTA are able to protect gDNA from degradation. C & D, Density plots of the agarose gel

signal intensity (3 h) against the molecular weight (kb) showing that gDNA and gDNA-BALF

controls remain as high molecular weight DNA >10 kb (top panel). Once gDNA is incubated

with naïve-(Fig. 2C) or PBS-BALF (Fig. 2D) in the presence of both Mg2+

and Ca2+

, gDNA is

significantly fragmented to a range of 0.25 – 3 kb DNA sizes (middle panel). The presence of

chelators protects gDNA as >10 kb (bottom panel). Images of agarose gels and signal intensity

plots are representative of two independent experiments. gDNA, genomic DNA; BALF, BAL

fluid, M, MgCl2 (5 mM), C, CaCl2 (5 mM), EGTA (20 Mm), EDTA (20 mM).

41

differential cell count of the BAL validated that neutrophils (96 %) were recruited to the airway

by LPS, post-24 h (Fig. 2.3A). Detection for CitH3 (~17 kDa), a NET marker, in cell-free

BALF by Western blot shows elevated levels of CitH3 in LPS-instilled mice compared to its

PBS control (Fig. 2.3B). To further confirm that NETs accumulate in the airways of LPS-

instilled mice, we next examined BALF NETs using agarose gel electrophoresis (Fig. 2.3C). PK

treatment of LPS-BALF NETs abolished proteins in existing DNA-protein complexes and

shifted high molecular weight DNA bands to low molecular weight DNA bands. This shift in

size suggests that NET-DNA, or fragments of it, were held together by proteins. Non-buffered

DNase and/or RNase treatment demonstrates that the nucleic acid in the gel is primarily

composed of DNA. Together, these studies show that in response to LPS, NETs are formed in

the airways of 4-week-old mice and exist as fragments of DNA-protein complexes.

Nucleases present in the airways of LPS-instilled mice also require cations for activity

The airways of non-inflamed naïve and PBS-instilled mice contained a basal level of nuclease

activity. We next investigated whether inflammatory airways of LPS-instilled mice contained

additional nucleases. Cell-free supernatant BALF was incubated with intact gDNA for 1 h and 3

h at 37 °C. gDNA controls remain as high molecular weight DNA as visualized by agarose gel

electrophoresis (Fig. 2.4A). Also, BALF alone controls show some background DNA (Fig.

2.4A), which was earlier described to be NETs (Fig. 2.3). Therefore, any additional increase in

intensity on the agarose gel can be attributed to the degradation gDNA by LPS-BALF nucleases.

Similar to the findings of non-inflamed lungs, electrophoretic analysis shows that gDNA is

degraded in a dose-dependent and time-dependent manner (Fig. 2.4A). Signal intensity plots (3

h samples) confirm the degradation of high molecular weight gDNA (~10 kb) (Fig. 2.4B, top

panel) to low molecular weight DNA (0.25 – 1 kb) (Fig. 2.4B, bottom panel). Also, as reaction

volumes and loading volumes were kept the same, a larger signal from the intensity plot

indicates that more gDNA is being degraded by LPS-BALF compared to naïve- and PBS-

BALF. Therefore, inflamed airways contain more nucleases.

Next, we examined whether these nucleases have similar cation preferences to nucleases

found in naïve and PBS-BALF. Indeed, LPS-BALF nucleases become active in the presence of

Mg2+

and not Ca2+

. Furthermore, nuclease activity is synergistically enhanced by adding both

Mg2+

and Ca2+

together, resulting in the mass cleavage of gDNA to ~0.25 – 2 kb DNA

42

Figure 2.3. Neutrophils recruited to the airways of 4-week-old mice form NETs.

A, Following LPS (1 µg) instillation into the airways of BALB/c mice for 1 day, neutrophils

represent 96 % of the total cell population compared to PBS control (n = 4 mice). AM, alveolar

macrophage; PMN, polymorphonuclear neutrophils. B, Detection of CitH3 in BALF of LPS-

instilled mice indicates that neutrophils recruited to the airways form NETs (n = 4 mice). C,

Treating the cell-free BALF with Proteinase K (PK) abolishes existing DNA-protein complexes

and releases NET-DNA fragments of low molecular weight (~0.25 kb) in the agarose gel.

Treatment with DNase (D) confirms that the nucleic acid is primarily DNA. RNase (R) addition

shows that there is little to no RNA in the BALF. Image of agarose gel is representative of three

independent experiments. PBS-BALF, BAL fluid from PBS-instilled mice (1 day); LPS-BALF,

BAL fluid from LPS-instilled mice (1 day).

Cit H3~17 kDa

A

B

C PBS LPS

3

10

0.25

0.75

2

kb

43

Figure 2.4. Nucleases are present in the BAL fluid of LPS-instilled mice.

A, Agarose gel showing gDNA degradation following the incubation with LPS-BALF, Mg2+

,

and Ca2+

at 37 °C for 3 h. This effect is dose-dependent and time-dependent, with more

degradation observed at higher BALF concentrations and a longer incubation time (3 h). LPS-

BALF alone controls show background DNA which were described as NETs in Fig. 2.3. B,

Density plots of agarose gel signal intensity (3 h) against molecular weight marker (kb) showing

that gDNA alone and gDNA with Mg2+

(“M”) and Ca2+

(“C”) remain as high molecular weight

(m.w.) structures (top panel). Once gDNA is incubated with LPS-BALF in the presence of both

Mg2+

and Ca2+

, gDNA becomes degraded as shown by the peak of low m.w. DNA at ~0.25 –

0.3 kb (bottom panel). Images of agarose gels and signal intensity plots are representative of

three independent experiments. gDNA, genomic DNA; LPS BALF, BAL fluid LPS-instilled

mice, MC, MgCl2 (5 mM) & CaCl2 (5 mM).

gDNA + + + + + - - -

Mg2+/Ca2+ - + + + + + + +

LPS BALF (ul) - -

t = 1 hr

t = 3 hr

A B

3

10

0.25

0.75

2

kb

10

kb

3

0.25

0.75

2

44

fragments (Fig. 2.5A, B, middle panel). EGTA or EDTA was able to block nuclease activity as

observed by the high molecular weight peak of gDNA (Fig. 2.5A, B, bottom panel). Therefore,

cation supplementation is required for nuclease activity in the airways of mice instilled with

LPS. Collectively, it is shown here that nucleases present in both inflamed and non-inflamed

lungs are similar enzymes that require cations to initiate DNA hydrolysis.

NET-DNA is degraded by nucleases in the BAL fluid of LPS-instilled mice

After NETs are formed, NETs exist as large web-like structures until it is cleared or removed.

As such, we investigated whether nucleases in the airways are able to degrade NETs into

fragments and whether this form of degradation also required cations. NET-containing BALF

from LPS-instilled mice was incubated with different combinations of Ca2+

, Mg2+

, EGTA,

and/or EDTA at 37 °C for 3 h. Agarose gel electrophoresis (Fig. 2.6A) and intensity plots (Fig.

2.6B, middle panel) show that BALF NETs are further degraded by nucleases in the presence of

Ca2+

rather than Mg2+

. Both ions together do not show any detectable synergistic effect on

nuclease activity. It was also observed that some NET-DNA near the top of the wells was

protected from nuclease degradation. Post-treatment of these reaction samples with PK releases

NET-DNA from its high molecular weight complexes (Fig. 2.6A, bottom gel). Chelating ions

with EGTA or EDTA prevented BALF NETs from being degraded by BALF nucleases (Fig.

2.6A, B, bottom panel). Here we show that NETs are degraded by LPS-BALF nucleases in a

Ca2+

-dependent manner. This is different from the earlier observation that showed gDNA being

degraded by LPS-BALF nucleases in a Mg2+

- and Mg2+

/Ca2+

-dependent manner.

Airway nuclease maximal activity has 2 unique pH ranges

Nucleases function optimally at specific pHs. To investigate the effect of pH on nuclease

activity from the airways of LPS-instilled mice, HEPES (pH 6.8 – 8) and MOPS (pH 6.0 – 6.6)

buffers were added to gDNA-BALF containing Mg2+

and/or Ca2+

and incubated at 37 °C for 3 h.

Samples were analyzed in an agarose gel. gDNA controls are not affected by pH changes (Fig.

2.7A and B). However, BALF controls containing NETs show some degradation near acidic pH

6.0 to 6.6 (Fig. 2.7C). Also, gDNA-BALF controls show that degradation can persist even in the

absence of exogenous cations between pH 6.0 to 7.0 (Fig. 2.7D). Therefore, we can only assess

the effect of cations on nuclease activity at and above pH 7.2. The specific addition of Mg2+

to

45

Figure 2.5. Nucleases in the inflamed airways of LPS-instilled mice require cations for

activity.

A, Agarose gel showing gDNA incubated with LPS-BALF in the presence of cations and/or

chelators at 37 °C for 3 h. gDNA controls remain as high molecular weight (m.w.) bands.

Addition of Mg2+

leads to gDNA degradation by LPS-BALF nucleases, whereas Ca2+

does not.

Nuclease activity is synergistically enhances by the presence of both Mg2+

and Ca2+

. EGTA and

EDTA addition maintains gDNA as high m.w. DNA. B, Signal intensity plots of the agarose gel

(3 h) showing gDNA and gDNA-BALF controls remain as high molecular weight DNA at >10

kb (top panel). Once gDNA is incubated with LPS-BALF, Mg2+

and Ca2+

, gDNA is

significantly fragmented from 10 kb to a range of 0.25 – 3 kb products (middle panel). The

presence of chelators protects gDNA as >10 kb (bottom panel). Images of agarose gel and signal

intensity plots are representative of three independent experiments. gDNA, genomic DNA; LPS

BALF, BAL fluid from LPS-instilled mice, M, MgCl2 (5 mM), C, CaCl2 (5 mM), EGTA (20

Mm), EDTA (20 mM).

LPS BALF

Ca2+

Mg2+

EGTA

- - + + + + + +- + - - + + + +

- + - + - + + +- - - - - - + -

EDTA - - - - - - - +

gDNA + + + + + + + +

A B

3

10

0.25

0.75

2

kb

46

Figure 2.6. Degradation of NETs in the BAL fluid of LPS-instilled mice.

A, Agarose gel showing that NET-DNA present in the airways of LPS-instilled mice is degraded

by BALF nucleases when Ca2+

is present, rather than Mg2+

. PK treatment abolishes the DNA-

protein complexes and releases the rest of the DNA fragments that was not cleaved. B, Plots of

the agarose gel signal intensity versus the molecular weight marker (kb) after 3 h. BALF

controls contain NET-DNA of >10 kb or 1 – 3 kb (top panel). With the supplement of Ca2+

to

BALF, the 1 – 3 kb fragments disappear (middle panel). The addition of chelators, EGTA and

EDTA, prevents nucleases from cleaving NET-DNA (bottom panel). Images of agarose gels and

signal intensity plots are representative of three independent experiments. gDNA, genomic

DNA; LPS BALF, BAL fluid from LPS-instilled mice, PK, Proteinase K (200 µg/ml); M,

MgCl2 (5 mM), C, CaCl2 (5 mM), EGTA (20 Mm), EDTA (20 mM).

t = 3 hr

LPS BALFCa2+

Mg2+

EGTA

+ + + + + + + +- + - + + + - -- - + + + + - -- - - - + - + -

EDTA - - - - - + - +

t = 3 hr + PK treated

A B

3

10

0.25

0.75

2

kb

3

10

0.25

0.75

2

kb

47

the reaction leads to gDNA degradation by BALF nucleases at pH 7.2 to 8.0 (Fig. 2.7E),

whereas the addition of Ca2+

had little effect (Fig. 2.7F). A similar synergistic relationship of

both ions on nuclease activity was detected between pH 7.2 – 8.0 (Fig. 2.7G).

Since the buffering capacity of HEPES and MES is limited near pH 6.8 and 6.6,

respectively, PIPES and MOPS were used to assess nuclease activity in these ranges. In a

similar experiment using PIPES, we noticed again that without the addition of any cations,

BALF controls containing NETs show some degradation near acidic pH 6.0 to 6.4 (Fig. 2.8C).

Without the addition of cations, gDNA-BALF controls also show significant degradation at

acidic pHs (6.0 to 6.4) (Fig. 2.8D). The effect of adding Mg2+

to the reaction initiates gDNA

degradation at and above pH 6.6 (Fig. 2.8E), whereas consistently, nucleases remain mostly

inactive in the presence of Ca2+

(Fig. 2.8F). It is confirmed once again that both Mg2+

and Ca2+

ions together enhance the function of LPS-BALF nucleases when buffered with PIPES (Fig.

2.8G).

In another set of experiments utilizing MOPS as the buffer, similar results for gDNA

controls (Fig. 2.9A) and BALF alone controls were obtained (Fig. 2.9B). Again, analysis of

gDNA-BALF controls shows significant degradation at acidic pHs (6.0 to 6.6). Addition of

Mg2+

(Fig. 2.9E), but not Ca2+

(Fig. 2.9F) promotes degradation. As anticipated, presence of

both ions enhanced the activity of nucleases based on the increase of low molecular weight

gDNA products (Fig. 2.9G). We further chelated ions using EDTA and found that, in doing so,

this chelator protected gDNA from enzymatic degradation between pH 6.4 to 8.0 (Fig. 2.9H).

Across the various buffers, BALF nucleases tend to degrade gDNA at acidic pH values.

However, nuclease activity peaks again at a very specific pH. This occurs specifically at pH 7.0

in HEPES and MOPS buffers, and pH 6.8 in PIPES buffer (Figs. 2.7D, 2.8D, 2.9D). Therefore,

these studies demonstrate that LPS-BALF nucleases work optimally at two different pH ranges,

one near acidic pH (6.6 or less) and one near neutral pH (6.8-7.0). The same experiments were

repeated using BALF from naïve and PBS-instilled mice and similar effects were observed

(Supplementary Figs. S2.1 and S2.2).

48

Figure 2.7. Nucleases are active with Mg2+

/Ca2+

and also without cations near acidic pH.

Using the HEPES and MES buffers, nuclease activity from the airways of LPS-instilled mice

were assessed on an agarose gel. LPS-BALF and gDNA samples with cations and/or chelators

were incubated at 37 °C for 3 h prior to electrophoretic analysis. A & B, gDNA controls remain

as high molecular weight (m.w.) bands. C, NET-DNA in LPS-BALF control is degraded in the

absence of cations at pH 6.0 to 6.6. D, gDNA incubated with LPS-BALF shows cleavage at pH

6.0 to 7.0. E-G, Unlike Ca2+

(F), the addition of Mg2+

leads to gDNA degradation by nucleases

in the BALF at all pHs (E). The presence of both ions synergistically enhances the activity of

BALF nucleases based on the abundance of low m.w. DNA bands (G). gDNA, genomic DNA;

LPS BALF, BAL fluid from LPS-instilled mice; D, non-buffered DEPC-H2O.

gDNA

LPS-BALF

gDNA+ Ca2+

+ Mg2+

gDNA + LPS-BALF

pH

D

8 7.8 7.6 7.4 7.2 7 6.8 6.4 6.2 6

HEPES MES

6.6 pH

D

8 7.8 7.6 7.4 7.2 7 6.8 6.6 6.4 6.2 6

HEPES MES

gDNA + LPS-BALF+ Ca2+

gDNA + LPS-BALF+ Mg2+

gDNA + LPS-BALF+ Ca2+

+ Mg2+

B

C

D

F

G

EA

0.75

3

10

0.25

2

kb

0.75

3

10

0.25

2

0.75

3

10

0.25

2

0.75

3

10

0.25

2

0.75

3

10

0.25

2

10

10

kb

49

Figure 2.8. Nucleases of inflamed airways have two pH optimums – PIPES buffer.

Using the HEPES, PIPES and MES buffers, nuclease activity from the airways of LPS-instilled

mice were assessed on an agarose gel. The conditions of the samples were prepared as did

before. A & B, High molecular weight (m.w.) bands for gDNA controls. C, NET-DNA in LPS-

BALF control is degraded in the absence of cations at pH 6.0 to 6.4. D, Cleavage of gDNA by

BALF nucleases occurs near acidic pHs 6.0 to 6.4 and also specifically at pH 6.8 (arrow). E-G,

Addition of Mg2+

causes slight gDNA degradation (E) compared to the null effect of Ca2+

,

except at acidic pH 6.0 to 6.4 (F). The presence of both ions synergistically enhances the activity

of BALF nucleases based on the disappearance of high m.w. bands and the abundance of low

m.w. bands (G). Images of agarose gels and signal intensity plots are representative of three

independent experiments. gDNA, genomic DNA; LPS BALF, BAL fluid from LPS-instilled

mice; D, non-buffered DEPC-H2O.

0.25

10

10

+ Ca2+

+ Mg2+

pH

D

8 7.5 7.0 6.8 6.6 6.4 6.2 6

HEPES MESPIPES

gDNA + LPS BALF

+ Ca2+

gDNA + LPS BALF

gDNA + LPS BALF

+ Mg2+

pH

D

8 7.5 7.0 6.8 6.6 6.4 6.2 6

HEPES MESPIPES

gDNA

+ Ca2+

+ Mg2+

gDNA

LPS BALF

gDNA + LPS-BALF

B

C

D

F

G

EA

10

10

kb

0.75

3

10

0.25

2

0.75

3

10

0.25

2

10

kb

0.75

3

0.25

2

0.75

3

0.25

2

0.75

3

2

50

Figure 2.9. Nucleases of inflamed airways have two pH optimums – MOPS buffer.

Using the HEPES, MOPS and MES buffers, nuclease activity from the airways of LPS-instilled

mice were assessed on an agarose gel. The conditions of the samples were prepared as did

before. A & B, gDNA controls have high molecular weight (m.w.) bands. C, NET-DNA in LPS-

BALF control is degraded in the absence of cations at pH 6.0 to 6.4. D, Cleavage of gDNA by

BALF nucleases occurs near acidic pHs 6.0 to 6.4 and also specifically at pH 7.0 (arrow). E-G,

Addition of Mg2+

causes slight gDNA degradation (E) compared to the null effect of Ca2+

,

except at pH 6.0 and 6.2 (F). The presence of both ions synergistically enhances the activity of

BALF nucleases based on the disappearance of high m.w. DNA and the appearance of low m.w.

DNA (G). H, Chelating ions with EDTA protects gDNA from being degraded by BALF

nucleases. Images of agarose gels and signal intensity plots are representative of three

independent experiments. gDNA, genomic DNA; LPS BALF, BAL fluid from LPS-instilled

mice; D, non-buffered DEPC-H2O.

3

10

0.75

10

10

HEPESpH

D8 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6

MESMOPS

+ Ca2+

+ Mg2+

gDNA + LPS-BALF

+ Mg2++ Ca2+gDNA

LPS-BALF

gDNA + LPS-BALF

HEPES

pH

D

8 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6

MES

gDNA

MOPS

A

B

C

D

F

G

E

gDNA + LPS-BALF+ EDTA

H

10

10

kb

0.75

3

0.25

2

0.75

3

0.25

2

3

0.25

2

10

gDNA + LPS-BALF

+ Mg2+

0.75

3

0.25

2

gDNA + LPS-BALF

+ Ca2+

0.25

10

0.75

3

2

10

0.750.25

2

kb

51

Supplementary Figure S2.1. Nucleases from naive airways of mice have two pH optimums

– MOPS buffer.

Using the HEPES, MOPS and MES buffers, nuclease activity from the airways of naïve mice

were assessed on an agarose gel. The conditions of the samples were prepared as did before. A

& B, gDNA controls have high molecular weight (m.w.) bands. C, Naïve-BALF control does

not contain any DNA. D, Cleavage of gDNA by naïve-BALF nucleases occurs near acidic pHs

6.0 to 6.4 and also specifically at pH 7.0 (arrow). E-G, Addition of Mg2+

causes slight gDNA

degradation (E) compared to the null effect of Ca2+

, except at pH 6.0 and 6.2 (F). The presence

of both ions synergistically enhances the activity of BALF throughout all pH values (G). H,

Chelating ions with EDTA protects gDNA from being degraded by naïve-BALF nucleases,

except at pH 6.0 and 6.2. Images of agarose gels and signal intensity plots are representative of

two independent experiments. DNA, genomic DNA; naïve-BALF, BAL fluid from naive mice;

D, non-buffered DEPC-H2O.

3

10

0.75

10

HEPESpH

D8 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6

MESMOPS

+ Ca2+

+ Mg2+

gDNA + naive-BALF

+ Mg2++ Ca2+gDNA

gDNA + naive-BALF

HEPES

pH

D

8 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6

MES

gDNA

MOPS

A

B

C F

G

E

gDNA +naive-BALF+ EDTA

H

10

10

kb

0.75

3

0.25

2

3

0.25

2

10

gDNA + naive-BALF

+ Mg2+

0.75

3

0.25

2

gDNA + naive-BALF

+ Ca2+

0.25

10

kb

0.75

32

10

0.75

0.25

2

kb

10

0.75

3

0.25

2

D

naive-BALF

52

Supplementary Figure S2.2. Nucleases from PBS-instilled airways of mice have two pH

optimums – MOPS buffer.

Using the HEPES, MOPS and MES buffers, nuclease activity from the airways of naïve mice

were assessed on an agarose gel. The conditions of the samples were prepared as did before. A,

Cleavage of gDNA by PBS-BALF nucleases occurs near acidic pHs 6.0 to 6.4 and also

specifically at pH 7.0. B & C, Addition of Mg2+

causes slight gDNA degradation (B) compared

to the null effect of Ca2+

, except at pH 6.0 and 6.2 (C). D, The presence of both ions

synergistically enhances the activity of BALF throughout all pH values. E, Chelating ions with

EDTA protects gDNA from being degraded by PBS-BALF nucleases, except at pH 6.0 and 6.2.

The agarose gels and density plots representative of two independent experiments. gDNA,

genomic DNA; PBS-BALF, BAL fluid from PBS-instilled mice; D, non-buffered DEPC-H2O.

3

10

0.75

10

HEPESpH

D8 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6

MESMOPS

+ Ca2+

+ Mg2+

gDNA + PBS-BALF

gDNA + PBS-BALF

C

D

B

gDNA +PBS-BALF+ EDTA

E

0.75

3

0.25

2

3

0.25

2

10

gDNA + PBS-BALF

+ Mg2+0.75

3

0.25

2

gDNA + PBS-BALF

+ Ca2+

0.25

10

kb

0.75

3

2

10

0.75

0.25

2

kb

Akb

53

2.5 Discussion

NETs are large, web-like structures made up of nuclear and/or mitochondrial DNA and

antimicrobial proteins which can be detrimental to the host tissue if not removed in a timely

manner72–74

. Exogenous DNase is generally used for dismantling NETs in experimental models.

Here we investigate whether endogenous nucleases are present in vivo and whether these

nucleases are able to cleave NETs. In our study we found that the BALF from healthy and

inflamed lungs of mice contain nucleases that degrade gDNA in a dose-dependent and time-

dependent manner (Figs. 2.1, 2.4). Degradation of gDNA is also dependent on the presence of

divalent cation Mg2+

, in which a synergistic effect on nuclease activity is observed with both

Mg2+

and Ca2+

together (Figs. 2.2, 2.5). On the other hand, degradation of NETs by BALF

nucleases was shown to be Ca2+

-dependent (Figs. 2.3, 2.6). BALF nucleases were further

characterized to have optimal activity near neutral pH (6.8-7.0) and acidic pH (6.6 and less),

which are properties similar to DNase I and DNase II, respectively (Figs. 2.7-2.9, S2.1-2.2).

Of the many characterized nucleases, DNases are the most widely studied195

. DNase I is

the best known Mg2+

/Ca2+

nuclease and is generally found in the pancreas and parotid

glands196,197

. Similar to the findings in our study (Figs. 2.2, 2.5), this enzyme is strongly

activated by Mg2+

, whose function is maximized by both Mg2+

and Ca2+ 198,199

. This synergistic

effect is the result of divalent cations stabilizing the DNase structure and modifying the

electrostatic fit of DNA to the hydrolytic pocket199

. CADs are another class of DNases which

only require Mg2+

for activity. CAD is activated by caspase 3 during apoptosis which generate

DNA fragments through internucleosomal cleavage. However, caspase 3 in neutrophils is not

activated during NETosis38,55,56

. Therefore, it is less likely that CADs represent the pool of

nucleases found in our study, as this enzyme is primarily localized to intracellular

compartments. To date, only a few studies have characterized DNase involvement in the

airways229–231

. One study showed that DNases are present in BALF of healthy and CF patients

whose function can be activated by exogenous addition of Mg2+

. In spite of this finding, the

effect of Ca2+

on DNase activity was not assessed231

. The presence of nucleases was also

documented in the airways of mice to interfere with gene therapy229,230

. Here, I not only show

that nucleases are present in the airways of mice, but show that these nucleases require Mg2+

for

activity and are enhanced synergistically by the addition of both Mg2+

and Ca2+

. Although the

54

preference for Mg2+

/Ca2+

is characteristic of DNase I as well as other DNase I-like enzymes202–

204, further validation experiments are required to identify this airway nuclease.

Currently, the mechanism of NET clearance in the airways is not well understood.

Farrera and colleagues found that NETs are taken up by HMDMs in an endocytic manner and

that this process is facilitated by C1q and DNase I159

. However, the effect of endogenous

DNases on airway NETs has not been established. Previously, we have shown that NETs

formed in the airways of 8-12-week-old mice are present as fragments25

. The presence of NET

fragments is likely due to the presence of airway nucleases. In our current study, we used a

similar model and corroborated the finding that NETs in the BALF of LPS-instilled 4-6-week-

old mice exists as fragments (Fig. 2.3). This BALF also contained more nucleases compared to

naïve mice and mice instilled with PBS (Fig. 2.4). Nucleases during inflammation can be

contributed in part by serum leakage or from some other source in response to LPS. Other

sources may include AMs, neutrophils and even epithelial cells as nucleases have been detected

in these cell types and/or cell lines223,225,227,228

.

Similar to the nucleases present in non-inflamed airways of mice, we found that

nucleases from LPS-instilled mice are activated by exogenous Mg2+

alone or by both Mg2+

and

Ca2+

together (Fig. 2.4). However, this Mg2+

/Ca2+

-dependent degradation of gDNA is different

from the degradation of NETs. We showed that NETs are cleaved by LPS-BALF nucleases in a

Ca2+

-dependent manner (Fig. 2.5). Our finding agrees with the Ca2+

requirement of a serum

nuclease for degrading NETs. In that study, the authors focused on NET degradation in the sera

of healthy and SLE patients, and particularly demonstrated that DNase I present in the serum of

healthy donors was able to cleave NETs in a Ca2+

-dependent manner. However, DNases from

SLE patients were inhibited by the presence of NET-proteins and anti-NET antibodies103

. The

presence of both Mg2+

/Ca2+

-dependent gDNA degradation and Ca2+

-dependent NET

degradation suggests that there are either two types of nucleases present or one type of nuclease

with the ability to cleave various forms of DNA based on the cation(s) it is bound to.

Nucleases localized to tissues are influenced by the conditions of the environment. For

this reason, nucleases operate at specific pH determined by the surroundings. In our study, we

assessed whether pH affects nuclease activity in the airways of mice. We found that without the

addition of divalent cations, nucleases in BALF were active at specific pH 6.8 (Fig. 2.8D) and

7.0 (Fig. 2.9D, supplementary Figs. S2.1D, S2,2A), but also at acidic pHs below 6.6 (Figs.

55

2.7D, 2.8D, 2.9D, supplementary Figs S2.1D, S2.2A). The finding of two optimal pH (near

neutral and acidic pH) for BALF nucleases could mean: i) the BALF itself may originally

contain diluted cations which have the ability to activate nucleases once adjusted to optimal

pHs; and/or ii) there is a subset of nucleases in the BALF that does not require cations to

function. We investigated these possibilities by adding chelators to the reactions and found that

both might be correct. Namely, EDTA was able to inhibit nuclease activity near neutral pH,

confirming that low concentrations of BALF cations were enough to drive DNA hydrolysis (Fig.

2.9H). It is noteworthy that other than Mg2+

and Ca2+

, divalent cations such as Mn2+

and Co2+

are also strong activators of DNases. However, these heavy metals usually exist in trace

amounts as elevated levels are associated with toxicity and lung pathologies282–284

. While Sr2+

and Ba2+

can replace Ca2+

, these ions poorly catalyze DNA hydrolysis when paired with Mg2+

200. Rather than serving as activators, cations such as Zn

2+, Ni

2+ and Cu

2+ are known to inhibit

DNase function207

. Monovalent cations Na+, K

+, and NH4

+ have too been shown to have similar

inhibitory effects205

. Nuclease activity in the airways is likely regulated by the balance of

activating and inhibiting ions. Although EDTA added to the reactions was able to inhibit

nuclease activity near neutral pH, this protective effect was not observed at pHs 6.0 and 6.2

(Fig. 2.9H). Therefore, it is possible that acidic nucleases are present in BALF. DNase II is an

acidic nuclease that functions independently of cations and exists in AMs and neutrophils as

described earlier225,285

. However, additional studies are required to identify whether these

nucleases are the same nucleases found in the airways. Generally, airway pH is slightly more

acidic than blood pH 7.4. The normal pH of ASL in humans and mice has been reported as 6.6-

7.4 and 7.1, respectively169,286–289

. Discrepancies in airway pH values are a result of different

methods of measurements. During an inflammatory response, the pH in the airways is thought to

drop slightly290,291

. This change towards a more acidic environment is thought to serve as a

titratable host defense that takes advantage of weak endogenous acids to fend against airborne

pathogens292,293

. Decreases in airway pH during airway inflammation might be related to the

production of metabolic acids by recruited leukocytes294,295

. In addition, activation of apical

proton channels on airway epithelial cells has been shown to increase acid secretion into the

ASL296

. In our study, we observed that NETs degraded near acidic pHs. Therefore, a lower pH

may facilitate the cleaving of NET DNA.

56

Airway acidification is only a transient state in the lungs as normal conditions are

presumably restored following the resolution of inflammation. However, persistent or severe

acidification is associated with respiratory diseases such as COPD, asthma and CF291,297,298

.

Adult military trainees diagnosed with asthma have lower mean exhaled bronchial condensate

(EBC) pH (6.39) than trainees without the condition (pH 6.64)299

. Individuals with stable CF

have EBC pH (5.88) lower than controls (6.15). Exacerbations of the CF disease further reduces

EBC pH (5.32) compared to those with stable CF300

. Low pH in CF airways have been shown in

a porcine model to foster bacteria growth and have impaired bacteria-killing ability when the pH

is reduced from 7.6 to 6.8 or 6.4298

. Conversely, by raising ASL pH back to normal values,

killing of bacteria was rescued in CF pigs298

. Long-term airway acidification may also alter

nuclease activity due to changes in airway ions and the release of inhibitors including actins,

proteases, and autoantibodies. In fact, low Mg2+

levels measured in the sputum of a subset of CF

patients have been associated with a low response to rhDNase treatment. By adding Mg2+

to the

sputum, degradation of the sample by rhDNase was restored232

. As we know now that NETs are

major contributors to lung disorders, therapeutic interventions targeting the breakdown of NETs

may need to focus on adjusting airway pH back to normal values or by the re-activation of

endogenous airway nucleases. The supplementation of elastase inhibitors while activating

airway nucleases could be considered as the release of elastases during NET degradation causes

tissue damage. These options would be ideal for restoring the balance between NET formation

and NET clearance.

57

Chapter 3

Surfactant protein D enhances the clearance of neutrophil extracellular traps

by alveolar macrophages

Lily Yip1,2

, David N. Douda1,2

, Hartmut Grasemann3,4

, Nades Palaniyar1,2,3

1Lung Innate Immunity Research Laboratory, Program in Physiology & Experimental Medicine

SickKids Research Institute

2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto,

Ontario, Canada

3Division of Respiratory Medicine, Department of Paediatrics, The Hospital For Sick Children

4Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada

Corresponding author: Nades Palaniyar, PhD, Lung Innate Immunity Research Laboratory, The

Hospital For Sick Children, 555 University Ave., Toronto, ON, Canada, M5G 1X8. Phone: 1-

416-813-7654 ext 302328. Fax: 1-416-813-5771. e-mail: nades.palaniyar@sickkids.ca

Experiments were contributed equally by both Lily Yip and David N. Douda.

The work presented in this chapter has been prepared as a manuscript for future submission.

58

3.1 Abstract

Excessive NETs or the inability to clear NETs has been shown to contribute to the pathogenesis

of several lung disorders. Although treatment with DNases can lead to NET degradation, this is

usually a temporary solution and leads to the release of other cationic proteases that cause lung

tissue damage. Our lab has previously identified a high affinity interaction between surfactant

protein D (SP-D) and NETs. SP-D is a lung protein known to opsonize apoptotic cells and DNA

in addition to enhancing their removal by alveolar macrophages. We show here that SP-D

knockout mice are defective in NET clearance and that supplementing SP-D enhances the

clearance of NETs ex vivo by AMs. We also show that NETs that are partially cleaved by

nucleases are present in the cytoplasm of macrophages while full-length NETs are superfluously

attached to macrophages. Airway SP-D is known for enhancing small particle clearance by

alveolar macrophages. Therefore, SP-D is a likely candidate for promoting clearance of NET

fragments by endocytosis in alveolar macrophages.

3.2 Introduction

SP-D is an innate immune collectin of the lung and contains a unique structure that allows the

protein to bind to several factors. The CRD is known to opsonise dying cells and bacteria for the

removal by lung phagocytes254,277,278,279,153,280

. Also, the collagen stems have been shown by our

lab to bind to DNA and enhances its uptake by alveolar macrophages266,242

. Interestingly, a

similar interaction to genomic DNA-derived NETs was recently discovered in our lab25

. NETs

are a result of a novel form of neutrophil cell death termed NETosis. Neutrophils entering a site

of inflammation undergo a series of modifications that result in the hypercitrullination of

histones (CitH3) and mixing of decondensed DNA with its own granular proteins (elastase and

myeloperoxidase), all to be released together outside of the cell body61,301,63

. While NETs are

effective in trapping and assisting the killing of bacteria25,18,302,45

, too many NETs can be

cytotoxic to airway epithelial and endothelial cells73

. Therefore the proper removal of NETs is

important in maintaining lung homeostasis. To date, the role of SP-D in mediating NET

clearance is unknown. Here, we propose that SP-D enhances NET clearance by alveolar

macrophages.

59

3.3 Materials and Methods

Reagents

Reagents were purchased from Sigma-Aldrich unless otherwise stated. SP-D was purified by

affinity chromatography from the BALF of an individual with pulmonary alveolar proteinosis

(PAP) as described previously25,242,266

. Briefly, BALF was centrifuged at 10,000 g for 60 min

and the resulting supernatant was incubated overnight with maltose-agarose beads in the

presence of calcium (10 mM) at 4 °C. SP-D-bound beads were collected and washed with a

buffer containing 20 mM Tris (pH 7.4), 1 M NaCl and 10 mM CaCl2. SP-D was competitively

eluted with a buffer containing MnCl2 (100 mM) and Tris (20 mM). The eluate was

concentrated and the protein was separated with a Superose 6 column (GE Healthcare, Baie

d’Urfe, Quebec, Canada). Quality and quantity of SP-D was verified by running the purified

sample on SDS-PAGE gels and Western blots.

Mice and bronchoalveolar lavage

WT litters were of BALB/c background purchased from Charles River. SP-D knockout (SP-D

KO) mice were generated by crossing heterozygous mice containing the sftpd deletion303

. Mice

were maintained at The Hospital for Sick Children Lab Animal Services facility. For all

experiments, mice aged 4 – 6 weeks were used to avoid lung complications that develop in SP-

D knockout mice at older ages257,303

. For the analysis of pulmonary NET clearance in vivo,

BALB/c and SP-D knockout mice were sedated using oxygen-carried isoflurane and 1 µg LPS

(E. Coli O111:B4) carried in 50 µl PBS or a PBS control was intranasally instilled for up to 5

days as described previously25

. Mice were sacrificed with 0.05 ml Euthanyl (Bimeda-MTC, ON,

CA) prior to bronchoalveolar lavage (BAL). BAL was performed with 1 ml of chilled calcium-

and magnesium-free Hanks Balanced Salt Solution (HBSS) (Invitrogen) three times to a total of

about 3 ml. BAL fluid (BALF) was centrifuged at 400 g for 10 min. to pellet the cells from the

supernatant. The cell-free supernatant was analysed for DNA using a Quant-iT™ PicoGreen

dsDNA reagent kit (Invitrogen). RBCs in the cell pellet were lysed with a hypotonic saline

solution (0.2 % (w/v) NaCl) for 30 seconds, followed by the addition of an equal volume of

buffered hypertonic saline solution (1.6 % (w/v) NaCl, 20 mM HEPES, pH 7.2) to achieve an

isotonic equilibrium. The resulting RBC-free cell pellet was resuspended in calcium- and

60

magnesium-free HBSS for cell counting with a haemocytometer. Cytospin preparations were

made and further stained with Hemacolor™ histology staining kit (EMD Chemicals,

Gibbstown, NJ) for cell differential analysis. Histology images were taken with a light

microscope and images were randomly chosen for determining percent populations of each cell

type. A minimum of 100 cells were counted for each condition.

Gel electrophoresis of BAL fluid NETs

BALF from PBS- and LPS-instilled WT and SP-D knockout mice were incubated without

treatment or with Proteinase K (PK; 200 µg/ml), MgCl2 (5 mM) and CaCl2 (5 mM) at 37 °C for

30 min. PK-treated BALF was then supplemented with DNase I (50 µg/ml) or RNase (250

µg/ml) and incubated at 37 °C for an additional hour. Twenty µl samples were separated in a 1.5

% (w/v) agarose gel at 115 V for 25 min and stained with SYBR Gold (Invitrogen) for 35 min in

TAE (40 mM Tris-Acetate, 1 mM EDTA) buffer. Gel images were taken with AlphaImager HP.

Ex vivo NET clearance by murine alveolar macrophages

WT mice were instilled with 5 µg LPS (E. Coli O111:B4) carried in 50 µl PBS for 1 day. BALF

was obtained and airway neutrophils were collected by spinning down the cell pellet at 400 g

for 10 min. RBC lysis was performed as did before and cells were resuspended in DMEM media

supplemented with 10 mM HEPES in 96-well special optics plate (Corning, Lowell, MA).

Neutrophils (5 105) were allowed to form NETs for 4 h. After 4 h, alveolar macrophages (2.5

105; AM) isolated from naïve mice were added to the NETs in the presence or absence of SP-

D (5 µg/ml) and incubated at 37 °C for 6 h and 18 h. AMs were pre-stained with PKH2 green

fluorescent general cell membrane dye according to the manufacturer’s protocol before

culturing with or without NETs. Cells/NETs were fixed with 2 % (w/v) paraformaldehyde

(PFA) overnight at 4 °C. NETs were stained with Sytox Orange and images were taken with a

20x objective by confocal microscopy. The microscope was Olympus IX81 inverted

fluorescence microscope equipped with a Hamamatsu C9100-13 back-thinned EM-CCD

camera, a Yokogawa CSU X1 spinning disk confocal scan head and 4 separate diode-pumped

solid state laser lines (Spectral Applied Research, 405 nm, 491 nm, 561nm, 642 nm). The

microscope was operated with Volocity software (Perkin Elmer, Waltham, MA). At least 50

61

NETs were quantified from 10 randomly selected images by two investigators who were blinded

for the identity of the samples.

In vitro NET clearance by macrophage cell line

Primary neutrophils were isolated from healthy blood donors under a signed, informed consent

approved by the ethics board at The Hospital for Sick Children. Peripheral blood was drawn into

K2 EDTA tubes (BD, Franklin Lakes, NJ), and neutrophils were isolated over a

PolymorphPrep™ gradient (Axis-Shield, PoC, Oslo, NO) according to the manufacturer’s

protocol with slight modifications. Specifically, red blood cells (RBCs) were lysed with a

hypotonic saline solution (0.2 % (w/v) NaCl) for 30 seconds, followed by the addition of an

equal volume of buffered hypertonic saline solution (1.6 % (w/v) NaCl, 20 mM HEPES, pH 7.2)

to achieve an isotonic equilibrium. Neutrophils were resuspended in RPMI 1640 (Invitrogen)

supplemented with 10 mM HEPES (pH 7.2) and total cell counts were determined by a

haemocytometer. Neutrophils (1.5 106) were left untreated or stimulated with 25 nM phorbol

12-myristate 13-acetate (PMA) for 4 h to form NETs in the presence of cell-impermeable DNA

dye, Sytox Green. After 4 h, supernatant was resuspended vigorously to lift NETs and

centrifuged at 1000 g to separate cell bodies/debris (pellet) from released NETs (supernatant).

An equal aliquot of NETs was digested with 50 µg/ml micrococcal nuclease (MNase) for 20

min at 37 °C. The fluorescence of the DNA dye, Sytox Green, was measured with a Gemini EM

fluorescence microplate reader (Molecular Devices, Sunnydale, CA). A ratio of 7.5:1 NETing

neutrophils or digested NETs to RAW 264.7 cells (macrophage cell line) were incubated

together in 8-well chamber slides (BD Biosciences) for 2 h and 18 h at 37 °C. Macrophage cell

line was pre-stained with lipid membrane fluorescent dye, Vibrant DiD (V-22887; Invitrogen),

according to the manufacturer’s protocol. Cells were fixed with 4 % (w/v) PFA for 10 min

before imaging for NET uptake by macrophages by confocal microscopy.

Western Blot

Equal volume of cell-free BALF from WT and SP-D KO mice were diluted with 5 loading

dye (250 mM Tris-HCL, pH 6.8, 0.1 % (w/v) SDS, 0.5 % (v/v) glycerol, 0.05 % (w/v)

bromophenol blue, 10 mM DTT) and incubated at 95 °C for 10 min. An equal volume of each

sample was separated in 12 % (w/v) polyacrylamide gels and transferred to nitrocellulose

62

membranes. The transferred membranes were blocked with 5 % (w/v) non-fat powdered milk in

PBS containing 0.05 % (v/v) Tween-20 (PBST) for 1 h, followed by three 10 min washes with

PBST. Primary antibodies were diluted in 1 % (w/v) non-fat powdered milk in PBST and

incubated for either 1 h, shaking at room temperature, or overnight, shaking at 4 °C. Primary

antibodies used included rabbit polyclonal anti-Histone H3 (citrulline 2 + 8 + 17) (ab5107;

Abcam, ON, CA) at 1:2000 dilution, rabbit polyclonal anti-SP-D (purified from serum) at

1:10000 dilution and rabbit polyclonal anti-SP-A (purified from serum) at 1:10000 dilution.

Membranes were washed five times, 5 min each, and incubated with secondary goat anti-rabbit

conjugated to horse radish peroxidise (BD, ON, CA) at 1:5000-1:10000 dilution in 1 % (w/v)

non-fat powdered milk in PBST for 1 h. After final washing for four times, 5 min. each,

membranes were coated with enhanced chemiluminescence substrate (PerkinElmer, ON, CA)

and exposed to radiographic films. Films were scanned with a Epson perfection 610 photo

scanner. Densitometry analysis was done with ImageJ software (version 1.45s; NIH, Maryland,

US).

Statistical Analysis

All data are presented as averages ± standard error of mean (s.e.m.). Statistical analysis was

done with GraphPad Prism software (version 4.03). Two groups were compared with Student’s

t-tests. When comparing more than two groups, ANOVA with Bonferroni post test was used. A

p-value of 0.05 or less was considered statistically significant.

3.4 Results

Neutrophils recruited to the airways by LPS undergo NET formation in both wild type and

SP-D-deficient mice

Similar to our previous studies25

and results in Chapter 2, intranasal instillation of LPS recruited

immune cells to the airways of WT as well as SP-D KO mice. PBS was used as a vehicle control

which did not recruit any immune cells (Fig. 3.1A). Cell differential analyses of samples from

LPS-instilled WT and SP-D KO mice (untreated or naïve and treatment of up to 5 days) shows

that neutrophils make up the majority of the total cells found in the airways (Fig. 3.1A).

Treatment of BALF with PK confirms the presence of DNA-protein complexes, such as NETs,

in the airways of SP-D KO mice. Specifically, PK degraded proteins found on NETs and

63

released low molecular weight NET-DNA fragments from its high molecular weight DNA-

protein complexes (Fig. 3.1B). These NETs are similar to the NETs found in the airways of WT

mice shown in our previous study25

and in Chapter 2. We also detected CitH3, a NET marker, in

WT and SP-D KO mice instilled with LPS and will be discussed more extensively below (Fig.

3.1B)

SP-D KO mice require an additional day to clear NETs from the airways

Pulmonary SP-D is known to bind to and enhance the phagocytosis of DNA and cell debris by

AMs. While SP-D also binds to NETs, NET clearance by AMs in the presence of SP-D is

unknown. Therefore, we assessed whether SP-D-deficient mice are impaired in their ability to

clear NETs. Cell-free BALF of WT and SP-D KO instilled with LPS for up to 5 days were

measured for DNA content as a surrogate marker of NETs. A comparison of DNA profiles

between these two groups of mice demonstrates that DNA clearance is altered in SP-D KO

mice. Namely, the resolution of DNA, as measured from the projected peak to baseline, is

accomplished within 2 days in SP-D KO mice. WT mice on the other hand, require only 1 day

to clear DNA from their airways (Fig. 3.2A). To ascertain that the DNA being cleared is

associated with NETs, we next probed for CitH3 in these mice. Protein analysis reveals that

CitH3 profiles of both mice parallels the findings of the DNA data. That is, SP-D KO mice

require an additional day to remove CitH3 from the airways compared to WT mice (Fig. 3.2B).

A delay in the kinetics of DNA and CitH3 clearance in SP-D KO mice suggests that SP-D is

required for normal NET clearance. The changes in collectin levels of SP-D and SP-A in

response to PBS or LPS was also assessed in WT and SP-D KO mice. SP-D measurements from

the BALF of PBS-instilled WT mice did not show any significant changes over a 5-day period.

On the other hand, WT mice instilled with LPS releases maximal SP-D into the airways by day

2 (Fig. 3.2C). The kinetics of SP-D also correlates with the rise and decrease of DNA measured

in the airways (Fig. 3.2D). However, SP-A measured from the BALF of both WT and SP-D KO

mice instilled with PBS or LPS show variable fluctuations of SP-A protein levels increasing and

decreasing (Fig. S3.1). Overall, this model demonstrates that SP-D-deficient mice are defective

in clearing NETs compared to wild type mice.

64

Figure 3.1. Neutrophils recruited to the

airways have NET-derived DNA-

protein complexes in SP-D KO mice.

Immune cells are recruited into the

airways of WT and SP-D KO mice

instilled with LPS (1 µg/ml). Cells are

isolated from BALF of both PBS- and

LPS-instilled mice and Cytospins stained

with Hemacolor™ were used for

differential cell count. A, Neutrophils

make up the majority of recruited immune

cells and are highest by day 1 for both WT

and SP-D KO mice. The proportion of

alveolar macrophages remains unchanged

for both mice. n=3-7 mice per condition;

*** p<0.001 by two-way ANOVA with

Bonferroni post test. B, An agarose gel

showing that NET-derived DNA-protein

complexes are present in the airways of

SP-D KO mice instilled with LPS (24 h).

NETs from BALF instilled with LPS are

originally present as large DNA-protein

complexes. Upon treatment with PK,

proteins are degraded and NET-DNA of

low molecular weight fragments are

released to the bottom of the gel. Nuclease

treatment confirms that DNA is the

primary nucleic acid. The image of the

agarose gel is representative of three

independent experiments. BALF,

bronchoalveolar lavage fluid; PK,

Proteinase K (200 µg/ml); D, DNase (50

µg/ml); R, RNase (250 µg/ml).

SP-D KOLPS

SP-D KO PBS

3

10

0.25

0.75

2

kb

A

B

0 1 2 3 4 50.0

0.5

1.0

1.5

2.0

Day(s) Post Instillation

Ab

so

lute

Cell C

ou

nt

10

6

Alveolar Macrophage

Neutrophil

0 1 2 3 4 50.0

0.5

1.0

1.5

2.0

WT PBS

SP-D KO PBS

WT LPS

SP-D KO LPS

Ab

so

lute

Cell C

ou

nt

(10

6)

***

65

Figure 3.2. SP-D-deficient mice are

defective in NET clearance.

Cell-free supernatant of BALF from

WT and SP-D KO mice instilled with

PBS or LPS (1 µg/ml) for up to 5 days

were assessed for DNA content, CitH3,

and SP-D. DNA was quantified with

Quant-iT™ PicoGreen dsDNA reagent

kit (Invitrogen) and CitH3 and SP-D

by Western blots. A, Quantification of

DNA shows that WT mice requires

one day to clear DNA (as measured

from peak to baseline) whereas SP-D

KO mice requires two days. B, The

airways of both WT and SP-D KO

mice instilled with LPS contains

CitH3, a marker of NETs, and follows

the same clearance kinetics as the

DNA data in A. C, SP-D measured in

the BALF of WT mice instilled with

LPS peaks by day 2. D, Kinetics of SP-

D and CitH3 in WT mice both peak at

day 2 post-LPS instillation. Statistical

analyses were performed by two-way

ANOVA with Bonferroni post test.

n=3-7 mice per condition. A-B, ***

p<0.001 compared between WT LPS

and SP-D KO LPS. C, ** p<0.01 and

*** p<0.001 compared between WT

PBS control and WT LPS.

0 1 2 3 4 50

2

4

6

8

***

*** WT PBS

SP-D KO PBS

WT LPS

SP-D KO LPS

Cit

H3

Rela

tive In

ten

sit

y

0 1 2 3 4 50

1

2

3

4

5

6

WT PBS

WT LPS

**

***

Day(s) Post Instillation

SP

-D

Rela

tive In

ten

sit

y

A

B

C

0 1 2 3 4 50

2

4

6

8

10 WT PBS

SPD KO PBS

WT LPS

SPD KO LPS

***

DN

A

g/m

l

D

0 1 2 3 4 50

2

4

6

8WT SP-D (LPS)

WT CitH3 (LPS)

0

1

2

3

4

5

6

Day(s) Post Instillation

SP

-D

Rela

tive In

ten

sit

y

CitH

3

Rela

tive In

ten

sity

66

Supplementary Figure S3.1. SP-A levels are variable in the BALF of both PBS- and LPS-

instilled WT and SP-D KO mice. Cell-free supernatant of BALF were used to assess SP-A

protein levels in PBS- and LPS-instilled mice of up to 5 days. A, Densitometry of SP-A levels in

WT and SP-D KO mice instilled with PBS. B, Densitometry of SP-A levels in WT and SP-D

KO mice instilled with LPS. Densitometry for Western blots is normalized to equal volume of

BALF loaded in each well. n=3-7 mice per condition.

BALF SP-A

0.25 1 2 3 4 50.0

250000.0

500000.0

750000.0

1000000.0WT PBS

KO PBS

Day(s) Post Instillation

Inte

nsit

y (

Arb

itra

ry U

nit

)

BALF SP-A

0.25 1 2 3 4 50.0

250000.0

500000.0

750000.0

1000000.0WT LPS

KO LPS

Day(s) Post Instillation

Inte

nsit

y (

Arb

itra

ry U

nit

)

A

B

67

SP-D enhances the clearance of NETs by alveolar macrophages ex vivo

Next we examined whether supplementing SP-D can enhance the phagocytosis of NETs ex vivo.

We tested this by adding murine AMs and SP-D to a culture of NETed neutrophils for 6 h and

18 h. Images taken by confocal microscopy after 18 h revealed that AMs are highly co-localized

with NETs. It was also noticeable that there were fewer NETs in the condition containing AMs

and SP-D compared to its control at 18 h (Fig. 3.3A). This was confirmed by quantifying the

amount of NETs leftover, which showed a significant reduction of NETs (>50 %, p<0.01) in

cultures containing both AM and SP-D compared to its control at 18 h (Fig. 3.3B). No

statistically significant difference was observed after 6 h of incubation (Fig. 3.3B). Therefore,

SP-D enhances the clearance of NETs by AMs ex vivo.

Digested NETs are ingested by macrophages

NETs are large elaborate structures that may be difficult to be cleared by macrophages. We next

tested if macrophages can clear NETs more efficiently if NETs were digested into shorter

fragments. To do so, undigested or partially-digested NETs isolated from PMA-activated human

neutrophils were added to RAW 264.7 macrophages for 2 and 18 h. An agarose gel of isolated

NETs confirms that large NETs are effectively cleaved by MNase to produce small DNA

fragments near 0.25 kb (Fig. 3.4A). A measure of fluorescence of Sytox Green DNA dye

validates that increased amounts of DNA are only found in conditions containing NETs or

MNase-digested NETs compared to its negative control (Fig. 3.4A). Immunofluorescence

images show that remnants of MNase-digested NETs stained with Sytox Green are found

localized to the cytoplasm of macrophages after 2 h. This is different from undigested NETs

which appear as thick DNA fibers attached to multiple macrophages at 2 h (Fig. 3.4B, left

panel). Interestingly after 18 h, we observe that MNase-digested NETs are no longer visible in

the cytoplasm of these macrophages (Fig. 3.4B, right panel). We also observe physical changes

to NET morphology when comparing ‘NETs only’ control between 2 and 18 h. That is, NETs

that initially appeared as thick DNA fibers at 2 h become much more dispersed and string-like

by 18 h (Fig. 3.4B, right panel). Therefore, as NETs spread out to form a larger meshwork of

DNA fibers, these structures are not the best substrates for clearance. However, nuclease-

mediated fragmentation of NETs promotes clearance by macrophages.

68

Figure 3.3. SP-D enhances the clearance of murine NETs by alveolar macrophages ex vivo.

Neutrophils isolated from WT BALB/c mice instilled with LPS (5 µg/ml for 24 h) were allowed

to undergo NET formation for 4 h before culturing with naïve alveolar macrophages (AMɸs) in

the absence or presence of SP-D (5 µg/ml) for 6 h and 18 h. A, Immunofluorescence images

showing that fewer NETs (red) are visible in the presence of SP-D after 18 h incubation with

AMɸ (green)-NET cultures. B, Manual quantification of remaining/uncleared NETs from

randomly acquired images at 6 h and 18 h. Counting was performed by two individuals in a

blinded fashion. n=3, ** p<0.01 by one-way ANOVA with Bonferroni post test.

69

Figure 3.4. Digested NETs are cleared by macrophages by 2 h.

Supernatant of PMA-activated human neutrophils contain NET-DNA that can be fragmented by

MNase. Isolated NETs (green) stained with Sytox Green is fed to RAW 264.7 macrophages

(DiD stain, purple) for 2 and 18 h at 37 °C and fixed for imaging. A, An agarose gel stained with

SYBR gold showing the absence (negative control) and presence of DNA in the supernatant of

human neutrophils untreated or treated with PMA (4 h). MNase treatment of PMA-activated

neutrophils digests NET-DNA into small 0.25 kb fragments. A measure of DNA-dye, Sytox

Green, confirms that high levels of DNA are present in NETs and MNase-treated conditions.

Image of agarose gel and Sytox Green readings are representative of three independent

experiments. B, MNase-digested NETs are found in the cytoplasm of macrophages after 2 h.

NETs alone or when incubated with macrophages appear as thick fibers at 2 h. By 18 h, NETs

appear as elaborate strings of DNA. Mɸ, RAW 264.7 macrophage; PMN, polymorphonuclear

neutrophil; MNase, micrococcal nuclease (50 µg/ml); n=1-3; scale bar=23 µm, 40 .

B

MФ+ PMN negative

control (no NETs)

NETs only

MФonly

MФ+ undigested

NETs

2 hr 18 hr

MФ+ Mnase-digested NETs

DNAMФ

15,173 85,138 67,044Sytox green reading

large NETs

fragmented NETs

A

3

10

0.25

0.75

2

kb

70

3.5 Discussion

The formation of NETs is a novel defense mechanism against microorganisms. While NETs are

beneficial in disarming pathogens, excessive NETs or impaired NET clearance is detrimental to

host cells such as pulmonary endothelial and epithelial cells in the airways. Many studies to date

have placed much emphasis on controlling or inhibiting pathways of NET formation as a

solution to restoring NET balance. Alternatively, the importance of elucidating pathways for

NET clearance has been undermined. Here in our research we focus on the role of SP-D, an

innate immune protein, in the clearance of NETs by macrophages. In our in vivo study, we

found that SP-D knockout mice exhibited slower clearance of NETs compared to WT mice (Fig.

3.1 and 3.2). We further corroborated our in vivo findings with an ex vivo assay in which the

addition of SP-D to a culture of murine NETs and AMs resulted in fewer remaining NETs

compared to its control after 18 h (Fig. 3.3). Therefore, SP-D enhances the removal of NETs by

AMs. We also show that NETs that are partially cleaved by nucleases are present in the

cytoplasm of macrophages while full-length NETs are attached to macrophages (Fig. 3.4).

In our study, we found that SP-D is important for the clearance of NETs in the airways

(Figs. 3.1-3.3). This finding is consistent with the ability of SP-D in enhancing the clearance of

DNA and other ligands by alveolar macrophages163,242,243

. Conversely, the airways of SP-D

knockout mice have increased DNA, dying cells, and anti-DNA antibodies of which can be

corrected by the treatment with recombinant SP-D (n/CRD)279

. Undoubtedly, a deficiency in SP-

D is linked to various other defects observed in older mice. Normally, SP-D is heavily involved

in the regulation of lipid homeostasis and macrophage function. Mice of 8-12 weeks old that are

deficient in SP-D have major increases in surfactant lipid pools, lipid-laden foamy macrophages

and hypertrophic type II alveolar epithelial cells and AMs303,304

. In our studies we used SP-D-

deficient mice 4-6 weeks old to avoid the severe lung phenotype observed in aged mice.

However, it is noteworthy that even younger mice show minor manifestations of foamy

macrophages and slight increases in phospholipid pools279,303

. SP-D is known to suppress

activation of AMs by binding to SIRP-1α234

. In contrast, AMs in mice lacking SP-D are highly

activated due to increases of NF-ĸB and metalloproteinase expression as well as overproduction

of ROS256,279,305

. By 6 weeks of age, apoptotic and necrotic AMs are found to be increased by 5-

to10-fold compared to their wild type counterparts. The pro-inflammatory state of SP-D-

deficient mice likely promotes neutrophil activation, thereby accelerating NETosis in the

71

airways. In addition, clearance processes are expected to be altered by aberrant AM activity and

AM hypertrophy. As such, NET clearance in SP-D-deficient mice is not simply affected by the

lack of SP-D during phagocytosis, but also by other confounding factors that arise from the null

phenotype. Despite the limitations of this mouse model, evidence from our ex vivo assay

together with our in vivo data supports a significant role for SP-D in mediating NET clearance in

the airways. We further report here that LPS changes the concentration of airway SP-D in WT

mice during inflammation and resolution. That is, airway SP-D peaks at the same time that

NETs are found most concentrated in BALF (day 2). Similarly, SP-D levels decrease during the

same time that NETs are being resolved (Fig. 3.2D). Synchronization of these events reinforces

a role for SP-D in mediating NET clearance and the resolution of inflammation. The changes in

the relative levels of SP-D correspond to our previous study25

, to which we derived our model

from. However, in the previous model, SP-D protein peaked closer to day 3. This difference

might be due to the use of older (8-12-week-old) mice and instillation of higher amounts of LPS

(5 µg) in our previous model25

.

Like SP-D, SP-A is categorized as an innate immune collectin with various roles in

immunomodulatory defense and clearance of apoptotic cells. SP-A, too, binds to and enhances

the clearance of DNA by macrophages, although less effectively than that of SP-D243,306

. Mice

deficient in SP-A have normal lung anatomy and function as well as normal macrophages and

unaltered surfactant lipid homeostasis307,308

. DNA and apoptotic cell clearance are also normal

in these mice242

. As SP-D levels remain unchanged in SP-A-deficient mice, normal clearance

pathways are likely maintained by the redundant properties of SP-D307

. SP-A-deficient mice are

mainly found to be more susceptible to infections, in addition to having defects in tubular

myelin formation308

. SP-A molecules are made up of the similar polypeptide chain as SP-D,

except that SP-As are present as hexamers of trimeric subunits which resembles a bouquet of

tulips254

. Although SP-D and SP-A are very similar proteins, whether SP-A binds to NETs is

unknown. SP-A may play a lesser role in NET clearance as SP-A binds with lower affinity to

DNA compared to SP-D. SP-A measured in the airways of WT and SP-D knockout mice show

various cycles of the protein increasing and decreasing after PBS and LPS instillation over the

course of time (Fig. S3.1). These fluctuating patterns may correlate with the translation activity

of SP-A by pulmonary cells, but the relevance of SP-A levels to NET clearance is not obvious.

72

NETs are large structures containing smooth stretches of DNA with a diameter of 15 to

17 nm and globular domains (DNA and protein) of around 25 nm. The globular domains of

NETs can further aggregate into thick bundles of fibers with diameters averaging up to 50 nm18

.

However, under flow conditions, NETs are present as elaborate networks of DNA that spread

out to be hundreds of nanometers in length and width. NETs form in the direction of the flow

and resists shear at 0.5 dyne/cm2, a physiologic shear rate expected in the capillaries of the

lung45

. Consequently, the full-length structure of NETs may cause challenges for airway

macrophages to restore balance by phagocytosis. Previously, we have noted that NETs isolated

from the airways of LPS-instilled mice are present as fragments of NETs25

. Using the same

method, except with mice instilled with 1 µg LPS, we reproduced similar findings in both WT

and SP-D knockout mice (Chapter 2, Fig. 2.3C and Chapter 3, Fig. 3.1B). We also found

nucleases to be present in the airways of WT mice through the use of genomic and NET

degradation assays (Chapter 2). Therefore, partially fragmented NETs may be a result of

endogenous airway nucleases. We further postulated that these cleaved NETs are taken up more

efficiently by macrophages. In a pilot study, we show that partially-digested NETs resided in the

cytoplasm of macrophages by 2 h. Full-length NETs, on the other hand, were largely attached to

the macrophages in addition to passing through the cell as analyzed by fluorescence microscopy

(Fig. 3.4). While NETs interacting with or attaching to macrophages is a commonly reported

feature in studies assessing NET clearance, the authors of these studies also use the same

observations as evidence for phagocytosis33,159

. AMs are specific to the lung and have been

documented as poor phagocytes of large particles. By substituting RAW 264.7 cells with MH-S

cells (a murine AM cell line) in future studies, we can more accurately determine whether

partially fragmented NETs are beneficial to NET clearance in the airways.

It was further noted in our studies that NETs that appeared as thick bundles of fibers at 2

h became much more dispersed and string-like by 18 h (Fig. 3.4B). These string-like NETs are

studded with globular domains (in ‘NETs only’ and ‘Mɸ and undigested NETs’ condition) and

are similar to the NETs shown by SEM by Brinkmann and colleagues18

and by our lab25

. The

‘spreading’ of NETs over time might indicate the need for degradation by nucleases and further

clearance by SP-D and AM to prevent damage to lung tissues. Whether this occurs in vivo is not

clearly established. Partially-digested NETs could no longer be visualized in the cytoplasm of

macrophages after 18 h. Farrera and colleagues showed that the inability to detect NETs in

73

phagocytes is due to the efficient processing of NETs within the engulfing cell159

. Using a

chloroquine inhibitor, which blocks lysosomal activity, and late endosomal marker Lamp-1,

ingested NETs were found localized to the endosomes of HMDMs post-endocytosis159

. Their

finding that NETs are localized to endosomes, combined with our data, solidifies the possibility

that fragmented NETs in the airways favours the endocytosis of NETs by AMs.

In summary, we identified a role for SP-D in facilitating NET clearance in the airways.

Our work suggests that NET-associated lung disorders might also be correlated with altered

levels of pulmonary SP-D. In fact, many clinical studies of lung disorders such as CF have been

shown to be commonly associated with low concentrations of airway SP-D309,310

. CF airway

inflammation is inversely related to pulmonary SP-D, yet positively correlated with the level of

NETs309,310

. The lack of SP-D may, therefore, be associated with ineffective clearance of

DNA/NETs in CF disease. Identification of the involvement of SP-D in mediating NET

clearance highlights the multifaceted functions of SP-D in maintaining lung tissue homeostasis.

74

Chapter 4:

Overall Discussion and Conclusions

75

4.1 Overall Discussion

Neutrophils and the newly identified neutrophil extracellular traps are major contributors to host

defense. Recently, the benefits of NETs have been overshadowed by the detrimental outcomes

of deregulated NET formation and/or impaired NET clearance. As a result, NETs have now

been placed at the forefront of several immunopathologies. Although an increasing number of

research groups have placed much emphasis on identifying pathways of NETosis, very little is

known about the processes that occur after NETs have been formed. Specifically, it is largely

assumed that NETs are removed by macrophage phagocytosis. Other studies have shown the

actions of serum nucleases in degrading NETs formed in circulation. Currently, the precise

mechanism(s) involved in airway NET clearance remains elusive. Therefore, the goal of my

Master’s project focuses on identifying clearance mechanisms to better understand how

NETosis is regulated in the lungs.

4.1.1 Airway nucleases degrade genomic DNA and NET DNA

We first sought to investigate whether endogenous nucleases in the airways are able to degrade

NETs. We identified BALF nucleases to have two specific properties: one that was dependent

on Mg2+

/Ca2+

for the degradation of genomic DNA and another that was dependent on Ca2+

for

the degradation of NETs (Chapter 2). Nucleases have been shown to exist in the airways and are

known to interfere with gene therapy by cleaving administered plasmids. The same authors of

these studies also demonstrated that plasmids are protected by the co-administration of ATA, a

direct DNase inhibitor, and enhances transfection activity in vitro and in vivo229,230

. In contrast,

EDTA and citrate, which are indirect DNase inhibitors that chelate divalent cations, exhibited

no protective effect in vivo229

. The consequence of this null effect by EDTA and citrate in vivo

might result from the inhibitors being diluted after instillation. However, in vitro, EDTA and

citrate are effective inhibitors of nucleases isolated from the BALF. Therefore, these findings

confirm the observations from our studies which show that airway nucleases are dependent on

available divalent cations for activity. The differences that we observe between genomic DNA

degradation and NET-specific degradation might suggest a physiological adaptation by the host.

That is, by having specific ion preferences, there is less competition for the same cofactor. This

could augment the efficiency of both DNA and NET clearance in the airways. Our finding that

76

NETs are degraded in a Ca2+

-dependent manner corroborates the results of another study that

investigated NET-degradation capabilities in the sera of healthy and SLE individuals. The

authors of this study found that serum DNase I augments NET-degradation in a Ca2+

-dependent

manner and that impaired degradation is correlated with disease progression in SLE individuals.

4.1.1.1 Maximal airway nuclease activity at neutral and acidic pH

In our work, we also found two pH optimums for airway nucleases from murine BALF.

Specifically, maximal activity was detected near neutral pH (6.8 and 7.0) as well as near acidic

pHs (6.6 or less). DNase I and DNase II are the most well known nucleases with optimum

activity at neutral pH and acidic pH, respectively. We additionally showed that the ion

requirements at neutral pH (Mg2+

/Ca2+

) matches those of DNase I, whereas lack thereof, at

acidic pHs, matches the profile of DNase II. Interestingly, DNase I has been reported to have

another pH optimum (5.88) when observed in the presence of Mg2+

and EGTA. The authors

found this to be a property of both serum as well as pancreatic DNase I311

. However, the use of

EGTA and EDTA in reactions with low pHs (less than 8.0) might lead to precipitation of these

chelators and/or to reduce their chelating abilities. As such, interpretation of results from low pH

reactions should be taken with caution. Nonetheless, additional studies are required to identify

whether DNase I and/or DNase II are the same nucleases in the airways. The origin of lung-

derived nucleases is not clearly established. Nucleases may be secreted by pulmonary cells such

as epithelial cells and AMs. DNase IIβ has been detected in an epithelial cell line. Yet, whether

DNase IIβ is produced and/or secreted by alveolar epithelial cells in vivo is not known. A more

likely source would be from AMs as their main role is to phagocytose material in the lung. AMs

contain DNase II and a DNase I-like nuclease referred to as DNase ɣ227

. Some authors have

described DNase ɣ to be a non-secretory protein found near the perinuclear space203

, whereas

others have found active secretion of the protein into the extracellular space227

. Thus far, it has

not been established whether DNase ɣ exists in the ASL. During inflammation, serum leakage

as well as recruited neutrophils might also contribute to the pool of airway nucleases. While

DNase II-like nucleases have been identified in neutrophils228

, the release of such an enzyme to

the ASL is not known.

Knowledge of the properties of airway nucleases could help us to understand how

pathologies arise from ineffective DNA and NET clearance. Lung pathologies might be

77

associated with an imbalance of ions, overproduction of nuclease inhibitors and/or changes to

airway pH. By restoring the conditions needed for optimal nuclease activity, the symptoms

associated with increased NETs might be alleviated. NETs have recently been detected in CF

sputum. Currently, rhDNase I is administered to destroy DNA content, and presumably NETs,

in the lungs of individuals with CF. However, rhDNase I is not effective for a subset of CF

patients, known as “non-responders”, who have decreased levels of Mg2+

in their sputum. When

Mg2+

levels are restored to normal physiological concentrations, degradation of sputum DNA by

rhDNase I was also restored. Thus far, the effect of supplementing Mg2+

/Ca2+

to the airways on

DNA degradation has not been established. Based on our work, supplementing airway Ca2+

to

degrade NETs is a possible option in treating NETs-related diseases. Furthermore, CF patients

have acidified airways with pH measured from EBCs to be as low as 5.32300

. This acidification

process reduces bacterial killing and fosters growth of P. aeruginosa298

. By raising the pH

slightly (from 6.8 to 7.4) in a porcine CF model, antimicrobial activity was restored298

. This

study, as well as data from our work, demonstrates the importance of a buffered

microenvironment for enzymes to function optimally. As we know now that NETs are major

contributors to lung disorders, the processes that regulate NET formation and NET clearance are

likely disturbed in these diseases.

4.1.2 SP-D enhances the clearance of NETs

With proper clearance mechanisms in place, the novelty of NET formation and the role of NETs

as a host immune defense is not compromised. NETs that are removed in a timely manner

prevent lung tissues from being damaged by cytotoxic NETs. Currently, there is only one study

identifying a specific pathway for NET clearance159

. The authors of this study suggest that

physiological DNase I does not completely degrade NETs and that this partial process requires

additional mechanisms to remove NETs from circulation. This study used HMDMs and found

that the removal of NETs was facilitated by C1q and DNases in an endocytic manner. However,

there are no established pathways for the clearance of NETs in the airways. In our work, we

sought to identify such a pathway and speculate that SP-D is required. SP-D is an innate

immune pattern recognition molecule that is present on mucosal surfaces248–251

. Previous studies

have shown that SP-D enhances microbial clearance by microagglutination and modulates

immune cell functions163,254

. Our lab also showed that SP-D mediates DNA clearance and that

78

the lack of SP-D leads to the generation of anti-DNA autoantibody production242

. Recently, our

lab characterized a novel interaction between SP-D and NETs in facilitating the trapping of

microagglutinated bacteria. Based on the role of SP-D in clearance pathways and the property

that SP-D binds to NETs, we predicted that SP-D will enhance the uptake of NETs by

macrophages in the airways. In our study, we find that SP-D-deficient mice are defective in

NET clearance compared to WT mice. We further corroborate this finding with an ex vivo assay

which showed that the supplementation of SP-D led to fewer remaining NETs in AM-NET

cultures. Therefore, we show that SP-D enhances the clearance of NETs by AMs (Chapter 3).

Although not yet established, it is likely that SP-D also enhances the removal of

microagglutinated bacteria trapped on NETs.

4.1.3 Clearance of NET fragments by macrophages

Largely owing to the nature of the lung environment, AMs are a unique set of cells that are

distinct from peritoneal macrophages. AMs are described as poor phagocytes of early (large)

apoptotic cells compared to peritoneal macrophages238–240

. Our lab showed that rather than

ingesting large particles, AMs are more efficient at phagocytising late (small) apoptotic cells,

especially in the presence of apoptotic binding agents, IgM and SP-D153

. AMs are constantly

burdened by microparticle invasion and it is likely that these cells prefer to clear small particles

over larger matter. NETs are large, elaborate structures that stretch up to hundreds of

nanometers in length and width under flow conditions. We postulate that NETs are cleaved into

smaller fragments in the airways and that NET fragments are phagocytosed more efficiently by

macrophages. Previously, our lab showed that airway NETs are present as fragments in the

airways of 8-12-week-old WT mice. Using a similar model in our present study, we reproduced

these findings in WT and SP-D knockout mice of a younger age (4-6-week-old) (Chapter 2 and

Chapter 3). We also found nucleases to be present in the airways of WT mice which are able to

cleave both genomic DNA and NETs (Chapter 2). Therefore, the observation of partially

fragmented NETs is likely due to the activities of endogenous airway nucleases. Incomplete

digestion of airway NETs in vivo might be due to the presence of NET proteins, proteases

and/or actin molecules known for blocking access or inhibiting DNase activity. Using a

phagocytic assay with isolated full-length or partially-digested NETs, we show that

macrophages are able to clear small NETs more efficiently than thick fibres of NETs. This

79

confirms the findings by Farrera and colleagues who demonstrated that DNase I involvement

significantly enhances C1q-mediated NET clearance by HMDMs. The authors further show that

NETs are endocytosed by macrophages. Their study, combined with our data, supports the

possibility that fragmented NETs in the airways favours endocytosis by AMs25

. C1q is a

complement protein found in circulation312

. During inflammation, C1q may infiltrate the lungs

from the serum and take part in processes involved in complement activation313,314

. While it has

been shown that C1q enhances the clearance of apoptotic cells through calreticulin/CD91 on

AMs, C1q is not a lung-specific protein151,268

. SP-D, on the other hand, is a lung protein. SP-D

not only binds to full-length NETs, but also to fragmented NETs25

. Therefore, we predict that

SP-D binding to fragmented NETs also facilitates their removal by AMs in an endocytic

manner.

In summary, we identified a role for pulmonary nucleases and SP-D in facilitating NET

clearance in the airways. Therapeutic interventions targeting the breakdown of NETs may need

to focus on adjusting airway pH back to normal values or the re-activation of endogenous

airway nucleases. From our study, we suggest that NET-associated lung disorders might also be

correlated with altered levels of pulmonary SP-D. The work that we have demonstrated here

illustrates the importance of regulating NET clearance in order to balance NET formation.

4.2 Conclusions

Excessive NET production or ineffective clearance is associated with several lung disorders. In

Chapter 2, we conclude that nucleases do exist in the airways of mice and are functionally active

at degrading gDNA and NETs in a Mg2+

/Ca2+

- and Ca2+

-dependent manner, respectively.

During inflammation, it is possible that two types of nucleases exist: divalent-dependent

nucleases that work optimally at neutral pH and divalent-independent acidic nucleases. SP-D is

highly involved in phagocytic processes, especially in the lungs. In Chapter 3, we further

demonstrate the importance of SP-D in enhancing the clearance of NETs ex vivo with AMs as

well as in vivo using SP-D knockout mice. We conclude that NET fragments are also

phagocytosed by macrophages more efficiently.

80

4.3 Future Directions

The exact identities of airway nucleases are not known. Therefore, future experiments should

focus on inhibition assays and protein work to identify different classes of nucleases present.

More importantly, the source of nuclease secretion may be determined in vitro by assessing the

supernatant of cultured cells (AMs, neutrophils, pulmonary epithelial cells) under activated and

non-activated conditions. We can further explore the idea of instilling Mg2+

and Ca2+

to the

airways of healthy and diseased mice as a new form of treatment for the destruction of

accumulated DNA. Isotonic saline solutions are normally used to deliver drugs through

nebulizers. Isotonic saline solutions may be exchanged against isotonic Mg2+

/Ca2+

solutions

containing additional buffers before being administered to the airways of mice. The degradation

of NETs by nucleases is one component of the clearance process which likely facilitates the

uptake of NET fragments by AMs. Therefore, additional studies should assess whether NET

fragments are preferentially phagocytosed over uncleaved NETs.

Our lab was the first to show that SP-D binds DNA266

and NETs25

. In chapter 3, we

identified a role for SP-D in mediating NET clearance. Future experiments should focus on SP-

D rescue studies to assess whether normal NET clearance can be restored in SP-D knockout

mice. As AMs are the main phagocytes of the lungs, immunohistochemical staining for NET

markers in lung sections can verify whether NETs are indeed taken up by AMs in these mice.

Thus far, only one study has attempted to elucidate the mechanism of NET clearance in

circulation. These authors found that C1q promotes the uptake of NETs by HMDMs via

endocytosis. However, the receptors involved in C1q-mediated clearance were not identified159

.

In the present study, we have data supporting that NETs exist as fragments in the airways and

that clearance of these fragments may be more efficient over full-length NETs. We postulate

that the clearance of NET fragments is further assisted by SP-D via the endocytic receptor,

CD91. The CD91 receptor, also known as the alpha-2-macroglobulin (A2M) receptor, is found

on AMs and binds to A2M ligands in the airways to promote several endocytic processes. A2M

is a serum protease inhibitor which fills the airways by up to 100-fold during

inflammation272,273

. While our lab has identified a strong interaction between SP-D and A2M in

the human ASL271

, the implication of this interaction in clearance pathways is unknown. It is

possible that NETs bound to SP-D-A2M enhances NET clearance via the CD91 receptor on

81

AMs. Overall, NET formation is only favourable to the host as long as functional clearance

mechanisms are in place. Perhaps the advent of partially fragmented NETs over complete

digestion permits pathogen trapping while promoting efficient clearance of the ensnared

pathogen. Complete degradation of DNA results in increased concentrations of nucleotides that

are highly pro-inflammatory and recruits additional immune cells315

.

82

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 David N. Douda, Lily Yip, Meraj A. Khan, Hartmut Grasemann and Nades Palaniyar neutrophil death to apoptosisAkt is essential to induce NADPH-dependent NETosis and to switch the

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the MCs of 5 of 19 FP-positive CEL patients (Figure 1). The KITD816V mutation was not detectable in DNA/RNA extracted fromwhole white blood cells derived from peripheral blood or BM samples,probably as a consequence of the very low burden of mutated KITD816V-positive cells, which was only detectable in DNA obtainedfrom microdissected MCs. Fluorescence Immunophenotyping andInterphase Cytogenetic as a Tool for Investigation Of Neoplasia(FICTION) using aVysis 4q12 tri-color rearrangementfluorescence insitu hybridization probe kit (Abbott Molecular, Wiesbaden, Germany)resulted in 1 green/aqua fusion signal with a deletion of the orangesignal of the CHIC2 gene in the nuclei of tryptase1 BM MCs ina patient with KIT D816V-positive MCs (Figure 1). The presence ofCD251-positive MCs with concomitant KIT D816V and serumtryptase levels .20 ng/mL support the diagnosis of SM-AHNMD/SM-FP–positive CEL in 2 patients. This discrepancy to the previouslypublished negative KITD816V mutational analysis of microdissectedMCs may be explained by a sampling effect because only 2 FP-positive SM-CEL cases were examined.6 It remains elusive whetherFP andKITD816V are present in the same clone or whether there are2 separate clones. We reported on the heterogeneity of molecularaberrations in KIT D816V-positive SM with $1 additional mutation,for example, TET2, SRSF2, ASXL1, and others in 24 of 27 patients.7 Ina murine model, expansion of eosinophils andMCs may result from aninteraction between FP, interleukin 5, the ligand stem cell factor, andKIT in the absence of a KITmutation.8 It can be speculated that the FPfusion gene favors secondary KIT mutations in MCs via growth andproliferation signals or that a yet unknownmechanism causes genomicinstability with independent evolution of FP and KIT D816V.

Annette Hildegard Schmitt-Graeff

Departement fur Pathologie, Universitatsklinikum Freiburg,

Freiburg, Germany

Philipp Erben

III Medizinische Klinik, Universitatsmedizin Mannheim,

Mannheim, Germany

Juliane Schwaab

III Medizinische Klinik, Universitatsmedizin Mannheim,

Mannheim, Germany

Beate Vollmer-Kary

Departement fur Pathologie, Universitatsklinikum Freiburg,

Freiburg, Germany

Georgia Metzgeroth

III Medizinische Klinik, Universitatsmedizin Mannheim,

Mannheim, Germany

Karl Sotlar

Pathologisches Institut, Ludwig-Maximilians-Universitat Munchen,

Munchen, Germany

Hans-Peter Horny

Pathologisches Institut, Ludwig-Maximilians-Universitat Munchen,

Munchen, Germany

Hans-H Kreipe

Institut fur Pathologie, Medizinische Hochschule Hannover,

Hannover, Germany

Paul Fisch

Departement fur Pathologie, Universitatsklinikum Freiburg,

Freiburg, Germany

Andreas Reiter

III Medizinische Klinik, Universitatsmedizin Mannheim,

Mannheim, Germany

A.H.S.-G., P.E., and A.R. contributed equally to this work.

Acknowledgments: This work was supported by the European Leukemia Net,

Work Package 9.

Contribution: A.H.S.-G., P.E., and A.R. designed the study; A.H.S.-G., P.E.,

J.S., B.V.-K., P.F., andA.R. performedexperiments andanalyzed data; A.H.S.-G.,

P.E., J.S. G.M., and A.R. collected patients’ samples; K.S. gave advice

concerning the KIT D816V mutational analysis; A.H.S.-G., K.S., H.-P.H., and

H.-H.K. critically reviewed patients’ BM biopsies; A.H.S.-G., P.E., G.M., P.F.,

and A.R. wrote the manuscript; and all authors critically reviewed and edited

the paper.

Conflict-of-interest disclosure: The authors declare no competing financial

interests.

Correspondence: Annette H. Schmitt-Graeff, Departement fur Pathologie,

Universitatsklinikum Freiburg, Breisacherstrasse 114a, D-79106 Freiburg,

Germany; e-mail: annette.schmitt-graeff@uniklinik-freiburg.de.

References

1. Swerdlow SH, Campo E, Harris NL, et al, eds. World Health OrganizationClassification of Tumours of Haematopoietic and Lymphoid Tissue, 4th ed. Lyon,France: IARC Press; 2008.

2. Pardanani A, Brockman SR, Paternoster SF, et al. FIP1L1-PDGFRA fusion:prevalence and clinicopathologic correlates in 89 consecutive patients withmoderate to severe eosinophilia. Blood. 2004;104(10):3038-3045.

3. Cools J, DeAngelo DJ, Gotlib J, et al. A tyrosine kinase created by fusion of thePDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathichypereosinophilic syndrome. N Engl J Med. 2003;348(13):1201-1214.

4. Metzgeroth G, Schwaab J, Gosenca D, et al. Long-term follow-up of treatmentwith imatinib in eosinophilia-associated myeloid/lymphoid neoplasms withPDGFR rearrangements in blast phase. Leukemia. 2013;27(11): 2254-2256.

5. Pardanani A, Lim KH, Lasho TL, et al. Prognostically relevant breakdown of 123patients with systemic mastocytosis associated with other myeloid malignancies.Blood. 2009;114(18):3769-3772.

6. Sotlar K, Colak S, Bache A, et al. Variable presence of KITD816V in clonalhaematological non-mast cell lineage diseases associated with systemicmastocytosis (SM-AHNMD). J Pathol. 2010;220(5):586-595.

7. Schwaab J, Schnittger S, Sotlar K, et al. Comprehensive mutational profiling inadvanced systemic mastocytosis. Blood. 2013;122(14):2460-2466.

8. Yamada Y, Sanchez-Aguilera A, Brandt EB, et al. FIP1L1/PDGFRalphasynergizes with SCF to induce systemic mastocytosis in a murine model ofchronic eosinophilic leukemia/hypereosinophilic syndrome. Blood. 2008;112(6):2500-2507.

© 2014 by The American Society of Hematology

To the editor:

Akt is essential to induce NADPH-dependent NETosis and to switch the neutrophil deathto apoptosis

Neutrophil extracellular traps (NETs) have been recently identified asmajor contributors of several hematological and vascular diseases.These disorders include thrombosis, small vessel vasculitis, systemic

lupus erythematosus, autoimmunity, pneumonia, sepsis, and bloodtransfusion–related acute lung injury.1-4 NETs are DNA-based extra-cellular traps that not only trap and kill invading microbes but also

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Figure 1. Akt shifts NETosis to apoptosis in human neutrophils. (A) Immunoblot analysis for the activation of Akt as determined by its phosphorylation. Cells were lysed

after 1 hour of PMA (25 nM) activation. DPI (20 mM) was used for inhibiting NOX2-mediated ROS production. Total Akt and glyceraldehyde-3-phosphate dehydrogenase were

used as loading control (n5 6 donors;2ve, negative control; DPI→ PMA, neutrophils were pretreated with DPI [20 mM] and then activated with PMA). (B) Analysis of cells for

their production of ROS by flow cytometry. Prior to the activation with PMA (25 nM), cells were preincubated for 30 or 60 minutes in the presence or absence of Akt inhibitor XI

(Akt-i XI; 10 mM; Akt-i XI → PMA) or DPI (20 mM; DPI → PMA) for 30 minutes. In all conditions, cells were also incubated with dihydrorhodamine 123 as a probe for ROS

production. Cells were gated using forward and side scatter and confirmed with Hoechst 3342 as a counterstain. Shown is a representative of 4 independent experiments with

cells from 4 individuals. (C-D) Fluorescence plate reader assay for NETosis. Cells were cultured in a 96-well culture plate (3 3 105 cells per well) in the presence of Sytox

Green (5 mM; Invitrogen), a cell impermeable DNA-binding dye, to monitor release of NET DNA. Varying concentrations (0-10 mM) of 2 different Akt-i, (C) Akt inhibitor XI

(Millipore), and (D) MK2206 (Sellekchem), were added to the cells 30 minutes prior to the activation of cells with PMA (25 nM). Numbers beneath the graphs represent the

concentration of Akt-i used prior to activation with PMA. Extracellular DNA release was monitored at t 5 0, 30, and 60 minutes and every hour for a total of 5 hours. Shown is

the fluorescence intensity at 5 hours. NETotic index was calculated as percentage of total fluorescence given off by PMA-only positive control. (n 5 4-7 donors). (E)

Differential quantification of live, NETotic, and apoptotic (pyknotic) nuclei. Cells were cultured in an imageable special optics 96-well plate in the presence or absence of Akt-i

(0-10 mM) for 30 or 60 minutes prior to the activation of PMA (25 nM). The numbers in parentheses represent the concentration of Akt-i used prior to activation with PMA. Live

and apoptotic nuclei are not stained by Sytox Green dye unless the cells are fixed. Thus, the cells from the plate reader assay were fixed at the end of the assay in the

presence of the dye, and cells were differentially quantified on the basis of their nuclear morphology. Representative images of live, NETotic, and apoptotic nuclei quantified

are shown below the graph. At least 100 cells were quantified in each condition (n5 4-5 individual donors). (F) Immunofluorescence staining for myeloperoxidase (MPO) and

cCasp3. Neutrophils were incubated with H2O2 (8 mM) to induce necrosis. In other conditions, cells were activated with PMA (25 nM) with or without preincubation for 30

minutes with Akt inhibitor XI. Cells were stained with MPO (mouse a-MPO, 1:250; Abcam) as a marker for NETs and cCasp3 (rabbit a-cleaved caspase 3, 1: 150; Cell

Signaling) as a marker for apoptosis. Arrows, NETotic DNA and nuclei; arrowheads, cCasp3-positive cells. Bar: 10 mm (n 5 4 donors). All data are expressed as mean 6

standard error of the mean where appropriate. *P , .05 and ***P , .001 compared with PMA only controls (C-D). Analysis of variance with (C-D) Dunnett’s or (E) Bonferroni

post-tests was used for determining statistical significance. (G) Proposed role of Akt in regulating the switch between NETosis and apoptosis.

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injure host tissues.1,5-7 Therefore, regulating NETosis is importantto prevent many pathological conditions.1 However, key moleculesthat switch neutrophil death from NETosis, which is proinflam-matory, to apoptosis, which is anti-inflammatory, have not beenclearly established.

Nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-dependent reactive oxygen species (ROS) production in neutrophilscan induce either NETosis or apoptosis. Phorbol 12-myristate 13-acetate (PMA) has been extensively used as an agonist to activateNOX2-mediated ROS production to study NETosis.6,8 A seminalstudy showed that PMA induces autophagy and that both autophagyand PMA-mediated ROS production are required for NETosis.8

This inference was made based on the inhibitory effect of a proteinkinasae C inhibitor (wortmannin) on PMA-mediated autophagy andNETosis. In another study, rapamycin was used for directly suppres-sing mammalian target of rapamycin, a well-established regulator ofautophagy. These studies show that mammalian target of rapamycinregulates NETosis via modifying hypoxia-inducible factor HIF1-a.9

However, the identities of other key kinases that regulate NETosis-apoptosis pathways remain elusive.

Akt is a well-known inhibitor of apoptosis.10 Inhibition of Aktusing pharmacological inhibitors promotes apoptosis in many celltypes. Hence, it is an excellent candidate to act as a direct molecularswitch for regulating the NETosis-apoptosis axis. Here we show thatPMA activates Akt during the induction of NETosis (Figure 1A, lanes1 and 2), whereas the NOX2 inhibitor diphenyleneiodonium (DPI)completely suppresses Akt activation (Figure 1A, lane 3). Therefore,Akt activation is dependent onNOX2-mediatedROSproduction. Flowcytometry analysis confirms the production of ROS in these cells andshows that DPI, but not Akt-specific inhibitor (Akt-i) XI, inhibitsPMA-induced ROS production (Figure 1B). In a Sytox Green platereader assay, 2 different Akt inhibitors, M2206 and XI, inhibit DNArelease by activated neutrophils in a dose-dependent manner(Figure 1C-D). Therefore, activation of Akt is essential for NOX2-mediated NETosis.

To assess whether Akt is involved in redirecting NETosis toapoptosis, immunofluorescence microscopy and quantitative analyseswere performed. The results show that preincubation of cells withAkt-i XI dose dependently increases the number of apoptotic cellscontaining pyknotic nuclei and a concomitant decrease in NEToticcells (Figure 1E). Immunofluorescence microscopy analysis ofMPO and cleaved caspase 3 (cCasp3) further confirms that theinhibition of Akt switches neutrophil death from NETosis toapoptosis (Figure 1F). H2O2 (8 mM) is known to induce necrosis inneutrophils.8 Necrosis in neutrophils neither activates apoptoticcaspase 3 nor precoats MPO on DNA before release (Figure 1F).Collectively, these data show that NOX2-mediated NETosis isdependent on Akt activation, and suppression of Akt switchesNETosis to apoptosis.

Based on the data presented in this study, we propose that Akt isa bona fide molecular switch that regulates the NETosis-apoptosisaxis (Figure 1G). Taken together, PMA-mediated NOX2-dependentactivation of Akt induces NETosis while suppressing apoptosis.Suppression of Akt, on the other hand, allows for the induction ofcaspase-dependent apoptosis. The finding that NETosis and apo-ptosis are 2 opposing pathways in neutrophils and that NETosis canbe redirected by targeting Akt could provide avenues for noveltherapeutic strategies to treat NET-related hematological and otherinflammatory disorders.

David N. Douda

Lung Innate Immunity Research Laboratory,

Program in Physiology and Experimental Medicine,

SickKids Research Institute,

The Hospital for Sick Children,

Toronto, ON, Canada

Department of Laboratory Medicine and Pathobiology,

University of Toronto,

Toronto, ON, Canada

Lily Yip

Lung Innate Immunity Research Laboratory,

Program in Physiology and Experimental Medicine,

SickKids Research Institute,

The Hospital for Sick Children,

Toronto, ON, Canada

Department of Laboratory Medicine and Pathobiology,

University of Toronto,

Toronto, ON, Canada

Meraj A. Khan

Lung Innate Immunity Research Laboratory,

Program in Physiology and Experimental Medicine,

SickKids Research Institute,

The Hospital for Sick Children,

Toronto, ON, Canada

Hartmut Grasemann

Division of Respiratory Medicine, Department of Paediatrics,

The Hospital For Sick Children,

Toronto, ON, Canada

Institute of Medical Sciences,

University of Toronto,

Toronto, ON, Canada

Nades Palaniyar

Lung Innate Immunity Research Laboratory,

Program in Physiology and Experimental Medicine,

SickKids Research Institute,

The Hospital for Sick Children,

Toronto, ON, Canada

Department of Laboratory Medicine and Pathobiology and

Institute of Medical Sciences,

University of Toronto,

Toronto, ON, Canada

Acknowledgments: Approval to obtain blood samples from healthy volunteers

for the study was approved by the Research Ethics Board of the Hospital for Sick

Children.

D.N.D. was supported by an Ontario graduate scholarship, the Ontario Student

Opportunity Trust Fund/SickKidsRestracomp, theDrGoranEnhorningAward in

Pulmonary Research, and the Peterborough K.M. Hunter graduate studentship.

L.Y. was supported by an Ontario graduate scholarship. M.A.K. received

a postdoctoral fellowship from the operating grants awarded to N.P. from Cystic

Fibrosis Canada (grant 2619) and Canadian Institutes of Health Research

(CIHR; MOP-111012). This work was funded by CIHR operating grant MOP-

111012 to N.P.

Contribution:D.N.D. designedand conducted experiments, analyzed the data,

and wrote the manuscript; L.Y. and M.A.K. did experiments, analyzed the data,

and participated in manuscript revisions and editing; H.G. participated in

experimental design; and N.P. conceived the project, supervised the experi-

ments, analyzed the data, and participated in manuscript revisions and editing.

Conflict-of-interest disclosure: The authors declare no competing financial

interests.

Correspondence: Nades Palaniyar, Lung Innate Immunity Research Labo-

ratory, The Hospital For Sick Children, 555 University Ave, Toronto, ON,

Canada M5G 1X8; e-mail: nades.palaniyar@sickkids.ca.

References

1. Cheng OZ, Palaniyar N. NET balancing: a problem in inflammatory lungdiseases. Front Immunol. 2013;4:1.

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2. Sangaletti S, Tripodo C, Chiodoni C, et al. Neutrophil extracellular trapsmediate transfer of cytoplasmic neutrophil antigens to myeloid dendriticcells toward ANCA induction and associated autoimmunity. Blood. 2012;120(15):3007-3018.

3. Thomas GM, Carbo C, Curtis BR, et al. Extracellular DNA traps are associated withthe pathogenesis of TRALI in humans and mice. Blood. 2012;119(26):6335-6343.

4. Chen K, Nishi H, Travers R, et al. Endocytosis of soluble immune complexesleads to their clearance by FcgRIIIB but induces neutrophil extracellular trapsvia FcgRIIA in vivo. Blood. 2012;120(22):4421-4431.

5. Douda DN, Jackson R, Grasemann H, Palaniyar N. Innate immune collectin surfactantprotein D simultaneously binds both neutrophil extracellular traps and carbohydrateligands and promotes bacterial trapping. J Immunol. 2011;187(4):1856-1865.

6. Brinkmann V, Zychlinsky A. Neutrophil extracellular traps: is immunity thesecond function of chromatin? J Cell Biol. 2012;198(5):773-783.

7. Yousefi S, Simon D, Simon HU. Eosinophil extracellular DNA traps: molecularmechanisms and potential roles in disease. Curr Opin Immunol. 2012;24(6):736-739.

8. Remijsen Q, Vanden Berghe T, Wirawan E, et al. Neutrophil extracellular trapcell death requires both autophagy and superoxide generation. Cell Res. 2011;21(2):290-304.

9. McInturff AM, Cody MJ, Elliott EA, et al. Mammalian target of rapamycinregulates neutrophil extracellular trap formation via induction of hypoxia-inducible factor 1 a. Blood. 2012;120(15):3118-3125.

10. Rane MJ, Klein JB. Regulation of neutrophil apoptosis by modulation ofPKB/Akt activation. Front Biosci (Landmark Ed). 2009;14:2400-2412.

© 2014 by The American Society of Hematology

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