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Distinct Oral Neutrophil Subsets Define Health and Disease States
by
Siavash Hassanpour
A thesis submitted in conformity with the requirements for the degree of Master of Science
Faculty of Dentistry University of Toronto
© Copyright by Siavash Hassanpour 2017
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Distinct Oral Neutrophil Subsets Define Health and Disease
States
Siavash Hassanpour
Master of Science
Faculty of Dentistry
University of Toronto
2017
Abstract
Neutrophils exit the vasculature and swarm to sites of inflammation and infection. Neutrophils
are also actively recruited to the healthy periodontium without eliciting clinical signs of
inflammation, suggesting a unique immune surveillance neutrophil function. We hypothesized
that oral neutrophils in the healthy periodontium exist in a “para-inflammatory” state that allows
neutrophils to symbiotically interact with the commensal oral microflora. Using the principles of
immunophenotyping, we identified four distinct neutrophil subsets: resting circulatory
neutrophils, two para-inflammatory and one pro-inflammatory tissue neutrophil phenotypes in
the absence and presence of chronic inflammation, respectively. This work is the first to
comprehensively characterize healthy oral neutrophils and identity the para-inflammatory
neutrophil phenotype, thereby demonstrating that not all neutrophils trafficking through
periodontium are fully activated. In addition to establishing possible diagnostic and treatment
monitoring biomarkers, the oral tissue neutrophil phenotype model builds on existing literature
suggesting that the healthy periodontium may be in a para-inflammatory state.
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Acknowledgments
I would like to start by thanking by wonderful and beautiful wife Kelley Hassanpour who has
endured a seemingly never ending (thirteen-year) journey. None of my accomplishments would
have been possible without her boundless love and support. Kelley, I want to thank you for being
a constant source of happiness and my greatest motivation. We finally did it!
I would like to thank my mentor and supervisor Dr. Michael Glogauer. In 2008, with my
rejection from dental school looming large over my head, Michael gave me an opportunity to
work towards a Master of Science Degree. His faith in me allowed me to not only gain my first
Master of Science degree in 2010 but also propelled me into dental school and later into the
graduate periodontology program. Michael, I want to thank you for always believing in me and
for pushing me to go further than I ever thought possible. The lessons that I have learned from
you will stay with me for a life time and I am forever grateful for the opportunities you have
given me. I would also like to extend a warm thanks to my advisory committee members Drs.
Howard Tenebaum and James Scholey who’s guidance and wisdom were instrumental in the
completion of this project.
I would like to thank my dear friends from the University of Toronto’s Dentistry Class of IT4.
Timur Shigapov, Shlomi Tamam, Nicholas Tong, Alice Chen and Emily Tichenoff, I want to
thank you all for helping me survive the rigors of dental school. I also want to thank all the
Glogauer lab members who have been an integral part of my journey. I want to thank Jan Kuiper,
Noah Fine, Chunxiang Sun, Yongqiang Wang, Roland Leung, Guy Aboodi, Carol Forster,
Michael Lim, Corneliu Sima, Morvarid Oveisi and Alon Borensetin. Thank you all for your help
and support through the years. A very special thank you to Flavia Lakschevtiz who has literally
been there with me every step of the way for the last six years.
Finally, I want to thank my parents, Shohre Mahdi and Hosein Hassanpour and my sister
Maryam Amini, who’s countless sacrifices have given me a once in a life time opportunity.
Thank you everyone!
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Table of Contents
Contents
Acknowledgments .......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Tables ................................................................................................................................. vi
List of Figures ............................................................................................................................... vii
Abbreviations ............................................................................................................................... viii
Chapter 1: Introduction ................................................................................................................... 1
Chapter 2: Literature Review .......................................................................................................... 2
2.1 The Inflammatory Spectrum and Pathogenesis of Periodontitis ......................................... 2
2.1.1 The Inflammatory Spectrum ................................................................................... 2
2.1.2 Inflammation and Periodontitis ............................................................................... 3
2.1.3 Pathogenesis of Chronic Periodontitis .................................................................... 3
2.2 Neutrophil Homeostasis and Lifecycle ............................................................................... 5
2.2.1 Neutrophil Differentiation ...................................................................................... 5
2.2.2 Circulatory Neutrophils .......................................................................................... 6
2.2.3 Neutrophil Extravasation ........................................................................................ 7
2.2.4 Neutrophil Clearance .............................................................................................. 8
2.3 Neutrophil Function ............................................................................................................ 9
2.3.1 Neutrophil Granules ................................................................................................ 9
2.3.2 Neutrophil Mediated Bacterial Killing ................................................................. 11
2.4 Neutrophil Plasticity and Heterogeneity ........................................................................... 12
2.4.1 Neutrophil Subpopulations ................................................................................... 13
2.4.2 Identification and Characterization and Cellular Subpopulations ........................ 14
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2.5 Oral Neutrophils as a Unique Population of Neutrophils ................................................. 16
Statement of the Problem .............................................................................................................. 20
Chapter 3 : Materials and Methods ............................................................................................... 21
3.1 Human Subjects ................................................................................................................ 21
3.2 Sample Collection and Processing .................................................................................... 21
3.3 Electron Microscopy ......................................................................................................... 22
3.4 Histopaque Density Centrifugation ................................................................................... 22
3.5 Multicolor Flow Cytometry .............................................................................................. 23
3.6 ROS Assay ........................................................................................................................ 23
3.7 NET Assay ........................................................................................................................ 24
3.8 Statistical Analysis ............................................................................................................ 24
Chapter 4 : Results ........................................................................................................................ 25
4.1 Clinical Description of Periodontal Status ........................................................................ 25
4.2 CP Oral Neutrophils Are More Degranulated and Have Increased Phagocytosis
Compared to Healthy Oral Neutrophils ............................................................................ 25
4.3 Flow Cytometric Gating Strategy ..................................................................................... 26
4.4 Characterization of Oral Neutrophil Populations in Health and CP ................................. 26
4.5 Pro-inflammatory Neutrophils Have Elevated ROS Production and NET Formation ..... 27
Chapter 5 : Discussion .................................................................................................................. 28
Conclusion and Future Directions ................................................................................................ 31
Figure Legend ............................................................................................................................... 33
Tables and Figures ........................................................................................................................ 36
Contributions to the Thesis and Manuscript ................................................................................. 49
References ..................................................................................................................................... 50
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List of Tables
Table 1: CD Marker Panels for Multicolor Flow Cytometry.
Table 2: Demographic and Periodontal Characteristics of Patients Cohorts.
Table 3: CD Marker Expression Levels on Healthy and CP Neutrophils.
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List of Figures
Figure 1. Neutrophil Recruitment to the Periodontium in Health and Disease.
Figure 2. Etiology and Pathogenesis of Chronic Periodontitis.
Figure 3. Multicolor Flow Cytometry Gating Strategy for Blood and Oral Neutrophils.
Figure 4. Oral Neutrophils Have Elevated Phagocytosis and Greater Degranulation in CP.
Figure 5. Identification of Para-inflammatory Neutrophils in Oral Health.
Figure 6. Para- & Pro-inflammatory Oral Neutrophil Populations Are Defined by Unique CD
Marker Expression Profiles.
Figure 7. CD Markers Can Be Used to Gate on Para1 and Para2 Oral Neutrophil Populations.
Figure 8. Pro-inflammatory Oral Neutrophil Populations Have Elevated NET Formation and
Increased ROS Production Compared to Para-inflammatory Neutrophils.
Figure 9. Model of Para- and Pro-inflammatory Neutrophil Phenotypes in Health and Disease.
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Abbreviations
AO – Antioxidant
BOP – Bleeding on Probing
CAL – Clinical Attachment Loss
CCL – Chemokine Ligand
CD – Cluster of Differentiation
CGD - Chronic Granulomatous Disease
CP – Chronic Periodontitis
CXCR - CXC Chemokine Receptor
DEL - Developmental Endothelial Locus
DHR - Dihydrorhodamine
DNA - Deoxyribose Nucleic Acid
E/A - Epon Araldite
FACS - Fluorescence-Activated Cell Sorting
fMLP – Formyl-methionyl-leucyl-phenylalanine
FMO – Fluorescence Minus One
FSC – Forward Scatter
GCF - Gingival Crevicular Fluid
G-CSF – Granulocyte - Colony Stimulating Factor
GI – Gastrointestinal
HD – High Density
HDN – High Density Neutrophils
HLA - Human Leukocyte Antigen
HTS - High-Throughput Screening
ICAM – Intercellular Adhesion Molecule
INF – Interferon
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IL – Interleukin
LD – Low Density
LDN – Low Density Neutrophils
LFA- Leukocyte Function Associate Antigen
LXR – Liver X Receptor
NAPDH - Nicotinamide Adenine Dinucleotide Phosphate
NET – Neutrophil Extracellular Trap
NRF2 - Nuclear Factor Erythroid 2-Related Factor
MFI - Mean Fluorescence Intensity
MMP - Matrix Metalloproteinases
MPO - Myeloperoxidase
MRSA - Methicillin-Resistant Staphylococcus aureus
PARA – Para-inflammatory
PBS – Phosphate-Buffered Saline
PD – Probing Depth
pDC - Plasmacytoid Dendritic Cells
PFA – Paraformaldehyde
PKC – Protein Kinase C
PMA - Phorbol 12-Myristate 13-Acetate
PMN – Polymorphonuclear Cells or Neutrophils
PO - Propylene Oxide
PRO – Pro-inflammatory
RNA – Ribonucleic Acid
ROS - Reactive Oxygen Species
SSC – Side Scatter
SLE - Systemic Lupus Erythematous
x
TAN - Tumor Associated Neutrophils
TNF - Tumor Necrosis Factor
TLR - Toll-Like Receptors
VAMP - Vesicle Associated Membrane Protein
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Chapter 1: Introduction
Neutrophils are essential primary innate immune responders that kill invading pathogens, but can
also cause substantial host tissue damage as part of the inflammatory response to those pathogens
[1]. Chronic periodontitis (CP), a highly prevalent, dysbiotic, inflammatory condition of the oral
cavity, is a model of tissue destruction mediated by the pro-inflammatory neutrophil response [2-
7]. CP is characterized by amplified neutrophil recruitment [8], gingival inflammation and
progressive, irreversible loss of periodontal attachment. Paradoxically, neutrophils are also
constitutively recruited to the oral cavity in health without inducing clinically evident
inflammation or tissue destruction [9,10] (Figure 1). Within tissues, the intermediary immune
state that allows the host to adequately respond to low grade noxious agents or tissue damage,
without clinical signs of inflammation, has been termed “Para-inflammation” [11-14].
Immunogenic events that induce a switch from a para-inflammatory to a pro-inflammatory state
are likely to be central to the pathogenesis of chronic inflammatory diseases associated with
biofilm-bearing tissues. Mechanisms that restrain oral neutrophil function in health and allow for
management and tolerance of the commensal microbiota while avoiding tissue damage may be
essential regulators of the para-inflammatory state. Normal circulating blood neutrophils are
naïve, becoming primed or activated during certain disease states, while oral neutrophils, which
have undergone extravasation and exposure to the oral biofilm, are activated [15]. Based on
reports of characteristic oral neutrophil phenotypes associated with periodontitis [16] and
neutrophil phenotype heterogeneity in other pathological conditions [17-19], we hypothesized
that oral neutrophils associated with periodontal health might have a distinct phenotype from
neutrophils associated with inflamed periodontal tissues. Using CP, a model of oral mucosal and
connective tissue inflammation, in conjunction with comprehensive cluster of differentiation
(CD) marker expression profile analysis we demonstrate that two unique tissue neutrophil
phenotypes are present in the oral cavity: a Para-inflammatory neutrophil in the healthy oral
cavity, and a Pro-inflammatory neutrophil, which occurs in CP.
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Chapter 2: Literature Review
2.1 The Inflammatory Spectrum and Pathogenesis of Periodontitis
2.1.1 The Inflammatory Spectrum
Inflammation is the adaptive response to a noxious stimulus. At its core, the inflammatory
machinery can be subdivided into four basic components: a stimulus, a sensor, a mediator and
the target tissue(s). Bacterial, fungal, parasitic or viral infections as well as non-infected tissue
injury all trigger unique sets of events aimed at controlling and eliminating the source of noxious
stimulus. Acute inflammation is resolved with the elimination of the triggering stimulus, repair
of damaged target and reestablishment of homeostasis [20]. The tapering and cessation of acute
inflammation is a regulated mechanism that involves a transition from pro-inflammatory to pro-
resolution mediators as well as a shift from neutrophil to macrophages as the predominant
inflammatory cell[21]. If acute inflammation is unresolved either through the persistence of the
triggering stimulus or the inability to resolve the acute inflammatory response, chronic
inflammation ensues resulting in tissue damage. The transition from the protective role of acute
inflammation into the damaging effects of chronic inflammation is the reason why inflammation
is associated with a myriad systemic disorders such as obesity, diabetes, cancer and
cardiovascular disease [20]. Para-inflammation, is an intermediary inflammatory state that allows
host tissues to adapt and tolerate a mild noxious stimulus with the physiological role of
maintaining or restoring tissue function and homeostasis [22]. The evolutionary origins of Para-
inflammation are though to arise from exposure to stimuli that were not present during early
human history such as over-feeding and aging [14]. The key mediator of para-inflammation in
the peripheral tissues are resident macrophages but depending on the severity of the stressor
stimulus can include other resident cells (e.g. mast cells and endothelial cells) and recruit
circulatory inflammatory cells (e.g. inflammatory monocytes). Much like unresolved acute
inflammation, unresolved chronic para-inflammation results in initiation and progression
pathological conditions such as type 2 diabetes mellitus, atherosclerosis and age-related
neurodegenerative diseases [14,23]. There is currently no comprehensive explanation of the
mechanisms that lead to an excessive, dysregulated inflammatory response that from shifts the
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beneficial reparative functions of inflammation into detrimental tissue damage role of
inflammation.
2.1.2 Inflammation and Periodontitis
The protective yet destruction duality of inflammation is best illustrated by the neutrophil
paradox in periodontal health and disease. The oral cavity is colonized early in life with plethora
of microbes [24]. Early colonization of the oral cavity allows for the induction and maintenance
of immunological microbial tolerance for the host. Microbial tolerance is crucial for our survival
considering each human cell in our body is out-numbered by ten microbial cells [25]. Our ability
to amicably tolerate a vastly diverse commensal flora suggests that our immune system is
capable of sensing beneficial microorganisms, while targeting and eliminating pathogenic
microbial organisms by activating the innate and adaptive immune system [26]. In return,
commensal organisms provide the host with unevolved traits and instruct and guide the proper
induction and functioning of the immunological system [27]. The major players in the sensing
and protection of commensal organisms have been reported to be gut epithelial cells, dendritic
cells and macrophages [28][29] [27]. The oral cavity, a direct extension of the GI is dominated
by a single myeloid lineage, the oral neutrophil. Despite the lack of the macrophages and
dendritic cells, the oral neutrophils are still capable of coexisting with the healthy commensal
oral microbiota without eliciting a true an inflammatory reaction, suggesting a previously
unreported immune surveillance role for the oral neutrophil.
2.1.3 Pathogenesis of Chronic Periodontitis
Periodontitis is a chronic inflammatory disorder that leads to the destruction of the supporting
apparatus of teeth (the periodontium), resulting in progressive loss of dental attachment that
culminates in the premature exfoliation of teeth. Periodontitis is global problem that affects
nearly half of the world’s population. In North America, 46% of the population has periodontitis,
with 8.9% of the populations suffering from severe chronic periodontitis [3]. The etiology of
periodontitis is multifactorial and relies on the microbial, host and systemic factors (Figure 2).
At the microbial level, periodontitis pathogenesis depends on the quality and quantity of the
microbial biofilm. Classically, the “red complex” bacteria (Porphyromonas gingivalis,
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Treponema denticola and Tannerella forsythia) were viewed as the causative agent of
periodontitis [30]. However, recent evidence suggests that the microbial pathogenesis of
periodontal disease goes beyond the mere presence of the “red complex” microbial species. In
fact, periodontal infections are rarely caused by a large biomass of a solitary specie. Rather,
complex, multi-species microbial populations called biofilms induce most oral and periodontal
infections. Therefore, the etiology of periodontitis on a microbial level periodontitis is best
described by an micro-environmental shift that disrupts a healthy and harmonious (“symbiotic”)
biofilm into a pathogenic and destructive (“dysbiotic”) microbial community [31-33]. In
periodontal health, there exists an equilibrium between commensal microorganisms and the host
immuno-inflammatory state. This is analogous to the harmonious relationship between the
commensal gut microbiota and mucosal immune cells that allows for a state of “controlled
inflammation” in the intestines [34]. By quickly colonizing multiple oral niches such as the
tongue, teeth and gingival sulcus, the symbiotic microbiome confers colonization resistance to
the host by impeding the colonization of oral niches with pathogenic bacteria. Further, the
symbiotic periodontal microbiota, composed primarily of facultative gram positive bacteria of
the Actinomyces and Streptococci species, protects the host by promoting proper tissue structure
and function [35], partly by regulating and shaping the mucosal immune response[35,36]. This is
in stark contrast to the dysbiotic microbial community which is predominated by gram negative,
anaerobic microorganisms rich with virulence factors that allow the biofilm circumvent the
adaptive and innate immune system thus allowing the pathogenic microbiota to flourish in a pro-
inflammatory environment[31-33]. In a dysbiotic biofilm, pathogenic bacteria, such as P.
gingivalis, subvert the immune system and induce major qualitative and quantitative changes in
the oral biofilm, triggering a robust inflammatory response that ultimately results in host tissue
damage [37].
Bacterial dysbiosis may be required to initiate periodontitis but dysbiosis alone often fails to
cause prolonged attachment loss. Many individuals who lack adequate professional and personal
sanative care present with heavy supra and sub-gingival dysbiotic biofilms fail to display the
clinical signs and symptoms of periodontal destruction. These individuals appear to tolerate the
dysbiosis due to a hypo-responsive immune-inflammatory reaction. Conversely, other members
of the population present with severe inflammatory response in the face of a slight pathological
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microbial shifts[38]. Although certain behavioral (e.g. smoking) [39], systemic (e.g. uncontrolled
diabetes) [40] and genetic (e.g Interleukin (IL)-1 polymorphism) [41] risk factors have been
described , nearly half of the variation in periodontal disease susceptibility is unaccounted for
[42]. It is postulated that some individuals may be less susceptible to periodontal disease and
have an inherent ability to resist the conversion of a symbiotic microbiota into a dysbiotic one
thanks to a yet to be identified undetermined quality of the immune system. We have proposed
that the missing susceptibility link in periodontitis susceptibility lies within the oral neutrophil
phenotypes of patients [43,44].
2.2 Neutrophil Homeostasis and Lifecycle
2.2.1 Neutrophil Differentiation
To better appreciate the role of neutrophils in the pathogenesis and resistance to periodontitis, it
is beneficial to first understand the neutrophil homoeostasis and function. Neutrophil
homoeostasis is a well-tuned machine with multiple mechanisms that tightly regulate neutrophil
production, trafficking and clearance [45]. Neutrophils are terminally differentiated, non-
proliferating cells that account for two-thirds of all leukocytes in blood [46]. This impressive
number of circulatory neutrophils is sustained through the daily differentiation of over 1011
neutrophils in the bone marrow [47][46]. Neutrophil differentiation can be divided into six stages
based on the basis of cell size, nuclear morphology, granule content and mitotic activity
(Reviewed in detail [48]). Neutrophil production and egress from the bone marrow is primary
controlled through the granulocyte colony-stimulating factor (G-CSF), as demonstrated by severe
neutropenia in animal models and human patients lacking the G-CSF receptor [49]. G-CSF hones
neutrophil progenitors to the bone marrow, helps commit progenitors to the myeloid lineage and
controls the release of mature neutrophils to the blood stream [49]. Interleukin (IL) -17 can also
promote granulopoiesis and promote the recruitment, activation and survival of neutrophils by
upregulating G-CSF expression [46,50,51]. Neutrophil egress is also mediated through the
expression of CXC Chemokine Receptor (CXCR) 4 and CXCR2 receptors on neutrophil
precursors. Neutrophil maturation in the bone marrow coincides with a progressive and gradual
decline in CXCR4 expression that is counterbalanced by an increased expression of CXCR2
expression in the latter stages of neutrophil maturation [52]. CXCR4 receptor signaling is
responsible for the initial recruitment of neutrophil progenitors to the bone marrow, while
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continued expression of CXCR4 receptor is required for the retention of the neutrophil
precursors in the bone marrow. The deletion of the CXCR4 gene results in a marked egress of
neutrophils into circulation from the bone marrow [53][54][1][55,56]. Immune cells can also
directly influence the egress of neutrophils from the bone marrow. For instance, dendritic cells
can influence the distribution of neutrophils between the bone marrow, peripheral organs and
blood [1], while phagocytosis of apoptotic neutrophils by macrophages results in a negative
feedback loop that depresses the production of the pro-inflammatory cytokine IL-23, which in
turn decreases the production of IL-17 by T-lymphocytes. A drop in IL-17 ultimately decreases
the production of G-CSF by fibroblasts, thus limiting the egress of neutrophil from the bone
marrow [57,58].
2.2.2 Circulatory Neutrophils
Once fully formed, mature neutrophils are released into the blood stream and recruited to tissues
to ward off infections and resolve tissue damage. Mature circulatory neutrophils possess the
entire complement of antimicrobial factors, respiratory burst machinery and receptors required to
mount an inflammatory response prior their egress from the bone marrow (see below). Mature
neutrophils have a limited lifespan in circulation that typically requires little to no functional
activity. To that end, circulatory neutrophils are largely considered to be “dormant” cells that
don’t readily respond to activating stimuli [46]. This dormancy should not be equated to
transcriptional inactivity as studies have shown that circulatory neutrophils constitutively express
over 12,000 transcripts, while other studies have shown mature circulatory neutrophils to
upregulate the expression of several transcription factors and increase ribonucleic acid (RNA)
biosynthesis in response to inflammatory challenges. [46,59].
Under physiological conditions, only mature neutrophils are released into circulation such that
immature neutrophil phenotypes represents less than three percent circulatory neutrophils [60].
Neutrophilia, induced by acute inflammation accelerates neutrophil recruitment from the bone
marrow, resulting in the depletion neutrophil reserves in the bone marrow and marginated pools
in the lungs, live and spleen [1][61,62]. To compensate for the increase in demand, neutrophil
maturation time is reduced resulting in the release of functionally competent yet morphologically
immature neutrophils [63]. This is supported by the presence of CD177+, immature neutrophils at
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sites of infection, presumably to assist in the local production of neutrophils and help in the
clearance of severe infections [64]. This process is most likely regulated through “emergency
granulopoiesis”. Unlike steady state granulopoiesis, emergency granulopoiesis is initiated by
microbial challenges, bacterial products such as endotoxins, inflammatory mediators such as IL-
1, Tumor Necrosis Factor (TNF)- and G-CSF [65]. Neutrophils with immature phenotype have
also been described in a number of pathological and physiological conditions ranging from SLE,
cancer, sepsis [17,18].
2.2.3 Neutrophil Extravasation
When a microbial challenge breaches the local physical barriers, neutrophils aggregate around
the infection in multiple waves. Rapid recruitment of neutrophils to tissues requires the
coordinated mobilization of neutrophils from bone marrow reserves, accelerated hematopoiesis
and recruitment from marginated pools [66,67]. Neutrophil extravasation is a cascading, stepwise
process that involves rolling, firm adhesion, polarization, and transmigration[47]. Neutrophil
rolling is the initial interaction between granulocytes and vascular endothelial cells and is
mediated through a family of C-type lectin glycoproteins known as selectins [68]. L-selectin
(CD62L) is key in regulating neutrophil rolling and is constitutively expressed on the neutrophil
membranes. The interaction of CD62L with its ligand on the surface of endothelial cells initiates
a low-affinity interaction that tethers and slows circulating neutrophils into “rolling” along the
vascular endothelial cells [69]. Selectin mediated bonds are weak and transient in nature but are
sufficient to activate and polarize captured neutrophils to activate a second phase of higher
affinity interactions mediated by Leukocyte Function Associate Antigen (LFA)-1 (CD11a/CD18)
neutrophil integrins and endothelial cell Intracellular Adhesion Molecule-1 (ICAM-1) [70,71].
This interaction can be inhibited by a recently identified glycoprotein, Developmental
Endothelial Locus (DEL)-1 thus suppressing firm adhesion and subsequent transmigration [72].
While in circulation, neutrophils only express relatively few low-affinity β2-integrins. However,
upon contact with the endothelial membrane 2-integrins quickly transform into a high affinity
or active state and increase dramatically in number and membrane density. Once activated
integrins mediate adhesion and outside-in signaling that culminate in changes in the actin
cytoskeleton at the leading edge of the cell that mediate neutrophil transmigration [73]. Once
firmly attached to the endothelial lining, neutrophils begin the process of transmigration out of
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the vessel lumen and into the underlying tissue either between endothelial cells or through them
[74]. During transmigration, neutrophils dynamically alter their membrane phenotype by
initiating the exocytosis of secretory vesicles that contain CD11, CD18, CD31, CD54, CD44 and
CD47 to the cell surface [1,46,75-79]. The transmigration cascade culminates as the elongated
uropod of the emigrating neutrophil detaches from the vessel wall and the neutrophil migrates
into tissue [80].
As neutrophils leave the circulation they must adapt and transform to suit their new function as
tissue phagocytes. To this end, emigration from circulation alters the functional state of
neutrophils characterized by enhanced Nicotinamide Adenine Dinucleotide Phosphate (NAPDH)
oxidase activity [81], increased production of pro-inflammatory cytokines such as IL-8 [82] and
enhanced anti-apoptosis mechanisms. In addition, tissue neutrophils are also more
transcriptionally active than circulatory neutrophils[16]. The burst of transcriptional activity in
tissue neutrophils promotes the antimicrobial capacity of neutrophils and generates and promotes
the release of immune active chemokines and cytokines that recruit additional inflammatory cells
[82]. The transition from a passive, dormant circulatory neutrophil to a more transcriptionally
active, antimicrobial, immune active tissue neutrophil is commonly referred to as activation.
2.2.4 Neutrophil Clearance
As neutrophils continue to age in circulation without being targeted to tissues a feedback circuit
involving L-selectin, chemokine receptors and integrins are activated [1]. Aging circulatory
neutrophils shed L-selectin (CD62L), upregulate the expression of the bone marrow homing
receptor CXCR4 as well as CD11b. Collectively these changes target old circulatory neutrophils
(CD62Llo, CXCR4hi) back to the bone marrow where they are cleared by macrophages. This
processes promotes the activation of Liver X Receptor (LXR) transcription factors in
macrophages, which in turn increases the expression of CXCL12 in the bone marrow. Enhanced
CXCL12 expression promotes the release of newly formed mature neutrophils (CD62Lhi,
CXCR4lo) back into circulation. This process repeats in a oscillating circadian rhythm and
ensures an equilibrium state between the introduction of newly formed neutrophils and removal
of aged neutrophils into and out of circulation [83]. Aging is accompanied by depressed
neutrophil function. Studies in elderly humans have shown that neutrophils maintain their
9
chemotactic ability but are inefficient in bacterial clearance and cause more host mediated tissue
destruction through the inadvertent spilling of protease contents [1].
Controlled neutrophil clearance is essential for the maintenance of tissue hemostasis and
resolution of inflammation. Deficiencies in controlled neutrophil clearance results inappropriate
neutrophil activation, in the progression of chronic inflammation [84,85] and ultimately ends in
host tissue destruction. Neutrophil clearance occurs either through programed cell death
(apoptosis) or retreat from inflammatory sites against the chemotactic gradient (reverse
migration) [21][86]. Once at the site of inflammation, neutrophils normally undergo apoptosis
after engaging the offending insult. However, recent evidence suggests neutrophils are capable of
migration against the chemoattractant gradient. Reverse neutrophil transmigration has been
shown to be critically important in inflammation resolution. The exact pathophysiology and
implications have yet to be fully elucidate [86]. Apoptosis on the other hand, allows for the
clearance of neutrophils while minimizing the release of cytotoxic and proteolytic granule
contents to the surrounding tissues[87]. Apoptosis is regulated through the engagement of death
receptors found on the surface of apoptotic neutrophils. During the early stages of neutrophil
maturation, apoptosis is only triggered in response to aberrant Deoxyribose Nucleic Acid (DNA)
replication. However, as neutrophils mature they pack their secretory vesicles with so called
death receptors that respond to extracellular death ligands commonly found in pro inflammatory
microenvironment [1]. To prolong their survival outside the bone marrow, neutrophils become
anti-apoptotic in response to various growth factors and cytokines and thus earn prolonged life
span following the egress from the bone marrow and during diapedesis [88].
2.3 Neutrophil Function
2.3.1 Neutrophil Granules
Throughout neutrophil maturation, undifferentiated cells acquire morphological and functional
features necessary for the neutrophil antimicrobial activity. During neutrophil development,
granules first appear in the transition from myeloblast to promyelocyte stage. Structurally, four
granule subsets, including azurophil (primary), specific (secondary), gelatinase (tertiary) and
secretory vesicles have been described in the literature [89]. Azurophil granule protein contents
10
are formed early during the promyelocyte stage. Azurophil granules are the lysosomes of
neutrophils and contain myeloperoxidase (MPO) as well as defensins and acid hydrolases.
Specific granules contents are synthesized at the myelocyte stage and consists of bacteriostatic
and bactericidal agents including proteolytic enzymes and complement activators. As their name
suggests, gelatinase granules are packed with gelatinases, phosphates and Matrix
Metalloproteinases (MMPs). Gelatinase granule contents are formed at the metamylelocyte stage
and neutrophils use their targeted release to facilitate tissue migration. Secretory vesicles are the
last neutrophil granules to be formed and are the main source of membrane receptors and CD
markers such as CD11, CD18, CD67, CD14, CD35, CD10 and CD13 [89,90]. Unlike other
granule subsets, the key to secretory vesicles lies within the membranes as opposed to the
granule contents. Again, unlike other granules, the release of secretary vesicles is not only
regulated by inflammatory signals such as IL-8 and Formylmethionyl-leucyl-phenylalanine
(fMLP) [55,90,91] but by transmigration via interaction of L-selectin on circulatory neutrophils
and endothelial P- selectin [55,92]. As secretory vesicles fuse with the plasma membrane, they
“prime” the neutrophil for antimicrobial activity by delivering a rich source of receptors and
other functional proteins to the neutrophil surface. Importantly, the fusion of secretory vesicles
enhances neutrophil functional capacity without releasing proteolytic and cytolytic granule
content[89]. Overall, neutrophil granules display a continuum with respect to their propensity for
exocytosis that is best described by the targeting-by-timing hypothesis [93]. The enhanced
exocytosis capabilities of gelatinase and secretory vesicles compared to specific and azurophil
granules is directly correlated to density of expression of the fusogenic proteins such as Vesicle
Associated Membrane Protein (VAMP)-2 on the granule membrane surface [94]. Following a
mild trigger, such as a transient rise in intracellular calcium ions, it is detrimental to neutrophils
and the host to indiscriminately and simultaneously release all granule contents. It is more
prudent for neutrophils to grade their response according to the situation. When neutrophils
initially transmigrate out of circulation and into the tissues, there is a specific need for the
targeted release of gelatinizes, MMPs, and proteases. This initial of matrix degradation
supersedes the need for complement receptors that mediate phagocytosis of microorganisms or
pro-antibacterial proteins. To that end secretory and gelatinase granules with the highest
concentration of VAMP-2 are released first. Only a sustained exocytic stimulation allows for the
release of the tissue destructive specific and azurophil granules [89].
11
2.3.2 Neutrophil Mediated Bacterial Killing
Neutrophils neutralize invading microorganisms through a three-pronged approach consisting of
an oxygen dependent pathway, an oxygen independent pathway and NETosis. The majority of
the antimicrobial activity of neutrophils occurs within phagosomes. During phagocytosis,
opsonized targets are ingested through the generation and extension of pseudopods that
encapsulate the organism [95]. The internalized particles are housed in membrane-bound
phagosomes, which fuse with granules containing proteolytic proteins [96]. Once ingested,
microbes trapped in phagosomes can be neutralized through the oxygen dependent pathway
revolving around the assembly of the multiprotein enzyme complex known as the reduced
NADPH oxidase. When activated neutrophils dramatically increase oxygen consumption and
activate the NADPH oxidase complex that is normally quiescent. Once the NADPH oxidase
complex is assembled, it functions as an electron transport chain that funnels negative charges
into the phagosome lumen and thereby depolarizes the phagosomal membrane, initiates the
respiratory burst and catalyzes the formation of superoxide anion [97][98]. The NAPDH oxidase
enzyme complex is composed of the oxidase proteins p22phox, p47hox, p67phox and gp91phox,
p40phox (suffix phox representing phagocyte oxidase) the GTPases Rac1/2 and Rap1A.
Activation of the electron transport machinery requires the translocation of cytoplasmic p47phox,
p67phox and p40phox to the phagosome membrane [99]. The importance of the NAPDH oxidase in
neutrophil function is best characterized by mutation in genes the encode the subunits of the
NAPDH oxidase collectively known as Chronic Granulomatous Disease (CGD) [100].
Neutrophils from CGD patients fail to produce normal amounts of superoxide. Superoxide (O2-)
is the primary product of the respiratory burst. Superoxide subsequently dismutates into
hydrogen peroxide (H2O2) [101]. Neutrophils can also release MPO which combines with H2O2
to produce the extremely cytotoxic hypoclorous acid. Superoxide and hydrogen peroxide impart
their cytotoxic effects thanks to their ability to generate additional Reactive Oxygen Species
(ROS) namely hydroxyl radicals (OH-), singlet oxygen (1O2) and ozone (O3). The generated
ROS contribute tissue destruction and pathogenesis of periodontal disease [102] and respiratory
levels are commonly used as an indicator of neutrophil activation [6].
12
Neutrophil inflammatory response was largely believed to be intracellular. However, the
discovery of Neutrophil Extracellular Traps (NET), shed light on the extracellular contribution of
neutrophils in innate immunity. NETs are composed of strands of DNA wrapped in proteolytic
proteins that are capable of entrapping and killing invading microbes, thus minimizing the extent
or rate of spread of an infection [1][103]. The formation of NETs (or NETosis) is a terminal
event for neutrophils and is an alternative death pathway to apoptosis. NETosis begins as
neutrophils dissolve their nuclei, chromatin and granules. Neutrophils then extrude large strands
of histone covered DNA coated with cytosolic and granular proteins [104,105]. NET associated
proteins are categorized as either bactericidal (such as histones, defensins, lactoferrin, and MPO),
pattern recognition proteins (such as Pentraxin 3) or cytosolic proteins (calportectin) [47]. In an
in vitro setting, NETosis induced by Phorbol 12-Myristate 13-Acetate (PMA), is dependent on
the presence of elastase, MPO and active NAPDH oxidase [106]. As expected, NETosis is
devoid or absent in patients that suffer from conditions that cause a deficiency in MPO, an
inactive NADPH oxidase or the lack elastase and the serine proteases [107-109]. NETs may also
be a major contributory factor to the onset and exacerbation of a number of autoimmune
diseases. NETs contribute to autoimmune disease such as systemic lupus erythematous and
Wegener’s granulomatosis as they lead to the extracellular release of multiple intracellular target
antigens such as DNA and MPO [110].
2.4 Neutrophil Plasticity and Heterogeneity
Despite the reported complexity, neutrophils are often discounted as a phenotypically
homogenous population with a suicidal mentality of swarming sites of infection to exuberantly
clear out invading pathogens only to perish in the infiltrated tissues. However, there has been a
recent paradigm shift in the understanding and appreciation of neutrophil diversity. A growing
body of evidence suggests that neutrophils should be viewed as diverse population with a broad
spectrum of phenotypes and functions and that are subject to microenvironmental modulation.
Recent investigation into neutrophil biology as changing our understanding of every aspect
neutrophil. For instance, we now know that circulatory neutrophils are longer lived than
originally thought, with a reported circulatory half-life of over five days [111,112]. We also
know that aging neutrophils not recruited to sites infection display a unique phenotype
characterized by diminished functional capacity (degranulation, respiratory burst, phagocytosis,
13
chemotaxis), increased expression of bone marrow homing signals, decreased sensitivity to
inflammatory signals and cytokines and increased apoptotic tendency [113]. Research has also
shown that neutrophils are not necessarily “dead end” cells and are capable of reverse
transmigration (migration from tissues – to blood). The process of reverse transmigration results
in distinct phenotypic changes in neutrophils characterized by increased expression of ICAM-1
and low expression of CXCR1. These ICAM-1highCXCR1low displayed reduced tendency to
migrate through an endothelial barrier, increased cell rigidity, increased ROS production and
decreased apoptosis. ICAM-1highCXCR1low neutrophils have been detected in the circulation of
patients with rheumatoid arthritis and atherosclerosis but their role in the pathogenesis of these
chronic inflammatory conditions has yet to be elucidated [86]. However, the process of reverse
transmigration does likely lead to the generation of potentially pathogenic subset of neutrophils
that may disseminate systematic inflammation from a localized site of injury to distant organs
[114]. Lastly, diverse and novel functions for neutrophils have been described in details in
complex interactions with the adaptive immune response (reviewed in detail in [115-117].
2.4.1 Neutrophil Subpopulations
Distinct neutrophil subpopulations have been identified in a number of pathological conditions.
In a murine cancer model, cancer was associated with specialized neutrophil subsets with a
unique transcriptional profile referred to as Tumor Associated Neutrophils (TAN) [118]. Within
the TAN family of neutrophils, two subsets were identified, namely N1 and N2 neutrophils or
PMNs. N1 neutrophils were created by the blockage of the transforming growth factor- (TGB-
) and had an immunostimulatory and anti-tumor phenotype. N2 neutrophils, which constituted
the majority of TANs, were in turn immunosuppressive and pro-tumorigenic. Two unique
neutrophil populations (PMN-I PMN-II) have also been identified in a murine Methicillin-
Resistant Staphylococcus aureus (MRSA) model [119]. PMN-I neutrophils are induced in
MRSA infected mice as a result of a mild secondary systemic inflammatory response while
PMN-II are induced in Methicillin-Resistant Staphylococcus aureus (MRSA) infected mice as a
result of a severe secondary inflammatory response. When compared to PMN-I neutrophils,
PMN-II neutrophils have a more immature nuclear morphology and express differing cytokines,
chemokines, Toll-Like Receptors (TLR) and adhesions molecules. These differences ultimately
lead to impaired activation of macrophages resulting in inefficient anti-bacterial response and
14
clearance of MRSA [119]. Neutrophil heterogeneity has also been identified in sepsis. Not
surprisingly sepsis is associated with profound neutrophilia. However, despite the increase in
circulatory neutrophils, septic patients are severely immune compromised, prone to infections
and often succumb to their illness [120]. Decreased immunity in the face of neutrophilia is
suggestive of an altered function of circulating neutrophils. In response to systemic endotoxins, a
heterogeneous neutrophil population can be identified with an increase in the proportion of
neutrophils characterized by high levels of CD markers, CD16, CD11b and CD11c and low
levels of CD62L [121]. These cells displayed reduced interactions with opsonized bacteria,
displayed decreased ROS production and were suppressive to T-cell proliferation.
A unique subset of Low Density Neutrophils (LDN) has been implicated in the pathogenesis of
early stage autoimmune diseases such as Systemic Lupus Erythematous (SLE) and type 1
diabetes. SLE is an autoimmune disorder characterized by the presence of autoantibodies against
nuclear antigens. LDNs are reported to directly participate the pathogenesis of SLE as they are
more prone to death by NETosis resulting in the expulsion of host DNA material in the blood
stream. The self-DNA-antimicrobial protein complex has been shown to activate plasmacytoid
Dendritic Cells (pDCs) which in turn increase Interferon (IFN) – type I production. The INF-I
release initiates a perpetual positive feedback loop that beings with the activation of B
lymphocytes to produce autoantibodies against self-DNA and antimicrobial peptides released by
neutrophils and further activate neutrophils to release eve more NETs thus exasperating the auto-
immune disorder [110,122]. Similar to SLE, neutrophil activation of pDCs has also been
implicated in the pathogenesis of murine Type I Diabetes Mellitus [123].
2.4.2 Identification and Characterization and Cellular Subpopulations
A CD marker cluster is defined as a specific cohort of antibody clones that define a single cell
surface protein. The CD marker system allows for unambiguous identification of a specific cell
type or subpopulation based on their unique set of cell surface proteins [124]. However,
individual CD markers are often expressed in more than one cell type, which is why the
comprehensive and systematic characterization of specific CD markers are required for the
identification of unique cellular phenotypes and subpopulations. The analysis of a heterogeneous
cell population with the ultimate goal of discerning the phenotypes and subpopulations is called
15
immunophenotyping [125]. Immunophenotyping requires the use of several CD markers and
unique gating strategies using a flow cytometer to accurately detect target populations [126]. The
identification of purified neutrophil populations and subpopulations relies determining cell size
and granularity, as well as on the expression profile of a number of CD markers. To date a
variety of CD markers including CD11b, CD14, CD16, CD62L, CD18, CD45 have been used for
the purification of neutrophils. Unfortunately, there has been little to no consistency in the use of
CD markers in the literature for the purification of neutrophils. For instance, Brooks et al. (2013)
used the combination of CD45mid (a marker of epithelial cells), CD14low (a marker of monocytes
and macrophages) and CD16+ (a marker of neutrophils) to select for neutrophils in fresh and
frozen sputum samples [127]. Fortunati et al. (2008) on the other hand, only used the
combination of CD45low and CD16high to select for neutrophils [128]. Conversely, in a
publication by Tak et al. (2015), circulatory granulocytes were isolated based on a combination
of forward-/side-scatter (FSC/SSC) and CD16+ while sputum neutrophils were identified as cells
that were CD11b+ (to exclude epithelial cells), 405/450mm-autflorescencelow (to exclude
macrophages) and CD16+ (to exclude eosinophils) [129]. Here we reviewed only three varying
examples of CD markers used to selectively isolate neutrophils from a heterogeneous population
using flow cytometry. To overcome this shortcoming and discrepancy in the literature, a
comprehensive High-Throughput Screening (HTS) assay was carried out to identify unique
neutrophil CD markers that are ubiquitously expressed in circulatory and tissue neutrophil
regardless of activation status. This project identified CD11b, CD66b, and CD16 as three
markers that are consistently expressed on neutrophils independent of the cell location, level of
activation and disease state. Cell sorting against CD11b, CD16 and CD66b allowed for the
enrichment of mature neutrophils, yielding neutrophil populations with up to 99% purity. These
findings suggest an ideal surface marker set for isolating mature neutrophils from humans.
In other publications, authors resort to using density gradients and centrifugation in varying
configurations to isolate specific cell populations. Density centrifugation relies on layering cells
with varying inherent densities onto a medium with a range of banding densities. Cells are then
spun at varying speeds such that cells will localize to a point where their density precisely
coincides with the density of the surrounding solution. Commonly used mediums in density
centrifugation include Percoll, Ficoll and Histopaque [130]. These media are generally used due
16
to their biocompatibility and densities that are ideal for isolation of basophils, 1.080 g/mL
(1.075–1.081 g/mL); eosinophils, 1.088 g/mL (1.085–1.100 g/mL); neutrophils, 1.090 g/mL
(1.080–1.099 g/mL); lymphocytes, 1.077 (1.066–1.077 g/mL); and monocytes, 1.065 g/mL
(1.059–1.068 g/mL). Although technically simple, density centrifugation does have major
limitations. A study by Kuijpers et al. (1991) showed that expression of neutrophil surface
molecules is influenced by density centrifugation purification procedures. Centrifugation of
isolated circulatory neutrophils resulted in activation demonstrated by the increase of surface
CD10. Also, surface expression of several antigens that were expressed on circulating
neutrophils increased significantly after density-gradient centrifugation. The isolation method
caused increased expression of CD13, CD16, CD18, CD45, and CD67 but did not alter CD32
(FcRII), CD54 (ICAM-I), CD58 (LFA-3), Leu-8 and Human Leukocyte Antigen (HLA) class I
antigen expression [131].
2.5 Oral Neutrophils as a Unique Population of Neutrophils
Neutrophils are the most common circulatory leukocytes, yet relatively little is known about the
functional and biological diversity of these important cells as they migrate out of the circulation
and into peripheral organs. This can partly be attributed to the difficulty of collecting and
harvesting pure population from peripheral tissues. The gastrointestinal (GI) system is an
excellent model system to study tissue neutrophils in the context of inflammation and
inflammatory resolution [132]. Neutrophil have been shown to play a unique role in mediating
mucosal inflammation in the GI tract [133]. For instance, neutrophil depletion worsens the
inflammatory bowel disease symptoms in mice suggesting that neutrophils play a protective role
in GI mucosal tract [134][132]. Unfortunately, harvesting, collecting and analyzing human gut
neutrophils is inherently challenging and invasive. The oral cavity on the other hand, a terminal
end point of the GI system, is an accessible and abundant source of tissue neutrophils. Much like
GI mucosal neutrophils, oral tissue neutrophils play a critical surveillance role to help maintain
local equilibrium.
Neutrophils are the predominant the leukocyte in the oral cavity. Each hour, 2x106 neutrophils
are recruited out of the circulation and into periodontal connective tissues towards the gingival
sulcus where they are eventually washed out into the oral cavity with the Gingival Crevicular
17
Fluid (GCF). Nearly all of the leukocytes that enter the oral cavity remain functional for many
hours [135] [136][137]. Once in the oral cavity, neutrophils can be readily harvested through
sequential oral saline rinses, with two minute rests to allow for steady-state recruitment of oral
neutrophils from the circulation. Oral rinses can then be pooled and neutrophils selectively
isolated through nylon mesh filtration [5,138]. Oral neutrophil counts in saliva are positively
correlated with the degree of inflammation in the oral cavity. Therefore, just as circulatory PMN
counts have been used a proxy for the determination of systemic health, oral neutrophil counts
can be used to assess the degree of periodontal disease and clinical inflammation and tissue
destruction in the oral [8]. That said, oral neutrophils do represent a unique neutrophil population
that does not always correlated or mimic circulatory neutrophils [139]. Oral neutrophils are
constantly recruited towards a healthy gingival sulcus in response to host and bacterial
inflammatory signals. Neutrophils are recruited to the oral cavity in germ free mice that lack
commensal oral bacteria suggesting that junctional epithelial cells alone are capable of
promoting neutrophil chemotaxis [140]. However, neutrophil recruitment to the oral cavity by
the healthy junction epithelial cells that are in direct contact with commensal microbiota is
significantly increased through the expression of an IL-8 gradient [141]. Again, significantly
more neutrophils are recruited oral cavity in the presence of periodontal disease [138]. While
traversing through the gingival sulcus, the neutrophil enriched GCF protects the dental junctional
epithelium from invasion and apical migration sub-gingival bacterial biofilm [142,143]. This is
supported by the observation that salivary leukocytes counts are lowest in completely edentulous
patients, intermediate in dentate patient lacking periodontal inflammation and highest in dentate
patients with clinical gingivitis [144]. Neutrophils play a protective role in the oral cavity
through phagocytosis, generation of ROS and degranulation. That said, neutrophils play a
doubled edged sword in oral cavity, particularly in the pathogenesis of periodontal disease. On
one hand, the absence of neutrophil in the periodontal tissues results in aggressive and highly
destructive forms of periodontal disease [145]. Conversely, excessive neutrophil recruitment and
hyperactive neutrophil response in periodontal tissues is directly correlated to disease severity
and pathogenesis [6,138][146].
Unfortunately, little effort has been devoted towards characterizing oral neutrophils in health
with the majority of published literature on oral neutrophils being aimed at characterizing the
18
function and phenotypes of oral neutrophils in patients with chronic inflammation, typically
chronic periodontitis. Lakschevitz et al. (2013) examined the gene expression profile of blood
and oral neutrophils of healthy patients and those with chronic periodontitis. This study
demonstrated that there is a major gene expression change in oral neutrophils as they traverse out
of circulation and into the tissues regardless of oral disease status. There was a six-fold increase
in the degree of transcriptome change in oral neutrophils of patients with chronic periodontitis
compared to periodontally healthy patients. The gene profile of oral neutrophil in periodontitis is
characterized up-regulation in genes in the TLR and other inflammation pathways such as IL-1
and Chemokine Ligand (CCL) 3. In addition, alterations in genes regulating apoptosis were
noted, which is corroborated by the pro-inflammatory nature and prolonged longevity of oral
neutrophils in patients with choric periodontitis [5,147]. Oral neutrophils isolated from
periodontally healthy patients also display reduced phagocytic activity but similar levels of
intracellular bacterial killing when compared to circulatory neutrophils. Compared circulatory
neutrophils, oral neutrophils isolated from patients with chronic periodontitis were characterized
by enhanced hydrogen peroxide and ROS generation, greater potential for NETosis, enhanced in
chemotaxis, enhanced expression of CCL3 and IL-1 [5,148]. Taken together these findings are
reflective of the need for neutrophils to arrive at the site of inflammation and to carry out its
physiological roles.
Blood neutrophils isolated from patients with refractory chronic periodontitis, which does not
respond to conventional therapy, display higher levels of ROS production compared to blood
neutrophils of periodontally healthy patients [2]. These findings were induced by the induction of
ROS using PMA, a physiologically irrelevant yet useful laboratory tool. PMA directly activates
intracellular Protein Kinase C (PKC) independent of any membrane receptor ligands [2]. Other
studies, which induced ROS through the Fc receptor pathway, showed that CP peripheral
neutrophils produced more ROS compared to healthy blood neutrophils suggesting that CP
neutrophils have increased ore more responsive [146]. The capacity of blood neutrophil to
produce ROS is less critical than the ROS production capacity of tissue neutrophils. When
examining the ROS production of tissue neutrophils, studies have shown that oral neutrophils
have elevated baseline ROS production levels when compared to circulatory blood neutrophils
and that patients with more severe disease were characterized by oral neutrophils that produced
19
the greatest levels ROS [6]. Concurrently, antioxidant (AO) levels in whole saliva of patients
with CP is significantly lower than in whole saliva of periodontally healthy patients [102].
Nuclear Factor Erythroid 2-Realted Factor (Nrf2) regulates the gene transcription of a group of
AO in response oxidative stress. Nrf2 binds to the promoter region of AO enzymes including
catalase and superoxide dismutase among others [149]. The Nrf2 pathway is down-regulated in
oral neutrophils of patients with severe CP compared to periodontally healthy patients. Ligature
induced periodontitis model in Nrf-/- null nice also displayed increased oxidative damage. The
Nrf2 pathway is key in the regulation and expression of cytoprotective factors (e.g. catalase,
superoxide dismutase), cell survival and tissue production during oxidative stress [7]. The
simultaneous down-regulation of the protective Nrf2 pathway in concert with increased
neutrophil recruitment to the oral cavity in CP provides a direct mechanism for tissue
destruction.
20
Statement of the Problem
Neutrophil accumulation at sites of tissue injury and infection is a hallmark of acute
inflammation. Based on their classic functional role, neutrophils are viewed as pro-inflammatory
members of the innate immune system that seek and destroy invading pathogens and help
propagate the inflammatory response. In fact, improper and/or prolonged activation of
neutrophils can result in autoimmune and chronic inflammatory diseases characterized by
progressive tissue damage. However, the mere presence of neutrophils does not necessarily
imply disease. Research in neutrophil biology during the last three decades has elevated our
understanding of neutrophils. We now recognize neutrophils as sophisticated cells with
widespread roles in immunity and inflammation that extend far beyond the elimination of
pathogens. We further appreciate that in addition to their pro-inflammatory roles, neutrophils
also play a key role in establishing and maintaining tissue health and homeostasis. The paradox
of the destructive yet protective neutrophil is best exemplified in the oral cavity in the presence
and absence of chronic inflammation in the form of periodontitis. The etiology of periodontitis is
multifactorial and relies on the microbial, host and systemic factors. It is postulated that some
individuals may be less susceptible to periodontal disease due to an inherent ability to resist the
conversion of a symbiotic microbiota into a dysbiotic one. We have proposed that the missing
susceptibility link in periodontitis susceptibility lies within the neutrophil phenotypes of patients.
Therefore, we hypothesize that oral neutrophils isolated from the oral cavity of periodontally
healthy patients are functionally and phenotypically different than oral neutrophils isolated from
patients with chronic severe periodontitis. We further, propose that the quality and quantity of
oral neutrophils may be key in preventing or resisting periodontal breakdown in the face of a
microbial challenge. Using principles of immunophenotyping, flow cytometry and a unique
developed set of neutrophil specific cluster of differentiation markers we aim to show that in
healthy oral tissues, there exists an intermediary “para-inflammatory” immune state that allows
the host to respond to noxious agents without clinical signs of inflammation.
21
Chapter 3 : Materials and Methods
3.1 Human Subjects
Participants were recruited from Toronto General Hospital’s Nephrology Center and University
of Toronto’s Graduate Periodontology Clinic. The study was approved by the University of
Toronto’s Research Ethic Board (#30044 & 29410) as well as the University Health Network
Research Ethic Board (#13-6896-AE). Signed informed consent was obtained from all
participants prior to inclusion. Following a preliminary complete dental examination, patients
with generalized severe CP [150] were recruited for the study. Volunteers with a healthy
periodontium and no history of disease, as defined by the absence of loss of clinical attachment,
were recruited as controls. Participants were excluded based on the following exclusion criteria:
1) pregnancy, 2) antibiotic or surgical periodontal therapy within the last six months 3) complete
edentulism and 4) other active oral disease including caries, endodontic lesions and
mucocutaneous diseases. After sample extraction, periodontal clinical parameters including
bleeding on probing (BOP), probing depth (PD), recession and clinical attachment level (CAL)
were measured at six sites per tooth. The degree of inflammation was determined by percentage
of sites that displayed bleeding upon probing [151]. A site was considered to have “active
disease” if it displayed bleeding on probing in combination with a probing depth of greater than
or equal to 5mm [152].
3.2 Sample Collection and Processing
Blood and oral samples were obtained as previously described [5]. Blood samples were drawn
into a vacutainer containing 0.1 volumes of sodium citrate as anticoagulant. With the exception
of electron microscopy and ROS assays, freshly drawn whole blood and oral rinse samples were
immediately fixed with 1.6% paraformaldehyde (PFA) for 15 minutes at 4oC, which we found
preserved native surface expression of CD markers without altering antigenicity. Erythrocytes
were eliminated by isotonic lysis. Oral samples were pelleted at 690 x g and resuspended in 10
ml of cold Phosphate Buffered Saline (PBS) and epithelial cells were removed by filtration as
previously described [153]. Cell sorting based on CD18 expression was performed to confirm
>98% purity of oral neutrophil populations.
22
3.3 Electron Microscopy
Fresh oral neutrophils were prepared as above, without PFA fixation. Sample preparation and
electron microscopy were performed as previously described [154]. Briefly, cells were pelleted
and fixed with Karnovsky’s style fixative (3.2% paraformaldehyde + 2.5% glutaraldehyde in a
0.1M Sorensen’s phosphate, pH 6.8 - 7.0) for one hour. Fresh fixative was replaced and samples
were left over night at 4oC. After removal of the fixative the pellets were washed with Sorensen’s
phosphate buffer (A mixture of 0.1 M monobasic and 0.1 M dibasic sodium phosphate salts in a
ratio to give the required pH) and then post-fixed with 1% osmium tetroxide for one hour.
Following another wash, the pellets were dehydrated using a graded series of ethanol/distilled
water at 30%, 50%, 70%, 95% and 100% ethanol. The pellets were then washed with the
transitional solvent propylene oxide. Infiltration was performed using Epon Araldite resin,
prepared as described previously [155], using a graded series of Epon Araldite (E/A) and
propylene oxide (PO) at 1:1 E/A and PO for half an hour, 3:1 E/A and PO for two hours, and
100% E/A overnight. Another infiltration using 100% E/A was performed the next day for
another two hours. The samples were then placed in polyethylene BEEM capsules with fresh
E/A, and placed in an oven for polymerization for 48 hours at 60°C. After complete
polymerization, the solid resin blocks containing the samples were sectioned on a Reichert
Ultracut E microtome to 60 to 90 nm thickness and collected on 200 mesh copper grids. The
sections were counter stained using saturated uranyl acetate for 15-20 minutes, rinsed in distilled
water, followed by Reynold’s lead citrate for 15-20 minutes and rinsed again in distilled water.
The sections were examined and photographed in a Hitachi H7000 transmission electron
microscope at an accelerating voltage of 75Kv. Image analysis was performed using ImageJ
software.
3.4 Histopaque Density Centrifugation
Discontinuous density gradients were generated by carefully layering equal volumes of
1.077g/ml and 1.119g/ml Histopaque centrifugation medium (Sigma). Whole blood and saliva
rinse samples were layered onto the gradient and centrifuged at 1000 x g for 35 minutes. High-
density (HD) cells were collected from the 1.077-1.119 interface, while low-density (LD) cells
were collected from the plasma-1.077 interface. Contaminating erythrocytes were eliminated via
hypotonic lysis.
23
3.5 Multicolor Flow Cytometry
Whole blood leukocytes and oral neutrophils (5 x 105) were resuspended in 50 µl of
Fluorescence-Activated Cell Sorting (FACS) buffer and labeled with two separate panels of
antibodies as detailed in Table 1. The markers were classified into four categories based on
function: degranulation/activation markers (CD10, CD63, CD64 and CD66a), immunoregulation
markers (CD16 and CD170), adhesion markers (CD11b, CD18 and CD177) and complement
regulators (CD55). Cells were labeled for 30 minutes on ice in the dark and washed three times
with FACS buffer. At least 2 x 104 gated events were acquired using an LSR Fortessa (BD
Biosciences) flow cytometer. For each CD marker, appropriate fluorescently tagged isotype
control antibodies were used to determine autofluorescent signals, which were subtracted from
Mean Fluorescence Intensities (MFI). Flow cytometer channel voltages were calibrated manually
using rainbow beads in order to normalize sample acquisition on different days. Compensation
was performed with single stained OneComp eBeads (eBioscience). Gating was performed as
described in (Figure 3A and B). Doublets were excluded by SSC-W x SSC-H. Data were
analyzed using FlowJo (vX) software. Samples from one healthy volunteer were run on multiple
occasions to confirm reproducibility on different days. Also, samples stained in duplicate on a
given day were found to yield identical results.
3.6 ROS Assay
ROS assays were performed essentially as described [6]. Unfixed oral neutrophils were prepared
and incubated for 20 minutes at 37°C in Hanks-/- containing Dihydrorhodamine (DHR) at a final
concentration of 2 µM, and stimulated for an additional 15 minutes with Phorbol 12-Myristate
13-Acetate (PMA) at a final concentration of 200 nM, or left unstimulated. The cells were placed
on ice and labeled with CD18-BV421. Flow cytometry, gating and analysis were performed as
described above. The use of DHR with flow cytometry to measure ROS production has
previously been validated and has been proven to be a fast, reliable and easy method to evaluate
ROS production [2].
24
3.7 NET Assay
Preparation of fixed, purified oral neutrophils was performed as above. Flow cytometric analysis
of NET formation was performed as described previously [156]. Oral neutrophils were labeled
sequentially with primary anti-Histone H3 (Citruline R2 + R8 + R17, Abcam) and secondary
goat anti-rabbit-AF488 (Abcam) antibodies, and then labelled with MPO-PE (Clone: 2C7, Acris)
and CD18-BV421. Each incubation was for 30 minutes, followed by one wash with FACS
buffer. Flow cytometry, gating and analysis were performed as described above.
3.8 Statistical Analysis
One-way ANOVA was performed with a post-hoc Tukey’s test for pairwise comparisons.
Clinical parameters were compared using Student’s t-test. P ≤ 0.05 were considered statistically
significant. Statistical analysis was performed using GraphPad software
25
Chapter 4 : Results
4.1 Clinical Description of Periodontal Status
Seventeen CP patients and 11 healthy controls were recruited in order to characterize blood and
oral neutrophil phenotypes. On average, patients with CP had 3.1 fewer teeth (24.9 ± 4.2) than
healthy controls (28 ± 1.7, p = 0.05), with BOP occurring at 62% of sites in CP patients
compared to 8.6% of sites in healthy controls (p < 0.001). In patients with periodontal disease,
25.5% of all sites were deemed to be undergoing active inflammation-mediated periodontal
tissue breakdown, while active disease (pocket depth ≥ 5 with BOP) was virtually absent (0.1%)
in healthy controls (p < 0.001). Consistent with previous observations [138], patients with CP
had an elevated oral inflammatory cell load as demonstrated by a five-fold increase in oral
neutrophil counts compared to healthy controls (p = 4 x 10-4). There were no significant
differences between the two groups with respect to age, age range or gender (Table 2).
4.2 CP Oral Neutrophils Are More Degranulated and Have Increased
Phagocytosis Compared to Healthy Oral Neutrophils
In order to characterize functional differences between blood and oral neutrophils in health and
disease, we evaluated granule content and phagocytosis in CP and healthy oral neutrophils by
electron microscopy (Figure 4). The mean number of granules per µm2 of cytoplasm was lower
in oral neutrophils compared to blood neutrophils (Figure 4B), and granule content was further
reduced in oral neutrophils from CP patients compared to healthy controls. There were no
differences in granule content between blood neutrophils of healthy and CP patients.
Phagosomes were commonly observed in oral neutrophils, but not in blood neutrophils (Figure
4C). Oral neutrophils from chronic periodontal disease patients contained, on average, more
early and late phagosomes than oral neutrophils from healthy controls. Elevated degranulation
and phagocytosis by CP oral neutrophils confirms that these are in a heightened inflammatory
state compared to healthy oral neutrophils.
26
4.3 Flow Cytometric Gating Strategy
Oral rinse samples contain neutrophils, epithelial cells, bacteria and debris. Epithelial cells were
removed by nylon mesh filtration, and we found that gating on CD18+ve was sufficient to exclude
debris (Figure 3A). Cytological analysis of sorted cells confirmed the purity of neutrophils in
CD18+ve populations, while CD18-ve populations contained debris but no cells (Figure 3A).
Neutrophils in whole blood were gated based on high CD16 expression in combination with SSC
(Figure 3B). Although a subset of circulating monocytes are known to be CD16+ve [157],
analysis of high and low density blood fractions confirmed that these monocytes had lower SSC-
A and lower CD16 expression than blood neutrophils and were excluded by our gating strategy
(results not shown).
4.4 Characterization of Oral Neutrophil Populations in Health and CP
We performed high-resolution multicolor flow cytometry to analyze CD marker signatures of
blood and oral neutrophils, and to identify and immunophenotype neutrophil subsets in health
and CP. Two panels of antibodies were developed based on an HTS assay [44] and literature
confirming the relevance of specific neutrophil CD markers in human inflammatory conditions
(Table 1). All of the markers that were selected had significantly altered expression on oral
neutrophils compared to blood neutrophils in the HTS screen. Analysis of FSC x SSC, which are
generally equivalent to cell size and granularity, indicated that oral neutrophils were shifted
towards the lower left quadrant compared to blood neutrophils (Figure 5). In healthy oral
neutrophil samples, we identified two populations with distinct epicenters based on FSC x SSC
(Figure 5A and C), while only one population was observed in CP (Figure 5B and D). The two
oral neutrophil populations in health, which occurred in roughly equal proportions, were: a
population with similar FSC-A x SSC-A properties to blood neutrophils (P1, 45.2% ± 3.2), and a
population that was shifted to the lower left quadrant of the scatterplots (P2, 50.3% ± 3.2)
(Figure 5A). The CP oral neutrophil population was consistently observed in the lower left
quadrant of scatterplots (P3) (Figure 5B). The mean geometric MFI of each CD marker was
determined for blood neutrophils and for each oral neutrophil population (Figure 6 and Table
3). There was no difference in expression of CD markers between blood neutrophils of healthy
volunteers and CP patients. All of the CD markers in our panels had elevated expression on each
27
of the oral neutrophil populations compared to blood, with the exception of CD16, which had
reduced expression. By comparing oral neutrophil populations, we found that seven CD markers;
CD10, CD11b, CD18, CD55, CD63, CD64 and CD66, had significantly elevated expression on
CP oral neutrophils compared to either of the oral neutrophil populations in health. Furthermore,
CD16 and CD170 had elevated expression on the CP oral neutrophils compared to the P2 healthy
oral neutrophils, but similar expression to the P1 healthy oral neutrophils. Comparing the two
healthy oral neutrophil populations, we found significant differences in expression of four CD
markers. Expression of CD55 and CD63 were elevated, while the expression of CD16 and
CD170 were reduced on the P2 population compared to the P1 population. Retrospectively, we
found that gating based on expression of CD16 and CD170 markers was sufficient to distinguish
between the two healthy oral neutrophil populations (Figure 7). Based on our observation of
higher degranulation, phagocytosis and expression of neutrophil markers of activation by CP oral
neutrophils, this population was designated as pro-inflammatory (Pro), while the two healthy oral
neutrophil populations, based on the para-inflammation hypothesis, were called Para1 (FSC-
Ahigh/SSC-Ahigh) and Para2 (FSC-Alow/SSC-Alow).
4.5 Pro-inflammatory Neutrophils Have Elevated ROS Production and
NET Formation
To assess the functionality of oral neutrophil populations, we examined baseline and PMA
stimulated generation of ROS by flow cytometry. By gating on the two healthy oral neutrophil
populations (Para1 and Para2), we found that the Para2 population produced more ROS than the
Para1 population at baseline, and both populations showed increased ROS production after
stimulation with PMA (Figure 8A). Pro-inflammatory neutrophils produced more ROS than the
para-inflammatory oral neutrophils. However, in contrast to the Para1 and Para2 populations,
PMA stimulation did not induce a significant increase in ROS production by the CP neutrophils,
suggesting that they have exhausted their potential for ROS production, while para-inflammatory
neutrophils retain their potential for further stimulation (Figure 8A). A flow cytometric NETosis
assay, which detects myeloperoxidase (MPO) and histone citrullination on the cell surface, was
used to detect neutrophil extracellular trap (NET) formation in oral neutrophil populations. We
found that Pro oral neutrophils from CP patients showed high levels of NET formation,
compared to the Para cells from healthy patients (Figure 8B).
28
Chapter 5 : Discussion
The present study is the first to characterize oral neutrophil subsets specific to health and chronic
inflammation of the oral mucosa. This reports indicates that oral neutrophils from CP patients are
in a pro-inflammatory activation state compared to healthy oral neutrophils, as determined by
elevated degranulation, phagocytosis, ROS production, NETosis and a characteristic signature of
cell surface markers of activation. We thus demonstrate an intermediate or para-inflammatory
state of oral neutrophils in health. Neutrophils are important primary innate immune responders
that migrate from the circulation to sites of inflammation in the tissue. Neutrophils contribute to
periodontal tissue homeostasis through microbial surveillance and direct modulation of the host
tissue response [158], however pathological neutrophil responses can result in tissue damage
[95,159,160]. While multiple circulating neutrophil subtypes are known [17,19,114,161], to our
knowledge, diversity of neutrophil populations in tissue has not been studied, nor is there an
understanding of how neutrophils maintain tissue homeostasis in the presence of commensal
bacteria. CD markers and their surface expression levels can be used for three main purposes: 1)
To label/define a specific population of interest, 2) as markers of functionality or 3) to gauge the
state of activation of a particular function [162,163]. Here we identified separate neutrophil
populations with unique phenotypes in health and disease using custom CD marker panels, and
compared the functional activity of these populations. We found CD markers indicative of cell
activation to be expressed at much higher levels on oral neutrophils in CP compared to healthy
oral neutrophils, constituting a multiparameter CD marker signature that can differentiate
between para- and pro-inflammatory tissue neutrophil populations, based on activation state. The
markers that were up-regulated on CP oral neutrophils fall into three categories: 1) markers of
activation/degranulation; CD10 [131], CD63 [164,165], CD64 [166,167] and CD66a [168], 2)
adhesion receptors: CD11b and CD18, and 3) complement inhibitor CD55 [169]. The pro-
inflammatory phenotype of CP oral neutrophils was confirmed by elevated degranulation,
phagocytosis, ROS production and NETosis.
Interestingly, we observed two different populations of oral neutrophils in health. The Para1 and
Para2 populations differed based on their size and granularity profile, expression of specific CD
markers, production of ROS and NET formation. The Para1 oral neutrophil phenotype, which is
present only in health, has a FSC and SSC profile that is similar to naïve blood neutrophils, and
29
these cells are in a lower state of activation relative to the Para2 oral neutrophils, based on the
relative expression levels of four CD markers. Compared to the Para1 population, Para2 oral
neutrophils had elevated expression of two markers of activation (CD55 and CD63) and reduced
expression of the inhibitory receptor, CD170 [170], and the low affinity Fc-receptor, CD16. A
drop in CD16 is likely a consequence of internalization as a result of phagocytosis, and therefore
is consistent with elevated phagocytic activity of the Para2 population compared to the Para1
population. Functionality, as determined by ROS production and NET formation, was also
reduced in the Para1-neutrophils compared to Para2-neutrophils. We surmise that the Para2
neutrophils may be the front line neutrophils within the healthy tissue that interact with the
commensal biofilm, or are coming from pockets with increased biofilm load, resulting in an
increased activation state that is still below the maximal activation state of the Pro-inflammatory
neutrophil population observed in CP. Similarly, Para1 neutrophils, which also show
upregulation of activation markers compared to blood, but have restrained activation compared
to Para2 neutrophils, could be oral neutrophils that are in a primed state.
In contrast to health, there is only one population of CP oral neutrophils, which have a similar
FSC/SSC profile to Para2-neutrophils, but are more activated than either of the Para-
inflammatory populations based on ROS production, NET formation and expression of CD
markers of activation. This suggests that periodontal tissue neutrophils lie on a spectrum of
increasing activation; Para1 < Para2 < Pro. Based on the fact that the Para1 and Para2
populations occur in equal proportions, and were absent in CP, it is possible that the balance
between the two populations contributes to the maintenance of tissue health in the presence of
commensal microorganisms. Another possibility is that Para2 neutrophils come from sites of
undetected subclinical inflammation in the mouth. While CD16 and CD170 expression are
reduced in Para2 populations compared to Para1, levels of these two markers remain high on
pro-inflammatory neutrophils in CP. It is unclear why CD16 and CD170 expression remain high
on the CP oral neutrophils, since this would predict that these cells are not fully activated. It is
possible that periodontal pathogens may be involved in the up-regulation of these markers as part
of their immune-modulatory actions, causing suppression of certain aspects of neutrophil
function.
Reduced SSC of Para2 and Pro neutrophils, relative to the Para1 population, are consistent with
increased degranulation in these populations, however, nuclear morphological changes including
30
those associated with NETosis might also alter the spectral properties of the cells. Upregulation
of CD markers associated with granules confirms that healthy and CP oral neutrophil populations
have undergone degranulation relative to blood neutrophils [91]. Furthermore, a relative increase
in degranulation markers on oral neutrophils in CP, compared to healthy oral neutrophils, is
consistent with increased degranulation observed by electron microscopy, and suggests that
reduced SSC is at least partly due to increased degranulation in these populations.
In CP there is a shift from two para-inflammatory populations to one pro-inflammatory
neutrophil phenotype in response to the dysbiotic microenvironment instigated by pathogenic
microbes. This conversion of para-inflammatory neutrophils into pro-inflammatory neutrophils is
likely to be a key aspect of pathogenesis in periodontal disease. Further research will be
necessary to elucidate the functional interplay between the two para-inflammatory subtypes.
Furthermore, experimental models of gingivitis will be useful to better understand the transition
from Para1 to Para2 to Pro neutrophils.
31
Conclusion and Future Directions
Inflammation is the physiological response to a noxious stimulus. Chronic inflammation can be
thought of as the extreme end of a spectrum of tissue states that ranges from steady state
homeostasis, to protective para-inflammation and finally to acute or choric inflammation. In the
healthy oral cavity there is a constant low-grade stress caused by repeated physical trauma and
presence of commensal microbiome. This is countered by a constant influx of neutrophils that
help establish and maintain tissue homoeostasis. Based on the outcomes of this study, we can
conclude that the neutrophils recruited to the healthy oral cavity are in an intermediate or Para-
inflammatory state and have not been activated to their full potential. Neutrophils are classically
thought to be in either a resting or activated state, however we have demonstrated that they
actually exist in four states: a resting basal state in blood, two para-inflammatory states in the
healthy oral cavity and a fully activated pro-inflammatory state in the diseased oral cavity. Oral
neutrophils from CP patients are in a Pro-inflammatory state when compared to healthy oral
neutrophils (Para-1 and Para-2), based on elevated degranulation, phagocytosis, ROS & NET
production and CD marker expression profile (Figure 9A). Para-1 oral neutrophils have a flow
cytometry profile that is similar to naïve blood neutrophils and show upregulation of activation
markers compared to blood neutrophils but have restrained activation compared to Para- 2
neutrophils. Therefore, Para-1 neutrophils represent oral neutrophils that have recently migrated
out of the circulation into the tissues and are in a primed state. Para-2 neutrophils have an
intermediary phenotypic and functional state between Para-1 and Pro-inflammatory neutrophils.
Para-2 oral neutrophils have a flow cytometry profile that resembles CP oral neutrophils. On a
functional level they produce intermediate level of NETs and ROS but can be further stimulated
towards Pro-inflammatory neutrophils. Para-2 neutrophils have elevated expression of some
activation markers (CD55 and CD63) and reduced expression of the inhibitory receptor CD170
and the low-affinity Fc receptor, CD16 relative to Para-1 neutrophils. These differences are
consistent with elevated phagocytic activity of the Para-2 population. Para-2 neutrophils likely
represent front-line neutrophils within the healthy tissue that interact with the commensal biofilm
resulting in an increased activation state that is still below the maximal activation state of the
pro-inflammatory neutrophil population observed in CP. Para-2 inflammatory neutrophil subsets
arise in response to localized inflammatory cues within the tissue and may serve to protect
tissues from host mediated tissue damage. The host protective role of para-inflammatory
32
neutrophils has yet to be clinically proven and requires further investigation. The current study
has made significant advancement in our understanding of oral neutrophils. However, there are
certain limitations that need to be addressed. First and foremost, the findings of this study need to
be repeated and validated using a large sample size of patients. Secondly, the presence and
function of Para-inflammatory neutrophils in patients with more intermediate levels of
periodontal disease as well as patient is gingivitis should be investigated. This study only focused
on the extreme ends of periodontal health to disease to facilitate the identification of key
differences between oral neutrophil phenotypes in health and disease. Lastly, the possible
presence of role of Para-inflammatory neutrophils should be investigated in other chronic
inflammatory conditions such as diabetes mellitus, inflammatory bowel disease, ulcerative colitis
and rheumatoid arthritis.
33
Figure Legend
Figure 1. Neutrophils in the Maintenance of Periodontal Health and Pathogenesis of
Chronic Periodontitis. Blood neutrophils are constitutively recruited to the periodontal tissue
and transition into the mouth. In periodontal health, there is a steady state recruitment of
neutrophils in the healthy oral cavity that is capable of co-existing with the commensal flora
without initiating frank inflammation and host tissue destruction (left panel). In CP, greater
number of neutrophils enter the gingival sulcus in response to pro-inflammatory mediators
generated by pathogenic bacteria and damaged host tissues. The resulting dysbiotic/pro-
inflammatory environment results in destruction of connective tissues in the periodontium, loss
of attachment and eventual tooth exfoliation (right panel).
Figure 2. Etiology and Pathogenesis of Chronic Periodontitis. The etiology of periodontitis is
multifactorial and relies on the microbial, host and systemic factors. At the microbial level,
periodontitis pathogenesis is driven by the quality and quantity of the biofilm. In the transition
from health to disease there is a micro-environmental shift that disrupts a healthy and
harmonious (“symbiotic”) biofilm into a pathogenic and destructive (“dysbiotic”) microbial
community that promotes periodontal breakdown. Bacterial dysbiosis may be required to initiate
periodontitis but often fails to cause periodontitis. Behavioural (e.g. smoking), systemic (e.g.
diabetes) and genetic (e.g IL-1B polymorphism) risk factors can account for half of the variation
in periodontal disease susceptibility.
Figure 3. Multicolor Flow Cytometry Gating Strategy for Blood and Oral Neutrophils.
Scatterplots of representative blood and oral neutrophil samples from a healthy individual are
shown to demonstrate the gating strategy used for multicolor flow cytometry. Doublets were
excluded based on SSC-H x SSC-W. (A) Oral neutrophils were gated based on expression of
CD18 to exclude debris. Cytospins of CD18+ve and CD18-ve sorted cells were analyzed by Diff-
Quik staining. Unfiltered oral cells are shown for reference. Bars are 10 µm. (B) Neutrophils in
whole blood were gated using CD16hi/SSC-A.
Figure 4. Oral Neutrophils Have Elevated Phagocytosis and Greater Degranulation in CP.
Blood and oral neutrophils were pelleted, fixed and imaged by electron microscopy. (A)
34
Representative images are shown. Arrowheads indicate granules and arrows indicate
phagosomes. Bar is 2 µm. Granule number normalized to cytoplasmic area (B) and phagosomes
per cell (C) were determined using ImageJ software. At least 25 cells were analyzed from paired
blood and oral neutrophils of controls (n = 5) and periodontal disease patients (n = 5). Significant
differences were determined by ANOVA with a post hoc t-test. *<0.05.
Figure 5. Identification of Para-inflammatory Neutrophils in Oral Health. Representative
contour plots of blood and oral neutrophils from a healthy (A) volunteer and a CP (B) patient.
Two oral populations were observed in health, in roughly equal proportions, and were gated
based on FSC x SSC profiles. In CP, only one population of oral neutrophils was observed with
low FSC and low SSC. Gates for the three oral neutrophil populations (P1, P2 and P3) are
shown. Conversely, only a single Pro-inflammatory neutrophil population was identified in oral
samples of patients with CP. FSC x SSC profiles of five representative oral neutrophil samples
from health (C) and CP (D) are shown. Bi-modal populations in health were gated as P1 and P2,
as indicated. Percentages of total neutrophils are indicated.
Figure 6. Para- & Pro-inflammatory Oral Neutrophil Populations Are Defined by Unique
CD Marker Expression Profiles. Blood and oral neutrophils of healthy controls (n=11) and CP
patients (n=17) were analyzed by multicolour flow cytometry. (A) Representative histograms
expression on blood neutrophils (red), P1 oral neutrophils (green), P2 oral neutrophils (orange)
and P3 oral neutrophils (blue) are shown. FMO isotype controls are indicated for each CD
marker. (B) The geometric MFI for each CD marker is shown for the healthy (P1, P2) and CP
(P3) oral neutrophil populations. Mean geometric MFI ± SEM are shown. ANOVA with a post-
hoc t-test was performed to determine statistical significance (**P < 0.01).
Figure 7. CD Markers Can Be Used to Gate on Para1 and Para2 Oral Neutrophil
Populations. Oral neutrophil populations were analyzed based on CD16 x CD170 (A) and CD16
x CD55 (B). Contour plots for a representative healthy subject are shown.
Figure 8. Pro-inflammatory Oral Neutrophil Populations Have Elevated NET Formation
and Increased ROS Production Compared to Para-inflammatory Neutrophils. (A) Oral
neutrophils from healthy controls and CP patients were labelled with DHR and treated with PMA
(+PMA) or left untreated (-PMA) at 37°C for 15 minutes. Representative histograms showing
levels of the DHR oxidation product, rhodamine 123, on Para1, Para2 and Pro-inflammatory
35
neutrophil populations, are shown. Fluorescence minus one (FMO) controls are indicated. At
least 2 x 104 events were acquired in three independent experiments. Bar graphs show basal
mean geometric MFI’s of rhodamine 123 signal and the fold-increase in MFI in response to
PMA stimulation for each oral neutrophil population. (* P < 0.05, n=3). (B) Fixed oral
neutrophils were labelled with H3Cit, MPO and CD18 antibodies and analyzed by flow
cytometry. Doublets were excluded and neutrophils were gated based on expression of CD18.
Representative histograms of H3Cit and MPO expression on Para1, Para2 and Pro-inflammatory
neutrophil populations, are shown. FMO controls are indicated. At least 2 x 104 events were
acquired in three independent experiments. Bar graphs show mean geometric MFI’s for H3Cit
and MPO for each oral neutrophil population ± SEM. (* P < 0.05, ** P < 0.01, n=7).
Figure 9. Model of Para- and Pro-Inflammatory Neutrophil Phenotypes in Health and
Disease. Para-inflammatory neutrophil subtypes are present in periodontal health while pro-
inflammatory neutrophils occur in CP. The two para-inflammatory populations in health differ
based on CD marker expression and functional activity. Para2 oral neutrophils produce more
ROS and NETs compared to Para1 populations. In addition to elevated neutrophil recruitment
during CP, Pro-inflammatory neutrophils are in a heightened inflammatory state based on high
expression of CD markers of activation, elevated production of ROS and more NET formation
compared to both para-inflammatory oral neutrophils.
36
Tables and Figures
Figure 1. Neutrophils in the Maintenance of Periodontal Health and Pathogenesis
of Chronic Periodontitis.
37
Figure 2. Etiology and Pathogenesis of Chronic Periodontitis
38
Table 1. CD Marker Panels for Multicolor Flow Cytometry
CD
marker
Protein - Function Flour Supplier Clone
Panel 1
CD16 Fc-γRIII – Low affinity Fc-
receptor
PE Biolegend 3G8
CD11b αM-integrin – Adhesion,
complement receptor
PerCP-Cy5.5 Biolegend ICRF44
CD177 NB1 - Adhesion APC Abcam MEM-
166
CD64 Fc-γRI – High affinity Fc-receptor Alexa700 BD 10.1
CD10 Neprylisin – Inflammatory
metalloproteinase
APC-Cy7 Biolegend HI10a
CD18 2-integrin – Adhesion,
complement receptor
BV421 BD 6.7
Panel 2
CD55 DAF – Complement inhibitor FITC BD IA10
CD170 Siglec-5 – Inhibitory receptor,
adhesion
PE R&D 194128
CD63 Granulophysin – Degranulation PerCP-Cy5.5 Biolegend H5C6
CD66a Ceacam – Adhesion, degranulation APC eBioscience CD66a-
B1.1
CD16 Fc-γRIII – Low affinity Fc-
receptor
Alexa700 BD 3G8
CD11b αM-integrin – Adhesion,
complement receptor
APC-Cy7 Biolegend ICRF44
CD18 2-integrin – Adhesion,
complement receptor
BV421 BD 6.7
39
A
FSC-A SSC-H CD18 FSC-A
SSC
-A
SSC
-W
SSC
-A
SSC
-A
Ora
l
Unfiltered Debris Neutrophils
Blo
od
B
FSC-A SSC-H CD16 FSC-A
SSC
-A
SSC
-W
SSC
-A
LDHD CD16+
SSC
-A
C
CD16
SSC
-A
Figure 3. Multicolor Flow Cytometry Gating Strategy for Blood and Oral
Neutrophils
40
Table 2. Demographic and Periodontal Characteristics of Patients Cohorts
*Mean ± STDev †Neutrophil yield was per 20ml of saliva
Characteristic Healthy (n=11)
Diseased (n=17)
Intergroup P-value
Age (years)* 49 ± 18 57 ±13 0.16
Female (%) 9 (58%) 9 (41%) 0.23
Smoking 0.07
Never 10 9 -
Former smoker (years ago) 1 (10) 5 (15-50) -
Current smoker 0 3 -
Number of teeth* 28 ± 1.7 24.9 ± 4.2 0.04
Number of sites* 168 ± 10.2 150.± 25.5 0.04
% Sites with BOP* 8.6 ± 10.4 62. ± 18.5 1.06 x 10-8
Active disease (PD ≥ 5mm + BOP)
Number of sites with active disease* 0.2 ± 0.6 39.5 ± 26.4 8.63 x 10-5
Percent sites with active disease* 0.1 ± 0.4 25.5 ± 15.5 2.74 x 10-5
Neutrophil yield (x 106)*† 1.1 ± 0.6 5.2 ± 3.8 4 x 10-4
41
Figure 4. Oral Neutrophils Have Elevated Phagocytosis and Greater Degranulation
in CP
42
Figure 5. Identification of Para-inflammatory Neutrophils in Oral Health
SSC
SS
C
FSC
C
D
FSC
43
Figure 6. Para- & Pro-inflammatory Oral Neutrophil Populations Are Defined by
Unique CD Marker Expression Profiles
44
45
Table 3. CD Marker Expression levels on Healthy and CP Neutrophils
Marker
BloodH
(n=11), Mean
geometric MFI
± SEM (x 103)
Para1
(n=11), Mean
geometric MFI ±
SEM (x 103)
Para2
(n=11), Mean
geometric MFI
± SEM (x 103)
BloodCP
(n=17), Mean
geometric MFI
± SEM (x 103)
Pro
(n=17), Mean
geometric MFI
± SEM (x 103)
CD10 0.12 ± 0.01 1.27 ± 0.17 1.26 ± 0.18 0.10 ± 0.01 2.06 ± 0.24
CD11b 0.18 ± 0.02 3.34 ± 0.78 3.42 ± 0.56 0.16 ± 0.01 6.70 ± 0.60
CD16 7.84 ± 0.29 4.92 ± 0.33 2.10 ± 0.18 7.13 ± 0.22 3.82 ± 0.45
CD18 1.72 ± 0.08 8.91 ± 0.62 8.54 ± 0.60 1.72 ± 0.11 13.06 ± 1.07
CD55 0.87 ± 0.05 3.13 ± 0.36 4.72 ± 0.70 0.84 ± 0.02 6.71 ± 0.65
CD63 0.37 ± 0.02 6.47 ± 0.98 9.14 ± 0.99 0.34 ± 0.02 19.66 ± 2.22
CD64 0.24 ± 0.01 0.61 ± 0.07 0.71 ± 0.09 0.23 ± 0.01 1.12 ± 0.10
CD66a 0.78 ± 0.04 9.00 ± 0.62 9.04 ± 0.70 0.73 ± 0.02 14.28 ± 1.16
CD170 2.69 ± 0.25 24.32 ± 1.91 12.54 ± 1.09 2.41 ± 0.12 24.01 ± 3.25
CD177 3.28 ± 0.26 7.19 ± 1.17 5.36 ± 0.91 3.13 ± 0.29 9.81 ± 2.27
46
Figure 7. CD Markers Can Be Used to Gate on Para1 and Para2 Oral Neutrophil
Populations
A
B
47
Figure 8. Pro-inflammatory Oral Neutrophil Populations Have Elevated NET
Formation and Increased ROS Production Compared to Para-inflammatory
Neutrophils
48
Figure 9. Model of Para- and Pro-Inflammatory Neutrophil Phenotypes in Health
and Disease
A
49
Contributions to the Thesis and Manuscript
Siavash Hassanpour is solely responsible for the entire content of this thesis.
The contents of this thesis is based on the manuscript entitled “Distinct Oral Neutrophils Subsets
Define Health and Periodontal Disease States” [43]. The contributions to the manuscript were as
follows:
Siavash Hassanpour contributed to the conception of the project, the design of the experiments,
sample preparation, data analysis, interpretation of the data and preparation of the manuscript.
Siavash Hassanpour was solely responsible for the screening and recruitment of all patients, as
well as sample and patient data collection.
Noah Fine contributed to the conception of the project, design of the experiments, sample
preparation, data analysis, interpretation of the data and preparation of the manuscript. Noah Fine
was solely responsible for all flow cytometry based experiments.
Alon Borensetin contributed by performing the electron microscopy experiments.
50
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