Inflammation and

Regeneration in the Dentin-

pulp Complex: Net Gain or
Net Loss?
Paul R. Cooper, BSc, PhD, Ilaria J. Chicca, BSc, PhD,
Michael J. Holder, BSc,and Michael R. Milward, BSc,
PhD
JOE - Volume 43, Number 9S, September 2017

Presented by – Manpreet Kaur

MDS 2nd year
 Cells of the dentin-pulp complex detect invading bacteria by
their expression of a range of pattern recognition receptors
(PRRs), which identify pathogen associated molecular
patterns.
 The PRRs reported as being present in the pulp include
 Toll-like receptors;
 Nucleotide-binding oligomerization domain proteins 1 and 2;
 Nod-like receptor family member pyrin domain containing 3
complex, also known as the inflammasome.
 The expression of many of these molecules has been shown on
odontoblasts, pulp fibroblasts, pulp stem cells, neurones, and
endothelial cells, and they are able to detect several
components of the invading bacteria ranging from their DNA
to outer membrane components, such as lipopolysaccharides
(LPSs).
 Once host cells have detected bacterial components, they induce the
expression of antimicrobial peptides (AMPs) as well as invoke the
inflammatory cascade ultimately eradicating, the infection.
 Initially, because of their location at the periphery of the pulp, it is the
odontoblasts that are the first responders; however, as the infection
advances, cells deeper in the pulp core, including pulp fibroblasts,
endothelial cells, and stem cells, also become involved in the defense
reaction.
 In addition, there are immune cells resident in healthy pulp tissue, such as
dendritic cells and mast cells, that act as sentinels and also orchestrate the
early local immune response.
 At the molecular level, the detection of bacterial components via the PRRs
results in the activation of intracellular signaling cascades, with the
primary effects being mediated via the nuclear factor kappa B (NF-kB)
proteins and p38 mitogen-activated protein kinases.
 These pathways ultimately activate the gene expression of
proinflammatory cytokines and chemokines, such as interleukin (IL)-1a
and IL-1b, tumor necrosis factor alpha, IL-4, IL-6, IL-8, and IL-10.
 These molecules exert both autocrine and paracrine effects that can
amplify the inflammatory and immune responses and, in
particular, generate chemotactic gradients that lead to the
recruitment of immune cells from the vasculature, including T and
B lymphocytes, plasma cells, neutrophils, monocytes, and
macrophages.
 During the early stages of disease or when the infection has been
minimized, either by the host’s immune response or clinical
intervention, the tooth tissue may evoke tertiary dentinogenic
responses.
 These responses can be relatively simple in the form of reactionary
dentinogenesis, which involves the direct activation of the existing
primary odontoblasts, or the response may be relatively more
complex in the case of reparative dentinogenesis, which involves
orchestrated stem cell responses culminating in the generation of
new odontoblast like cells.
 Recently, a novel bacterial killing mechanism termed
neutrophil extracellular traps (NETs) has been described
that uses ROS signaling and results in cellular DNA extrusion.
The NETs are decorated with antimicrobial peptides (AMPs),
and their interaction with bacteria results in microbial
entrapment and death.
 This review considers the potential role of NETs within pulpal
infections and how these structures may influence the pulp’s
vitality and regenerative responses.
INNATE IMMUNE RESPONSE TO
DENTAL TISSUE INFECTION
 Similar to wound infections occurring at other sites in
the human body, it is neutrophils (polymorphonuclear
leukocytes) that are abundantly recruited and provide
the first line of defense in the innate immune
response in pulpal tissue.
 Because of their role and the increased demand
placed on the immune system during infection, their
levels released into the bloodstream increase; the
cells also become primed, and their longevity
increases.
 When circulating and surveying for microorganisms,
neutrophils reportedly have an average life span of
5.4 days, following which point they subsequently
undergo apoptosis and are removed by macrophages.
 Their priming, before reaching the site of infection, is
important because it aids in their rapid response for pathogen
clearance.
 This peripheral priming is achieved by activation of various
cytokines, growth factors, complement, or bacterial
components which instruct the neutrophil to leave the
circulation and traverse to the site of infection.
 The process of neutrophil recruitment involves the steps of
tethering, rolling, adhesion, crawling, and, finally,
transmigration.
 Although neutrophils aim to combat the invading bacteria, it is
known that they can also be one of the most significant
mediators of local host tissue damage because of their release
of ROS and proteolytic enzymes as they traverse the tissue and
combat the infection.
Neutrophil antibacterial
mechanisms
 After contact with bacteria, the neutrophil can undertake
phagocytosis and encapsulation into phagosomes with wide
range of antimicrobial proteins and molecules.
 The neutrophil then destroys the pathogens by intracellular
release of ROS (via nicotinamide adenine dinucleotide
phosphate oxygenase dependent mechanisms) or AMPs,
such as cathepsins, defensins, lactoferrin, and lysozyme.
 Notably, these AMPs are not only released by the
neutrophil granules into phagosomes but also into the
extracellular milieu.
 Hence, degranulation can provide an extracellular killing
mechanism; however, it may also cause further host
collateral tissue damage.
 Human neutrophils consecutively form 3 types of granules
packed with pro-AMPs and inflammatory proteins during their
cellular maturation.
1. Azurophilic (primary) granules contain myeloperoxidase
(MPO and azurocidin)
2. Specific (secondary) granules contain lactoferrin
3. Gelatinase (tertiary) granules contain matrix
metalloproteinase 9 (also known as gelatinase B).
 Notably, azurophilic granules can be further subdivided
depending on peroxidase and defensin content.
 Specific granules can also be further subdivided based on
lactoferrin, cysteine-rich secretory protein 3, gelatinase, and
ficolin content.
 Neutrophil
extracellular traps
(net)
 Neutrophil Extracellular Trap
Biology
NETs are weblike structures containing
decondensed nuclear chromatin adorned with
antimicrobial molecules, including histones
and the AMPs derived from azurophilic,
specific and gelatinase granules.
 Proteins shown as being associated with NETs
include
• AMPs, such as lactoferrin, cathepsin G,
defensins, LL37, and bactericidal/permeability-
increasing protein;
• Proteases, such as neutrophil elastase,
proteinase 3, and gelatinase; and
• Enzymes responsible for ROS generation, such
as MPO.
 Signaling of NET release
1. ROS signaling pathway uses nicotinamide adenine
dinucleotide phosphate oxidase assembly, superoxide and
hydrogen peroxide production, and subsequent conversion to
hypochlorous acid by MPO for NET release .
 The importance of ROS signaling in regulating NET release
is further highlighted in chronic granulomatous disease.
 Previously, only patients’ lack of ROS production was
thought to be responsible for susceptibility to infection;
however, more recently, their impaired NET production has
also been shown to be implicated in their impaired infection
control.
2. Decondensing of the nuclear chromatin, which is
achieved by enzymatic action. Knockout mice for the
calcium dependent enzyme peptidylarginine
deiminase 4 (PAD4) cannot make NETs under normal
physiological conditions, indicating the essential activity
of this enzyme.
 This citrullination process transforms positively charged
arginine residues in histones into neutrally charged
citrulline, leading to the loss of electrostatic attraction
between the DNA and its packaging proteins.
3. Primary granule derived neutrophil elastase enters the
nucleus and partially degrades histones enabling subsequent
binding of MPO derived from the same granules, resulting in
decondensation of the chromatin
 These series of events lead to nuclear morphologic changes
and nuclear membrane breakdown and are associated with the
neutrophil granules releasing their cargos .
Subsequently, the DNA and antimicrobial proteins and
enzymes combine with the chromatin in the cytoplasm
before the rupturing of the neutrophil outer cell membrane
and expulsion of these constructs.
 NET stimuli
The stimuli for NETs are complex and varied.
Furthermore, the temporality and context of NET
induction and release are crucial because aberrant
release can impede other immune functions, such as
chemotaxis and phagocytosis.
 Currently, a range of disease-relevant stimuli for
NET production have been reported and include
nitric oxide, cytokines, bacteria, and their
components, such as LPS and bacterial toxins,
yeasts, fungi, protozoa, AMPs etc.
(A) (i) ROS and (ii) NETs induced at 4 hours stimulated with heat-killed endodontic infection–
associated bacteria, P. gingivalis (MOI 1:100) and F.nucleatum (MOI 1:100), along with the
PMA (50 nm) positive control. Statistically significant differences from negative phosphate-
buffered saline control are shown with P values * = .05, ** = .01, and **** = .0001 (n = 4). (A)
(iii) An example fluorescent image of NETs induced by PMA stimulation. Nuclei and extrude
NETs are stained with Sytox Green (Thermo Fisher Scientific, Rugby, UK). (B) Fluorescent
images of (i) control (unstimulated) and (ii) F. nucleatum–induced NETs stained with Sytox
Green. (iii) A fluorescent image showing some F. nucleatum bacteria associating with a NET
DNA strand
Many in vitro studies use phorbol 12-myristate
13-acetate (PMA) as a stimulus for NETs.
 PMA works efficiently and directly by bypassing
receptor ligand binding on the neutrophil surface
and activates cytoplasmic protein kinase C
signaling, which leads to intracellular ROS
generation, stimulating approximately one third
of neutrophils to release NETs by 4 hours.
 Microbial evasion of NETs
 Deoxyribonuclease (DNase) enzymes expressed by bacteria have now
been shown to confer resistance to this antimicrobial strategy.
 Indeed, studies in mice using Streptococcus pneumoniae showed that the
strain expressing the wild-type EndA DNase exhibited 20% increased
resistance compared with the EndA deletion mutant. The wild-type strain
also showed an increasing spread of infection into the lungs and
bloodstream compared with the EndA deletion mutant strain .
 Role of DNase expression in NET evasion in group A streptococci has
shown DNase deletion mutants exhibited increased susceptibility to
neutrophil killing in vitro, and in vivo they showed significantly less
virulence in causing necrotizing fasciitis compared with the wild-type
strain.
 These data indicate the importance of NETs in limiting bacterial invasion
and dissemination.
Expression of a bacterial polysaccharide
capsule, a feature often associated with increased
virulence. This bacterial outer covering
modification has been shown to significantly
decrease pneumococci entrapment by NETs
compared with the nonencapsulated strains.
Cell wall lipoteichoic acid modification on
Gram-positive bacteria is a further adaptation that
enables the evasion of NET killing.
 As early as 1974, Porschen and Sonntag reported DNAse
activity in the endodontic infection-associated Gram-negative
bacteria Fusobacterium nucleatum; however, at the time, the
purpose of this enzymatic activity was not known.
 Jackubovics et al 2015 showed that this bacterial DNase was
able to degrade NETs in vitro.
 Clearly, within a biofilm, such as that growing within root
canals, expression by certain bacteria at particular stages of
growth may confer benefits to the whole microbial community
in terms of NET killing evasion.
 This virulence trait of the biofilm as a whole may then enable
further propagation and dissemination of bacteria within the
endodontic tissues.
 Interestingly, bacterial DNase expression may also be
important in modifying the biofilm matrix to enable plaque
development and maturation.
 Host response to NETs
 Excess and aberrant NET release or clearance may provide a source of
inflammatory and cytotoxic molecules.
 Notably, in the autoimmune disease systemic lupus erythematosus,
neutrophil derived granular proteins associated with NETs, including MPO
and proteinase 3, can cause autoantibody responses in patients, and NET
clearance by sera from some patients is impeded.
 This impaired removal of NETs has been attributed to the presence of
DNase inhibitors in patients’ sera or increased levels of anti-NET antibodies
or complement factors, which may protect NETs from DNAse degradation .
 Furthermore, delayed removal of NETs may provide a reservoir of PAD4
hypercitrullinated proteins, which provides antigens and contributes to
disease.
 Interestingly, P. gingivalis, which is frequently found in endodontic
infections, possesse its own PAD enzyme which PAD may citrullinate its
own or host proteins, and this may drive inflammation locally within the
infected root canal network.
 Recent studies in mouse models of lung disease have shown
that NETs and their components cause significant damage
to lung epithelial and endothelial cells. This effect was
particularly evident in animals with an influenza virus and
Streptococcus pneumoniae dual infection and was associated
with compromised macrophage clearance of NET.
 Data indicated that the viral infection led to neutrophil
priming, which was subsequently hyperactive in terms of
NET release. These dual-infected animals exhibited
increased lung tissue damage, which was associated with
exaggerated inflammation and damage to the alveolar-
capillary barrier.
 A recent review has highlighted the potentially
important role of histones in the pathobiology of
several diseases (Holder et al, unpublished data,
2018) histones have shown antibacterial activity,
including killing of endodontic infection–associated
bacteria but also reported to act as damage-associated
molecular patterns when they are released into the
extracellular space.
 There are 5 histone types responsible for the
packaging of nuclear DNA, and they are categorized
into 2 groups: the core histones (H2A, H2B, H3, and
H4) and linker histones (H1 and H5).
 Several stress-associated mechanisms have
been shown to result in their release including
apoptosis, necrosis, and now NETosis.
High concentrations of serum histones are
detected in animals or patients with cancer,
inflammation, and infection, and inadequate
clearance of this cellular debris by
macrophages may lead to accumulation of
these damage or disease markers.
 These published reports (Holder et al, unpublished
data, 2018) relating to the proinflammatory and
cytotoxic nature of NETs and their components have
driven to explore their effects on pulpal cell
populations.
 Preliminary in vitro data are consistent with those
seen in other diseases and indicate that single
histones, such as H2A or combinations of histones,
are cytotoxic to human pulp cells and can drive IL-8
release, which potentially could perpetuate the
chronic inflammatory cycle within the pulpal tissue.
 conclusion
 It is interesting to postulate that NETs and their components, such as
histones, may provide novel prognostic markers for pulp pathologies.
 Such a concept could underpin the development of novel antibacterial
therapeutic approaches because it is clear that infecting bacteria, such
as P. gingivalis, aim to perpetuate chronic inflammation within the
host tissue to increase nutrient availability and enable themselves and
the biofilm to thrive.
 Although some indirect data exist that indicate the presence of NETs
in infected pulpal tissue, there is now considerable scope for further
research to characterize their roles in endodontic pathobiology.