DR.

JOERG MEYLE (Orcid ID : 0000-0003-0495-6926)

Accepted Article
Article type : Other

Corresponding author mail id:- Joerg.Meyle@dentist.med.uni-giessen.de

Title Page

The innate host response in caries and periodontitis

Joerg Meyle1, Henrik Dommisch2, Sabine Groeger1, Rodrigo A. Giacaman3, Massimo

Costalonga4, Mark Herzberg5

Affiliations:
1
Department of Periodontology, University of Giessen, Giessen, Germany
2
Department of Periodontology and Synoptic Dentistry, Charité – Medical University Berlin,
Berlin, Germany
3
Cariology Unit, Department of Oral Rehabilitation and Interdisciplinary Excellence
Research Program on Healthy Aging (PIEI-ES), University of Talca, Talca, Chile.
4
Department of Developmental and Surgical Sciences, University of Minnesota, Minneapolis,
Minnesota, USA
5
Department of Diagnostic and Biological Sciences, University of Minnesota, Minneapolis,
Minnesota, USA

Statement of sources of funding for the study

No funding from any source was provided or used.

Research on this topic performed in the Herzberg lab has been supported by NIH/NIDCR
grants R01DE11831, R21DE015056, R01DE015503, and R01DE021206.

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/jcpe.12781

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 Disclosure of any conflicts of interest
Accepted Article The authors declare that there are no conflicts of interest to be reported.

Abstract

Innate immunity rapidly defends the host against infectious insults. These reactions are of
limited specificity and exhaust without providing long-term protection. Functional fluids and
effector molecules contribute to the defence against infectious agents, drive the immune
response, and direct the cellular players.

Aim. To review the literature and present a summary of current knowledge about the
function of tissues, cellular players and soluble mediators of innate immunity relevant to
caries and periodontitis.

Methods. Historical and recent literature was critically reviewed based on publications in
peer-reviewed scientific journals.

Results. The innate immune response is vital to resistance against caries and periodontitis
and rapidly attempts to protect against infectious agents in the dental hard and soft tissues.
Soluble mediators include specialized proteins and lipids. They function to signal to immune
and inflammatory cells, provide antimicrobial resistance and also induce mechanisms for
potential repair of damaged tissues.

Conclusions. Far less investigated than adaptive immunity, innate immune responses are
an emerging scientific and therapeutic frontier. Soluble mediators of the innate response
provide a network of signals to organize the near immediate molecular and cellular response
to infection, including direct and immediate antimicrobial activity. Further studies in human
disease and animal models are generally needed.

Clinical Relevance

Scientific rationale. Dental caries and periodontitis are among the most prevalent diseases
of bacterial origin affecting humans. As we come to understand the scope and impact of the
complex immune response to these infections, we may be able to design better and more
specific preventive and therapeutic methods.

Prinicipal findings. The innate immune response is vital to our resistance to caries and
periodontitis. Soluble mediators of innate immunity include certain proteins and lipids, which
regulate the orderly march of immune and inflammatory cells, provide antimicrobial
resistance, and also signal mechanisms for potential repair of damaged tissues. The roles of
soluble innate immune mediators in caries are less well charted than for periodontal
diseases.

Practical implications. A more complete picture of innate immunity and how this aspect of
the immune system impacts caries and periodontitis will refine our diagnostic tools and
therapeutic armamentarium.

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 Introduction
Accepted Article Innate immunity reflects the capacity of the host to defend rapidly against infectious insults.
The innate response is of limited specificity, responsive to generalizable types of agents,
and exhausts without providing long-term protection. This paper reviews innate defense
mechanisms of the host towards infectious agents in the oral bacterial biofilm. The innate
defense mechanisms tend to protect the host against caries and periodontitis. Part of this
review has been incorporated into the consensus report of group 1 of the joint EFP/ORCA
workshop on the boundaries between caries and periodontal diseases (Jepsen et al., 2017,
Sanz et al., 2017)

During caries onset, the enamel surface and dentin, and cementum and dentin, form innate
barriers against cariogenic microbes protecting the dentin-pulp complex. Soluble mediators
contained in saliva include many antimicrobial molecules, which likely modulate the onset of
disease. In deep carious lesions, the role played by cells in the pulp is more apparent. In
periodontitis, the innate response to infection is formed by soft tissue barriers and cells,
soluble antimicrobial proteins including the complement system, and certain immune cells.
Innate immune cells include phagocytes such as neutrophils, macrophages, dendritic cells,
natural killer cells and γδ T cells. These cells migrate to the site of infection in response to
chemotaxins and cytokines.

This review presents a summary of current knowledge about the function of tissues, cellular
players and soluble mediators of innate immunity relevant to caries and periodontitis.

Material and Methods

A comprehensive search strategy was implemented as each author interrogated the
PubMed database, focusing on a subset of peer-reviewed literature characterizing innate
immune barrier, cells or molecules in relation to caries or periodontitis. As available,
systematic and narrative reviews were included and the conclusions incorporated into the
manuscript. The following set of keywords and mesh terms (listed in alphabetical order) was
used in different combiantions to screen the data base:

antimicrobial paptides; caries; gingival connective tissue; defensins; dendritic cells;

fibroblast; gingiva; gingival crevicular fluid; junctional epithelium; Langerhans cells; lipoxins;
monocytes; macrophages; mast cells; mucins, mucosa; neutrophils OR polymorphonuclear
leukocytes; NK cells; odontoblast; oral mucosal epithelia; pattern recognition receptors;
periodontitis OR periodontal diseases; periodontal ligament; plasma cells; prostaglandins;
pulp disease; pulpitis; resolvins; saliva; salivary proteins; Th17 cells; Treg.

In general, the search was restricted to papers that were published from 2000 onwards.
Some earlier landmark papers were also included. Some search combinations failed to
generate any outcomes, including “prostaglandin AND caries” and “Treg AND caries”,

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 Results
Environmental zones presenting differential innate immunity
Accepted Article
Liquid “barrier”: saliva and gingival crevicular fluid (GCF)
As part of the innate immune response of the oral cavity, saliva is a key factor in protecting
dental enamel, gingiva and mucosa by flushing microbes and foodstuffs, buffering acids,
remineralizing the tooth, providing antimicrobial activity, and permitting selective adhesion of
commensal microorganisms to maintain a symbiotic environment in the dental biofilm (Van
Nieuw Amerongen et al., 2004).

Saliva contains macromolecules that bind microbes, including mucins, salivary agglutinin
and secretory IgA. One or more of these glycoproteins can bind simultaneously to a single or
multiple microbial cells. Collectively, the complex of bound microbes and large glycoproteins
forms an agglutinated mass that is regularly and harmlessly swallowed, which clears the
infectious cells and viruses from the oral cavity while minimizing the retained microbial
populations.

The salivary flow rate – high or low – is characteristic of each individual and could contribute
to protection (Stookey, 2008) (de Almeida Pdel et al., 2008) (Leonor et al., 2009, Tenovuo,
1997) and inter-individual variability in caries risk. Higher flow rates may promote salivary
clearance of microbes from the oral cavity and contribute to the anticaries effect of the fluid.
Reduced salivary flow, as in Sjögren's syndrome, is associated with a higher incidence of
dental caries (Scully, 1986),(Scully, 1986). Salivary constituents have anticaries properties
(Lenander-Lumikari and Loimaranta, 2000) and may contribute to anticaries activity but the
relative contributions of individual components remains largely under-investigated. In
addition to their antimicrobial properties, salivary statherin, proline-rich proteins (PRP),
cystatins and histatins importance in the acquired salivary pellicle mediate the homeostasis
of the supersaturated state of calcium and phosphate salts (Hay et al., 1982). This
proteinaceous film reduces demineralization of the enamel surface, augmenting this innate
barrier’s resistance against acidogenic bacteria. The acquired pellicle is also an adhesion
substrate for pioneer colonizing bacteria in the dental biofilm. How the flow rate and
composition of saliva, and the supersaturated pellicle environment collectively affect the
supraginigval plaque microbiome and the cariogenicity of plaque bacteria remain topics for
active investigation (Cunha-Cruz et al., 2013).

Salivary innate immunity also controls the growth of plaque biofilm community members.
Canonical host defense antimicrobial proteins and peptides (AMPs) such as defensins,
calprotectin, cathelicidins (LL-37), and histatins (Diamond et al., 2008), and peroxidase
systems, lysozyme, lactoferrin, and secretory immunoglobulin A (sIgA) are suggested to
collectively provide anticaries activity (Castro et al., 2016) and reduce the risk of periodontitis
(van 't Hof et al., 2014). Caries-active children, for example, show lower levels of α-defensin
(Tao et al., 2005) and LL-37 (Davidopoulou et al., 2012) than caries-free children. Clinical
caries have not been associated, however, with salivary levels of β-defensins. Yet, a
polymorphism in the gene encoding human β-defensin 1 (DEFB1) may be associated with
higher caries experience (Navarra et al., 2016) (Navarra et al., 2016). Whereas AMPs show
in vitro activity against a broad spectrum of oral microorganisms, data are needed to show
activity in vivo.

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 Saliva is rich in gel-forming mucins, which bind many bacterial species, provide a potential
source of carbohydrates and aminoacids for bacterial metabolism and promote clearance
Accepted Article by swallowing (Tabak, 1995). Major constituents of the dental pellicle, mucins also promote
initial adherence of microbes to the tooth. The competing balance between saliva-mediated
adhesion to the tooth and clearance of microbes by swallowing may differ from person-to-
person. One member of the mucin family of glycoproteins, Muc19, may specifically
contribute to the innate immune response against caries (Culp et al., 2015)(Culp et al.,
2015). After infection with Streptococcus mutans and cariogenic challenge with sucrose,
Muc19-/- mice develop two-fold more carious lesions of greater severity than wild-type mice.
Muc19 heterotypic complexes with other salivary constituents and are suggested to bind S.
mutans to promote clearance by swallowing.

Saliva and GCF contain at least 45 different AMPs that appear to act to control oral
microbes and protect dental hard tissues and mucosal surfaces, maintaining healthy
homeostasis in the oral cavity (Gorr and Abdolhosseini, 2011). In general, the AMPs in these
biological fluids are produced by epithelial cells of the salivary ducts and the proximal
gingiva, and neutrophils (Wenghoefer et al., 2008, Dale et al., 2001, Dunsche et al., 2002,
Lehrer, 2004, Gursoy et al., 2011). Saliva also contains varying amounts of
immunomodulatory interleukin-1β, interleukin-17 and interleukin 23, although it is not known
whether they contribute to innate immunity on mucosal surfaces of the oral environment
(Liukkonen et al., 2016).

Hard tissue barrier: enamel surfaces

Coated by salivary pellicle, the enamel forms a nearly impenetrable barrier against microbial
invasion of the dental pulp (Niviethitha et al., 2016). Resistance of enamel to bacterial
attachment, acidogenesis and erosion is affected by the protein composition of the salivary
pellicle (Cheaib et al., 2015). Salivary pellicle cystatin-B, for example, may increase acid
resistance (Delecrode et al., 2015). On enamel smooth surfaces, dietary sucrose and highly
processed starch increase the acidogenicity of oral biofilms in vitro (Botelho et al., 2016),
whereas free unsaturated fatty acids may limit demineralization (Giacaman et al., 2016).

Anatomic variations in the thickness and organization of the enamel barrier and underlying
dentin occur on the tooth, reflecting differences in vunerability to caries. The thickness of the
enamel is significantly greater on distal than mesial surfaces (Fernandes et al., 2011). After
exposure to a cariogenic experimental S. mutans biofilm, cervical enamel and dentin
demineralization was analyzed using swept-source optical coherence tomography.
Independent of other oral factors, enamel near the CEJ showed significantly more
demineralization than other enamel regions (Tezuka H, 2016). Vulnerability to caries is also
modified by the quality of the salivary pellicle, access to acidogenic dietary substrates, and
pathogenic differences in the proximal microbial biofilm. Although the enamel can remain
unaffected, root caries occurs when the cementum and dentin are demineralized and
breached.

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 Soft tissue barriers: mucosal epithelia
As an innate immune barrier, the oral mucosa partitions the inside of the body from the
Accepted Article
environment to exclude environmental pathogens, exogenous substances, and resist
mechanical stress (Presland and Jurevic, 2002). As a barrier, the gingival epithelium is
formed by interconnecting keratinocytes bridged one to another by cell adhesion molecules
(CAMs) (Groeger and Meyle, 2015). The CAMs of the multilayered syncytium are
susceptible to digestion by gingipains from P. gingivalis, which could increase tissue
permeability (Groeger et al., 2010, Hintermann et al., 2002, Katz et al., 2000, Nisapakultorn
et al., 2001). The protective function of the gingival epithelium is enhanced by keratinization,
which helps resist abrasion, and saliva, which physically lubricates the surfaces and
provides antimicrobial protection.

Proximal to the tooth surface, the specialized junctional epithelium (JE) is non-keratinized
and modified to attach to the hard tissue using hemi-desmosomes. Like other gingival
tissues, JE cells express CAMs including integrins, which mediate cell interactions with the
extracellular matrix, basement membranes, and contribute to cell-cell adhesion (Danen and
Sonnenberg, 2003, Larjava et al., 2014, Larjava et al., 2011) as well as cadherins, which
form tight contacts between cells (Juliano, 2002).

In periodontitis, αvβ6 integrin expression in the pocket epithelium is strongly down-regulated,

simulating a localized αvβ6 integrin deficiency. In mice expressing an αvβ6 integrin defect,
periodontal pockets, inflammation and alveolar bone loss occur spontaneously (Ghannad et
al., 2008, Gursoy et al., 2016). The signs of experimental periodontitis appear to be induced
by activation of transforming growth factor β1 (TGFβ1) in the absence of αvβ6 integrin.
Hence, TGFβ1 suppression in the presence of αvβ6 integrin are determinants of periodontal
health.
Epithelial proliferation, wound repair, anti-apoptosis, regeneration, mucin interactions, and
neovascularization are mediated at least in part by trefoil factors (TFFs) (Choudhary et al.,
2015). The TFFs, members of the mammalian growth factor peptide family, are soluble
proteins secreted by oral mucosal epithelium (Hoffmann et al., 2001). TFF3 regulation may
contribute to the etiopathogenesis of chronic peridondontitis (Chaiyarit et al., 2012).

Soft tissue barriers: gingival connective tissue and periodontal ligament

Periodontal pathogens stimulate the release of proinflammatory cytokines in gingival

connective tissue as well as in the periodontal ligament, which induces osteoclastogenesis
and bone resorption. Fibroblasts are the predominant cells and produce collagens and other
connective tissue proteins to form the extracellular matrix, which provides tensile strength to
the tissues and invests blood vessels. During inflammation the same cells participate in the
inflammatory response and release tissue degrading enzymes (see below “cellular players”)
. (Dommisch et al., 2012, Hosokawa et al., 2005).

Soft tissue barriers: the dental pulp

Once the carious process disrupts the integrity of the hard surfaces, bacterial biofilms can
advance deeply beyond the dentin into the pulp. The dentin is an innate barrier covering the

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 pulp. Dentinal tubules and fluid may be a conduit for soluble bacterial virulence factors to
enter the pulp, initiating innate pulpal immune responses (Hahn and Liewehr, 2007). At the
Accepted Article advancing caries front, dentinal fluid flow increases, nerve fibers secrete neuropeptides
(Awawdeh et al., 2002), and neurogenic inflammation develops. Within the pulp, the
odontoblast layer protects against invading microorganisms and insipient dentin caries. The
inflamed pulp contains immune cells, including immature dendritic cells (DCs), natural killer
cells (NKs), and T cells, producing cytokines and chemokines (Dommisch et al., 2005).
Nerve fibers express CD14 and Toll-like–receptor-4 (TLR-4) receptors, which can bind
proinflammatory lipopolysaccharide (LPS) and also stimulate repair (Byers and Taylor,
1993). Neurogenic inflammation may be seminal to caries-induced pulp injury.

Cellular players in innate immunity

Oral epithelial cells

Epithelial cells lining the oral mucosal sufaces represent the first line of response to bacterial
challenges including pathogen-associated molecular patterns (PAMPs). To engage PAMPs,
oral epithelial cells express pattern recognition receptors (PRRs) such as Toll-like receptors
(TLRs) and proteinase-activated receptors (e.g. PAR-2) (Giacaman et al., 2009) (Beklen et
al., 2008) (Lourbakos et al., 2001). In reponse to bacterial challenge, oral epithelial cells
express several families of AMPs that may control mucosal microorganisms, and
autoregulatory cytokines such as IL-1α, which signal to upregulate AMPs (Sorenson et al.,
2012). With other factors, AMPs may mediate periodontal health and disease as reviewed
elsewhere (Dommisch and Jepsen, 2015b, Chung et al., 2007, Komatsuzawa et al., 2007,
Dommisch and Jepsen, 2015a).

Gingival fibroblasts

Fibroblasts are central to the pathogenesis of gingivitis and periodontitis. To respond to

invasive pathogens, fibroblasts express protease-activated receptors and extracellular and
intracellular toll-like receptors (Hosokawa et al., 2010). Bacterial proteins and
lipopolysaccharides (LPS) activate fibroblasts in vitro and in vivo. Activated fibroblasts
produce extracellular matrix degrading enzymes like matrix metalloproteinases (MMP-1,
MMP-3) and stromelysin and proinflammatory mediators including interleukin-6, interleukin-
8, prostaglandin E2 and chemokine ligand 8 (CXCL-8) (Takashiba et al., 2003, Palm et al.,
2015, Jung et al., 2016, Baek et al., 2013, Belibasakis and Guggenheim, 2011, Belibasakis
et al., 2007, Belibasakis et al., 2005b, Belibasakis et al., 2005a). Bacterial proteins and
microvesicles are negatively affect the viability of fibroblasts in vitro, while ultimately
suppressing production of proinflammatory CXCL8 and IL-6 and inducing production of
transforming growth factor beta (TGFβ) (Scheres and Crielaard, 2013, Palm et al., 2013,
Bengtsson et al., 2015).

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 Upon stimulation with proinflammatory cytokines like interleukin-1 and tumor necrosis
factor–α, fibroblasts also produce matrix-metalloproteineases and other proinflammatory
Accepted Article cytokines like interleukin-33, and interleukin-34 (Beklen and Tsaous Memet, 2014, Bostrom
and Lundberg, 2013). Much is yet to be learned about the the control of fibroblast physiology
in periodontal health and disease.

Odontoblasts

Odontoblasts are the first cells to respond to bacterial factors permiating the dentinal tubuli.
In response to PAMPs, odontoblasts engage by regulating the expression of TLRs, including
TLR-2, TLR-3, TLR-5 and TLR-9 (Durand et al., 2006). TLR signaling results in upregulation
of AMPs, which appear to form an antimicrobial “shield” (Veerayutthwilai et al., 2007). The
AMPs, including human β-defensins (hBD-2), may mediate early immune responses in the
dental pulp (Dommisch et al., 2007, Horst et al., 2011).

In teeth with vital dental pulps, caries stimulates upregulation of pro-inflammatory

mediators, including interleukin 1β (IL-1β), which then is suggested to induce expression of
hBD-2 mRNA (Horst et al., 2011). Conversely, recombinant hBD-2 increases expression of
pro-inflammatory IL-6 and IL-8 in odontoblast-like cells and dentin sialophosphoprotein
(DSPP) by dental pulp cells (Shiba et al., 2003, Dommisch et al., 2007). DSPP is essential
to dentinogenesis (Kim and Simmer, 2007). In response to PAMPs that diffuse through
dentinal tubules into the pulp, tertiary dentine formation may be regulated by hBD-2
induction of DSPP.

Polymorphonuclear leukocytes (PMNs)

In response to pro-inflammatory, chemotactic mediators such as IL-1, IL-8 or bacterial

peptides (i.e., fMLP), neutrophils emigrate from the blood into tissues within thirty minutes to
clear invading microorganisms (Kolaczkowska and Kubes, 2013, Marki et al., 2015,
Herrmann and Meyle, 2015). In the tissues, neutrophils phagocytose microorganisms and
produce reactive oxygen species (ROS) to kill within the cells; extracellular microbes are
thought to be controlled by neutrophil extracellular traps (NET-formation, =netosis) that form
when PMNs lyse and release antimicrobial peptides and DNA (White et al., 2016, White et
al., 2014, Hirschfeld et al., 2015, Hirschfeld, 2014, Campbell and Colgan, 2015). NETs are
webs of complexed nuclear and mitochondrial chromatin/DNA and antimicrobial molecules
such as histones and AMPs (Brinkmann et al., 2004, Yousefi et al., 2009). In cooperation
with neutrophils, NETs are thought to protect the tissues against invading microbes during
early gingival inflammation, but the precise role in periodontal health and disease remains to
be characterized (White et al., 2016, Hirschfeld et al., 2015). In caries, pulpal PMNs,
monocytes and macrophages (mononuclear phagocytes) and natural killer cells (NK) are the
likely cellular effectors of innate immunity. During a canonical innate immune response,
neutrophils play a major role. In early caries, however, neutrophils are unlikely to protect
against the pathogens since the dentin is interposed until the pulp is exposed (Izumi et al.,
1995).

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 Neutrophils in the gingival crevice engage microbial biofilms on the tooth surface but are
generally overwhelmed and cannot clear the mass of periodontal microorganisms (Alfakry et
Accepted Article al., 2016). In established lesions, “frustrated” neutrophils release toxic superoxides, free
oxygen radicals, and tissue degrading enzymes contributing to local inflammation and tissue
damage (Cortes-Vieyra et al., 2016). Contributing to an exuberant inflammatory response in
generalized aggressive periodontitis, neutrophil-platelet aggregates form, perhaps as an
antimicrobial response, and release platelet granules containing AMPs and abundant
proinflammatory mediators (Zhan et al., 2016, Manne et al., 2016). The proinflammatory
response may be augmented by release of neutrophil stores of calprotectin into the tissues
and gingival crevicular fluid. In the tissue fluid, calprotectin appears to function as an
“alarmin”, a strong proinflammatory signal for monocytes and other inflammatory cells
(Austermann et al., 2014).

Neutrophils are critical to the occurrence of periodontitis, when there are either too many
activated cells or too few (Hajishengallis et al., 2015, Hajishengallis and Hajishengallis,
2014). For example, leukocyte adhesion deficiency (LAD) syndrome, which is accompanied
by generalized early-onset periodontitis, is characterized by abundant circulating neutrophils
that fail to localize in the periodontal tissues (Moutsopoulos et al., 2014, Hajishengallis and
Moutsopoulos, 2014, Hajishengallis et al., 2016, Hajishengallis et al., 2015). The severe
tissue destruction is associated with dysregulation of the immune response and the IL-23/IL-
17 axis, which signals for aggressive inflammation and bone resorption. Conversely, excess
“hyperactivated” neutrophils in the tissues produce high levels of tissue toxic, reactive
oxygen species, which characterizes localized aggressive periodontitis (Karima et al., 2005,
Hasturk and Kantarci, 2015, Kantarci et al., 2003). Hence, in the different forms of
periodontitis, neutrophils can contribute to antimicrobial defense or tissue destruction.

Monocytes and macrophages

Little is known about the role of macrophages in the response against caries. When the
carious process progresses through the dentin, the number of macrophages increases in the
dental pulp (Izumi et al., 1996, Izumi et al., 1995), reflecting increased vascular permeability
and favoring removal of bacterial antigens. Upon exposure to Gram-positive lipoteichoic acid
(LTA), pulpal macrophages up-regulate vascular endothelial growth factor (VEGF) promoting
neovascularization (Telles et al., 2003). In the pulp microenvironment, IFN-γ produced by
NK cells activate macrophages to assume a phagocytic phenotype (Hahn et al., 2000).

In inflamed periodontal tissues, monocytes appear upon extravasation from gingival

capillaries. The monocyte chemoattractants can include bacterial proteins and
lipopolysaccharides. In response, monocytes produce proinflammatory cytokines like IL-1
and TNFα. Conversely, monocytes, resident macrophages and mast cells function to resolve
inflammation by switching production of lipid mediators from pro-inflammatory to resolving in
the tissues (Freire and Van Dyke, 2013). Failure to clear the infection or resolve
inflammation is associated with an impaired host immune response leading to chronic
inflammation. In this environment, monocytes may differentiate into osteoclasts and activate
bone destruction (Di Benedetto et al., 2013). During periodontitis, the peripheral blood
monocyte Th1-promoting phenotype can change to Th2 ex vivo in response to LPS

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 (Fokkema, 2012). After periodontal treatment in non-smokers, the Th2-promoting cells
appear to be replaced by Th1. In smokers, however, the Th2 phenotype in peripheral blood
Accepted Article persists. The phenotype of monocytes appears to be plastic in response to environmental
factors, which may manifest in different disease outcomes.
The destruction of alveolar bone in periodontitis is generally associated with Th17/IL-17
responses. These responses can be simulated in vitro by incubating P. gingivalis or A.
actinomycetemcomitans with monocytes and CD4+ T-cells. When activated by these
pathogens, monocytes increase expression of CD40, CD54 and HLA-DR, secrete TNF-α, IL-
1β, IL-6 and IL-23, and differentiate into macrophages. P. gingivalis invades macrophages
and lipid A non-canonically inactivates the inflammasome, supporting intracellular bacterial
survival (Zenobia and Hajishengallis, 2015b, Zenobia and Hajishengallis, 2015a).

Monocyte-derived tissue macrophages ingest particulate antigens, microbes and apoptotic

cells. Tissue macrophages are antigen-presenting cells (APCs) presenting antigens to
effector/memory CD4+ T cells. The APCs ultimately link the innate and adaptive immune
responses. Macrophages may differentiate into M1 and M2 phenotypes (Hasturk et al.,
2012). M1 cells are activated by IFN-γ and lipopolysaccharide (LPS). M2 tend to respond to
IL-4 or IL-13 and appear to participate in resolution of inflammation with reduced cytokine
production. These phenotypes may reflect developmental plasticity, show overlapping
functions, and may be useful descriptors in limited situations (Martinez and Gordon, 2014).

Mast cells

In mast cells the cellular ligand CD117 regulates growth, migration, and specific effector
functions in response to the microbial environment and may modulate innate and adaptive
immune responses (Galli et al., 2011). Mast cells express pattern recognition receptors
(PRRs) including TLR-2 and TLR-4 (Trinchieri and Sher, 2007). In response to PAMPs, mast
cells synthesize TNFα, IL-8, C-X-C motif chemokine ligand 8, (CXCL8), C-C chemokine
ligand 20 (CCL20), and histamine, which is released from intracellular granules (Johnzon et
al., 2016). In the gingiva, mast cells appear to release histamine, enhancing expression of
CCL20 and IL-8 by gingival fibroblasts (Dommisch et al., 2015, Minami et al., 2007). In
experimental periodontitis, bone loss was reduced by local application of a histamine
receptor antagonist (Hasturk et al., 2006). In humans, however, topical administration of
cimetidine increased phagocytosis and bacterial killing by crevicular fluid neutrophils,
consistent with proposed interactions between mast cell activation and attenuation of the
signs of periodontitis (Van Dyke et al., 2005). Cimentidine, however, did not appear to
change gingival inflammation during the 28 day course of the study . Hence, it is unclear to
what degree mast cells might contribute to human gingivitis and periodontitis.

Dendritic cells

Dendritic cells (DCs) in the epithelial and subepithelial layers survey the microbiota on the
mucosal surfaces. In response to microbial challenge, DCs as antigen presenting cells drive
immune responses through cognate interactions with T cells and bystander release of

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 cytokines (Banchereau and Steinman, 1998). Of the groups or subtypes of mucosal DCs,
intraepithelial Langerhans cells (LCs) are the first immune cells to sample antigens of the
Accepted Article periodontal biofilm and elicit regulatory or proinflammatory immune responses (Malissen et
al., 2014). In mice, mucosal DC subsets are identified by anatomical location, migration
kinetics, and expression of cell surface markers. The subsets include the intraepithelial LCs,
subepithelial CD207 (Langerin+) DCs, which can be CD103 positive or negative, and
conventional DCs. Antigen-loaded DCs of all four subsets prime naïve T cells while
migrating to draining lymph nodes (Teng et al., 1999, Gao et al., 2013, Stashenko et al.,
2007, Cardoso et al., 2009, Eskan et al., 2012, Moutsopoulos et al., 2012, Jin et al., 2014).
LCs may play a regulatory role in experimental periodontitis. In a mouse model of chronic
periodontitis, mucosal LCs appear to drive P. gingivalis-specific Th17 differentiation without
affecting the extent of bone destruction or the frequency of P. gingivalis-specific iTregs
(Bittner-Eddy et al., 2016). When LCs and subepithelial Langerin+ DCs are both ablated in
mice, alveolar bone destruction and IFN-γ production are significantly increased, whereas IL-
17A and IL-10 expression are unaffected (Arizon et al., 2012). Langerin+ DCs induce retinoic
acid-dependent differentiation of IL-10 producing inducible regulatory T cells (iTregs)
(Coombes et al., 2007, Iwata et al., 2004) and regulate RANK-L expression (Ernst et al.,
2007). The model of bystander activation of subepithelial Langerin+ DCs and conventional
DCs by mucosal LCs is novel and warrants further investigation. In humans, the cellularity of
the marginal gingiva and oral mucosa has recently been dissected and described (Dutzan et
al., 2016) but the relative contribution of each DC subset to the pathogenesis of periodontitis
remains unclear.
In the dental pulp, immature DCs are greater in number in carious than healthy pulp tissue
(Yoshiba et al., 2003). Their role in caries etiology is unclear.

Th17 cells

Natural, interleukin-17 (IL-17) producing gamma-delta T cells (γδT, Th17) cells are abundant
innate immune cells; Th17 cells can be effectors without explicitly inducing an immune
response (Chien et al., 2013, Zhu and Qian, 2012). During an immune response, antigen
encounters with γδ T cells induce maturation and differentiation to produce IL-17. Natural
and antigen-stimulated Th17 cells contribute to inflammatory diseases (Cauli and Mathieu,
2012, Monteleone et al., 2012). Indeed, IL-17+ cell counts in biopsies are greater in
periodontitis patients than healthy controls (Cheng et al., 2016, Allam et al., 2011). IL-17 and
Th17 cells in human periodontal lesions appear to contribute to gingival inflammation and
bone destruction (Hienz et al., 2015, Cheng et al., 2014). Animal studies support these
findings. IL-17 enhances RANK-L expression by osteoblasts and CD4+ T cells to promote
osteoclastogenic activity (Di Benedetto et al., 2013, Ohlrich et al., 2009, Gaffen and
Hajishengallis, 2008). Using Th17 cells from gingival and alveolar bone samples from
healthy and chronic periodontitis patients, cytokine expression (mRNA) was analyzed
(Cardoso et al., 2009). When compared to healthy samples, gingiva from periodontitis
patients showed elevated IL-17, TNF-β, IL-1β, IL-6, and IL-23 mRNAs and contained Th 17
cells. Similarly, bone from periodontitis sites showed increased expression of IL-17 and the
bone resorption factor receptor activator of NF-κB ligand when compared to healthy (control)
bone (Cardoso et al., 2009). Yet, the relationship between IL-17/Th17 levels and the severity
of periodontitis is not definitive (Gaffen and Hajishengallis, 2008).

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 There is no evidence at this time in the literature for a role of IL-17 in caries.
Accepted Article
Discussion and Conclusions

We describe innate immune mechanisms that protect hard and soft tissues from the
microbial environment and against diseases of the oral hard and soft tissues. Innate
protection against caries and periodontitis is provided by saliva. Saliva contributes bulk
clearance of microorganisms by swallowing; salivary pellicle constituents helps to prevent
demineralization and promotes remineralization of dental hard tissues, important in dental
caries. Innate immune molecules in pellicle may limit microbial colonization of the tooth
surface.

Hard tissues (enamel, cementum, and dentin) and the gingival architecture (i.e., intercellular
junctions, cell differentiation), including the gingival epithelium and connective tissues, resist
mechanical abrasion and hinder bacterial invasion. AMPs produced in saliva, mucosal
epithelial cells and neutrophils serve as innate protective mechanisms to help the gingiva to
resist infection. As gingival epithelial cells proliferate and desquamate, microorganisms are
cleared from the periodontal soft tissues. High cell turn-over is crucial to eliminate infected
cells. Proximal to the cemento-enamel junction (CEJ), the junctional epithelium (JE) is
structurally adapted to seal to the tooth and prevent infection of the connective tissues. JE
cells also desquamate to help prevent infection of the epithelium and underlying connective
tissues, and eliminate putative pathogens that may demineralize proximal hard tissues or
invade the periodontal tissues. Dental hard and soft tissues are also home to different cell
types (tissue and immune cells) that resist or control intrusion from the oral microbial
environment and regulate pro-inflammatory reactions. Thus, when these four levels of innate
protective mechanisms are homeostatic, health tends to be maintained.

In many cases, the defense systems are not, however, able to prevent caries processes or
avoid periodontal destruction. In comparison to innate defense mechanisms, which evolved
during millions of years, changes in lifestyle and nutrition during the last centuries have been
too rapid to facilitate co-evolution of alternative defense and protection mechanisms.

To understand the complexity of the oral microbiome and the innate immune responses,
researchers have followed a reductionist approach. Progress to elucidate key inflammatory
defense mechanisms has been hindered by our lack of understanding of the many biological
circuits that might describe host innate immune interactions with the microbiomes (Kornman,
2008, Meyle and Chapple, 2015). As we learn more about these complex curciuts, the
intersections between innate and adaptive immune reponses are blurring and the biological
curcuits are increasing in complexity (Paul, 2011). Resolution of the complexity of innate
immune-microbiome curcuits is the next frontier. What factors are the strongest drivers?

Tools continue to be developed to enable high-through-put sequencing and analyses of

genomes and transcriptomes, proteomes and metabolomes. Bioinformatics needs to keep
pace so that these big datasets and be interrogated in an integrated manner. Key findings
can be validated by use of genetic knockouts in experimental animals and silencing or
elimination of single genes in cell culture coupled with functional screens and assays. In the
near future, scieintists will work largely in silico – on their computers - to answer questions

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 by interrogating big data sets. Computer models will be created that will inform future
experiments at the bench and in the clinic. The future has just begun.
Accepted Article
Acknowledgements

The contents of this manuscript have been discussed during the European Workshop on
Caries and Periodontal Diseases, which was held in La Granja, Spain in November 2016.

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