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9. HOST-MICROBIAL INTERACTIONS IN PERIODONTAL DISEASES

For a comprehensive reading on this topic, please refer to CHAPTER 7 - HOST-MICROBIAL

INTERACTIONS IN PERIODONTAL DISEASES in Nitin Saroch. PERIOBASICS. A
Textbook of Periodontics and Implantology, First edition, Sushrut Publications, 2017

1. Evolution of the understanding of periodontal disease progression

The microbial biofilm that forms around the teeth is the main cause of periodontal disease initiation
and progression. This biofilm is a complex community of microorganisms which produces various virulence
factors that initiate an inflammatory response. The enzymes released by bacteria in the biofilm include
proteases that are capable of disintegrating collagen, elastin, fibronectin, fibrin and various other
components of the intercellular matrix of both epithelial and connective tissue. Other proteases are
leukotoxins which are capable of killing leukocytes. Endotoxins are produced by Gram-negative bacteria
and are strong inducers of cytokine production. The term lipopolysaccharide (LPS) is often used
interchangeably with endotoxin. As the subgingival biofilm is majorly composed of Gram-negative bacteria,
endotoxins produced by them leads to the induction of cytokine production by host cells, which causes
inflammatory changes, like increased vascular permeability and engorgement of blood vessels. The present
chapter describes the various interactions that take place between the plaque microbiota and the host
immune response.

2. Models of disease progression

In the past, several patterns of disease progression have been described (42-45). The patterns of
tissue destruction in the progression of periodontal diseases have been explained with the help of various
models:
a. Continuous (linear) model: the periodontal disease progression occurs in a slow, steady
and progressive manner. However, this model did not explain the rapid progression and periodic remission
of the disease activity.
b. Episodic burst model: irregular periods of exacerbation and remission. Disease
progression occurs as an episodic burst of activity with periods of remission.
c. Synchronous burst model: periodontal diseases progresses with periods of exacerbation
and remission during a defined period.
d. Epidemiologic model: disease progression is continuous with aging and it depends only
on the duration of the process.
e. Brownian motion or stochastic model: random periods of sharp bursts and/or remission
can occur, but the underlying disease activity remains constant.
f. Random walking model: when observed at regular intervals, this model is similar to the
Brownian motion model.
g. Fractal model: a multi-factorial model simulating disease advancing with age with burst
and remission.
Although many models have been proposed, it has now been recognized that linear disease
progression can occur and there may be episodes of tissue destruction (46, 47).
Recently, Kornman (28) (2008) proposed a biologic systems model of the pathogenesis of
periodontal disease. Although the non-linear model of disease progression still holds well, this model
incorporated the role of contributing factors in the pathogenesis of periodontal diseases. According to this
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model, disease activity depends on an ecological shift in the plaque biofilm that can lead to the emergence of
a specific set of microbial pathogens (29, 30). Various environmental factors can contribute strongly to the
individual patient differences in the susceptibility to periodontitis, which includes diabetes, genotype and
smoking (23-25).
Recent studies have strongly advocated the relationship between periodontal diseases and systemic
diseases like coronary heart disease (31-36), chronic obstructive pulmonary disease (37-39), all of which
involve a common inflammatory mechanism. And lastly, there is further enhancement of our knowledge
about specific bacterial mechanisms and immunoinflammatory mechanisms in periodontitis (40, 41).

3. Our current understanding of host-microbial interaction

To better understand the host-microbial interactions in periodontal diseases, one should have the
basic knowledge of innate and adaptive immune response which. The initiation of inflammatory response
occurs with microbial insult derived from microorganisms in the dental plaques.

Microbial insult
The bacterial species in the plaque biofilm may or may not be pathogenic to the host. Various
bacterial species which are not associated with the periodontal disease progression are designated as
commensals. On the other hand, bacterial species which have been shown to possess virulence factors that
cause damage to the host tissues are designated as periodontal pathogens. To produce insult to the host, the
microorganism should be able to enter the host, should establish itself in the host, should be able to evade
the host response and should be able to produce virulence factors that cause tissue damage. The
microorganisms that have been shown to be associated with the periodontal disease progression and
virulence factors produced by them have been dicussed in the Microbiology chapter.

Host response
The host immune response against microbial insult consists of innate and adaptive responses. The
first defense against the invading periodontal pathogens and their products is junctional epithelium. The
cells of the junctional epithelium have fewer desmosomes as compared to the normal epithelial cells, which
account for its remarkable permeability. This permeability allows the ingress of bacteria and their products
and outwards flow of the gingival fluid and transmigration of neutrophilic granulocytes between the
epithelial cells. Thus, the junctional epithelium and subjacent connective tissue becomes the battlefield for
host-microbial interactions.
The cells involved in the first line of defense against many invading microorganisms are
macrophages and neutrophils, which are important components of the innate immune response. However,
these cells may not always eliminate infectious organisms, and some pathogens may not be recognized by
them. The adaptive immune response which is specifically directed against these organisms is then
generated to eliminate them. Cells involved in adaptive immune response are lymphocytes. Both innate and
adaptive immune responses play a vital role in dealing with these infections.

Clinical indicators of an active periodontal lesion

• Bleeding on probing.
• Increase in sulcular temperature.
• Exudation.
• Presence of tissue destruction markers in GCF.
• Increased Gram-negative anaerobic species in the bacterial culture.
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4. Initiation of host response in junctional epithelium

The junctional epithelium is constantly exposed to microbial flora which can initiate the
inflammatory response. This inflammatory response is primarily mediated by neutrophils, which are the key
components of the host defense against bacterial infection. Phagocytic macrophages then play an important
role of recognition of invading microorganism and to initiate the adaptive immune response. The innate
immune response primarily consists of neutrophil’s response, complement system, and Toll-like receptors.
The adaptive immune response consists of antigen presentation by antigen presenting cells (APC’s) and
generation of T-cell and B-cell response.

5. The innate immune response

Gingival epithelium provides a physical barrier to infection and has an active role in the innate host
defense because the epithelial cells are in constant contact with the bacterial products (51). Along with this,
it is now well recognized that epithelium produces a diverse range of antimicrobial peptides which have
been found to be belonging to at least four families (α-defensins, β-defensins, cathelicidins, saposins) in
humans. It has been shown that oral, sulcular, pocket and junctional epithelia of the gingiva are associated
with the expression of defensin, more specifically β-defensins (hBD-1, hBD-2 and hBD-3). α and β
defensins have an important role in host immune response. Neutrophils are rich in a-defensins and
cathelicidin LL37 (proteolytic peptides). The primary role of B-defensins may be to signal other innate and
acquired immune responses, while LL37 and a-defensins may be more important for their antimicrobial
properties in the gingival sulcus. These peptides are capable of activating the classical complement pathway
and appear to upregulate IL-8 production by epithelial cells, which may enhance neutrophil recruitment to
the site of infection.
The polymorphonuclear leukocyte (PMN) appears to play a key role in the maintenance of
periodontal health, as molecular defects in PMNs, with a variety of functional consequences, result in
accelerated periodontal destruction.
The antimicrobial factors of saliva, such as histatins, lysozymes, and salivary immunoglobulins, also
have a specific role in the innate host defense in response to an infection.

6. Neutrophil response
Neutrophils are the primary cells involved in the initial immune response against invading
microorganisms. They are very important for the maintenance of periodontal health. Individuals suffering
from neutrophil disorders (i.e., leukocyte adhesion deficiency syndrome, Chediak-Higashi syndrome, cyclic
neutropenia, etc.) have an increased susceptibility to and severity of periodontal tissue destruction.
Neutrophils are the most abundant type of leukocytes within the periodontal tissues in acute and
chronic periodontal lesions. Healthy tissue also demonstrates infiltration of neutrophils in junctional
epithelium but their numbers are highly increased in the inflamed tissue (8). The neutrophil recruitment is
along the gradient which is created by the pro-inflammatory cytokines, secreted in response to bacterial
products. The most potent and abundant chemoattractants for neutrophils are CXC chemokines. The CXC
chemokines are a unique family of cytokines, which participate in the regulation of angiogenesis. IL-8 is the
most potent human CXC chemokine. IL-8 is secreted by various cells, including leukocytes, fibroblasts,
endothelial cells, and keratinocytes, in response to both endogenous and exogenous stimuli (56). In response
to IL-8 secreted by the cells in the junctional epithelium, the neutrophils migrate along the chemoattractant
gradient towards the surface of the junctional epithelium. The density of neutrophils within the junctional
epithelium has been found to be increased towards more superficial layers of the epithelium, which are in
close relationship with the subgingival plaque bacteria (57).
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7. Complement system
The complement system is a very important component of the innate immune response. It causes the
destruction of the microorganisms by the formation of a membrane attack complex. The classical,
alternative and lectin pathways of complement system have been well described.

8. Role of Toll-like receptors (TLRs) in host-microbial interaction

When microorganisms enter the tissue after penetrating the epithelial barrier, they are encountered by
tissue macrophages, mast cells and immature dendritic cells (58). These cells must be able to distinguish
between apoptotic particles generated by normal tissue turnover and particles that are indicative of infection.
The molecules mainly responsible for making this pivotal distinction are those of the family of TLRs. Along
with tissue macrophages, mast cells, and immature dendritic cells, these receptors are also expressed on
lymphocytes, osteoclast precursors, osteoblasts and stromal and epithelial cells, each of which has different
TLR expression profiles (59-62).
Members of the TLR family are responsible for the recognition of pathogen-associated molecular
patterns (PAMPs), expressed by a wide spectrum of infectious agents. The main effect of the stimulation of
TLRs is the synthesis and secretion of pro-inflammatory cytokines and lipid mediators, thereby initiating the
inflammatory response that recruits both soluble immune components and immune cells from the blood
(63).

9. Adaptive immune response

The adaptive/acquired immune response is activated when the epithelial barrier, with its
antimicrobial peptides and other components of innate systems, is breached. The pathogenic species present
in the subgingival biofilm evade the anti-bacterial host defense mechanisms by releasing an array of
virulence factors, which causes damage to the host tissue by immune/inflammatory interactions, which
typically consist of neutrophils, monocytes/macrophages, dendritic cells (DCs), T-cells, and predominantly
IgG-producing plasma cells. The majority of virulence factors include enzymes and endotoxins.
a.T-cell activation in adaptive immune response:
The T-cell adaptive immune response is activated by processing and presentation of bacterial
antigens by lymphocytes, macrophages, and dendritic cells. After phagocytosis of bacteria, its recognizable
surface antigens are presented on the surface of APC’s.

b.Antigen presenting cells (APC’s):

The APC’s have antigenic peptide with a major histocompatibility complex (MHC) molecule located
at their surface. Cytotoxic T- lymphocytes (CTL) expressing the CD8 co-receptor recognize the peptide
bound to MHC Class I molecules, whereas helper T-cells (Th) expressing the CD4 co-receptor do so with a
peptide associated with MHC Class II molecules. Co-stimulatory molecules such as B7-1 (CD80) and B7-2
(CD86) are also present on APC’s that interact with CD-28 on T-cells. Adhesion molecules such as ICAM-1
present on APC’s are involved in the formation of strong immunological synapses to facilitate the proper
activation of T-cells.
c. Interactions between APC’s and T-cells:
To understand the interaction between T-cells and antigen-presenting cells, it is important to
understand the receptors present on these cells and their interactions. There is a series of intracellular
signaling cascade that is activated when a receptor is activated, which ultimately leads to the synthesis and
secretion of biochemical mediators like cytokines, etc. The interface between lymphocytes and targets is
termed “immunological synapse” (IS).
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d. Steps in the activation of T-cells:

1. Antigen which is phagocytosed by a macrophage is cleaved into polypeptides which are then
transported to the surface for presentation to T-cells (only foreign particles consisting of proteins are
activated and processed, no other molecules like fatty acids etc. are presented).
2. The APC complex consists of both antigen and MHC. If MHC Class-II is associated with
presenting cells, CD4 or helper T-cells are activated, and if MHC Class-I associated with presenting cells,
CD8 or cytotoxic T-cells are activated. MHC complex is encoded by a group of genes, so different
polypeptides are presented on the surface of the MHC which is responsible for its diversity of antigen
presentation. MHC presents only proteins which may be derived from foreign or self-proteins. It depends on
the selection of T-cells in the thymus.
3. Macrophage which is attached to the antigen, produces IL-1 which activates CD4 cells.
4. CD4 interacts with MHC Class-II on APC surface. This union is stabilized by other proteins LFA-
1 on T-cells and ICAM 1 on APC.
5. A co-stimulatory signal is formed by B7 protein on APC and CD28 on CD4 cells which result in
the secretion of IL-2 by the helper Tcells. This is the step that is useful in the execution of all the functions
i.e., regulator, effector, and memory functions.
Production of IL-2 is the most crucial step in T-cell activation. If this co-stimulatory signal is not
formed, anergy takes place.

10. Cell-mediated immune response in periodontal diseases

T-lymphocyte response to antigenic challenges is called as a cell-mediated immune response. T-
lymphocytes can be functionally divided into CD4 (T-helper lymphocytes) cells and CD8 (cytotoxic T-
lymphocytes) cells by the type of antigen receptors and a small number of accessory markers on their cell
surface. Before going into details of the cell-mediated immune response in periodontal diseases, let’s first
try to understand Th1 and Th2 helper T-cells.

a.Th1 and Th2 helper T-cells:

Helper T-cells can be further differentiated into T helper 1 (Th1) and T helper 2 (Th2)-cells. They are
distinguished by the cytokines they produce and respond to, and are involved in different immune responses.
Th1 and Th2 cells are produced by differentiation from a non-antigen exposed precursor cell type, Thn
(naive T-cells). Differentiation of Thn cells into Th1 or Th2 cells depends on the cytokines they are exposed
to. When Thn cells are exposed to antigen by APC’s, it results in the differentiation of Thn cells into Th0
cells. The stimulation of Thn cells by exposure to APCs induces the proliferation of undifferentiated cells
and their expression of IL-2 and IL-2 receptor. The differentiation of Thn to Th1 or Th2 cells depend on the
cytokines they are exposed to. IL-12 causes Th1 differentiation and blocks Th2 cell production, while IL-4
causes Th2 differentiation and antagonizes Th1 development. IL-18 also induces Th1 differentiation.

11. B-cell activation in adaptive immune response

The B-cell mediated or the humoral immune response is mediated by the production of antibodies by
cells of B-lymphocyte lineage. B-cells express a unique B-cell receptor (BCR) on their surface that
recognizes and binds to only one particular antigen.
The primary differences between B-cell and T-cell mediated immune response is in the mechanism
by which they recognize the antigen. T-cell recognizes the antigen through MHC interactions, once it is
presented by the APC’s. The B-cell recognizes antigens in their native form. After a B-cell recognizes the
antigen and receives appropriate signals from helper T-cells, it further differentiates into an effector cell,
known as a plasma cell. The plasma cells produce antibodies against that antigen. These cells are short
lived cells (2-3 days). However, around 10% of plasma cells become long-lived and are referred to as
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antigen specific memory B-cells. The antibodies thus produced, bind to antigen, making it an easier target
for phagocytes.
The B-cell response may be T-cell dependent or T-cell independent, but it is primarily regulated by
T-cells.
a.T-cell independent activation of B-cells: T-cell independent response occurs by the cross-
linking of the IgM by antigen receptors on the B-cell, responding with IgM synthesis in the absence of T-
cell help.
b.T-cell dependent activation of B-cells: when a pathogen is ingested by an APCs such as a
macrophage or dendritic cell, the pathogenʼs proteins are then digested to peptides and attached to a Class II
MHC protein. This complex is then moved to the outside of the cell membrane. The APC, then interacts
with the helper T-cell (which recognizes MHC II). This activation leads to the secretion of cytokines by
helper T-cell, which causes B-cell proliferation and maturation. Activated B-cells subsequently produce
antibodies, which assist in the inhibition of pathogens (Figure 7.6).

12. Humoral Immune response in the periodontal diseases

Although the precise role of humoral immunity in the periodontal disease progression has not been
fully elucidated, however, it has been suggested that the humoral immune response has a protective role in
the pathogenesis of periodontal disease (105). B-lymphocytes contribute to immunity in multiple ways,
including the production of antibodies, presentation of antigen to T-cells, organogenesis of secondary
lymphoid organs and secretion of cytokines (106).
The antibodies protect bacterial colonization primarily by two mechanisms. The direct binding of the
antibodies to the bacterial surface antigens blocks adherence, colonization and aggregation of bacteria.
Furthermore, these also detoxify the bacterial products. The second mechanism involves opsonization of
bacteria by constant regions of antibodies which makes them susceptible to phagocytosis and also enables
activation of the complement cascade. Elevations in the humoral immune response are associated with
increasing severity of the disease
After encountering the invading pathogens (in particular, extracellular microorganisms), the antigen
(Ag)-specific naïve B-cells undergo affinity maturation via clonal selection, somatic hypermutation, and Ig
receptor editing (111). The B-cells bearing the higher or highest affinity for Ag are rescued from apoptosis
and subsequently, these B-cells with mutated B-cell receptor genes, located in the germinal centers of
regional lymph nodes, differentiate into effector or memory B-cells (112). Certain cytokines such as
interferon-y (IFN-Y), IL-4, IL-5, IL-10, IL 12, transforming growth factor-B (TGF-B), anti-CD40
Ligand/CD40, and bacterial LPS are potent stimulators of antibody class-switching (113, 114).
Immunoglobulins arriving at the periodontal lesion are from both systemic and local tissue sources.
The salivary IgA is involved in trapping of the antigen in a mucin layer with the subsequent disposal
(elimination) of the antigen. The local production of IgG, which is protective in nature and is found to be
increased in inflamed gingival and periodontal tissues.