Host microbial interaction

Contents
 Introduction
 Definitions
 Microbiologic aspects of the microbial-host interaction
 Immunologic aspects of the microbial host interaction
 Defects in neutrophils funtion
 Toll like receptors in microbial host interaction
 Signalling Pathways
 Mechanism of alveolar bone destruction in periodontitis.
 Connective tissue destruction: Healing process in periodontitis
 Conclusion
 References
3

Introduction
4
• The mouth, like all external surfaces of the body and the gut,
has a substantial microflora living in symbiosis with a healthy
host.
• The microflora of the mouth contains hundreds of species of
aerobic and anaerobic bacteria.
• Cultural studies indicate that more than 500 distinct microbial
species can be found in dental plaque.

5
• Although bacteria are necessary for periodontal disease to
take place, a susceptible host is also needed.
• The immune-inflammatory response that develops in the
gingival and periodontal tissues in response to the chronic
presence of plaque bacteria results in destruction of
structural components of the periodontium leading,
ultimately, to clinical signs of periodontitis.

6
• The host response is essentially protective, but both
hyporesponsiveness and hyper-responsiveness of certain
pathways can result in enhanced tissue destruction (Bruce
Pihlstrom 2005 ).
• Closer investigations of the destructive pathway of periodontal
disease began to focus on the relation-ship between bacteria
and the host response in the initiation and progression of
periodontal disease.

7
• This shift in etiological theory produced a paradigm that
called attention to the fact that although microorganisms are
the cause of periodontitis, the clinical expression of the disease
depends on how the host responds to the extent and virulence
of the microbial burden.
• It was found that degradation of host tissue results from this
bacterial-host interaction.

WHAT IS HOST ?
An organism which
harbors the parasite.
8

WHAT IS BACTERIA?
Extremely small—usually 0.3
to 2.0 micrometers in
diameter—and relatively
simple microorganisms
possessing the prokaryotic type
of cell construction.
9

INTERACTION
• The combined effect of two or more independent variables
acting simultaneously on a dependent variable.
10

Microbial Virulence
The properties of a microbe that enable it to cause disease are
referred to as virulence factors.
To function as pathogen, bacteria must:
1. Colonize at appropriate host tissue site.
2. Cause destruction of the host tissues.
11

Host Response
 The manner in which body responds to periodontal pathogen is
known as host response.
12

13
Microbial aspects of
the microbial host
interaction

14
• Gram negative anaerobes are
pathogenic organisms associated
with disease.
• The properties of microorganism that enable it to cause
disease is called Virulence Factor.
• Bacteria must colonize the host tissue and then cause
destruction of the host tissue.

15
Bacteria Evading Mechanism Action
A.actinomycetemcomitans ~ Leukotoxin
~Cytolethal distending
toxin
Kill lymphocytes; inhibit PMN
function
Impair lymphocyte function
P.gingivalis ~Protease/Gingipains
~Capsule
~Inhibition of IL-8
Degrade IgA & IgG
Evade phagocytosis
Inhibit neutophil chemotaxis
T.forsythia Cytotoxin Apoptosis of lymphocytes
F.nucleatum Heat Sensitive surface
protein
Apoptosis of mononuclear
cells
Capnocytophaga Chemotactic inhibitors Chemotactic defect in
neutrophils

16
Bacterial Colonization and Survival in Periodontal
region
• Bacterial Adherence
– GCF flows outwards from the base of pocket
– Bacterial species that colonize this region must attach to
available surfaces to avoid displacement
Eg: A viscosus and P gingivalis attaches through fimbrae on
bacterial surface to proline rich proteins found on saliva-coated
tooth surface.

17
• P gingivalis binds to epithelial cells and fibroblasts
• The ability of Porphyromonas gingivalis to attach to other
bacteria, epithelial cells, and connective tissue components
fibrinogen and fibronectin add to virulence of this
periopathogen

19
Host tissue Invasion
• Both gram-positive and gram- negative bacteria, including
cocci, rods, filaments and spirochetes have been seen in
gingival connective tissue and alveolar bone
• Presence of bacteria in periodontal tissue in gingivitis,
advanced chronic periodontitis and Aggressive
Periodontitis
• Presence of bacteria in host tissue in NUG

20
• Ulceration in epithelium of gingival sulcus or
periodontal pocket
• Observed in intercellular spaces of gingival tissues
• Direct penetration of bacteria into hosts epithelial or
connective tissue cell
Eg: A actinomycetemcomitans, P gingivalis, F
nucleatum, Treponema denticola to invade tissue cell
directly.

21
• Bacterial species that have been identified as capable
of tissue invasion are strongly associated with disease.
• Ability to invade has been proposed as a key
mechanism that distinguishes pathogenic from non-
pathogenic gram negative species.
• Localization of bacteria to the tissue provides an ideal
position from which organism can deliver toxic
molecules & enzymes to host tissue.

22
• “Burst of disease activity” may be related to phases of
bacterial invasion of the tissue.
• Mechanical debridement alone is insufficient, and
systemic antibiotic in combination with surgical therapy is
required to eliminate A actinomycetemcomitans from lesion
in aggressive periodontitis

23
Bacterial Evasion of Host Defense System
• To survive in periodontal environment bacteria neutralize
or evade host mechanism involved in bacterial clearance
and killing.
• Bacterial adherence and invasion are strategies through
which bacteria accomplish this task.
• Ability to adhere allows bacteria to avoid displacement
by host secretions.

24
Immunological
Aspects of
Microbial Host
Interaction

25
Involves following factors in response to bacterial infection
 Innate factors
 Acute inflammatory cells (neutrophils)
 Chronic inflammatory cells

26
 Periodontal disease is a well-regulated response to
protracted bacterial infection directed by inflammatory
cells of the host immune system
 Neutrophils primarily function as antimicrobial cells, and
chronic inflammatory cells orchestra adaptive responses
 Neutrophils function to contain microbial challenge
through phagocytosis and killing and may contribute to
local tissue changes by release of tissue-degrading enzymes

27
PMN’s form a protective layer in regions where
epithelium of the gingival sulcus has been
disrupted in Gingivitis

28
Innate factors and initiation of inflammation
• Onset of inflammation there is edema and erythema
leading to vascular changes
• Complement activation in response to bacterial infection
result in generation of C3a and C5a
• Degranulation of Mast cell
• Mast cell constitutely transcribe TNF-ά, TNF-β, IL-4, IL-6
• When stimulated induce produce proinflammatory
cytokines such as IL-1, IL-6, INF-γ

29
Well characterized interactions involve release of
1. IL-1
2. TNF
3. Prostaglandins
These host derived mediators have the potential to
stimulate bone resorption and activate or inhibit
other host immune cells.
From monocytes,
macrophages and PMNs
exposed to bacterial
endotoxin (LPS)
[Roberts FA, 1997.
Yoshimura A, 1997]

31
Host Defense Processes
• Innate Responses (Non-specific)
• Innate reactions include the inflammatory response and
do not involve immunological mechanisms.
• Adaptive Responses (Specific)
• Adaptive reactions that include immunological
responses tend to be very effective as the host response
is specifically “tailored” to the offending pathogen(s).

32
Transepithelial migration
• 1-2% neutrophils migrate across the junctional
epithelium daily through chemotactic gradient of IL-8
and ICAM-1 expressed by junctional epithelium.
• Porphyromonas gingivalis impedes transepithelial
migration of neutrophils by preventing epithelial cells
to secrete IL-8 in response to bacterial challange.

33
Opsonization
• Coating of bacteria by host proteins to facilitate
phagocytosis
• Bacteria are coated with complement components (iC3b,
C3b)
• Recognized by CR3 neutrophil receptor
• Antigen presenting cells such as Peripheral Dendritic
cells (langerhans,macrophages,B cells) are abundant in
gingival cells

35
Phagocytosis
• Bacterial cell ingestion by neutrophil results in
formation of Phagosome.
• Bacteria is killed by Oxidative or Non oxidative
mechanism.

36
Scanning Electron Micrograph of PMNs passing through
epithelium.

37
High power view of PMN and bacteria attached
undergoing phagocytosis

38
Oxidative Killing by
– NADPH Oxidase,
– Myeloperoxidase,
– Nitric oxide synthase
Non-oxidative Killing
by
– Defensin,
– Lysozyme,
– Neutral serine
proteases
– Bacterial
permeability
increasing protein

39
DEFECT IN NEUTROPHIL
FUNCTION AT ANY STAGE LEADS
TO AGGRESSIVE PERIODONTITIS

41
The clinical photo and the X-Ray of this 28 year-old man
show the advanced alveolar bone loss in the absence of
significant gingival inflammation, typical of the localized
aggressive periodontitis

43
These photos show the Palmo-plantar Hyperkeratosis
present in patients with the Papillon Lèfevre Syndrome.
These lesions remain for life but improve when treated with
retinoic acid

44
Connective tissue alteration: Tissue
destruction in Periodontitis
• The fundamental event in the transition from
gingivitis to periodontitis is the loss of the soft
tissue attachment to the tooth and subsequent loss
of bone.
• Mediators produced as part of host response
contribute to tissue destruction include Proteinase,
Cytokines, Prostaglandins.

45
Proteinases
• Matrix Metalloproteinases(MMP) are primary proteinases
involved in periodontal tissue destruction by degrading
extracellular matrix molecules.
• MMPs are family of Proteolytic enzymes found in
neutrophils, macrophages,fibroblasts, epithelial cells,
osteoblasts and osteoclasts.
• MMPs degrade extracellular matrix molecules, such as
collagen, gelatin, and elastin.

46
• MMP-1 is expressed by resident periodontal tissue
such as fibroblasts, monocytes, macrophages and
epithelial cells
• MMP-8 is released by infiltrating neutrophils
• MMP are activated by chymotrypsin-like protease
produced by Treponema denticola as well as host
enzymes such as neutrophil cathepsin G

47
• MMPs are inactivated by ά-macroglobulin found in
serum and GCF and by Tissue inhibitor of MMP’s
(TIMP) produced by many cell types and common in
host tissue and fluids
• Tetracycline also inactivates MMPs and have significant
therapeutic role

48
• Other Proteinases associated with Periodontitis include
neutrophil serine proteinases, Elastase and Cathepsin G
• Cathepsin G is elevated in gingival tissue and GCF
• Elastase degrades a wide range of molecules including
Elastin, Collagen, and Fibronectin
• Elevated Elastase level are associated with active
Periodontal attachment loss

49
Cytokines
• Three proinflammatory cytokines IL-1, IL-6 and TNF-ά
have a central role in Periodontal tissue destruction
• IL-1 is produced primarily by activated Macrophages or
Lymphocytes
• Bacterial LPS is potent activator of Macrophage IL-1
production
• TNF-ά is also produced by activated macrophages in
response to Bacterial LPS

50
• Also TNF-ά and IL-1 can activate macrophage IL-1
production
• TNF-β is primarily produced by Th1 subset of CD4+ T
cells that have been activated by antigen or mitogen

51
Proinflammatory effect of IL-1 and TNF-α are
• Stimulation of endothelial cells to express
selectins that facilitates recruitment of leukocytes
• Activation of Macrophage IL-1 Production
• Induction of Prostaglandin E2 (PGE2 )

52
• IL-1 is a potent stimulant of osteoclast
proliferation, differentiation, and activation
• TNF-ά have same effects on osteoclasts but less
potent

53
Prostaglandins
• Arachidonic metabolite generated by cyclooxygenase
(COX-1, COX-2)
• Arachidonic acid is a 20-carbon polyunsaturated fatty
acid found in plasma membrane of most cells
• COX-2 is upregulated by IL-1β, TNF-β and bacterial
LPS leading to formation of PGE2 associated with
inflammation

54
• PGE2 is increased in periodontal sites demonstrating
inflammation and attachment loss
• PGE2 also induces MMPs and osteoclastic Bone Resorption
• PGE2 is elevated in gingivitis and Periodontitis in active
disease
• PGE2 is partly responsible for bone loss associated with
periodontitis

55
Macrophages produce prostaglandin E
(PGE) and (IL-1) and lymphocytes
produce Interleukin-1 (IL-1) which
activate osteoclasts by interacting with
osteoblasts

56
• PGE2 is released from monocytes of patients with severe
or aggressive periodontitis
• High risk patient display “Monocyte Hyper secretory trait”
leading to exaggerated response both locally and
systemically to bacterial LPS
• Use of NSAID as an inhibitor of Prostglandins synthesis
has therapeutic role in preventing bone loss in
Periodontitis

58
Toll Like Receptors (TLRs)
• Toll-like receptors are structures evolved to detect bacterial
challenge and are present on all human cells, including epithelial
and endothelial cells, and may bind microbial cell molecules,such
as lipopolysaccharides, microbial fimbriae, and lipoteichoic acid.
• This suggests that even innate responses of the host may be
tailored to particular bacteria.

59
• Toll was initially discovered in Drosophila (Underhill DM,
2002)as an important gene for a trans-membrane receptor in
dorso-ventral development pattern.
• Toll mutants refers to the fact that these mutants could not
establish a proper dorsal-ventral axis.
• Toll in German means ‘great’, apparently this was one of the
words describing the scientists’ enthusiasm after observing the
mutant flies.

60
• LeMaitre B, 1996, showed that absence of Toll in
genetically deficient Drosophilia resulted in severely
impaired defense against fungii and gram positive bacteria.
• Hoffman and colleagues showed that Toll-mutant flies
were susceptible to fungal infections.
• Mammalian homologues were discovered and designated
as Toll-Like Receptors (TLRs).
• TLRs recognize specific patterns in pathogens which are
not observed in mammals.

61
Toll Molecular Structure (Akira,2003)

63
Toll Like Receptor
Expression and Microbial
Recognition in
Periodontal Tissues

64
• Within periodontal tissues, TLR2 and TLR4 expression appears
to be severely increased in diseased states.
• TLR-4 is expressed in equal levels by gingival and periodontal
fibroblasts.
• TLR-2 is expressed in higher levels by PDL fibroblasts.
• Human gingival and periodontal fibroblasts are known to
produce various inflammatory cytokines such as IL-1,6,8 when
stimulated by bacterial LPS.

65
• CD-14 is responsible for pattern recognition of common
bacterial cell surface components such LPS and
PGN.(Ulevitch RJ, 2003)
• Cementoblasts also express TLR-2,4 as well as CD-14
(Nociti FH,2004).
• It has been suggested that TLR4 mediates the response to
LPS while TLR2 is involved in response to other bacterial
cell wall components.

66
• TLRs are thus a major class of signaling receptors, recognizing
conserved bacterial structures. (Janeway,2002)
• TLR-2 : PGN, Bacterial lipoproteins, Zymosan and Atypical LPS.
• TLR-3: Double stranded RNA.
• TLR-4: LPS and HSP.
• TLR-9: CpG motifs of bacterial DNA.

67
Toll Like Receptor Expression and
Microbial Recognition in
Periodontal Tissues
• Within periodontal tissues, TLR2 and TLR4 expression appears to be
severely increased in diseased states.
• TLR-4 is expressed in equal levels by gingival and periodontal
fibroblasts.
• TLR-2 is expressed in higher levels by PDL fibroblasts.

68
• Human gingival and periodontal fibroblasts are known to produce
various inflammatory cytokines such as IL-1,6,8 when stimulated by
bacterial LPS.
• CD-14 is responsible for pattern recognition of common bacterial
cell surface components such LPS and PGN. (Ulevitch RJ, 2003).
• Cementoblasts also express TLR-2,4 as well as CD-14 (Nociti
FH,2004).
• It has been suggested that TLR4 mediates the response to LPS while
TLR2 is involved in response to other bacterial cell wall components.

69
Signaling Pathways
1. MyD88-dependent pathway (common to all TLRs)
2. MyD88-independent pathway (peculiar to TLR3 and
TLR4)

71
• Upon stimulation , MyD88 recruits IL-1 receptor-associated
kinase (IRAK) to TLRs.
• IRAK is activated by phosphorylation, then associates with
TRAF6.
• TRAF6 interacts with TAK1,TAB1 and TAB2 that further
forms a larger complex with Ubc13 and Uev1A, that
activates TAKI.
• Activated TAK1 phosphorylates the IKK complex,
consisting of IKKα, IKKβ, NEMO/IKKγ and MAP kinases
and thereby induces the activation of the transcription factors
NF-Κb and AP-1, respectively.

72
MyD88- independent pathway
• TLR3 signaling occurs mainly via MyD88-independent
pathway.
• In TLR3 and TLR4 mediated signaling pathways, a
MyD88-independent pathway exists that leads to the
activation of IRF-3 via TBK1 and IKKε / IKKi.
• The domain-containing adaptor TRIF mediates this
MyD88-independent pathway.

74
Mediators of Bone Resorption
(McCauley LK, 2001)

75
Pathobiology of Periodontal Diseases

76
Connective tissue alteration: Healing
Process in Periodontitis
• The chronic immune system plays an important role in
healing process, which consists regeneration and repair
• Regeneration involves the replacement of tissue with
new, identical tissues that function same as the orignal
tissue
• Repair involves replacement of one tissue with another
tissue, such as fibrous connective tissue

77
• Periodontal repair occurs in overlapping phases of
inflammation shutdown, angiogenesis and fibrogenesis.
• In postinflammatory healing process, the shutdown of
inflammatory processes and initiation of postinflammatory
healing is orchestrated by leukocytes.
• Some of the important antinflammatory signals generated by
leukocytes including IL-1ra, TGF-β.
• Other cytokines that depress an inflammatory response
includes IL-4, IL-10, IL-11.

78
• Angioenesis and fibrogenesis as well as cytokine as (IL-
1β, IL1-α, TNF-α) that help to induce these
processes,participate in both inflammation and healing.
• IL-1β, IL1-α are indirectly involved in inducing
fibroblastproliferation and collagen synthesis by
stimulating theproduction of PGE2 or release of PDGF and
TGF-β.

79
• PDGF activates fibroblasts and osteoblasts resulting in
the induction of protein synthesis.
• TGF-β is a multifunctional peptide that stimulates
osteoblasts, fibroblasts and inhibits osteoclats, epithelial
cells and most immune cells.

80
Conclusion
Periodotal diseases are inflammatory diseases where virulent microbial
species induces a series of host responses events that results in the
clinically visible inflammatory changes in the periodontal tissues. In the
process of host - microbial interaction, there is a damage to the host
tissue, which is clinically manifested as gingivitis, which may, layer on,
give rise to chronic or aggresive periodontitis. In a susceptible host,
there is dysregulation of inflammatory and immune pathways which may
result in excessive damage to the host tissues.

81
References
 The host-bacterial interaction theory and the risk continuum: casey hein,:
contemporary oral hygiene December 2004
 Text book of periodontology: Carranza 10 edn
 Text book of microbiology: Ananthnarayana 7 edn
 Mechanisms tissue matrix of connective destruction in periodontitis; john j.
 Reynolds & murray c. Meikle; periodantology 2000, vol. 14, 1997,144-157
 Oringer rj. American academy of periodontology, research, science, and
therapy committee. Modulation of the host response in periodontal therapy.
J periodontol. 2002;73(4): 460-470.
 The role of reactive oxygen and antioxidant species in periodontal tissue
destruction periodontology 2000;2007;vol43
 Toll-like receptors and their role in periodontal health and disease.
Periodontology 2000;2007;vol43;41-50

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