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GOOD MORNING

Host Microbial Interaction

Part – I

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Contents

1. Introduction.

2. Microbial Virulence.

3. Bacterial adherence in periodontal environment.

4. Host tissue invasion.

5. Bacterial evasion of host defense mechanism.

6. Bacterial enzymes capable of tissue destruction.

7. Immunological aspects of host microbial interaction.

8. Host defense processes

9. Host responses to bacterial invasion.

10. Inflammatory responses

11. Molecules, Cells and Processes in Host Response.

12. Connective Tissue Alterations

13. Bone Destruction

14. Healing processes in periodontitis.

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1. Introduction

• Periodontal disease is initiated and sustained

by factors (substances) produced by the

subgingival microbiota (the biofilm).

1. Direct injury to host cells and tissues.

2. Activation of inflammatory or cellular and

humoral immune systems that cause damage

to the periodontal tissues.

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2. 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.

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3. Bacterial Adherence in

Periodontal Environment

• Attachment to Acquired pellicle or saliva

coated tooth surfaces.

• Critical process in tissue invasion

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Bacterial Adhesins and Target

Surfaces (SS Socransky, 1991)Attachme

nt

Surface

Bacterial

Species

Substrate Bacterial

Adhesion

Substrate

Receptor

Tooth A. viscosus Saliva

coated

mineralized

surfaces

Fimbriae 1. Saliva

treated

HAP.

2. Proline

rich

proteins

S. mitis Saliva

coated

surfaces

70-90kD

protein

Sialic acid

residues

F. nucleatum 300-330 kD

outer

membrane

protein

Galactosyl

residues

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Bacterial Adhesins and Target Surfaces

(SS Socransky, 1991)Attachment

Surface

Bacterial Species Substrate Bacterial

Adhesion

Substrate

Receptor

Tissue P.gingivalis Epithelial cells Fimbriae Galactosyl

residues

A. viscosus

A. naeslundii

Fimbriae Galactosyl

residues

T. denticola Fibroblasts Surface protein Galactosyl or

Mannose residues

A.viscosus

A. naeslundiiPMNs Fimbriae Galactosyl

residues

F. nucleatum Protein Galactosyl

residues

P.gingivalis Connective

Tissue

Components

Membrane protein Fibrinogen,

Fibronectin

P. Intermedia Membrane protein Fibrinogen03-03-2015 8

Bacterial Adhesins and Target

Surfaces (SS Socransky, 1991)Attachment

Surface

Bacterial

Species

Substrate Bacterial

Adhesion

Substrate

Receptor

Pre-

Existing

Plaque

Mass

A. viscosus S.Sanguis Fimbriae Repeating

heptasaccharide

on

polysaccharide.

C. Ochraceus S.Sanguis

A.Naeslundii

A.Israelli

Heat sensitive

protein

Rhamnose,

Fructose.

N-acetyl-

muramic acid

residues

P. loescheii S.Sanguis

A.Israelli

75-45 kD fimbrial

proteins

Galactosyl

residues

F.nucelatum P.Gingivalis

T.Denticola

P.Micros

Heat and Protease

sensitive protein.

Galactosyl

residues

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4. Host Tissue Invasion

1. Bacteria may enter host tissues through ulcerations in the epithelium of gingival sulcus or pocket that are visible in the intercellular spaces of the gingival tissues.

2. Direct penetration of bacteria into host epithelial or connective tissue cells.

(Aac: Sreenivasan PK, 1993;

P.gingivalis: Sandros J, 1993;

F.nucleatum: Wang B, 1993;

T.denticola: Han YW,2000)

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Host Tissue Invasion

3. This ability of the bacteria to invade the host

tissue is one of the key characteristic in

distinguishing pathogenic from non pathogenic

strains.

4. Localization of bacteria in the tissues provides

an ideal position from which the bacteria can

deliver toxic molecules and enzymes to the host

tissue cells and this may be the significance of

invasion as a virulence factor.

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• The bursts of disease activity in periodontal

diseases may be related to active phases of

bacterial invasion of the tissues. (Saglie FR,

1988)

• Bacteria in the tissue may enable persistence

of the species in the pocket by providing a

reservoir for colonization.

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5. Bacterial Evasion of Host

Defense Mechanisms

• Bacterial adherence avoids displacement by

host secretions.

• Cell invasion disrupts natural barriers formed

by the host tissues.

• Bacterial Properties in Evasion of Host

Defense Mechanism (SS Socransky, 1991):

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Bacterial Evasion of Host Defense Mechanisms (SS Socransky, Haffajee A D, 1991)

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Host Defense

Mechanism

Bacterial Species Bacteria Property Biologic Effect

1. Specific

Antibody

P.gingivalis

IgA and IgG

degrading proteases

Degradation of

specific antibodyP.intermedia

P.Melanogenica

Capnocytophaga sp.

2. PMNsAac Leukotoxin Inhibition of PMN

function

F.nucleatum Heat Sensitive

Surface Protein

PMN Apoptosis

P.Gingivalis Capsule Inhibition of

phagocytosis

T.denticola Inhibition of

superoxide

production

Decreased bacterial

killing.

Host Defense

Mechanism

Bacterial

Species

Bacterial Property Biologic Effect

3.

Lymphocytes

Aac Leukotoxin 1. Killing of mature B

and T cells.

2. Non lethal

suppression of

lymphocyte activity.

Cytolethal Distending

Toxin

Impairment of function

by arresting lymphocyte

cell cycle.

F. nucleatum Heat sensitive surface

protein

Apoptosis of

mononuclear cells

T. Forsythia Cytotoxin Apoptosis of

lymphocytes

P. Intermedia

T. denticola

Aac

Suppression Decrease response to

antigens and mitogens

4. Release of

IL-8

P.Gingivalis Inhibition of IL-8

production by epithelial

cells.

Impairment of PMN

response to bacteria.

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6. Bacterial Enzymes Capable of Degrading

Host Tissues (Curtis MA, 1999; Kuramitsu HK,

1998)Bacterial Enzyme Species

Collagenase P. Gingivalis

Aac

Trypsin like enzyme P. Gingivalis

Aac

T.Denticola

Arylsulfatase Neuraminidase C.Rectus

P. Gingivalis

T.Forsythia

P.Melanogenica

Fibronectin degrading enzyme P.Gingivalis

P. Intermedia

Phospholipase A P. Intermedia

P.Melanogenica03-03-2015 16

7. Immunological Aspects of

Microbial Host Interaction

• 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.

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From monocytes,

macrophages and PMNs

exposed to bacterial

endotoxin (LPS)

[Roberts FA, 1997.

Yoshimura A, 1997]

Bacterial Species Bacterial

Component

Target Host Tissue Effect On Cytokine

Level

Aac

F.nucleatum

LPS PMNs Increased release of

Interleukin – 1

Aac 37 kD protein Macrophages

P.Gingivalis LPS Monocytes

Aac 37-kD protein Macrophages Increased release of

IL – 6 Aac

C.Rectus

Whole cells Gingival fibroblasts

E.Corrodens Whole cells Epithelial cells

Aac

F.nucelatum

E.Corrodens

Whole cells Epithelial cells Increase release of

IL – 8

Aac

C.Rectus

Whole cells Gingival fibroblasts

Aac

F.nucelatum

LPS PMNs

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Bacterial Species Bacterial

Component

Target Host Tissue Effect On

Cytokine Level

Aac

F.nucleatum

LPS PMNs Increased TNF

release

Aac 37-kD protein Macrophages

C.Rectus

Aac

P.intermedia

P.gingivalis

LPS Monocytes Stimulated release

of PGE2

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8.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).

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9. Host responses to Bacterial

Infection• Innate factors such as complement, resident leukocytes

especially mast cells play an important role in signaling endothelium and thus initiating inflammation.

• Acute inflammatory cells (Neutrophils) protect local tissues by controlling the periodontal microbiota within the gingival crevice and junctional epithelium.

• Chronic inflammatory cells ; macrophages and lymphocytes protect the entire host from within the subjacent connective tissues and do all that is necessary to prevent a local infection from becoming systemic and life threatening, including the sacrifice of local tissues.

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• Neutrophils function as antimicrobial cells.

• Chronic inflammatory cells organize adaptive responses.

• Neutrophils function to contain the microbial challenge through phagocytosis and killing and may contribute to local tissue changes by release of tissue degrading enzymes.

• The chronic inflammatory cells, the lymphocytes and monocytes, orchestrate connective tissue changes associated with both periodontal infection and repair and healing.

• They also function to assist the neutrophils in controlling bacterial infection by forming specific opsonic antibodies.

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10. Inflammatory Processes

• Redness

• Heat

• Swelling

• Pain

• Loss of function

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11. Molecules, Cells, and

Processes in Host Response

• PMNs

• Proteinases

• Proteinase inhibitors

• MMPs

• Cytokines

• Prostaglandins

• Bone Destruction

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Neutrophils / Polymorphonuclear

Leukocytes

• Elastase, a serine protease, is contained in the primary granules of the PMN; may cause tissue breakdown and is present with increased activity at sites of gingival inflammation.

• Lactoferrin is contained in the secondary granules of the PMN, and is released during PMN migration and is associated with PMN activation.

• A greater proportion of lactoferrin to elastase was found in advanced periodontitis lesions than in gingivitis sites.

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• TNF – A plays a major role in development of

inflammation by stimulating the release of

cytokines, including IL-1B from neutrophils.

(Price TH, 1994).

• Lipoxin A4 is a cytokine regulating lipid

mediator that can reduce the inflammation

induced by TNF – A.

• 1% - 2% Neutrophils migrate across the

junctional epithelium daily; which requires a

chemotaxin gradient.

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• P.gingivalis impedes transendothelial

migration of neutrophils (Madianos PN, 1997)

and prevents epithelial cells from secreting IL-

8 in response to bacterial challenge. (Huang

GT-J, 1998; Darveau RP, 1998)

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Cathepsin G

12. Connective Tissue Alterations:

Tissue Destruction in Periodontitis

• Remodeling of connective tissues that leads to

a net loss of local soft tissues, bone and

periodontal attachment apparatus.

• The fundamental event in the transition from

gingivitis to periodontitis is the loss of soft

tissue attachment to the tooth and subsequent

loss of alveolar bone.

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Components of

Periodontal ECM

Tropocollagen

Proteoglycans

ElastinTenascin

Osteocalcin,

Osteopontin,

Osteonectin

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All of these matrix components are constantly in a state of turnover and thus there is much matrix

enzyme activity in health, disease, and tissue repair and remodelling (Kinane 2001).

Proteinases/Proteases

• Proteinases (collagenase, elastase - like and trypsin-like, as well as serine and cysteine proteinases) cleave proteins by hydrolyzing peptide bonds.

• Depending on the location of activity of the enzyme on its substrate:

1. Endopeptidases

2. Exopeptidases,

• A reduction of protease levels following treatment was obtained in several studies.

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Proteinase Inhibitors

Alpha-2 macroglobulin (A2-M)

• Inhibits gingival collagenase

Alpha1 antitrypsin (A1-AT)

• Inhibits PMN collagenase

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• MMPs are a family of proteolytic enzymes found in neutrophils, macrophages, fibroblasts, epithelial cells, osteoblasts and osteoclasts. (Ryan ME, 2000) that degrade matrix molecules such collagen, elastin and gelatin.

• MMP1 and MMP8, both are collagenases.

• MMP 8 is released by infiltrating neutrophils whereas MMP1 is expressed by resident cells including fibroblasts, monocytes/macrophages and epithelial cells.

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Matrix Metalloproteinases

• MMPs are also produced by Aac and Pg which

is not considered a major factor in disease

progression. (AAP, 2002)

• MMPs are secreted in the inactive/latent form.

• Activation by proteolytic cleavage of a portion

of the latent enzyme.

• Proteases capable of MMP activation:

1. Chymotrypsin like protease by T .denticola

2. Host cell enzyme – Neutrophil Cathepsin G.

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• MMPs are inactivated by:

1. Alpha-macroglobulins found in serum and

GCF.

2. Tissue Inhibitors of MMPs (TIMP) produced

by cells of host tissue and fluid.

3. Tetracycline appears to inactivate neutrophil

MMP (Graves DT, 1999)

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Other Proteinases

1. Elastase : elastin, collagen, fibronectin.

2. Cathepsin – G: Bacterial proteinase and

MMP – 8 activation.

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• Neutrophil (PMN) Collagenase is found in higher

concentrations in inflamed gingival specimens

than in clinically healthy gingiva.

• The increased presence of these MMP enzymes in

diseased over healthy sites (Ohlsson et al. 1973),

their increase during experimental gingivitis

(Kowashi et al. 1979), and decrease after

periodontal treatment (Haerian et al. 1995, 1996)

suggest that MMPs from PMNs are involved in

periodontal tissue breakdown.

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Cytokines• Cytokines are soluble proteins, secreted by cells involved in both the

innate and adaptive host response, and act as messenger molecules that transmit signals to other cells.

• Actions:

1. Initiation and maintenance of immune and inflammatory responses.

2. Regulation of growth and differentiation of cells.

• Interleukins are important members of the cytokine group and are primarily involved in communication between leukocytes and other cells, such as epithelial cells, endothelial cells, and fibroblasts engaged in the inflammatory process.

• Interleukin (IL)-1a, IL-1b, and tumor necrosis factor (TNF)-alpha stimulate bone resorption and inhibit bone formation.

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• IL-1, IL-6and TNF have the central role in periodontal destruction. (Graves DT, 1999; Page RC, 1976)

• IL1a and IL1b are the 2 active forms.

• Main constituent of IL-1 : Osteoclast Activating Factor.

• IL-1 is produced by activated macrophages or lymphocytes but may also be produced by mast cells, fibroblasts, keratinocytes and endothelial cells.

• Bacterial LPS is a potent activator of macrophage IL-1 and TNFa secretion.

• TNFa and IL-1 can upregulate their own production.

• IL-6 also results in Bone remodeling. (Manolagas SC, 1999)

• TNF-a and TNF-b are active forms.

• TNFa shares similar biologic activity of IL-1a including stimulation of bone resorption. 03-03-2015 45

• Proinflammatory effects of IL-1 and TNF-a

(Page RC,1991):

1. Stimulation of endothelial cells to express

selectins that facilitate recruitment of

leukocytes.

2. Activation of macrophage IL-1production.

3. Induction of PGE2 by macrophages and

gingival fibroblasts.

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Prostaglandins

• Arachidonic acid derivative.

• Mediators of inflammation.

• IL-1b, TNFa and bacterial LPS upregulate COX-2.

• PGE2:

1. Potent vasodilator

2. Inducer of cytokine production by various cells.

3. PGE2 acts on fibroblasts and osteoclasts to induce production of MMPs.

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13. Bone Destruction

• Bone resorption is mediated by osteoclasts.

• Concomitant with the breakdown of the connective tissue attachment during disease progression.

• The mechanisms involved in bone resorption respond to signals from inflammatory cells in the lesion and initiate degradation of bone in order to maintain a “safety” distance to the periphery of the inflammatory cell infiltrate.

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Osteoclasts

• Osteoclasts are multinucleated cells that

develop from osteoclast progenitor

cells/macrophages.

• Mediators such as IL-1 beta, PGE2 and TNF

alpha, IL-6, IL-11 and IL-17 may act as

activators on osteoclasts.

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The Receptor Activator Of Nuclear

Factor-kappa Beta (RANK), the

RANK ligand (RANKL) and Osteoprotegrin (OPG)

• RANK is a receptor expressed by osteoclast progenitor cells.

• RANKL and OPG are cytokines that belong to the TNF family and are produced by osteoblasts and bone marrow stromal cells.

• While RANKL promotes activation of osteoclasts, OPG has the opposite effect.

• Thus, the binding of RANKL to the RANK will result in the differentiation of osteoclast progenitor cells into active osteoclasts, while OPG that binds to RANKL will inhibit the differentiation process.

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• Analyses of human biopsy specimens revealed that levels of RANKL were higher and levels of OPG were lower in sites with periodontitis than in sites representing healthy gingiva (Crotti et al. 2003; Liu et al. 2003).

• The RANK/RANKL/OPG system is also involved in bone degradation processes that are induced by pro-inflammatory cytokines such as PGE2, TNF alpha, IL-1 beta, IL-6, IL-11, and IL-17.

• The role of T cells, however, is unclear given that this cell not only produces RANKL but also inhibitors of RANKL, such as interferon (IFN)-gamma and IL-4 (Takayanagi 2005).

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14. Healing Process in Periodontitis

• Chronic immune system:

1. Regeneration: the replacement of tissues with new, identical tissues that function the same as the original tissues.

2. Repair: replacement of one tissue with another tissue.

• Under normal conditions, a platelet rich clot forms at the site of injury which facilitates in healing.

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• In periodontal infections, the platelet rich clot does not form.

• The periodontal healing cycle during the pathogenesis of periodontal diseases is primarily post inflammatory and cellular elements other than platelets provide important signals in this process.

• Periodontal repair occurs in overlapping phases of

1. Inflammation shutdown,

2. Angiogenesis

3. Fibrogenesis.

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Inflammation Shutdown• Orchestrated by leukocytes.

• Anti-inflammatory signals generated (Genco RJ,1991):

1. IL-1 receptor antagonist (IL-1ra)

2. Transforming Growth Factor Beta (TGF-b)

3. IL-4

4. IL-10 Manolagas SC, 2000.

5. IL-11

• In the inflamed periodontal tissues:

1. Macrophages produce IL-1ra. (Kabashima, 1996)

2. Mast cells and lymphocytes produce TGF-b. (Steinsvoll 1999)

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Angiogenesis and Fibrogenesis

• IL1b and TNF-b participate in both

inflammation and healing.

• IL1a and IL1b are involved indirectly in in

inducing fibroblast proliferation and collagen

synthesis by stimulating the production of

PGE2 or the release of PDGF and TGF-b.

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Angiogenesis and Fibrogenesis

• PDGF – AA, PDGF-AB, PDGF – BB, PDGF-CC, PDGF-DD.

• PDGF activates fibroblasts and osteoblasts and results in induction of protein synthesis. (GiannobileWV, 1996).

• TGF-b is a multifunctional peptide that stimulates osteoblasts and fibroblasts and inhibits osteoclasts, epithelial and immune cells.

• TGF-b activates in low pH/acidic conditions.

• It promotes the elaboration of fibroblast extracellular matrix adhesion.

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Angiogenesis and Fibrogenesis

• Basic fibroblast growth factor (bFGF)

produced primarily by PDL cells and

endothelium. (Gao J,1996)

• TGF – a (monocyte)

• TNF – a (monocyte)

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Healing of Alveolar Bone

• Activation of osteoblasts

• Inactivation of osteoclasts

• TGF-b is a potent inhibitor of osteoclast formation.

• Osteoclast differentiation and activation are inhibited by:

1. Interferon – g secreted by NK cell, Th1Tcells and macrophages. It inhibits IL-1 and TNF-a induced osteoclast activation.

2. IL-1ra is also effective in blocking IL-1 and TNF-a induced osteoclast activation.

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Conclusion

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Thank You!!!03-03-2015 60