GOOD MORNING

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Host Microbial Interaction Part I

Presented by: Dr Ashish Bisane Post Graduate Student Dept. of Periodontics & Implantology Swargiya Dadasaheb Kalmegh Smruti Dental College & Hospital, Nagpur.
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Contents
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Introduction. Microbial Virulence. Bacterial adherence in periodontal environment. Host tissue invasion. Bacterial evasion of host defense mechanism. Bacterial enzymes capable of tissue destruction. Immunological aspects of host microbial interaction. Host defense processes Host responses to bacterial invasion. Inflammatory responses Molecules, Cells and Processes in Host Response. Connective Tissue Alterations Bone Destruction 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 Bacterial nt Species Surface Substrate Bacterial Adhesion Substrate Receptor 1. Saliva treated HAP. 2. Proline rich proteins Sialic acid residues Galactosyl residues

Tooth

A. viscosus

Fimbriae Saliva coated mineralized surfaces

S. mitis

Saliva coated F. nucleatum surfaces

70-90kD protein 300-330 kD outer membrane protein

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Bacterial Adhesins and Target Surfaces (SS Socransky, 1991)

Attachment Surface Bacterial Species P.gingivalis A. viscosus A. naeslundii T. denticola Substrate Bacterial Adhesion Fimbriae Fimbriae Substrate Receptor Galactosyl residues Galactosyl residues Galactosyl or Mannose residues Galactosyl residues Galactosyl residues

Tissue

Epithelial cells

Fibroblasts PMNs

Surface protein

A.viscosus A. naeslundii F. nucleatum P.gingivalis P. Intermedia

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Fimbriae Protein

Connective Tissue Components

Membrane protein Fibrinogen, Fibronectin Membrane protein Fibrinogen

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Bacterial Adhesins and Target Surfaces (SS Socransky, 1991)

Attachment Surface Bacterial Species Substrate Bacterial Adhesion Substrate Receptor

PreExisting Plaque Mass

A. viscosus

S.Sanguis

Fimbriae

Repeating heptasaccharide on polysaccharide. Rhamnose, Fructose. N-acetylmuramic acid residues Galactosyl residues
Galactosyl residues
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C. Ochraceus

S.Sanguis A.Naeslundii A.Israelli

Heat sensitive protein

P. loescheii
F.nucelatum

S.Sanguis A.Israelli
P.Gingivalis T.Denticola P.Micros

75-45 kD fimbrial proteins

Heat and Protease sensitive protein.

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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)
Host Defense Mechanism Bacterial Species Bacteria Property Biologic Effect

1. Specific Antibody

P.gingivalis
P.intermedia P.Melanogenica Capnocytophaga sp. IgA and IgG degrading proteases Degradation of specific antibody

2. PMNs

Aac
F.nucleatum P.Gingivalis T.denticola

Leukotoxin
Heat Sensitive Surface Protein Capsule Inhibition of superoxide production

Inhibition of PMN function

PMN Apoptosis Inhibition of phagocytosis Decreased bacterial killing.
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Host Defense Mechanism

Bacterial Species

Bacterial Property Leukotoxin

Biologic Effect 1. Killing of mature B and T cells. 2. Non lethal suppression of lymphocyte activity. Impairment of function by arresting lymphocyte cell cycle.

3. Lymphocytes

Aac

Cytolethal Distending Toxin

F. nucleatum
T. Forsythia P. Intermedia T. denticola Aac 4. Release

Heat sensitive surface protein

Cytotoxin Suppression

Apoptosis of mononuclear cells

Apoptosis of lymphocytes Decrease response to antigens and mitogens

of

P.Gingivalis

IL-8
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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
Collagenase Trypsin like enzyme

Species
P. Gingivalis Aac P. Gingivalis

Aac
T.Denticola Arylsulfatase Neuraminidase C.Rectus P. Gingivalis

T.Forsythia
P.Melanogenica Fibronectin degrading enzyme P.Gingivalis P. Intermedia

Phospholipase A
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P. Intermedia
P.Melanogenica
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7. Immunological Aspects of Microbial Host Interaction

Well characterized interactions involve release of : 1. IL-1 From monocytes, macrophages and PMNs 2. TNF exposed to bacterial endotoxin (LPS) 3. Prostaglandins [Roberts FA, 1997.
Yoshimura A, 1997]

These host derived mediators have the potential to stimulate bone resorption and activate or inhibit other host immune cells.
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Bacterial Species Aac F.nucleatum Aac P.Gingivalis Aac Aac C.Rectus E.Corrodens Aac F.nucelatum E.Corrodens Aac C.Rectus Aac F.nucelatum
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Bacterial Component LPS 37 kD protein LPS 37-kD protein Whole cells Whole cells Whole cells

Target Host Tissue PMNs Macrophages Monocytes Macrophages Gingival fibroblasts Epithelial cells Epithelial cells

Effect On Cytokine Level Increased release of Interleukin 1

Increased release of IL 6

Increase release of IL 8

Whole cells LPS

Gingival fibroblasts PMNs

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Bacterial Species

Bacterial Component

Target Host Tissue Effect On Cytokine Level

Aac F.nucleatum Aac

LPS

PMNs

Increased TNF release

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 IL8 in response to bacterial challenge. (Huang GT-J, 1998; Darveau RP, 1998)

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Cathepsin G

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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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Tropocollagen

Osteocalcin, Osteopontin, Osteonectin

Proteoglycans

Components of Periodontal ECM

Tenascin

Elastin

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).
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Proteinases/Proteases
Proteinases (collagenase, elastase - like and trypsinlike, 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 (A1AT) Inhibits PMN collagenase

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Matrix Metalloproteinases
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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 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 23-09-2012 stimulation of bone resorption.

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 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 COX2. 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 proinflammatory 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!!!

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