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Good

Morning

Host Microbial Interaction – II

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Barrier Function

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Gingival CreviceFirst site for contact & attachment of bacteria

Mechanical cleansing by saliva and GCF

Inhibits bacterial growth by antibody

and phagocytic secretion.

Barrier Function

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Gingival Epithelium Bacterial Invasion

Antimicrobial peptides that kill

bacteria.

IL-1b to enhance local inflammatory

action.

IL8 attracts PMNs and Macrophages to

reduce microbial insult.

The Innate Immune Response

• The host may respond through inflammation to a

range of challenges, from bacteria to cholesterol.

• However, the nature of the response differs and its

character depends on the microbial triggering of

specific receptors, the signal transduction

pathways, and the way cells and tissues respond

to these signals in terms of cytokine and defensive

protein production.

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• The challenge to overcome escaping recognition by microbial mutation was overcome by innate system by Pattern Recognition Receptors (PRRs).

• These molecular motifs known as Pathogen Associated Molecular Patterns (PAMPs) have essential role in pathogen’s ability to evade host defense and thus are not subject to high mutation rates. (Medzhitov R,2001)

• Toll Like Receptors (TLRs) are critical for recognition of microbes by the immune system and for bridging the innate and acquired immune systems.

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• Innate immunity represents the inherited resistance to microbial infection,

which is detected by Pattern Recognition Receptors (PRRs).

• PRRs are strategically located at the interface between the mammalian host and

the microbes, and have evolved to recognize conserved microbial motifs,

known as Microbe Associated Molecular Patterns (MAMPs).

• Toll Like Receptors (TLRs) constitute an evolutionarily ancient PRR family,

which plays a central role in the induction of innate immune and inflammatory

responses.

• TLRs are expressed predominantly in cells which mediate first-line defense,

such as neutrophils, monocytes/ macrophages, and dendritic cells, as well as

epithelial cells. Distinct members of the TLR family respond to different types

of MAMPs, endowing the innate response with a relative specificity.

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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.

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• 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.

• 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.

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Toll Molecular Structure (Akira,2003)

Toll receptor has an extracellular region which contains leucine rich repeats motifs (LRRs)

Toll receptor has a intracellular cytoplasmic tail which contains a Toll interleukin-1 (IL-1) receptor (TIR) domain. (similar to the intracellular

domain of IL-1R)

The interaction between PLR-PAMP interaction results in recruitment of specific adapter molecules such as MyD88 which then bind to IRAK -1.

TIR

Domain

Toll (will become TLRs)

LRRsIg-like

domain

Box 1

Box 2

Box 3

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Types of TLRs

Receptor Cell Type

TLR1 Ubiquitous

TLR2 DCs, PMLs, and monocytes

TLR3 DC and NK cells, upregulated on epithelial and endothelial cells

TLR4 Macrophages, PMLs, DCs, ECs, but not on lymphocytes

TLR5 Monocytes, immature DCs, epithelial, NK, and T cells

TLR6 High expression in B cells, lower on monocytes and NK cells

TLR7 B cells, plasmacytoid percursor DCs

TLR8 Monocytes, low in NK cells and T cells

TLR9 Plasmacytoid percursor DCs, B cells, macrophages, PMLs, NK

cells, and microglial cells

TLR10 B cells, plasmacytoid precursor DCs

TLR11 Not Determined

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Mode of Action of TLRs

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MyD88= Myeloid

Differentiation Associated

Primary Response Protein 88.

IRAK=IL-1R Associated

kinase.

TRAF-6= TNFa Associated

Factor -6.

TAK1TGFb associated kinase.

IkB=Inhbitor of Nuclear Factor

k b kinase complex.

MKK: Mitogen Activated

Protein Kinase kinases.

phosphorylates

• The MyD88 dependent pathway leads to activation of IRAK, TRAF6 and ultimately NFkBand cytokine production.

• The MyD88 independent pathway does not activate IRAK1 and leads to NFkB activation with delayed kinetics.

• This pathway requires different adapter proteins and doesn’t lead to cytokine production.

• It is related to Interferon beta secretion and indirect upregulation of IFN dependent genes.

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Toll Like Receptor Expression and

Microbial Recognition in

Periodontal Tissues

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• 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.

• 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.

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• 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.

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TLRs and Receptors in Periodontal

DiseasesPRR Expressed in PAMP Periodontal

Pathogen

Pathogen

TLR-2 Monocytes,

Neutrophils,

Epithelial Cells,

Fibroblasts,

Macrophages, T

and B

Lymphocytes

Lipoproteins Tannerella forsythia

Atypical lipopolysaccharide

(LPS)

Porphyromonas

gingivalis,

Capnocytophaga

ochracea

Outer membrane proteins Oral treponemes

Fimbriae P. gingivalis

Non-endotoxic glycoprotein Prevotella intermedia

Whole bacteria Treponema denticola

Cell-surface BspA protein Tannerella forsythia3/3/2015 22

TLRs and Receptors in Periodontal

Diseases

PRR Expressed in PAMP Periodontal

Pathogen

TLR-4 Monocytes,

Macrophages,

Epithelial Cells,

Fibroblasts,

Neutrophils, T

And B

Lymphocytes

LPS Porphyromonas

gingivalis,

Capnocytophaga

ochracea

Atypical

lipopolysaccharide (LPS)

Porphyromonas

gingivalis,

Capnocytophaga

ochracea

Heat shock protein (HSP) Porphyromonas

gingivalis,

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TLRs and Receptors in Periodontal

DiseasesPRR Expressed in PAMP Periodontal

Pathogen

TLR-9 Epithelial cells, dendritic

cells,

T and B lymphocytes

CpG-containing DNA Aggregatibacter

actinomycetemcomita

ns,

P. gingivalis, P. micros

Nod1 Cytotoxic T cells,

dendritic

cells, epithelial cells

fibroblasts, osteoblasts,

macrophages

Meso-diaminopimelic acid

(meso-

DAP) containing

peptidoglycan

(PGN) fragments (mostly

gramnegative

bacteria)

Nod2 Neutrophils, monocytes,

dendritic cells, epithelial

cells,

fibroblasts, osteoblasts,

macrophages

Muramyl dipeptide (MDP)

found in

PGN of both gram-

positive and gramnegative

bacteria3/3/2015 24

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Effects on Resident cells

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Epithelial Cells

• Express TLR-2, 9. (Mempel M, 2003)

• LPS from Aac increases expression of ICAM-1 and LFA-1 due to bacterial LPS.

• However, P.gingivalis LPS down regulates ICAM1 andLFA1 secretion. (Huang GT, 2001)

• Increased PMN migration towards the pocket.

• Increased IL-8 production in response to LPS production by various pathogens. (Huang GT, 2001)

• Activate endothelial cells, macrophages, dendritic cells and PMNs to produce MMPs. (Warner R L, 2004)

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Dendritic Cells or Langerhans Cells

• DCs express TLR-9.

• Recognize PAMPs and initiate maturation process.

• Mature DCs present antigens to MHC Class-II.

• Produce cytokines and co-stimulatory molecules (CD-40,54,80,86) that induce activation of T-lymphocytes

• DCs + P.gingivalis fimbriae + T lymphocytes = Th1 type response.

• DCs + P.gingivalis LPS = Th2 type response. (Jotwani R, 2004)

• Because P.gingivalis activates TLR-2 and not TLR – 9. (Dillon S, 2004)

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Macrophages

• Produce cytokines and MMP-1, NO when

stimulated by CpG and LPS by periodontal

pathogens.

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Fibroblast

• Gingival fibroblast: Produces proinflammatory cytokines and also express adhesion molecules in response to PAMPs such as LPS, PGN and DNA of various pathogens.

• PDL fibroblast : Produces Alkaline Phosphatase activity similar to osteoblasts.

• These cells can also liberate proteinases causing tissue destruction.

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• The capsular polysaccharide from Aac can inhibit

expression of IL-6 and IL-8 induction in gingival

fibroblasts by LPS from the same micro-organism

and modulate an immune response in blocking

bone resorption which is supported by an increase

in OPG expression by LPS stimulated Gingival

fibroblasts. (Nagasawa T, 2002).

• On stimulation with PGN, PDL fibroblasts

express higher levels of IL-8 than gingival

fibroblasts, perhaps because of more TLR-2

expression. (Hatakeyama, 2003)

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Cementoblasts

• Stimulated by LPS exhibit decreased levels of

expression of RANKL and similar to gingival

fibroblasts, increased expression of both OPG

and OPN.

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Endothelial Cells

• May be stimulated by IL-8 secreted by other cells and also directly by LPS through TLR-4 and p-38 MAPK pathway (Fa1ure E,2000) ultimately leading to activation and increased adhesion of monocytes (Doherty DE,1989).

• This is due to the induction of cytokine production and increased expression of adhesion molecules like ICAMs and VCAM-1 by PAMPs. (Galdiero M, 1997)

• Also function as APCs.

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Osteoblasts

• P.intermedia LPS inhibits osteoblast differentiation and mineralization. (Pelt P, 2002)

• Capsular polysaccharide from Aac promote osteoclast differentiation of bone marrow cells. (Ito HO, 1996).

• In absence of stromal cells and osteoblasts, LPS inhibits RANKL induced differentiation of osteoclast precursors as a result of decreased M-CSF and RANK receptor.

• If osteoclast precursors are primed with RANKL, LPS synergistically increases differentiation influenced by autocrine stimulation with LPS induced TNFa and PGE2. (Nishihara T,1995; Zou W, 2002).

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Effects on Resident Cells

• Neutrophils:

1. PAMPs may directly stimulate PMNs

inducing chemotaxis, shedding of the

adhesion molecule L-selectin and cytokine

production (effects mediated by TLR-2

expression) ( Trevani AS, 2003)

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Monocytes

• Stimulated by PAMPs produce inflammatory cytokines and also increase proliferation and adhesion to endothelial cells.

• LPS induced differentiation of monocytes into osteoclasts even in absence of osteoblasts and the induction of RANKL expression is the key mechanism in it. (Jiang Y, 2002)

• In the presence of IL-12, P.gingivalis LPS significantly increases IFNg production by T cells and also augments production of IL-12 by monocytes. (Yun PL, 2002)

• Without co-stimulatory factors, Pg LPS fails to induce proinflammatory cytokines on monocytes ; on the contrary, it induces expression of anti-inflammatory IL-10 that can down regulate IL-12 levels.

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B Lymphocytes

• Directly stimulated by PAMPs specifically

CpG DNA because they lack TLR-2 while

simultaneously expressing TLR-9. (Wagner

M,2004)

• This leads to proliferation, antibody production

and production proinflammatory cytokines.

(Zugel U, 1999)

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T Lymphocytes

• Activation by LPS is species specific.

• LPS from E.coli was shown to induce both CD4+ and CD8+ T cells to produce IFNgproduction.

• P.gingivalis LPS was shown to induce Th2 cells. (Pulendran B, 2001)

• CpG DNA induces differentiation and activation of T cells as also inhibits CD4+ apoptosis.

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Bone Remodeling Cycle

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Mediators of Bone Resorption

(McCauley LK, 2001)

Stimulators Inhibitors

1. IL-1

2. IL-6

3. TNF

4. PTH

5. PTH Related Protein

6. PGE2

7. M-CSF

8. RANK

9. RANKL

10. Vitamin-D

1. Interferon gamma

2. OPG

3. Estrogens

4. Androgens

5. Calcitonin

6. Cyclosporin

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Pathobiology of Periodontal

Diseases

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• An inappropriate inflammatory response is the cause of many common diseases, including periodontitis.

• The local inflammatory reaction, in response to bacteria in the dental biofilm, is characterized by an initial increase in blood flow, enhanced vascular permeability, and the influx of cells from the peripheral blood to the gingival crevice.

• These initial events are triggered by bioactive molecules (such as histamine, bradykinin, PGE2, and nitric oxide) and produced by innate immune cells and resident, nonimmunecells present in the periodontal tissues.

• PMNs attracted to the area by other bioactive molecules (e.g., IL-8) migrate through the epithelial lining of the gingival sulcus to be the initial defense against invading plaque bacteria and their by-products.

• These cells are nonspecific phagocytes responsible for an acute and rapid defense.

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• Subsequently, there is an increase in the number of monocytes/macrophages, as well as an influx of T and B cells to the area.

• Once activated by cytokines, bioactive molecules, and MAMPs present in the area, infiltrating cells produce other inflammatory mediators that modulate the activity of other cells and affect homeostasis of non -mineralized and mineralized tissues in the periodontium.

• Patients with periodontal inflammation have high concentrations of TNF-α, IL-1β, RANKL, and MMP-13in the gingival crevicular fluid (GCF).

• Increased levels of IL-1β, IL-2, IL-6, IL-17, TNF-α, and IFN-γ in gingival tissues are also associated with destructive periodontal disease.

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• A characteristic cytokine profile has been associated with each type of periodontal disease (i.e., inflammation of marginal soft tissues without active bone resorption [gingivitis] or with active bone resorption [periodontitis]).

• Thus expression of Th1-type cytokines has been associated with gingivitis, whereas Th2 cytokines were found in higher levels in periodontitis-affected tissues; even though this distinction was not clear because both Th1 and Th2 cytokines were produced in gingivitis-and periodontitis-affectedissues; and the predominant profile may actually represent the current activity of tissue destruction

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Thank You !!!