host microbial interaction part 2
TRANSCRIPT
3/3/2015 1
Good
Morning
Host Microbial Interaction – II
3/3/2015 2
Barrier Function
3/3/2015 3
Gingival CreviceFirst site for contact & attachment of bacteria
Mechanical cleansing by saliva and GCF
Inhibits bacterial growth by antibody
and phagocytic secretion.
Barrier Function
3/3/2015 4
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.
3/3/2015 6
• 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.
3/3/2015 7
• 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.
3/3/2015 8
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.
3/3/2015 10
• 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.
3/3/2015 11
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
3/3/2015 13
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
3/3/2015 14
Mode of Action of TLRs
3/3/2015 15
3/3/2015 16
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.
3/3/2015 17
Toll Like Receptor Expression and
Microbial Recognition in
Periodontal Tissues
3/3/2015 19
• 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.
3/3/2015 20
• 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.
3/3/2015 21
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,
3/3/2015 23
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
3/3/2015 25
Effects on Resident cells
3/3/2015 26
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)
3/3/2015 27
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)
3/3/2015 28
Macrophages
• Produce cytokines and MMP-1, NO when
stimulated by CpG and LPS by periodontal
pathogens.
3/3/2015 29
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.
3/3/2015 30
• 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)
3/3/2015 31
Cementoblasts
• Stimulated by LPS exhibit decreased levels of
expression of RANKL and similar to gingival
fibroblasts, increased expression of both OPG
and OPN.
3/3/2015 32
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.
3/3/2015 33
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).
3/3/2015 34
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)
3/3/2015 35
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.
3/3/2015 36
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)
3/3/2015 37
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.
3/3/2015 38
Bone Remodeling Cycle
3/3/2015 39
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
3/3/2015 40
Pathobiology of Periodontal
Diseases
3/3/2015 41
• 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.
3/3/2015 42
• 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.
3/3/2015 43
• 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
3/3/2015 44
3/3/2015 45
3/3/2015 46
Thank You !!!