The host response and the interaction between the host and the microbes

1. Practical Molecular
Biology Of Host-
Microbe Interaction

2. Contents
 Microbe Associated Molecular Pattern
 Toll Like Receptors
 Nucleotide Binding Oligomerization Domain
like Receptors
 Complement System
 Antimicrobial Peptides
 Immunomodulatory Therapies
 Conclusion
 References

3. Introduction
 The host response is defined as the defense
mechanism of the host against exogenous
microorganisms.
 Periodontium is a dynamic remodelling tissue
challenged by bacterial biofilms colonizing
proximal odontogenic and mucosal surfaces.
 Extensive molecular research has elucidated
that the host immune response is the primary
mediator of periodontal tissue destruction.

4. Microbe Associated Molecular
Patterns (MAMPs)

5.  Evolutionary conserved molecular motifs
present in microorganisms. Found in higher
eukaryotes.
 Include microbial cell wall macromolecules,
nucleic acids and flagellin which functions as
ligands.
 Contributes to pathophysiologic tissue
destruction in chronic inflammatory disease
states such as periodontitis

6. TABLE 1
Host Cell Pattern-Recognition Receptor Ligand Binding of
Periodontal Bacteria-Derived Microbe-Associated Molecular
Patterns

7. Host Cells PRRs MAMPs Periodontal
Bacteria
References
(Carranza)
Neutrophils, monocytes,
macrophages, epithelial
cells, fibroblasts,
cementoblasts,
osteoblasts, dendritic
cells,T and B
lymphocytes
TLR-2 Lipoproteins Porphyromonas
gingivalis
61, 66, 115
Lipoproteins Tannerella
forsythia
60, 105
Lipoproteins Actinomyces
viscosus
152
Peptidoglycan Actinomyces
naeslundii
146
TLR-4 LPS Porphyromonas
gingivalis
13, 67, 117,
147
LPS Aggregatibacter
actinomycetem
comitans,
Fusobacterium
nucleatum
63, 137,
156,
190

8. Ss
iE-DAP-Gamma-D-glutamyl-mesodiaminopimelic acid;
LPS-Lipopolysaccharide;
MAMP-Microbe-associated molecular pattern;
NOD-Nucleotide-binding oligomerization domain;
PRR-Pattern-recognition receptor;
TLR-Toll-like receptor
TLR-9 CpG-DNA Porphyromonas
gingivalis,
Tannerella
forsythia
28, 44, 86,
110, 143
NOD1 iE-DAP Porphyromonas
gingivalis,
Aggregatibacter
actinomycetemc
omitans,
Fusobacterium
nucleatum
107, 119,
157, 162,
175–177,
182NOD2 MDP

9.  Periopathogenic Bacteria Microbe Associated Molecular Pattern
Induction of Biologic Mediators in Host Periodontal Tissue Cells
Host Cells MAMPs Biologic Mediators References
(Carranza)
Epithelial Cells LPS, fimbriae,
bacteria cell wall
extracts, gingipains
IL-8,G-CSF, GM-CSF,
β-
defensin-2, MMPs-
3/9/13,
MIP-1α, IL-1β
27, 32, 49, 57, 63–
65, 112,
120, 141, 144, 159,
163,
183
Dendritic Cells LPS, CpG-DNA,
fimbriae
IFN-α, IL-6, IL-8, IL-
10, IL-12, TNF-α,
GM-CSF
68, 77, 78, 128, 158
Endothelial Cells LPS IL-6, GM-CSF, ICAM-1 31, 40, 63, 75, 98
Gingival
Fibroblasts
LPS, CpG-DNA,
gingipains,
peptidoglycan
IL-1β, IL-6, IL-8,
TNF-α,
PGE2, MCP-1, MMP-2
5, 15, 35, 62, 63,
110, 112,
116, 118, 131, 164,
171,
183, 184

10. PDL
fibroblasts
LPS IL-6, IL-8, MMP-13,
RANKL
62, 63, 138, 139,
189
Cementoblasts LPS OPN,OCN, RANKL,
IL-6
107, 109
Macrophages LPS, CpG-DNA,
leukotoxin
IL-1α/1β, IL-6, IL-
12, TNF-α,
MMP-1, NO
63, 83, 87, 103,
110, 116, 168,
187
Osteoblasts LPS IL-1β, IL-6, TNF-α,
RANKL,
PGE2, NO, MMP-2,
MMP-9
63, 85, 124, 161,
185–188,
196
Neutrophils LPS, CpG-DNA IL-8, MIP-1α 141, 172, 173

11. A
G-CSF-Granulocyte colony-stimulating factor
GM-CSF-Granulocyte-macrophage colony-stimulating factor
ICAM-Intercellular adhesion molecule IFN-Interferon
IL-Interleukin LIF-Leukocyte inhibitory factor
LPS-Lipopolysaccharide MAMP-Microbe-associatedmolecular pattern
MCP-Monocyte chemo-attractant protein MMP-Matrix metalloproteinase
NO-Nitric oxide OPN-Osteopontin
PDL-Periodontal ligament PGE2-Prostaglandin E2
RANKL-Receptor activator of nuclear factor-κB ligand
TNF-Tumor necrosis factor CpG-DNA-Cytosine phosphate guanine DNA
MIP-Macrophage inflammatory proteins OCN-osteocalcin
Monocytes LPS, CpG-DNA,
fimbriae
IFN-γ, IL-1α/1β, IL-
6, IL-8,
IL-12, TNF-α, LIF,
RANKL,
PGE2
10, 11, 16, 31, 34,
40, 51,
59, 63, 71, 94, 97,
106,
131, 134
B lymphocytes CpG-DNA, cell
sonicate extracts
IL-6, IL-10, IL-12,
TNF-α
23, 114, 181, 193
T lymphocytes LPS, CpG-DNA,
peptidoglycan
IFN-γ, IL-4, IL-10,
IL-13
43, 99, 100, 128,
166, 193

12.  Recognition of MAMPs by innate immune cells
stimulates the secretion of proinflammatory
cytokines
 Examples: IL-1β, IL-6, TNF and type 1 interferons
(IFN-α ,IFN- β)
 Upregulates the production of co-stimulatory
molecules.
 PRRs are considered the bridge between the innate
and adaptive immune systems.

13. PRRs
(Pattern recognition
receptors)
DAMPs
(Damage associated
molecular patterns)
NLRs
(Nucleotide binding
oligomerization domain
like receptors)
TLRs
(Toll like receptors)

14. Toll Like Receptors

15. TLR
FAMILY
Endolysosomal
Membrane
Plasma
Membrane
TLR 1
TLR
10
TLR 6TLR 5
TLR 4TLR 2
TLR 9TLR 8
TLR 7TLR 3

16.  Plasma membrane TLR signalling induces the
expression of proinflammatory cytokines
 Endolysosomal TLR signalling predominantly induces
the expression of type 1 IFNs.
 TLRs localized to the plasma membrane recognize
extracellular microbial cell wall components (TLR 1,
TLR 2, TLR 4, TLR 6) or flagellin (TLR 5).
 TLRs localized to the endolysosomal membrane
recognize microbial nucleic acids (TLR 3, TLR 7, TLR
8,TLR 9)

17.  TLR 2 and TLR 4 recognises the extracellular
bacterial cell wall components at the cell surface
 TLR 9 is addressed with regard to recognizing
bacterial nucleic acids within endosomes.
 TLRs are single pass transmembrane proteins
characterized-
1. N-terminal leucine rich recognition domain
2. C-terminal (intracellular) Toll/IL1 receptor
signalling domain (TIR).

18. Myeloid
differentiation
primary response
protein 88 (MYD88)
TRIF related adaptor
molecule (TRAM)
MYD88 adaptor like
protein (MAL)
TIR domain
containing adaptor
protein inducing IFN-
β (TRIF)
TIR domain of TLRs act as a scaffold to recruit
various TIR domain containing adaptor proteins

19. Toll Like Receptor-4 Lipopolysaccharide
Recognition
 Oral bacterial interactions with host TLRs are
largely dependent on the exposed macromolecules.
 Lipoproteins are common constituents of the outer
membranes of both gram –ve and +ve bacteria.
 LPS is the major macromolecule composing the
outer surface envelope of gram negative bacteria.

20.  It induces a host immune response through recognition
of lipid A.
LPS is typically made up of three domains
Lipid A
O-Antigen
A short core
oligosaccharide

21.  Mammalian cells recognize LPS through-
 LBP processes and delivers LPS to CD14 which
sensitizes cells for LPS binding by MD2-TLR-4 receptor.
TLR-4
homodimer
protein
complex
consisting of
TLR-4
Coreceptor
myeloid
differentiati
on factor 2
(MD2)
Accessory
proteins
CD14 and
LBP

22. Toll Like Receptor-2- Lipoprotein/Lipoteichoic
Acid/Peptidoglycan Recognition
 TLR-2 has the capacity to recognize diverse
microbial macromolecules.
 Forms heterodimer protein complexes with other
TLR family members (TLR-1, TLR-6)
 TLR-2 ligands are highly relevant to oral flora
interactions with host cells include lipoproteins,
LTA and peptidoglycan.

23.  Triacylated lipoproteins are recognized by TLR-
2/TLR-1 heterodimer complex.
 Diacylated lipoproteins are recognized by TLR-
2/TLR-6 heterodimer complex.
 LTA and peptidoglycan are recognized by
incompletely characterized TLR-2/TLR-6
heterodimer complexes.

24. Toll Like Receptor-9-CpG-DNA
Recognition
 TLR-9 is different from TLR-2 and TLR-4 and
recognizes MAMPs in endosomes.
 TLR-9 is the endosomal TLR family member that
recognizes intracellular microbial nucleic acids.
 During infection, microbe derived nucleic acids are
sensed by endosomal TLRs.
 This sensing facilitates mounting a host immune
response to clear the invading microorganisms.

25.  TLR-9 recognizes both viral and bacterial CpG-DNA.
 CpG-DNA localized within lysosomal compartments
induces-
1. Trafficking of TLR-9 from the ER to the
endolysosome
2. Activates TLR-9 signal transduction.

26. Role Of Toll Like Receptors In
Periodontitis
 Gram–ve bacteria-induced periodontal destruction
is mediated through TLR-2 signal transduction.
 Gram–ve bacteria-induced catabolic actions are
1. Differential signalling at the TLR-2 receptor
2. Concomitant activation of TLR-4 receptor by LPS.
 P. gingivalis has the ability to stimulate TLR signal
transduction through various MAMPs.

27.  P. gingivalis coactivation of TLR-2 and TLR-4 is
critical in stimulating host immune response
mechanisms driving alveolar bone loss.
 TLR-9 expression up regulates in clinical
periodontitis tissues when compared with gingivitis
tissues and healthy gingival biopsy samples.

28. Nucleotide Binding Oligomerization Domain
Like Receptors
 Currently 22 family members comprise the
intracellularly expressed NLRs in humans.
 Localized to the cytosol and play a critical role in
sensing invading microorganisms and prompting the
immune response.
 Characterized by-
1. C-terminal leucine rich repeats that act as a
sensing domain(i.e., a NOD)
2. N-terminal effector domain that mediates
downstream signalling.

29. NOD1/NOD2-Peptidoglycan Recognition
 NOD1 recognizes gamma-D-glutamyl-
mesodiaminopimelic acid (iE-DAP), a component of
peptidoglycan.
 NOD2 recognizes muramyl dipeptide (MDP) which is
found in peptidoglycan.
 Peptidoglycan binding at NOD1 and NOD2 receptors
causes their oligomerization.

30.  This results in-
1. Recruitment of a serine/threonine kinase adapter
protein.
2. RIP-2/RICK to a caspase activation
3. Recruitment domain (CARD) at the N-terminus
 RIP-2/RICK recruitment at the N-terminus
activates NF-κB and MAPK-dependent up
regulation of proinflammatory cytokine genes.

31. NLRP3-Inflammasome Complex
 Inflammasomes are multiprotein complexes that-
1. Recognize diverse inflammation-inducing stimuli
including exogenous MAMPs and endogenous DAMPs
2. Control the production of proinflammatory
cytokines
3. Regulate pyroptosis (an inflammatory form of cell
death)
 Several PRR families act as components in the
inflammasome complex

32.  Recognition of cytosolic MAMPs and DAMPs induces
NLRP3 to act as a scaffold for inactive zymogen pro
caspase-1.
 Pro caspase-1(which has a CARD)is recruited to
inflammasome complex through-
1. Homotypic binding of CARD via a pyrin domain
(PYD)
2. Adaptor apoptosis-associated speck like protein
containing a CARD (ASC).

33.  Oligomerization of pro-caspase-1 proteins in the
inflammasome leads to their autoproteolytic
cleavage into active caspase-1.
 Activated caspase-1 functions to cleave pro IL-1β
and pro-IL-18 into their biologically active form.

34. TABLE 3:
Pattern-Recognition Receptor-Microbe Associated
Molecular Pattern Ligand Recognition in
Periodontitis

35. PRRs Localization MAMP Ligand Ligand Origin
TLR-2/TLR-1 Plasma Membrane Triacylated lipoproteins G- Bacteria
TLR-2/TLR-6 Plasma Membrane Diacylated lipoproteins G+ Bacteria
LTA G+ Bacteria
Peptidoglycan G+ Bacteria
TLR-4 Plasma Membrane LPS G- Bacteria
Endolysosome
TLR-9 Endolysosome CpG-DNA Bacterial and Viral
NOD1 Cytoplasm iE-DAP G+ Bacteria
G- Bacteria
NOD2 Cytoplasm MDP G+ Bacteria
G- Bacteria

36. Role of NOD-Like Receptors in
Periodontitis
 NOD1 and NOD2 are expressed in human oral
epithelium, gingival fibroblast cells and periodontal
ligament fibroblast cells.
 The knockout mice models found-
1. Mice deficient in NOD2 showed comparable levels
of alveolar bone resorption.
2. Mice deficient in NOD1 demonstrated reduced
levels of alveolar bone loss

37.  NOD1 knockout mice had fewer osteoclasts and
lower proinflammatory cytokine expression levels
in gingival tissue isolates.
 Another experiment:
1. NOD1 had exacerbated alveolar bone loss,
increased osteoclast numbers.
2. Up-regulated proinflammatory cytokine
expression levels in cultured bone marrow
macrophages.
 Hence it is unclear whether NOD1 or NOD2
critically regulates periodontal bone loss

38.  MAMPs present in biofilm can activate TLRs and
NOD1/2 signalling which converge at the MAPK and
NF-κB signalling pathways.
 NLRP3 and NLRP2 are found to be increased in
human gingival tissues.
 IL-1β and IL-18 mRNA expression levels were
increased in gingival tissues affected by periodontal
disease states.

39. Complement System
 Periodontal host immune response is dependent on
a functional complement system, which notably
coordinates the recruitment and activation of
immune cells, bacterial opsonisation, phagocytosis
and lysis.

40. Complement-Pattern-Recognition Receptor
Signalling
 Some soluble PRR families are also secreted into the
plasma as humoral proteins.
 Soluble PRRs represent the functional ancestors of
antibiotics including pentraxins, mannose-binding
lectin (MBL), ficolins and properdin
 Soluble PRRs interact with circulating MAMPs and
DAMPs to activate the complement system

41. Classical/Lectin/Alternative Pathways
The sequential activation and proteolytic cleavage of a
series of serum proteins by three distinct mechanisms
Classical
Pathway
Lectin
Pathway
Alternative
Pathway

42.  Classical Pathway:
1. Occurs in response to antigen-antibody complexes
that are recognized by the C1q subunit of C1.
2. C1q activates compliment by functioning as a PRR
to recognize distinct MAMPs and DAMPs
3. Activates through other soluble PRRs such as
pentraxins (i.e. C-reactive protein)
 Lectin Pathway:
1. Similarly triggered through soluble PRRs, including
MBL and ficolins which predominantly recognize
carbohydrate groups.

43.  Both the pathways then proceed through C4 and C2
cleavage for the generation of the classical/lectin
C3 convertase (C4bC2b)
 Alternative Pathway:
1. Initiated by the hydrolysis of C3 to C3(H₂O) which
is a C3b analogue that forms the initial alternative
pathway for C3 convertase.
2. Possesses a PRR based initiation mechanism via
properdin which recognizes MAMPs and DAMPs.
3. Serves as a positive feedback loop for the other
two pathways.

44.  Three pathways converge at the third component of
complement (C3)
 On activation by pathway-specific C3 convertases
leads to the generation of key effector molecules.
 Include C3a and C5a anaphylatoxins which activate
specific G-protein coupled receptor
 Mediate the mobilization and activation of
leukocytes.

45.  C3b opsonins which promote phagocytosis through
complement receptors
 C5b-9 membrane attack complex which can lyse
targeted pathogens

46. Role Of Complement In Periodontitis
 Dysregulation of complement activities may lead to
a failure to protect the host against pathogens.
 Activated components are found-
1. Higher levels in the GCF of periodontitis patients.
2. Chronically inflammed gingiva
 Complement components are non detectable at
lower levels in healthy gingival biopsy specimens.

47.  Local complement activation may promote
periodontal inflammation predominantly via
1. C5a induced vasodilation
2. Increases vascular permeability
3. Flow of inflammatory exudate
4. Chemotactic recruitment of inflammatory cells,
especially neutrophils.

48. Antimicrobial Peptides
 Components of the innate immune response in
eukaryotes.
 Provides defence against a wide spectrum of
gram+ve and gram–ve bacteria, viruses and fungi.
 In the oral cavity, 45 different antimicrobial
peptides are found in the saliva and GCF.

49. Defensins and Cathelicidin LL-37
 Cationic peptides that bind to negatively charged
molecules on the microbial surfaces.
 Examples: LPS in gram-ve bacteria and LTA in
gram+ve bacteria.
 Depolarize the cell membrane and render it
permeable with resulting bacterial cell death.
 Defensins are modulated by immune response
mediators

50. Based on structural distinctions in the connecting
patterns of three disulphide bonds and in the spacing of
cysteine residues, defensins are
α-Defensins
Β-Defensins

51.  6 human α-defensins and 4 human Β-defensins are
characterized
 α-defensins (1-4) present in the oral cavity are
known as human neutrophil peptides due to their
expression in neutrophils.
 α-defensins (5 and 6) are localised to the mucosal
Paneth cells of the small intestine.

52.  Β-defensins 1-4 are:
1. Produced by a variety of epithelial cells throughout
the body.
2. Produced abundantly by epithelial tissues within the
oral cavity
3. Found in the GCF and saliva.
 Cathelicidin LL-37 are
1. Human defense peptide residing in neutrophils
2. Found in the gingival epithelium.

53. Role of Antimicrobial Peptides in
Periodontitis
 Specific Β-defensins are located in different
anatomic regions of the periodontal epithelium.
 Β-defensins 1 and 2 are observed in :
1. Upper layers of the gingival and sulcular epithelium
2. Adjacent to the microbial biofilm and external
environment
3. Consistent with the innate immune “barrier”
function of the epithelium.

54.  Neither Β-defensins 1 or 2 are found in the
junctional epithelium
 Protection of JE provided by the higher
concentration of α-defensins 1-3 and LL-37.
 Expression of neutrophil derived α-defensins 1-3 and
LL-37 are elevated in the GCF of chronic
periodontitis patients.
 Expression of defensins induced by whole
periopathogenic bacteria is largely dependent on
TLR signalling.

55. Immunomodulatory Therapies
 MMP inhibitors have been used in combination with
scaling and root planing or surgical therapy.
 High risk patient(eg-diabetic patients) have
benefited from the systemic administration of MMP
inhibitors.
 Use of soluble antagonists of TNF-α and IL-1β
delivered locally to periodontal tissues in non
human primates has shown good results.

56.  Pharmacologic inhibitors of NF-κB and p38 MAPK
pathways are developed to manage rheumatoid
arthritis and inflammatory bone diseases
 With the use of this novel strategy, inflammatory
mediators (eg- IL-1, TNF, IL-6) are necessary for
inflammatory gene expression or mRNA stability.
 C3 is a central component of all three activation
pathways, blockade at this level is a reasonable
approach for treating periodontitis.

57.  CR3 antagonism through topical small molecules
inhibitors has been shown to reduce P. gingivalis –
induced alveolar bone loss
 C5a functions as a potent mediator of complement
signalling and neutrophil recruitment that may
protect and mediate excessive neutrophil activation
 Has the potential to augment tissue damage during
periodontal disease progression
 C5aR inhibitor is used for the treatment and
management of periodontal diseases.

58. Conclusion
Host response contributes to the progression
of the disease. Majority of periodontal
breakdown is caused by host derived
destructive enzymes & inflammatory
mediators. They are released during cascade
of destructive events of inflammatory
response. Paradoxically inflammatory response
which is essentially protective in design is
responsible for much of breakdown of the
periodontium.

59. References