immunity, Toll like receptors

1. Innate immunity in periodontal
diseases and Significance of
Toll- like receptors
Dr.T. Hudson Jonathan

2. Contents
• Introduction
• Classification
• Innate immunity
• Adaptive immunity
• Immune responses in
periodontal pathogenesis
• Pathogen recognition and
activation of cellular
innate response
• Toll-like receptors ligands
• Cell signalling pathways
• Positive edge of Toll like
receptors
• Negative edge of Toll like
receptors
• Clinical significance
• Conclusion

3. Introduction
• Immunity is defined as the capacity of the body to resist the
pathogenic agents. It is the ability to resist the entry of different
types of foreign bodies like bacteria, virus, toxic substances etc.
(Essentials of physiology – K S Sembulingam - 1st edition)

4. Person
becomes
immune as a
result of
primary
immune
response
Classificaton

5. Innate immunity
• Innate immunity is the inborn capacity of the body to
resists the pathogens.
• By chance if the organism enter the body, innate immunity
eliminates them before the development of any disease.

6. • This type of immunity represents the first line of defense against
any type of pathogens. Therefore it is also called non specific
immunity.
Cells of innate
immunity • Monocytes
• Macrophages
• PMNL’s
• Natural killer cells
• Dendritic cells
• Basophils
• Mast cells
• Eosinophils

7. Primary functions

8. Acquired immunity
• Acquired immunity is the resistant developed in the body
against any specific foreign body like bacteria, viruses,
toxins, vaccines, or transplanted tissues. So this type of
immunity is also known as the specific immunity.
• It is the most powerful immune mechanism that protects the
body from invading micro-organisms or toxic substances

10. • Innate immunity is required for the activation of a more specific
adaptive immune response, but it also plays an important role in
managing host microbial interactions

11. Immune responses in periodontal pathogenesis
• The immune system is essential for maintenance of periodontal
health and is central to the host response to periodontal
pathogens.
• However if the immune response is dysregulated,
inappropriate, persistent and/or excessive, then damaging
chronic inflammatory responses such as those observed in
periodontal disease can ensue. (Fraser ID et al 2009)

12. • Defenses against infection comprise a wide range of
mechanical, chemical and microbiologic barriers that prevent
pathogens invading the cells and tissues of the body.
• Saliva, GCF, and the epithelial keratinocytes of the oral
mucosa all protect the underlying tissues of the oral cavity and
in particular the periodontium.
• Recognition of pathogenic microorganism and recruitment of
effector cells (eg: neutrophils) and molecules (eg: the
complement system) are central to effective innate immunity.

13. • If innate immune responses, fail to eliminate infection (eg: in
susceptible host), then the effector cells of adaptive immune
response (lymphocytes) are activated.
Saliva:
• Saliva secreted from the three major salivary glands (parotid,
submandibular and sublingual) as well as from the numerous
minor salivary glands, has important role in maintaining oral
and dental health. (Carranza 11th ed)

14. • The action of shearing forces associated with saliva flow is
important in preventing the attachment of bacteria to the
dentition and the oral mucosal surfaces.
• Human saliva also contains numerous molecular components
that contribute to host defenses against bacterial colonization
and periodontal disease.

15. Carranza’s clinical periodontology – 11th edition

16. Epithelial tissues:
• The epithelial tissues play a key role in host defenses as they are
the main site of initial interaction between plaque bacteria and the
host and also are the site of invasion of microbial pathogens.
• The keratinized epithelium of the sulcular and gingival epithelial
tissues not only provides a protection for the underlying
periodontal tissue but also acts as a barrier against bacteria and
their products (Bartold PM et al 2000)

17. • In contrast, the unique microanatomic
structure of the junctional epithelium
has significant intercellular spaces,
and exhibits a higher cellular turnover
rate.
• These properties render the junctional
epithelium permeable, allowing for
inward movement of microbes and
their products and outward movement
of GCF and the cells and molecules of
innate immunity.

18. • Furthermore the spaces between the cells of the junctional
epithelium widen with inflammation, resulting in increased
GCF flow. (Schroeder HE et al 1997)
• Some species of periodontal bacteria invade host epithelial
tissues; at the molecular level the process of adhesion and
invasion are coupled.

19. Gingival crevicular fluid:
• GCF originates from the post capillary venules of the gingival
plexus. It has a flushing action in the gingival crevice but also
likely functions to bring the blood components (eg: neutrophils,
antibodies and complement components) of the host defenses
into the sulcus. (Griffiths GS 2003).
• The flow of GCF increases in inflammation and neutrophils are
an especially important element of GCF in health and disease.
(Kornman KS et al 1997).

20. Pathogen recognition and activation of
cellular innate response
• If plaque bacteria and their products penetrate the periodontal
tissues, then specialized “sentinel cells” of the immune system
can recognise their presence and signal protective immune
responses.
• Thus macrophages and dentritic cells express a range of Pattern
recognition receptor(PRRs) that interact with specific molecular
structures on microorganism called Microbe associated
molecular patterns (MAMPs) to signal immune responses.

21. • Thus innate immune responses are activated that provide
immediate protection and adaptive immunity is also activated
with the aim of establishing a sustained antigen-specific
defense.
• Excessive and inappropriate immune responses lead to chronic
inflammation and the concomitant tissue destruction
associated with periodontal disease. (Carranza 11th ed)
• The best studied of the signaling system involved in
recognition of plaque bacteria is the interaction of bacterial
LPS with TLRs.

22. BACTERIA
LPS
TLR
MYELOID IMMUNE CELLS
INTERACT
ACTIVATE
• However individual species of plaque bacteria have a wide variety of
MAMPs, which may interact with PRRs.
• Studies of P.gingivalis have served as a pattern for investigations of host
bacteria interactions in periodontal disease at molecular level.

23. • Thus p.gingivalis LPS signal via TLR (predominantly TLR-2)
and fimbriae, proteases and DNA from P.gingivalis are all
recognised by host cells through interaction with specific
PRRs.
• A number of non-immune cells in the periodontium (Epithelial
cells, fibroblasts) also express PRRs and may recognize and
respond to MAMPs from plaque bacteria.

25. Toll-like receptors ligands
• Bacterial plaque stimulates the host inflammatory response
leading to tissue damage .
• It is now known that the immune response applies a family of
pattern recognition receptors called Toll like receptors as a tool
to trigger an inflammatory response to microbial invasion
(Takeda K et al 2005).
• These data suggest the emerging role of Toll like receptors in
periodontitis.

27. • In 1991, the sequence of the cytoplasmic domain of the Toll
protein and the interleukin 1-receptor were reported to be
similar, which is consistent with involvement in immune
response (Gay NJ et 1991). This cytoplasmic domain is called
the Toll-IL-1 receptor domain.
• The discovery of mouse Toll like receptor 4 acting as a receptor
for Lipopolysaccharide linked Toll like receptor 4 to the innate
immune system. (Poltorak A et al 1998)

28. • Now it is clear that Toll like receptors function as key pattern
recognition receptors of the innate immune system. (Janeway
CA et al 2002)
• They recognize and distinguish highly conserved structures
present in large groups of micro-organisms. The structures are
referred to as Pathogen-associated molecular patterns.

29. • Examples of pathogen-associated molecular patterns are bacterial
lipopolysaccharide, peptidoglycan, lipoproteins, bacterial DNA,
and double-stranded RNA.
• In the innate immune system, Toll-like receptors sense invasion by
microorganisms such as bacteria, viruses, fungi, and protozoa, and
trigger immune responses to clear such pathogens.
• To date, 11 Toll-like receptors have been identified in human
periodontal tissues, and their expression, distribution and ligand
specificities have been characterized (Liu YC et al 2010).

32. Cell signalling pathways
• Upon interaction with pathogen-associated molecular patterns,
Toll-like receptors transmit this information through
intracellular signaling pathways, resulting in activation of
innate immune cells.
• The Toll-like receptor-mediated innate immune response is
also critical for the development and direction of the adaptive
immune system.

33. • The main distinction between the innate and adaptive immune
system lies in the receptors used for immune recognition.
• The antigen receptor of T and B cells are generated
somatically, whereas the innate pattern-recognition receptors
are encoded in the germ line (Akira S et al 2004).

34. • Historically innate immunity has been suggested to mediate
non-specific immune responses as a consequence of ingestion
and digestion of microorganism and foreign substances by
macrophages and neutrophils.
• However innate immunity is now recognized as showing
remarkable specificity by means of discriminating between the
host and pathogens through a sophisticated Toll-like receptor
based system.

35. • Although Toll like receptor and interleukin-1 receptor
cytoplasmic domains are homologous, the Toll like receptor
extracellular domains differ.
• The interleukin 1-receptor contain three immunoglobulin–like
domains, whereas the Toll-like receptor extracellular domains
are characterized by the frequency of leucine rich repeats.

36. • The number of leucine rich repeats in each of the 10 known
human Toll like receptors respond to distinctive pathogen
associated molecular patterns that characterize a microbial
infection.
• Specificity for pathogen associated molecular patterns is
provided by a relatively limited collection of Toll like receptors;
combination of Toll like receptors are generally required for
recognition of certain pathogen-associated molecular patterns
(Hajjar AM et al 2001)

37. • Upon ligand binding, Toll-like receptor-mediated signaling
activates signal transduction, leading to transcription of pro-
inflammatory cytokines that initiate innate immune responses
critical for the induction of adaptive immunity.
• Investigation of the Toll-like receptor signaling pathway is an
important step to understanding how Toll-like receptor
stimulation determines the outcome of immune responses.
• Toll-like receptor ligation initiates the interaction between Toll-
IL-1-receptor domains and cytoplasmic adaptor molecules.

38. • Toll-like receptor signaling cascades are separated into two
groups: the Myeloid differentiation primary response protein
88-dependent pathway and the Myeloid differentiation
primary-response protein 88- independent pathway.
• The Myeloid differentiation primary-response protein 88-
dependent pathway is essential for most Toll-like receptor
mediated cell activation.

39. • Myeloid differentiation primary-response protein 88 (MyD88), a
key adaptor molecule, is used by most Toll-like receptors. MyD88
mediates the Toll-like receptor-signaling pathway that activates
interleukin 1-receptor associated kinase.
• Interleukin 1-receptor-associated kinase then associates with
tumor-necrosis-factor receptor- associated factor 6, leading to the
activation of two distinct signaling pathways.
• One pathway leads to activation of activator protein-1 through
activation of mitogen-activated protein kinase.

40. • The other pathway activates the transforming growth factor-b-
activated kinase/transforming growth factor-b-activated kinase-1-
binding protein complex, which enhances activity of the inhibitor
of nuclear factor-kB kinase complex.
• Once activated, this complex phosphorylates and induces
subsequent degradation of the inhibitor of nuclear factor-kB and
releases nuclear factor-kB, which translocates into the nucleus
and induces expression of cytokines and chemokines (Akira S et
al 2001).

42. • A database search for molecules that are structurally related to
Myeloid differentiation primary-response protein 88 led to
identification of other adaptors including:
I. Toll-IL-1 receptor domain-containing adaptor protein/Myeloid
differentiation primary-response protein 88-adaptor-like
(TIRAP ⁄ MAL) (Fitzgerald KA et al 2001).
II. Toll-IL-1 receptor domain-containing adaptor inducing
interferon-b (TRIF) (Yamamoto m et al 2002).
III. TRIF-related adaptor molecules (TRAM) (Yamamoto M et al
2003).

43. • Current information suggests that specific Toll-like receptor-
mediated signaling pathways differentially select adaptors to
initiate the Myeloid differentiation primary-response protein 88-
dependent or Myeloid differentiation primary-response protein
88-independent pathway.
• A limited number of Toll-like receptors, acting through a
restricted portal of only four adaptors, might differentially signal
in response to a wide array of microbial products. As a result,
clusters of many genes are selectively regulated to control
needed immune processes (Akira S et al 2001).

44. Positive edge of Toll like receptors
• It has been suggested recently that the oral mucosa develops
tolerance after repeated exposure to bacterial products.[Muthukuru
M,et al 2005]. Down-regulation of TLR expression and inhibition
of intracellular signaling may be the underlying mechanisms of
tolerance.
• However, recent research has indicated that under steady-state
conditions, activation of TLRs by commensal bacteria is critical for
the maintenance of oral health.
• Gingival epithelial cells express TLR 2, 3, 4, 5, 6 and 9 and
recognize various microorganisms with the help of these
receptors.[Hirano H,et al 2004].

45. • These TLRs expressed on the gingival epithelium continually
interact with oral microorganisms that form biofilms on tooth
surfaces.
• This TLR signaling results in innate immune responses
involving the release of the antibacterial β-defensins cathelicidin
and calprotectin, as well as neutrophil chemoattractant (IL-8).
• Therefore, TLR signaling limits microbial invasion and prevents
commensal organisms from breaching the epithelial barrier,
thereby maintaining gingival health.

46. Negative edge of Toll like receptors
• When the epithelial barrier is breached, microorganisms and
their products access gain to the underlying connective
tissue and directly activate the cells present there.
• Once stimulated by PAMPs, gingival fibroblasts produce
pro-inflammatory cytokines leading to tissue destruction and
bone resorption.
• Periodontal ligament fibroblasts, on the other hand, produce
proteinases on TLR stimulation, resulting in direct
degradation of periodontal tissues.[Hatakeyama J,et al
2003]

47. CLINICAL SIGNIFICANCE
TLRs as potential biomarkers for CP:
• As TLRs play a pivotal role in periodontitis and abnormal
expression of TLRs can be observed in subjects with
periodontitis, TLRs have the potential to serve as diagnostic or
prognostic biomarkers for periodontitis.
• Saliva can serve as a diagnostic fluid in CP (Sahingur and
Cohen, 2004), and both TLR2 and TLR4 have been detected in
saliva.

48. • Prakasam et al (2014) found reduced soluble TLR2 (sTLR2) in
the saliva of CP subjects. After these subjects received scaling
and root planning, their sTLR2 levels increased.
• Buduneli et al (2011) revealed that salivary TLR4 was elevated
in a CP group relative to a control group. In addition, TLR2 and
TLR4 levels in the plasma of subjects with CP were markedly
higher than those of periodontally healthy subjects.
• Banu et al (2015) also showed that the levels of TLR4 and IL-18
in the plasma and saliva of CP patients were significantly higher
than those in healthy individuals.

49. • Measuring the TLRs levels in saliva or plasma to diagnose CP
and to determine prognosis might be simple and appropriate;
however, conclusions from various studies are not consistent.
• Detecting TLR levels in gingival crevicular fluid might be
another alternative. In short, TLRs are a potentially sensitive
biomarker for periodontitis that might be used to diagnose and
predict the course of periodontal disease.

50. TLRs in CP with systemic diseases
Association between RA and CP:
• Hao et al (2015) revealed that RA and periodontitis mouse
models with cathepsin K knockout demonstrated reduced
expression of TLR4, TLR5, and TLR9 and bone loss inhibition.
• Immune-associated cells and levels of proinflammatory
cytokines were decreased in both groups. These results indicated
that both RA and CP may result from a dysregulated immune
response and that the two diseases might share similar modes of
pathogenesis.

51. • Th17 cells as well as IL-17 were shown to be necessary mediators
of cartilage and bone damage (Kotake et al, 2012), and the
number of Th17 lymphocytes was significantly enhanced in RA
(van Hamburg et al, 2011; Samson et al, 2012).
• An in vitro study (de Aquino et al, 2014) showed that a lower
percentage of differentiated Th17 cells and IL-17 secretion
activated by Porphyromonas gingivalis and Prevotella nigrescens
were associated with TLR2, indicating that TLR2 might also be
involved in RA and that Th17 differentiation is TLR2 dependent.

52. Association between DM and CP:
• An association between DM and CP has been reported, and
DM is regarded as a risk factor for periodontitis (Kim et al,
2014; Soboku et al, 2014; Trivedi et al, 2014). Dysregulated
TLR expression might be involved in DM, which can
exacerbate CP.
• Sprague Dawley rats with DM showed higher expression
levels of TLR2 and TLR4 in gingival tissue. Furthermore, after
HGECs(human gingival epithelial cells) were treated with high
glucose, TLR4 mRNA levels were markedly enhanced.

53. • Then, when these high glucose-treated HGECs were
stimulated with LPS, IL-6, IL-8, and monocyte
chemoattractant protein- 1 (MCP-1) levels were increased.
• Promsudthi et al (2014) analyzed the TLR2 and TLR4 levels
in gingival tissue of CP patients and CP-free subjects with or
without DM by immunohistochemistry.

54. • Data indicated that TLR2 and TLR4 levels in the sulcular
epithelium and gingival connective tissue underneath the
sulcular epithelium in both CP and DM might be dysregulated.
• Rojo-Botello et al (2012) discovered that the levels of TLR2,
TLR4, and TLR9 were significantly higher in patients with CP
and DM than in patients with CP alone.

55. • Arce RM et al.2009, demonstrated increased TLR4
expression in a murine pregnancy model in response to oral
infection with the human periodontal pathogens
Campylobacter rectus and Porphyromonas gingivalis .
• Chaparro A et al.2013, proved an association in the placental
tissue between the presence of T. denticola and P. gingivalis
and preeclampsia leading to preterm labour and he also
observed increased TLR-2 expression.
TLR IN PERIODONTITIS AND PRE-TERM LABOUR

56. • These studies demonstrated that pathogens associated with
maternal periodontitis can stimulate placental inflammation
involving TLR 2 and 4 activation leading to down-stream of
molecular signalling that causes production of various
mediators of preterm births.

58. TLR blockade:
• A preliminary study in blockade of LPS - induced immune
responses by a Toll-like receptor 4 antagonist (TLR4A) which
would prevent elevations in amniotic fluid (AF) cytokines,
prostaglandins, and uterine contractility was conducted in
rhesus monkeys.
• They demonstrated that compared to saline controls, LPS
induced significant elevations in AF IL-8, TNF-α, PGE2,
PGF2α, and uterine contractility.

59. • In contrast, TLR4A pre-treatment inhibited LPS-induced
uterine activity and was associated with significantly lower AF
IL-8, TNF-α, PGE2, and PGF2α versus LPS alone
[Michalowicz BS et al 2013].
• Hence, the role of TLR in pathogenesis raises the possibility
that TLR blockade may become a critical component in
treating preterm labour induced by gram-negative bacteria.
• The blocking of TLR, the earliest step in the immune response
may down-regulate cytokine and prostaglandin synthesis
which is the cornerstone for infection-induced preterm labour.

61. Conclusion
• There are still gaps in our knowledge of the mechanism by
which TLRs maintain periodontal health and what leads to
bacterial immune evasion and disease progression.
• At present it is uncertain which specific signalling pathways
need to be blocked to attenuate the pathology or enhanced to
promote host defense, So further investigations are required in
this field to understand the initiation and progression of
periodontal disease

62. • A critical goal of the host is to develop a dynamic state of
health in which a continued bacterial challenge is countered
with an appropriate innate host response that leads to bacterial
clearance.
• Clearance of bacteria can occur due to the specifying within
the innate immune system that results from the consensus of
the variety of receptors and their specific ligands.

63. • Although the innate response is inherently rapid, multiple host
proteins are required and are equisitely invloved with these
interactions with microbial components, each host protein-
ligand complex possessing different specificities.

64. References
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Aug 2013 .Journal of Interdisciplinary Dentistry, Vol-3
Issue-2,

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66. Thank you