DENTAL PLAQUE IS A MICROBIAL BIOFILM

1. DENTAL PLAQUE IS A MICROBIAL BIOFILM • Microbial biofilm: a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. • Dr. Bill Costerton

2. Periopathogenic plaque (mostly gram negative anaerobes) Healthy plaque (mostly gram positive bacteria) Innate host response results in tissue protection Innate host response results in tissue and bone destruction There is a strong clinical correlation between the bacterial plaque composition and the innate host defense status Commensal and periopathogenic bacteria result in a significantly different innate host response

3. The oral microbial community is the best characterized microbial consortium associated with the human host. microbial shift disease Socransky (Forsyth) highly organized biofilm consortium (Kolenbrander, NIDCR)

4. Two major forms of periodontal disease gingivitis: inflammation that leads to redness and swelling of the periodontal tissue surrounding the tooth root surface reversible without professional treatment periodontitis: inflammation that leads to periodontal tissue destruction and alveolar bone loss, requires periopathogens and host susceptibility factors

5. GingivitisGingivitis is a milder and reversible form of periodontal disease that only affects the gums. It develops as toxins in plaque that irritate gums, making them red, tender, swollen, and likely to bleed easily. It can usually be eliminated by daily brushing, cleaning between your teeth, and regular dental cleanings.

6. Classification of gingival diseases • Dental plaque-induced gingival diseases • associated with dental plaque only • 2. hormonal changes associated gingivitis • puberty • pregnancy • 3. medication induced gingivitis (gingival overgrowth) • Phenytoin (taken to control seizures), • cyclosporine (taken by people who have had organ transplants) • nifedipine (taken to control blood pressure and heart rhythm abnormalities) • 4. malnutrition induced gingivitis • Vitamin C deficiency (scurvy); • Niacin deficiency (pellagra)

7. Classification of gingival diseases B. Non dental plaque-induced gingival lesions 1. specific bacterial origin (gonorrhoeae) 2. viral origin (herpes, HIV) 3. fungal origin (Thrush, candidiasis) 4. genetic origin (hereditary gingival fibromatosis) 5. gingival manifestations of systemic conditions (allergic reactions) 6. traumatic lesions

8. PeriodontitisGingivitis may lead to more serious, destructive forms of periodontal disease, called Periodontitis. There are several forms of periodontitis, with the most common being chronic adult periodontitis. The gum line recedes, which can expose the tooth's root. Exposed roots can become susceptible to decay and sensitive to cold and touch. The sulcus deepens into a pocket in the early stage of periodontal disease. Plaque that collects in these pockets can be difficult to remove during regular brushing and interdental cleaning. In some cases, so much ligament and bone are destroyed that the tooth becomes loose. Usually, your dentist can still treat the disease at this point.

9. Classification of Periodontal Diseases • Chronic periodontitis localized and generalized • Aggressive periodontitis localized and generalized • Periodontitis as a manifestation of systemic disease • (ex. neutropenia, Down’s syndrome) • Necrotizing periodontal diseases • necrotizing ulcerative gingivitis (NUG), necrotizing ulcerative periodontitis (NUP) • (trench mouth fusiform-spirochete variety) 1-week course of Metronidazole • Abscesses of the periodontium • gingival, periodontal

10. Site specific consortium patterns will be defined by microbial biogeography Questions: Site specific recolonization patterns Frequency of specific bacteria at different sites (F. nucleatum on molars) Adult type chronic periodontitis is a highly localized disease Horizontal and vertical transmission Reservoirs Correlations to health and disease Nature Reviews Microbiology 4, 102-112 (February 2006) | Microbial biogeography: putting microorganisms on the map

11. Bacterial factors associated with periodontitis sufficient numbers of bacteria are present to exceed disease threshold (ie 108 bacteria/pocket) periopathogens are of a virulent clonal type Invasion: P. gingivalis can invade epithelial cells and gain access to the connective tissue of the periodontium Toxins: Aa produces a leukotoxin that kills neutrophils strong correlation To disease Enzymes: P. gingivalis produces a protease that degrades host tissues including antibodies and other host defense proteins Toxic products: gram negative bacteria release LPS that can induce bone resorption

12. Localized juvenile aggressive periodontitis (LJP) There is good evidence for a bacterial etiological agent: Aggregatibacter actinomycetemcomitans strain JP-2 A.a. strainJP-2 has a deletion in the leukotoxin promoter region which results in high levels of leukotoxin secretion. Leukotoxin lyses neutrophils Longitudinal 2 year study examining Jp-2 colonization and susceptibility to disease JP-2 is endemic in Morocco and African Americans

13. Risk Factors for Periodontitis People who smoke or chew tobacco are more likely to have periodontal disease. And it's more likely to be more severe than in those who do not use any tobacco products. Some systemic diseases, such as diabetes, can lower your body's resistance to infection, making periodontal diseases more severe Many medications, such as steroids, some types of anti-epilepsy drugs, cancer therapy drugs, some calcium channel blockers, and oral contraceptives can affect the gums. In addition, medications that reduce your salivary flow can result in a chronically dry mouth, which can irritate your oral soft tissues. Let your dentist know about your medications and update your medical history files at the dental office when any changes occur Bridges that no longer fit properly, crooked teeth or fillings that have become defective can contribute to plaque retention and increase the risk of developing periodontal disease. Pregnancy or use of oral contraceptives increases hormone levels that can cause gum tissue to be more sensitive to the toxins and enzymes produced by plaque and can accelerate growth of some bacteria. The gums are more likely to become red, tender and swollen, and bleed easily Heredity. Sometimes you may do everything right and still develop periodontal disease. In that case, you — along with close to one-third of the population — may have inherited a predisposition to gum problems.

14. There is a characteristic shift in the microbial population from mostly gram positive bacteria in supra-gingival plaque to mostly gram negative bacteria in sub-gingival plaque supra-gingival plaque sub-gingival plaque Streptococcus sanguis Fusobacterium nucleatum Streptococcus mitis Eikenella corrodens Porphyromonas gingivalis Actinomyces naeslundii Actinomyces viscosus Prevotella intermedia

15. Health: Gingivitis Periodontitis Streptococcus sanguis Actinomyces species Porphyromonas gingivalis Bacteroides forsythus Streptococcus mitis Streptococcus species Veillonella species Treponema dentacola Veillonella parvula Fusobacterium species Actinomyeces naeslundii Prevotella intermedia Actinomyces viscosus Rothia dentocariosa Bacterial species associated with different periodontal clinical states

16. Periodontal tissue is highly vascularized giving the tissue access to serum soluble and cellular components of the innate host defense system

17. Paradigm for bacterial interactions with the periodontium Bacteria live on the tooth and tooth root surface and release components that interact with host tissue (PRR ligands) Composition of bacterial polymicrobial community contributes to the type of host tissue responses (PRR)

18. Non-specific* Minutes to hours Receptors are germline origin Complement Inflammation Specific 2-3 days Receptors are generated randomly with clonal expansion T, B, and dendritic cells Antibody and cellular immunity Innate Immunity Adaptive Immunity “buys time” for and Initiates adaptive immunity

19. Lipopolysaccharide is a potent stimulator of the host inflammatory response • Over 200 years ago spontaneous remission of “neoplasms” was found to be associated with acute infection • The discovery that infections were caused by bacteriainitiated therapeutic studies with bacterial fractions and led to the discovery of endotoxin and “hemorrhagic necrosis” of tumors • Lipopolysaccharide from Gram-negative bacteria was found to be the active anti-tumor component and led to the discovery of the potent cytokine TNF-a • The complete chemical synthesis of E. coli lipid A in 1985 confirmed LPS as a peotent activator of host defenses

20. The pattern recognition hypothesis provides a conceptual framework to understand host responses to LPS

21. Characteristics of Pattern Recognition Receptor Ligands • Shared by large group of pathogens • Conserved and essential structures, attempts to modify result in death of microorganism or loss of virulence • Distinct from self antigens The immune system evolved to discriminate infectious nonself from noninfectious self. Charles A. Janeway, Jr., Immunology Today 13:11-16, 1992.

22. The host does not recognize bacteria; it records bacterial colonization and movement by their structures.

23. Bacterial Recognition by the Innate Host Response System • Lipopolysaccharide (LPS) • Lipoteichoic Acid (LTA) • Peptidoglycan

24. GRAM POSITIVE CELL ENVELOPE secreted exoenzymes to perform extracellular digestion Lysozyme Lipoteichoic acid Teichoic acid Cytoplasmic membrane Cytoplasm

25. GRAM NEGATIVE CELL ENVELOPE Lysozyme Lipopolysaccharide Porin Outer Membrane (Braun) Lipoprotein Periplasmic space p e p t i d o g l y c a n degradative enzyme Inner (cytoplasmic) membrane Cytoplasm

26. Structure of Lipopolysaccharide The ENDOTOXIN vary conserved different bacteria have different lipid A structures

27. Pattern Recognition Receptors Functions: opsonization activation of complement and coagulation cascades phagocytosis activation of inflammation induction of apoptosis

28. Examples of pattern recognition receptors serum soluble C-reactive protein (CRP) CRP and SAP bind phosphorylcholine on bacteria and act as opsonins, also bind C1q to activate complement serum amyloid protein (SAP) MBL is member of the collectin family, has a C type lectin domain, binds mannose residues and activates complement by the alternate pathway mannan-binding lectin (MBL) lipopolysaccharide binding protein (LBP) LBP and sCD14 shuttle LPS and other microbial and host components to activation and neutralization pathways sCD14

29. Examples of pattern recognition receptors cell associated MMR has a C type lectin domain, binds mannose on microorganisms and mediates phagocytosis Macrophage mannose receptor (MMR) Macrophage scavenger receptor (MSR) MSR binds polyanionic ligands (RNA, LPS, LTA), mediates phagocytosis mCD14 mCD14 and TLR’s function in a complex to deliver activating signal to cells in response to binding microbial components Toll like receptors (TLR)

30. LPS interactions with TLR4 Host cells respond to LPS

31. TLR -2 TLR -4 TLR -5 TLR -9 p e pti d o g ly ca n E . c ol i ty p e L PS fl a g e lli n b a c t e r i a l DNA b a c t e r i a l P. g i ng i v a li s L PS li p op ro te i n s li p ot e i c h o ic a c id diff ere nt c o m bi n ati o n s o f TLRs m a y au g m e nt o r i n hib i t r e s p o n se s to o th er b a c t e r i a l c o m p o n e nt s Examples of Bacterial Components “recognized” by the Toll Like Receptor component of the innate host defense system

32. TLRs recognize molecular pattern associated with bacterial pathogens. TIR domain-containing adaptors define the specificity of TLR signaling.

33. LPS peptidoglycan lipoproteins LBP sCD14 cell membrane TLR6 or TLR1 TLR2 TLR4 TLR5 TLR9 MD-2 MyD88 Cytoplasm IRAK TRAF6 The type of bacterial component will influence the type of response. MKK p38 NF-kB AP-1 NF-kB Nucleus Inflammatory mediators

34. inflammatory cytokines cell adhesion molecules chemokines antimicrobial peptides costimulatory molecules MHC molecules Toll Signaling Pathways Activate:

35. How may commensal bacteria influence the periodontal innate host response ? Two approaches are being employed: Immunohistochemical analysis of healthy tissue Studies in germ free animals

36. Immunohistochemical analysis of clinically healthy periodontal tissue mCD14 LBP Co-expression of LBP and mCD14 peptides in gingival tissues. LBP expression is confined to the cytoplasm in gingival epithelium mCD14 is mainly confined to the cells in the epithelium-connective tissue interface. Dr. Jin University of Hong Kong

37. Expression of TLR 2 and TLR 4 in healthy and diseased periodontal tissue chronic periodontitis TLR 2 expresses in both gingival epithelium and connective tissues Negative control chronic periodontitis TLR 2 is found in gingival epithelium chronic periodontitis TLR 2 is found in connective tissue clinically healthy tissue TLR 2 is found in the gingival epithelium clinically healthy tissue TLR 2 is found in the gingival epithelium clinically healthy tissue No TLR 4 is found chronic periodontitis TLR 4 is found in connective tissue

38. Healthy Tissue Diseased Tissue sCD14 + + + + + + mCD14 + + + + + + LBP* + + + + + + IL-1b + + + + + + + + + + + + One contribution of commensal oral flora to innate immunity may be to induce innate defense detection mechanisms Detection Amplification TNFa + + + + + + TLR 2 & 4 Immunohistochemical and in situ analysis of diseased and healthy human periodontal tissue. Laboratory of Dr. Jin Li Jian, Hong Kong University

39. The innate host response can discriminate between commensals and pathogens by recognition of different microbial patterns Pattern recognition specificity due to a host and microbial regulated dialogue LBP, CD14, and TLR microbial ligand binding affinities TLR 1-9, different activation pathways with different microbial components TLR expression patterns epithelial, endothelial, monocyte Ex. epithelial cells do not have TLR 4

40. Germ free mice contain No bacteria Keeping germ-free mice in an isolator. germ free mice are born by sterile Caesarean section breed in a sterile isolator with sterile food, water and bedding . It is important to note that germ-free animals have no bacteria in the intestine or on other body surfaces, whereas specific-pathogen-free (SPF) mice are only devoid of known mouse pathogens and do contain intestinal bacteria.

41. Germ free mice have been employed to determine effects of commensal colonization of the intestine Early studies in germ-free mice demonstrated that: commensal bacteria have a direct impact on the morphology of the intestine (the villi of the small intestine are longer and the crypts are shorter and contain fewer cells in germ-free animals) commensal bacteria have a direct impact on the development and function of the intestinal immune system (commensal bacteria are required for the complete development of Peyer’s patches, the lamina propria, and the intraepithelial spaces, all three of the main immune elements found in the intestine) commensal bacteria are responsible for degradation of mucus glycoproteins commensal bacteria have effects on intestinal motility

42. Rapid microbial induction of angiogenesis in small intestinal villi of adult ex-germ-free mice A, B, and C are confocal scans of capillary network present in the upper third of small intestinal villi; D is the quantitation of villus capillary network density Thaddeus S. Stappenbeck, Lora V. Hooper, and Jeffrey I. Gordon* 2002 Proceedings National Academy of Science: 15451–15455

43. Do commensal bacteria actively interact with the innate host defense system? A study examining innate host defense mediator expression in germ free and conventionally reared mice was performed mouse gingival tissue was removed for RT-PCR analysis Dixon, D., Reife, R., Cebra, J., and Darveau, R. 2004 Commensal bacteria influence innate status within gingival tissues: A pilot study J. of Periodontology 75(11):1486-1492

44. Germ free mice display significantly lower IL-1b protein, consistent with the innate host response system responding to commensal bacteria.

45. Expression of IL-1β in periodontal ligament cells. a): 10×; b) and c): 40×. Immunohistochemical analysis confirms IL-1b expression in clinically healthy mouse periodontal tissue IL-1b was expressed in periodontal fibroblasts We suspect IL-1b may represent an global “indirect regulator of innate defense mediator expression

46. Summary of Microbial Colonization and Innate Defense Status Dental plaque is a highly organized site specific consortium of microbial species that evolved to live with each other and the host in the oral cavity There is a strong correlation between the type of microbial consortiums found in sub gingival plaque and the innate defense mediator expression In both clinically healthy and diseased periodontal tissue Germ free mice can be employed to identify innate host defense mediators that are expressed in response to commensal colonization compared to those that are developmentally expressed by the host.

47. Pathogens disrupt commensal bacterial interactions subverting normal innate host responses invade and gain access to protected compartments alter existing commensal / host innate defense dialogue

49. P. gingivalis:invades gingival epithelial and microvascular endothelial cells, produces proteases including hemolysins, induces bone loss in animal models of infection