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Innate Immunity of the oral cavity

Innate Immunity of the oral cavity. Dr. Aaron Weinberg DMD, PhD Department of Biological Sciences. Outline of Lecture. Innate vs adaptive immunity Oral mucosal strategy Mucin Lysozyme Lactoferrin Salivary peroxidase Histatins PRPs Statherin Cystatins

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Innate Immunity of the oral cavity

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  1. Innate Immunity of the oral cavity Dr. Aaron Weinberg DMD, PhD Department of Biological Sciences

  2. Outline of Lecture • Innate vs adaptive immunity • Oral mucosal strategy • Mucin • Lysozyme • Lactoferrin • Salivary peroxidase • Histatins • PRPs • Statherin • Cystatins • Epithelial cell derived antimicrobial peptides • “good bug” vs “bad bug”

  3. Oral Mucosal Wounds

  4. Innate Immunity • Evolutionarily ancient and conserved •Body’s first line of defense against infection • Mechanical barriers at body surfaces •Nonspecific/ no memory • Rapid response • Antibacterial substances in secretions -lysozyme & lactoferrin -low pH of stomach contents • Alternate Complement Pathway

  5. Epithelial Innate Immunity • Recognition of bacteria • PAMPs • Lipopolysaccharide, peptidoglycan, lipoteichoic acid, flagella, mannans, bacterial DNA, glucans • Toll-like Receptors (TLRs) • TLR-2: peptidoglycan • TLR-4: lipopolysaccharide • TLR-9: bacterial DNA • TLR-5: bacterial flagella • Homodimers/Heterodimers • Intracellular signaling • NF-kB signal Inler and Hoffman, Trends Cell Biol, 2001

  6. sugar alcohol phosphate polymers sugar alcohol phosphate polymers NAM-NAG kdo

  7. Christiane Nüsslein-Volhard, Nobel Laureate (Medicine, 1995) Toll mutant

  8. Lemaitre, B. et.al. 1996. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 86:973.

  9. Host responses to infection

  10. Innate vs Adaptive Immunity

  11. Oral Mucosal Strategy fluid phase defense static phase defense recruitable defense Parotid Submandibular Sublingual EAPs

  12. Salivary constituents and their functions defensins defensins Mucins defensins

  13. Mucins • Important in formation of lubricating biofilm to protect underlying mucosa and tooth surfaces from chemical and physical harm • Important for physiological processes: speech, swallowing, clearance of microbes • Glycoproteins, 30-90% carbohydrate w/serine or threonine-galactosylamine glycopeptide linkage • Two major salivary mucins: • MG1 (from mucous acini of seromucous salivary glands) • MG2 (from serous cells of seromucous salivary glands + parotid gland) • Distinguished by differences in size, carbohydrate content, sulfation (and charge), sialic acid, subunit structure (see Table) •  size: MG1 (MUC5B) > MG2 (MUC7) •  carbohydrates, sulfation:MG1 > MG2 •  MG1 bears ABH and Lewis blood group antigens for microbial adherence

  14. Mucins

  15. Mucins and mucosal surfaces • MG1 is tightly associated with mucosa • Serves as barrier against toxins, hydrolytic enzymes, acids, carcinogens • Traps various host defense factors, providing high concentrations of these factors near surface • sIgA concentrated w/i mucin layer overlying epithelium • In solution, MG1 forms complexes with various proteins: amylase, PRPs, statherin, histatins

  16. Mucins and dental surfaces • Mucins play important role in generation of the dental pellicle • “Pellicle” 1-2 m layer containing lipids + salivary proteins/glycoproteins: albumin, lysozyme, PRPs, lactoferrin, statherin, bacterial debri etc.. • MG1 covers outer layers of pellicle while MG2 more inside(Kajisa et al, 1990) • Pellicleimportant for colonization of first line of bacteria; “good guys”

  17. Mucins and fluid phase defenses • Mucins exert several antimicrobial activities in fluid phase. • MG2 prevents bacterial colonization of the pellicle coated-tooth by presenting identical surface carbohydrates in the fluid phase (Levine et al, 1985: Reddy et al, 1993) • MG1 believed to protect mucosa by preventing viral infections; ex. HIV and herpes simplex (Mandel and Ellison, 1985)

  18. Serous secretions • Provided by the parotid gland • Contain innate immune factors • Lysozyme, histatins, salivary peroxidase, lactoferrin, cystatins, PRPs, calprotectin • Contain adaptive immune factors • sIgA

  19. Lysozyme • Muramidase, N-acetylmuramide glycanohydrolase • Encoded on chromosome 12 • Cationic protein • Mol wt. 14.3 kD • Produced by myeloid cells and glandular epithelium • Parotid saliva: up to 10 mg/L (unstimulated); down to 1 mg/L (stimulated) • Activity: cleaves -1,4 linkage between NAM-NAG in bacterial cell wall peptidoglycan. • Protein contains deep groove capable of binding 6 sugar units of the NAM-NAG structure causing the backbone structure to snap. • Due to its charge, has some non-enzymatic microbicidal activity against bacteria and oral fungi (Laible and Germaine, 1985; Tobji et al, 1988)

  20. Lactoferrin (lactotransferrin) • LF iron chelating glycoprotein • 10-20 mg/L saliva; 1 g/L in milk • Produced by neutrophils (not other myeloid cells) and glandular epithelium • Encoded on chromosome 3 • LF single polypeptide; MW 80 kD; 2 homologous domains that each binds one Fe+2 ion • Activity: blocks growth of iron dependent organisms (Ca, Pg) • Apolactoferrin (iron-less) can kill certain oral bacteria (S. mutans, A.a.) by binding to metal and destabilizing outer membrane (conjecture; Ellison et al, 1988) • “Lactoferricin” microbicidal peptide domain released from LF by pepsin (gastric hydrolase) (Yamauchi et al, 1993)

  21. Salivary peroxidase • 78 kD enzyme • Produced by salivary gland epithelium • Catalyzes reduction of H2O2 to H2O and oxidation of electron donor • Main donor is thiocyanate (SCN-), halide ion, 1-2 mM in saliva; • H2O2 + SCN- H2O + OSCN- (hypothiocyanite) • Activity: • potentiates antimicrobial activity of fluoride against S. mutans(Lenander-Lumikari et al, 1997) • potentiates activities of lysozyme and lactoferrin • may function against H2O2 generating bacteria (Thomas et al, 1983) • neutralizes H2O2 along the mucosa (antioxidant effect) • H2O2 oral releasing bacteria can induce ulceration of the mucosa (conjecture) • Acatalasemia (deficiency in catalase; enzyme catalyzes H2O2) has been associated with extensive ulceration of oral tissues • Strep. H2O2 release is strain dependent; 0-165 nmol/min/mg bacterial protein in presence of glucose (Miyasaki et al, 1988) • Tissue destructive effects of H2O2 is probably indirect, requiring further reduction of H2O2 to •OH ? aphthous ulcerations respond to salivary peroxidase enhancement therapy; clinical study, 45 of 64 aphthous patients reported symptomatic relief; ? Oral strep connection??(Hoogendoorn and Piessens, 1987)

  22. Histatins (HRPs) • Basic, histidine rich, -helical peptides (7-38 aa) • Up to 12 different HRPs • Produced by salivary gland epithelium • Found in parotid and submandibular secretions • 50-425 g/ml saliva (Edgerton et al, 1998) • Strong anticandidal peptides;some anti- S. mutans and anti-P. gingivalis activity • 2 families of HRPs based on sequence analysis

  23. Antifungal activity of Histatins • Most important antifungal agents in saliva • Anti-histatin immunoaffinity adsorption of saliva removes candidacidal activity • Decrease in salivary histatins associated with increased incidence of candidiasis secondary to HIV (Mandel et al, 1992) • Topical histatins have been shown to prevent denture stomatitis (DS) • Dentures cover palate and prevent access of parotid saliva • DS presents as a superficial fungal problem; little fungus is found in mucosa •  Clinical observations reveal the significance of histatins in preventing superficial oral candidiasis.

  24. Mouse model of oral candidiasis Normal mouse tongue Candida/hyphae

  25. Proline-rich proteins (PRPs) • Acidic, with 25-40% proline content • 150-170 aa • Multigene complex on chromosome 12 • Multifunctional • In solution PRPs maintain salivary calcium phosphate in a supersaturated state (Gibbons and Hay, 1988) • Are a significant fraction of the acquired pellicle; important in dictating microbial attachment. • Pleomorphism • Some variants may be associated with greater susceptibility to dental caries • Bacteroides thetaiotomicron in mice (Jeff Gordon, et al)

  26. Statherin • 43 Kd phosphoprotein • Encoded on chromosome 4 • Like PRPs, maintains salivary calcium phosphate in a supersaturated state • Found in acquired pellicle • Involved in microbial attachment • May be evolutionarily related to the histatins

  27. Paul Kolenbrander

  28. Cystatins • Inhibitors of cysteine proteinases; widely distributed in tissues • MW ~14 kD, 120 aa, chromosome 20 (family 2), chromosome 3 (family 1, 3); derived from submandibular secretions • Most common in saliva are family 2 cystatins: S, (pI, 4.7), SA (pI, 4.3), SN (neutral), and C (pI, 7.5) • Myeloid cells are source of cystatin C • Cystatin C appears to increase in saliva from periodontitis and gingivitis cases • Importance: • neutralizing against microbial-derived cysteine proteinases • cystatin SN may exert an anti-adhesive effect by binding to bacterial pili (Reddy, 1998)

  29. Question *Why is the mouth so healthy in spite of constant trauma occurring in a very septic environment? Michael Zasloff Magainin Host Defense Peptides

  30. Oral Mucosal Strategy fluid phase defense static phase defense recruitable defense S. sanguis antagonizes A.a. and competes with C.a. for biotin EAPs

  31. Host defense peptides in humans • Adrenomedullin:52 aa vasoactive, cationic antibacterial peptide(Allakar RP, Kapas S, ’99, ’01, ’03) • Calprotectin:Two-subunit protein (, MRP8; , MRP18), anionic(Eversole, ’93; Ross and Herzberg, ’01) • SLPI: 12kDa non-glycosylated protein(Wahl, ’97; Shugars, ’97) • LL37: cathelicidin; PMNs, lymphocytes, macrophages, some epithelial cells (Lehrer and Ganz, ’02) • Human -defensins:beta sheeted; PMNS, Paneth cells(Kagan et al, 1994; Ouellette and Selsted, ‘96) • Human -defensins:beta sheeted; epithelial cells (Weinberg et al, ’98; Zasloff, ’02; Krisanaprakornkit et al, ’98, ’00; Quinones et al, ’03; Feng et al, ’05; Feng et al, ’06)

  32. Human -Defensins Produced by epithelial cells • Cationic, amphipathic peptides - hBD1, constitutive - hBD2, inducible - hBD3, inducible - hBD4, inducible ? - antibacterial, antifungal, antiviral • Mechanism of action anionic targets: LPS, LTA, phospholipids (phosphatidylglycerol) form pores in bacterial membrane • Cross-talk with adaptive immunity ++ ++ + + ++ ++ C C C C C C (Hancock, Lancet, 1997)

  33. Gram positive rod and hBD-3

  34. Defensins in innate and adaptive immunity_______________________________________ Ganz, Science 286:420, 1999 Yang et al, Science 286:525, 1999

  35. -defensins in innate and adaptive immunity • - defensins recruit iDCs and T cells via CCR6(Yang et al, 1999) • -defensins promote maturation of dendritic cells via TLR4(Biragyn et al, 2002) • Recruitment of monocytes is hBD3 isoform dependent(Wu et al, 2003) • hBD2 is chemotactic towards human neutrophils via CCR6(Niyonsaba et a, 2004) • hBD2 and -3 interact with CXCR4(Quinones-Mateu et al, 2003; Feng et al, 2006) • hBD3 induces co-stimulatory molecule expression in human monocytes/mDCsvia TLR1/2(Funderberg et al, 2007)

  36. IMF ofhBD-2andhBD-3in human oral epithelium Ge Jin, CWRU

  37. carcinoma in situ normal hBD-2 hBD-2/hBD-3 hBD-3 nucleus H&E staining

  38. Gingiva Skin B. Dale-Crunk, U. Washington

  39. Questions *Why is the mouth so healthy in spite of constant trauma occurring in a very septic environment? *Why are hBDs constitutively expressed in oral mucosa? *The answers may lie with some of the ubiquitous bacteria of the mouth that induce epithelial cell derived antimicrobial peptides.

  40. Homo sapiens or Homo bacteriens? 1013 eukaryotic cells 1014 bacteria  Henderson and Wilson, JDR, 1998

  41. Fusobacteriumnucleatum • gram negative fusiform • found in healthy sites • involved in dental plaque formation Porphyromonas gingivalis • black pigmented gram negative rod • found in diseased sites • associated with periodontal disease

  42. microbial challenge hBD-2 - Fn Pg F. nucleatum vs P. gingivalis induction of  defensins RT-PCR

  43. A. A. 200 200 200 180 180 180 160 160 160 140 140 140 - - - FN FN FN 120 120 120 100 100 100 + FN + FN + FN hBD3 mRNA (Fold Increase) hBD3 mRNA (Fold Increase) hBD3 mRNA (Fold Increase) 80 80 80 + PMA + PMA + PMA 60 60 60 40 40 40 20 20 20 0 0 0 2 hr 4 hr 8 hr 2 hr 2 hr 4 hr 4 hr 8 hr 8 hr 0 hr 0 hr 0 hr 48 hr 48 hr 48 hr 24 hr 24 hr 24 hr B. B. Collection Time Collection Time Collection Time - - - 2 2 2 4 4 4 8 8 8 24 24 24 48 48 48 + + + at 48h at 48h at 48h at 48h at 48h at 48h + + + m m m 10 10 10 g Fn cell wall g Fn cell wall g Fn cell wall rhBD3 rhBD3 rhBD3 F. nucleatuminduction of hBD-3 in NHOECs

  44. Can the “good guy” protect us from the “bad guy”

  45. Are these “good guy” bacteria sensitive to  defensins

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