Innate Immunity DENT 516 by dfhdhdhdhjr


									Innate Immunity of the oral

   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
•   Epithelial cell derived antimicrobial peptides
    – “good bug” vs “bad bug”
Oral Mucosal
           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
         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
      sugar alcohol phosphate polymers


              Christiane Nüsslein-Volhard,
              Nobel Laureate (Medicine, 1995)

Toll mutant
Lemaitre, B. 1996. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 86:973.
Host responses to infection
Innate vs Adaptive Immunity
              Oral Mucosal Strategy
phase           Submandibular



Salivary constituents and their functions

    defensins                defensins

FACTOR                   EFFECT

Antibodies               Bind bacteria, Neutralize and inactivate viral particles

                         Perform general antimicrobial activity; inhibit cysteine
Cystatins                    proteases

Defensins                Perform general antimicrobial activity; carry out charge
                             mediated disruption of bacterial membranes

Histatins                antifungal

Lactoferrin              Binds iron to inhibit bacterial growth

Lysozyme                 Lyses bacteria

Mucins                   Entrap and aggregate microbial particles

Proline-Rich Peptides    Bind bacteria

Statherin                Binds bacteria
• 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
Characteristic/Function              MG1               MG2
Molecular weight (daltons)           >106              2-2.5x105
Protein (composition)                14.9%             30.4%
Carbohydrate (composition)           78%               68%
   Size of oligosaccharides          4-17 residues     2-7 residues
   Number of oligosaccharides        46                170

   Number of sialic acid units       14                67
Sulfate                              7%                1.6%
Fatty acids                          Yes               negligible
Subunits                             Multiple SS-linked 1
Conc. in submand/submax saliva       6-8 mg/L          16-18 mg/L
Proposed protective function         Pellicle          Bacterial
  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
    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)
• Pellicle important for colonization of first line
  of bacteria; “good guys”
    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)
           Serous secretions

• Provided by the parotid gland
• Contain innate immune factors
  – Lysozyme, histatins, salivary peroxidase,
    lactoferrin, cystatins, PRPs, calprotectin
• Contain adaptive immune factors
  – sIgA
• 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)
            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)
                               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)
                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
    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
• 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.
                      Mouse model of oral

Normal mouse tongue

     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)
• 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
Paul Kolenbrander
• 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)
*Why is the mouth so healthy in spite of
constant trauma occurring in a very septic

Michael Zasloff   Magainin

Host Defense Peptides
              Oral Mucosal Strategy
                      S. sanguis antagonizes A.a. and competes with C.a. for
phase                 biotin


  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)
             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     (Hancock, Lancet, 1997)

   - form pores in bacterial membrane         C        C        C           C    CC

• Cross-talk with adaptive immunity
Gram positive rod and hBD-3
 Defensins in innate and adaptive immunity

                        Ganz, Science 286:420, 1999
                        Yang et al, Science 286:525, 1999
- 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,

• 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,

• hBD3 induces co-stimulatory molecule expression in human
  monocytes/mDCs via TLR1/2 (Funderberg et al, 2007)
IMF of hBD-2 and hBD-3 in human
         oral epithelium

                          Ge Jin, CWRU
                         normal   carcinoma in situ



          H&E staining


          B. Dale-Crunk, U. Washington
*Why is the mouth so healthy in spite of
constant trauma occurring in a very septic

*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.
Homo sapiens or Homo

    1013 eukaryotic cells

     1014 bacteria 

     Henderson and Wilson, JDR, 1998
     Fusobacterium nucleatum
• 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
         F. nucleatum vs P. gingivalis
           induction of  defensins

microbial challenge   RT-PCR
                                   Fn    Pg
F. nucleatum induction of hBD-3 in NHOECs

       hBD3 mRNA (Fold Increase)
                                   120                                                         - FN
                                   100                                                         + FN
                                    80                                                         + PMA
                                                   2 hr

                                                              4 hr

                                                                       8 hr

                                                                                       48 hr
                                         0 hr

                                                                              24 hr
      B.                                                  Collection Time

                    -                    2       4        8          24 48
                                                                     24 48      +
       at 48h     at 48h                                                      at 48h
                                                                              at 48h
                                                + 10 g Fn cell wall                   rhBD3
Can the “good guy” protect
 us from the “bad guy”
Are these “good guy” bacteria
sensitive to  defensins
                                 F. nucleatum and P. gingivalis sensitivity to recombinant hBDs

                                                      Pg 33277                                         Pg W50                                                       Pg 49417
                                 120                                                 120                                                120

                                                                                                                     % live organisms
              % live organisms

                                                                      % live organisms
                                 100                                                 100                                                100
                                     80                                                  80                                                 80
                                     60                                                  60                                                 60
                                     40                                                  40                                                 40
                                     20                                                  20                                                 20
                                     0                                                   0                                                      0
                                              0   1      5       10                            0   1    2   5   10                                      0       1        2       5       10
                                          recombinant hBD (uM)                                recombinant hBD (uM)                                      recombinant hBD (uM)

                                                  Fn 25586                                          Fn 11326                                                            Fn 49256
                   140                                                                                                                          120
% live organisms

                                                                                                                             % live organisms
                                                                      % live organisms

                   120                                                                                                                          100
                   100                                                                                                                           80
                                                                                  60                                                             60
                                                                                  40                                                             40
                                                                                  20                                                             20

                                                                                   0                                                                0
                                          0       1      5   10                                0   1    2   5   10                                          0       1        2       5    10
                                      recombinant hBD (uM)                                    recombinant hBD (uM)
                                                                                                                                                        recombinant hBD (uM)

2 X 105 bacteria incubated with rhBD-1 , -2        , or -3   , anaerobically, 3 h, followed by
serial dilutions and plating on sheep red blood agar plates.
Immunogold TEM of P. gingivalis after rhBD-2 incubation

Immunogold TEM of F. nucleatum after rhBD-2 incubation

Immunogold TEM of F. nucleatum after rhBD-2 incubation

     F. nucleatum vs P. gingivalis
• F. nucleatum induces hBD2 and hBD3 in NHOECs and
protects cells from P. gingivalis invasion
• P. gingivalis does not induce hBD expression in NHOECs

• F. nucleatum is resistant to -defensins ; P. gingivalis is
• F. nucleatum resistance may be due to “fimbrial" extensions
that sequester the hBD away from the outer membrane
• F. nucleatum factor(s) isolated from the cell wall
promotes  defensin expression; 4 candidate peptides;
12-13 kDa, pI 4-5

To top