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• Epithelial cell derived antimicrobial peptides

– “good bug” vs “bad bug”

Oral Mucosal Wounds

Innate Immunity

• 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

• Evolutionarily ancient and conserved

• 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

kdo

sugar alcohol phosphate polymers

NAM-NAG

sugar alcohol phosphate polymers

Toll mutant

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

Lemaitre, B. et.al. 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

fluid

phase

defense

static

phase

defense

recruitable

defense

EAPs

Parotid

Submandibular

Sublingual

Salivary constituents and their functions

defensins

defensins

defensinsMucins

SALIVARY FACTORS WITH ANTI-MICROBIAL ACTIVITY

FACTOR EFFECT

Antibodies Bind bacteria, Neutralize and inactivate viral particles

CystatinsPerform general antimicrobial activity; inhibit cysteine

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

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

MucinsCharacteristic/Function MG1 MG2Molecular 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 clearance

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

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)

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 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.

Mouse model of oral candidiasis

Normal mouse tongue

Candida/hyphae

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)

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

Paul Kolenbrander

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)

Question

*Why is the mouth so healthy in spite of constant trauma occurring in a very septic environment?

Michael Zasloff Magainin

Host Defense Peptides

Oral Mucosal Strategy

fluid

phase

defense

static

phase

defense

recruitable

defense

EAPs

S. sanguis antagonizes A.a. and competes with C.a. for 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

(phosphatidylglycerol)- form pores in bacterial membrane

• Cross-talk with adaptive immunity

(Hancock, Lancet, 1997)

+ + ++

++

++ ++

C C C C C C

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, 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/mDCs via TLR1/2 (Funderberg et al, 2007)

IMF of hBD-2 and hBD-3 in human oral epithelium

Ge Jin, CWRU

carcinoma in situnormal

H&E staining

hBD-2

hBD-3

nucleus

hBD-2/hBD-3

B. Dale-Crunk, U. Washington

Gingiva

Skin

Questions

*Why is the mouth so healthy in spite of constant trauma occurring in a very septic environment?

*The answers may lie with some of the ubiquitous bacteria of the mouth that induce epithelial cell derived antimicrobial peptides.

*Why are hBDs constitutively expressed in oral mucosa?

Homo sapiens or Homo bacteriens?

1013 eukaryotic cells

1014 bacteria

Henderson and Wilson, JDR, 1998

• found in healthy sites

• involved in dental plaque formation

Porphyromonas gingivalis

• black pigmented gram negative rod

• found in diseased sites

• associated with periodontal disease

• gram negative fusiform

Fusobacterium nucleatum

F. nucleatum vs P. gingivalis induction of defensins

hBD-2

- Fn PgRT-PCRmicrobial challenge

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F. nucleatum induction of hBD-3 in NHOECs

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

Are these “good guy” bacteria sensitive to defensins

Pg 49417

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F. nucleatum and P. gingivalis sensitivity to recombinant hBDs

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.

1.

Immunogold TEM of P. gingivalis after rhBD-2 incubation

2.

Immunogold TEM of F. nucleatum after rhBD-2 incubation

3.

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

• F. nucleatum factor(s) isolated from the cell wall promotes defensin expression; 4 candidate peptides; 12-13 kDa, pI 4-5

• F. nucleatum is resistant to -defensins ; P. gingivalis is not

• F. nucleatum resistance may be due to “fimbrial" extensions that sequester the hBD away from the outer membrane

• P. gingivalis does not induce hBD expression in NHOECs