L5 Etiopathogenesis Part 2

Etiopathogenesis: Host Responses to Oral Biofilms¹

Etiopathogenesis -2 Host responses to oral biofilms

Associate Professor Leticia A Miranda* DDS, Specialist (Perio), MSc (Perio), PhD (Perio, Medicine)

Dr. Pradeep Koppolu* BDS, MDS (Perio), PhD (Malaysia), FICOI, FPFA, PDCR Discipline Lead & Program Convenor Periodontics and Implantology

*notes

Reading Resources²

BOOK Periodontology at a Glance Clerehugh, Valerie. ; Tugnait, Aradhna. ; Genco, Robert J. ; Somerset : Wiley; 2013 [Available Online] →

Clinical Periodontology and Implant Dentistry, 2 Volume Set by Niklaus P. Lang, Jan Lindhe, and Niklaus P Lang

PUBLISHER John Wiley & Sons, Incorporated

DATE 2015-03-25

NEWMAN AND CARRANZA’S CLINICAL PERIODONTOLOGY AND IMPLANTOLOGY 14th EDITION

CHAPTER 11 Host-Microbe Interactions and the Inflammatory Response Keith L. Kirkwood | Carlos Rossa Jr. | George Hajishengallis | Ann Decker | Yvonne L. Hernandez-Kapla

Learning Outcomes³

• Recognize periodontal health and disease;
• Understand the peculiarities of the periodontal niche;
• Identify the key components of the host response in the dento-gingival area;
• Understand the histopathology of periodontal diseases.

Periodontal Health and Disease Overview⁴⁵⁶⁷

Periodontium

Periodontal health and diseases

THE UNIVERSITY OF WESTERN AUSTRALIA

Periodontal Area Peculiarities⁸

Biofilms always present

Health Disease

• Intact enamel

• Supragingival eubiotic biofilm

• Gingival crevice

• Subgingival eubiotic biofilm

• Homeostatic inflammation

• Intact bone

• Supragingival dysbiotic biofilm

• EPS matrix and acidogenic–aciduric environment

Periodontitis

• Periodontal pocket, attachment loss
• Subgingival dysbiotic communities on root surface, in GCF and in and on the epithelium
• Severe, destructive inflammation
• Resorbed bone

Caries

• Demineralized enamel leading to cavitation

Eubiotic: in a healthy balance with the host. Dysbiotic: When balance with the host is disrupted

• Supragingival biofilm = Gingivitis
• Subgingival biofilm = Periodontitis

Periodontitis peculiarities

• Tooth: non-shedding surface, partially outside the body (=oral cavity)
• Bacterial load is located generally “outside” the body
• Challenge for the immune-inflammatory response to take action
• Endogenous/opportunistic infection
• Dysbiosis
• Plaque as a biofilm

Hajishengallis et al 2015

Microbial Dysbiosis and Host Defense Balance⁹¹⁰

Dysbiosis

Dysbiosis is any perturbation of the normal microbiome content that could disrupt the symbiotic relationship between the host and associated microbes, a disruption that can result in diseases, such as inflammatory bowel disease and other gastrointestinal (GI) disorders, including gastritis, peptic ulcer disease, irritable bowel syndrome, and even gastric and colon cancer [3–6].

From: The Microbiota in Gastrointestinal Pathophysiology, 2017

Kinane, D. F. et al. (2017) Periodontal diseases Nat. Rev. Dis. Primers doi:10.1038/nrdp.2017.38

Host defences

(a) Host defences effective against microbial challenge in a conducive environment for periodontal health

Conducive environment

(b) Reduced or defective host defences are ineffective against normal microbial challenge

Host defences ineffective

Balance upset

Environment NOT conducive to health

(c) Increased microbial challenge overwhelms normal host defences

Host defences

Balance upset

Environment NOT conducive to health

Key:

• Reduced
• Normal
• Increased

Health -> Disease

Microbial challenge

Health -> Disease

Increased microbial challenge

Figure 7.1 Concept of balance between host defences, microbial challenge and environment. (a) Balance and periodontal health. (b) Host defences have a defect or are ineffective against microbial challenge, tipping the balance to periodontal destruction. (c) Microbial challenge overwhelms the host defences leading to an upset balance and periodontal destruction - this may relate to the environment not being conducive and/or changes in quality, quantity or virulence of microorganisms.

Current Etiopathogenesis Model¹¹

Current PD etiopathogenesis model

The University of Western Australia

• Behavourial risk factors absent

• Environmental risk factors absent

• Genetic risk factors absent

• Epipenetic effects not evident

• Behavourial risk factors present

• Environmental risk factors evident

• Genetic risk factors present

• Enzigenetic effects evident

Clinical Health Gingivitis Periodontitis

Health Promoting biofilm = Symbiosis

Incipient Dysbiosis (Quorum Sensing Bacteria)

Frank Dysbiosis (Pathogenic Biofilm)

• Low biomass
• High biomass
• High biomass

Complement PMNs

Proportionate Host response

Antibody PMNs ++ T & B cells

Proportionate Host response

Antibody PMNs +++ Plasma cells

Acute Resolution of inflammation

Chronic Resolution of inflammation

Faled Resolution of inflammation

Antigens Bact’1 DNA fMLP

High biomass

Antigens Virulence Factors LPS

Gingivapinas LPS

Connective Tissue & Zone Damage

Dis-proportionate Host response (hyper-inflammatory)

DAMPs Haem GCF

Chronic non-Resolving inflammation

Cytokines Prostanoids MMPs Oxidative Stress

FIGURE 1 Contemporary model of host–microbe interactions in the pathogenesis of periodontitis, in which the host response drives an incipient dysbiosis (gingivitis). If the biofilm is not disrupted/removed, frank dysbiosis results and perpetuates a chronic nonresolving and destructive inflammation. DAMPs, damage-associated molecular patterns; fMLP, N- formylmethionyl- leucyl- phenylalanine; GCF, gingival crevicular fluid; LPS, lipopolysaccharide; MMPs, matrix metalloproteinases; PMNs, polymorphonuclear neutrophils. This figure is referred fromref. 106.

Murakami S, Mealey BL, Mariotti A,Chapple ILC. Dental plaque–induced gingival conditions.J C i n Periodontol. 2018;45(Suppl 20):S17–S2

Periodontal Microbiota and Virulence Factors¹²¹³

Periodontal microbiota

• P. gingivalis
• B. forsythus
• T. denticola
• C. gracilis
• C. rectus
• C. showae
• E. nodatum
• F. nuc. nucleatum
• F. nuc. polymorphum
• P. intermedia
• P. micros
• P. nigrescens
• S. constellatus
• Actinomyces species
• V. parvula
• A. odontolyticus
• E. corrodens
• C. gingivalis
• C. sputigena
• C. ochracea
• C. concisus
• A. actino. a
• S. mitis
• S. oralis
• S. sanguis
• Streptococcus sp.
• S. gordonii
• S. intermedius

Periodontal Health

• Actinomyces
• other

Periodontitis

• Supragingival
• Subgingival

SOCRANSKY, S.S. and HAFFAJEE, A.D. (2002), Dental biofilms: difficult therapeutic targets. Periodontology 2000, 28: 12-55.

Virulence factors – perio microbiota

• Enzymes
• Waste products
• Proteinases
• Leukotoxins
• Lipopolysaccharides

EVADE HOST RESPONSES

Bacterial Biofilm on tooth surface with pellicle

Dento-Gingival Host Defenses

Innate and Adaptive Immunity Systems¹⁴¹⁵

Immunity

Innate Immunity (Immediate response)	Adaptive Immunity (Delayed response)
Barriers, Complement system, PMNL’s, Macrophages	B cells, T cells

Innate immune system

• Dendritic cell
• Eosinophil
• NK cell
• Basophil
• Monocyte
• Neutrophil
• Mast cell
• Macrophage
• Complement protein
• Common pathway
• γδ T cell
• NK T cell

Adaptive immune system

• T cell
• CD8+ T cell
• CD4+ T cell
• B cell
• Antibodies
• Cytokines

Innate immunity

• Virus -> Interferons -> Block
• Some Bacteria -> Defensins -> Lysis
• Some Bacteria -> Lysozyme -> Lysis
• Injury -> Tissue_damage -> Acute_inflammation -> Healing
• Some bacteria -> Tissue_damage -> Chronic_inflammation
• Complement -> Mast_cell -> Acute_inflammation
• Complement -> MAC -> Phagocytosis
• Complement -> Acute_inflammation
• PMN -> Phagocytosis
• Dendritic_cell -> Phagocytosis
• NK_cell -> Cytotoxicity

Adaptive immunity

• Specific antigens (All bacteria, virus, etc.) -> B_cell -> Antibody -> Entry_block/neutralization toxin
• Specific antigens -> T_cell

Interactions

• Antibody -> Adherence -> MAC
• Antibody -> Activation -> Complement
• MAC -> Activation -> T_cell
• Dendritic_cell -> Presentation -> T_cell
• T_cell -> Help -> B_cell

Saliva and Complement Pathways¹⁶¹⁷

Dento–gingival host defenses

THE UNIVERSITY OF WESTERN AUSTRALIA

Fig 7.2 Host defences against microbial plaque.

Innate immune response & Adaptive immune response	Mediators
Saliva	Epithelium	Inflammatory response
Prevents drying of gingiva and teeth	Physical barrier	Fluid component
Antimicrobial effects via:	Inflammatory response via keratinocytes	Gingival crevicular fluid
Swallowing bacteria	Immune response via Langerhans’ cells	Cellular components
Salivary IgA		Neutrophils
Salivary peroxidase		Macrophages
Lysozyme and lactoferrin

Clerehugh et al 2013

Classical pathway Via Ag-Ab complexes Microbe -> C1 -> IgG antibody

Lectin pathway Via MBL-MASP complexes Microbe -> Mannose -> MASP1 -> MBL -> MASP2

Alternative pathway Via spontaneous C3 hydrolysis Microbe -> C3 >> C3b

C3 Self-amplification loop

• C3a
• C3b -> Opsonization for phagocytosis
• C5 -> C5a -> C5b -> C6 -> C7 -> C8 -> C9 -> Membrane attack complex

TLR C3aR or C5aR -> Cross-TALK -> Inflammatory cell activation -> Inflammatory mediators, Degradative enzymes, Reactive oxygen species, Ag presentation & adaptive immunity

Dysbiotic microbiome -> Periodontitis -> Tooth -> Bone

Fig. 11.3 Complement activation and periodontal disease. The complement system can be activated by distinct initiation mechanisms: The classical pathway is triggered by antigen-antibody complex-mediated activation of the C1 complex. The lectin pathway is initiated when complexes of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) recognize and adhere to microbial surfaces. The alternative pathway is triggered by a “tick-over” mechanism that involves spontaneous C3 hydrolysis, which occurs in the absence of complement regulatory molecules (as is typically the case with foreign surfaces such as microbial cells). In the so-called alternative pathway-amplification loop, additional C3 is cleaved into even more C3b which further fuels the loop, thereby amplifying complement activation irrespective of the initiating mechanism. All three mechanisms of complement initiation and amplification converge at C3. The downstream effects of C3 activation include the generation of effectors that promote inflammation (C3a and C5a), opsonization for phagocytosis (C3b) and the generation C5b-C9 membrane attack complex (MAC). MAC can lyse susceptible targeted bacteria but has also been implicated in destructive inflammation. Whereas the role of MAC in periodontitis is uncertain, C3a and C5a activate specific G-protein-coupled receptors (C3aR and C5aR1), which cross-talk with Toll-like receptors (TLRs). This cross-talk interaction between complement and TLRs activates synergistically inflammatory leukocytes, which directly or indirectly mediate destructive inflammation that leads to periodontal tissue breakdown and alveolar bone loss in periodontitis.

Epithelial Barriers and Gingival Crevicular Fluid¹⁸¹⁹

Epithelium

Dento-gingival junction

1. Junctional epithelium (JE)
2. Sulcular epithelium
3. Oral epithelium
4. Epithelial rests of Malassez

JE functions

• attachment to tooth
• barrier
• rapid turnover
• antimicrobial defence
• GCF flow

Physical barrier: turnover + peeling

Biological barrier: defensins, IL-8, PMN

Gingival crevicular fluid (GCF)

Inflammatory exudate released through the crevice, resulting from the increase in permeability of vessels next to the JE and SE.

Composed of: plasma derived substances (antibodies, cytokines, enzymes), epithelium and immune cells, bacteria.

GCF volume and flow increase with increasing inflammation.

Cellular Components of the Immune Response²⁰

THE UNIVERSITY OF WESTERN AUSTRALIA

Innate immunity (rapid response)

• Dendritic cell
• Mast cell
• Macrophage
• Natural killer cell
• Complement protein
• Basophil
• Eosinophil
• Neutrophil
• Granulocytes

Adaptive immunity (slow response)

• γδ T cell
• Natural killer T cell
• B cell
• Antibodies
• T cell
• CD4+ T cell
• CD8+ T cell

Neutrophil Function and Phagocytosis²¹²²

First defense cells (innate immunity)

Figure 7.3 The role of neutrophils in phagocytosis or the killing of microorganisms.

If bacteria are in plaque matrix:

• Neutrophils attach to plaque matrix
• Neutrophils secrete externally:
  • Enzymes
  • Hydrogen peroxide and hypochlorous acid
• Neutrophils kill bacteria without phagocytosis
• Plaque matrix dissolved
• Washed away by gingival crevicular fluid
• Bystander damage caused

If bacteria are unattached:

• Neutrophils recognise and bind to bacteria which are phagocytosed into phagocytic vacuole
• Neutrophils produce and release:
  • Antibacterial granules
  • Hydrogen peroxide and hypochlorous acid
• Enzymes digest microorganisms
• Remnants expelled
• Bystander damage caused

Figure 7.4 The role of macrophages in inflammation and immunity.

INFLAMMATION

• Scavenger - responsible for phagocytosis of dead and dying cells
• Modulates fluid and cellular components of inflammation
• Secretes tissue-degrading enzymes
• Secretes mediators, e.g. IL-1, TNF-α, prostaglandins

IMMUNITY

• Traps and presents antigens in the connective tissue while CD44 acts as anchor
• Secretes IL-1 and TNF-α

Clerehugh et al 2013

The text on the page only makes sense in the context of the diagram (labels such as “Complement,” “Antibody,” “T-cell,” “MMP-8,” etc.), and its meaning is entirely dependent on the visual elements. Per the instructions, the entire content should be treated as a single figure.

Humoral and Cell-Mediated Responses^23242526

Adaptive immunity

Neutralization

Opsonization and Fc receptor-mediated phagocytosis

Bacterial lysis

Inflammation

Phagocytosis of C3b-coated bacteria

Antibody response

Macrophage activation ⇒ Phagocytosis and bacterial killing

Inflammation

Figure 15–1 Adaptive immune responses to extracellular microbes. Adaptive immune responses to extracellular microbes, such as bacteria, and their toxins consist of antibody production and the activation of CD4+ helper T cells. Antibodies neutralize and eliminate microbes and toxins by several mechanisms. Helper T cells produce cytokines that stimulate B cell responses, macrophage activation, and inflammation. APC, antigen-presenting cell; INF-γ, interferon-γ; TNF, tumor necrosis factor.

INNATE & ADAPTIVE IMMUNITY

HUMORAL RESPONSE

1. Plaque antigens diffuse through the junctional epithelium
2. Langerhans cells within the epithelium capture and process the antigens
3. Antigen-presenting cells (Macrophages and Langerhans cells) leave the gingiva in the lymphatics
4. Antigen-presenting cells reach the lymph node and begin to stimulate lymphocytes to produce a specific immune response
5. Periodontal microbe specific antibodies are produced by plasma cells within the lymph nodes and travel back to the gingiva via blood vessels
6. Antibodies leave the circulation and are carried to the crevice in the transudate from the inflamed and dilated blood vessels
7. Antibody action on microbes in the crevice can result in killing, aggregation, precipitation, detoxification, opsonization and phagocytosis of bacteria

The University of Western Australia

Adaptive - Cell-mediated response

Antigen penetration of JE => APC + T cell naïve =>

Different subsets of T-helper cells (Th1, Th2, Th17, T reg) proliferate and release different cytokines profiles

• Th1: IL-2, IFN-g
• Th2: IL-4, IL-5
• Th17: IL-17
• T reg: IL-10, TGF-b

Cytokines act on other cells (PMN, macrophages, B or T cells) to stimulate, inhibit or kill

T-helper cells on re-exposure proliferate and produce cytokines (memory)

Fig. 1 The potential role of distinct T cell subsets in inflammatory responses and alveolar bone resorption in periodontitis lesions. As a consequence of sustained infection by periodontopathic bacteria, an adaptive immune response is established. NKT cells are involved in shaping the course of the immune response. Th1, Th2, and Th17 cells contribute to infection control in different ways because of their distinct cytokine profiles. However, their action also enhances inflammatory responses that lead to periodontal tissue destruction. Particularly, Th17 cells have a high potential to facilitate osteoclastogenesis through the production of IL-17 to induce RANKL expression on osteoblasts, enhancement of local inflammation, and RANKL expression on themselves. However, Tregs attenuate the inflammatory responses by suppressing other immune cells and inhibit osteoclastogenesis; therefore, they could protect against tissue destruction. Enhanced inflammation may convert a fraction of Tregs to IL-17-producing cells. Mφ macrophage, Ob osteoblast

Mechanisms of Tissue Destruction²⁷

Immune response = cells+mediators

• Main aim is to control infection in different ways
• Connective tissue destruction in periodontitis occurs as a “side-effect” of the immune-inflammatory response
• Perio ligament and bone destruction in periodontitis:
  • Biofilm essential, but not sufficient
  • High levels of pro-inflammatory cytokines (IL1b, TNFa), prostaglandins, MMP’s, RANKL
  • Low levels of anti-inflammatory mediators (IL-10, TGF-b, TIMPs and OPG

Pro-inflammatory Mediators and Bone Resorption²⁸²⁹³⁰

Periodontal bacteria -> LPS -> Activated B-cell Periodontal bacteria -> Peripheral dentitic cell -> T-cell -> B-cell -> Plasma cell -> Antibody -> Complement Periodontal bacteria -> Phagocytosis -> MMP-8 -> PMN

LPS -> Macrophage -> IL-1β TNF-α -> Fibroblast -> MMP-1 -> PMN LPS -> Macrophage -> IL-1β TNF-α -> Osteoclast activation -> Bone

T-cell -> B-cell B-cell -> T-cell

IL-8 ICAM-1 -> PMN

332 Current Osteoporosis Reports (2023) 21:330–343

Macitentan

Denosumab

Osteoclast Activation and RANKL Regulation³¹³²

OSTEOCLASTS ACTIVATION

RANKL & OSTEOPROTEGERIN OPG STIMULATE OSTEOCLASTOGENESIS AND BONE RESORPTION.

RANKL /OPG HIGH RATIO PRORESORPTIVE.

RANKL /OPG LOW RATIO ANTIRESORPTIVE.

ACTIVATED T AND B CELLS CELLULAR SOURCE FOR RANKL

IFN-γ AND IL-17 INCREASE RANKL

IL-4 & IL-10 REDUCE RANKL/OPG RATIO

LYMPHOCYTES SUBSETS TH1 & TH17 PRO RESORPTIVE

TH2 & Tregs (Regulatory T) antiresorptive

Histopathology of Periodontal Diseases³³

PRISTINE GINGIVA x Histologic Perfection

The Initial Lesion and Gingival Health³⁴³⁵

• Clinically healthy gingiva with a limited coronal infiltrate (5-10% of the connective tissue area)
• Neutrophils and monocytes in JE
• Lymphocytes in the connective tissue
• Increase in vascular structures near JE
• Exudative fluid from vessels to tissues = GCF

INITIAL LESION histologically = gingival health clinically

• 24 HOURS PLAQUE ACCUMULATION
• CHANGE IN MICROVASCULAR PLEXUS JE
• ARTERIOLAS, CAPILLARIES AND VENULES DILATION
• HYDROSTATIC PRESSURE INCREASE
• INCREASED PERMEABILITY
• EXUDATE OF FLUIDS & PROTEINS
• INCREASED GCF
• ENHANCED PMNS MIGRATION
• PMNS ACCUMULATE IN JE AND SULCUS

The Early Lesion and Early Gingivitis³⁶

EARLY LESION x Early Gingivitis (The University of Western Australia logo)

• One week after plaque accumulation
• JE blood vessels remain dilated
• Increased in number and size of vasculature
• Infiltrate predominant PMNs and lymphocytes = now 15-20% volume
• Very few plasma cells
• Initial fibroblast degeneration and collagen destruction
• Proliferation of basal and JE cells
• Coronal rete pegs
• Inflammation clinically detected – bleeding – due to pocket epithelium ulceration

Figure 8.4 An early lesion. (Labels pointing to diagram text):

• Increased plaque biofilm formation
• Neutrophil migration continues
• Rete peg proliferation in coronal junctional epithelium
• Lymphocytes appear
• Early damage to fibroblasts and loss of gingival collagen
• Intact alveolar bone
• Base of junctional epithelium still at cement-enamel junction

pocket epithelium ulceration

The Established Lesion and Chronic Gingivitis³⁷³⁸³⁹

• Increased fluid and leukocyte migration
• More edematous swelling clinically
• Plasma cells 10-30 % on coronal connective tissue
• Collagen loss in apical and lateral directions
• Inflammatory cell infiltrate expands
• Extension of rete pegs into connective tissue
• JE deattached to tooth surface
• Pocket epithelium with heavy cell infiltrate PMNs
• Permeable and ulcerated pocket epithelium

TABLE 15.2 Hallmarks of Gingivitis

Feature	Healthy Gingiva	Gingivitis
Color	Coral pink	Red
Contour	Knife-edged and scalloped	Rolled with bulbous papillae
Consistency and texture	Firm and resilient with stippling of the attached gingiva	Edematous and with loss of stippling

CHAPTER 15 Dental Biofilm-Induced Gingivitis and Its Management 205

TABLE 15.1 Stages of Gingivitis

Stage	Time (Days)	Blood Vessels	Junctional and Sulcular Epithelia	Predominant Immune Cells	Collagen	Clinical Findings
I. Initial lesion	2−4	Vascular dilation Vasculitis	Infiltration by PMNs	PMNs	Perivascular loss	Gingival fluid flow
II. Early lesion	4−7	Vascular proliferation	Same as stage I Rete pegs Atrophic areas	Lymphocytes	Increased loss around infiltrate	Erythema Bleeding on probing
III. Established lesion	14−21	Same as stage II, plus blood stasis	Same as stage II but more advanced	Plasma cells	Continued loss	Changes in color, size, texture, and so on

PMNs, Polymorphonuclear leukocytes (neutrophils).

The Advanced Lesion and Periodontitis⁴⁰

ADVANCED LESION x Periodontitis

Advanced lesion

• Apical plaque growth
• Apical migration of JE from CEJ
• Lateral & apical extension of infiltrate
• Alveolar bone loss starts
• Extensive collagen fiber damage
• Plasma cells predominantly (>50%)

Conclusion⁴¹

Thank you!!!

THE UNIVERSITY OF WESTERN AUSTRALIA

SEEK WISDOM

Footnotes

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