Microbial interaction with the host in the periodontal diseases.

Dear colleagues I referred to 2009 scripts because the record was so bad , and there were no slides ! -----------------------------------------------------------------------------------------Today we'll talk about the microbial interaction with the host in the periodontal diseases. Getting back to talk about pathogenesis; but this time we want to stress on the host-.. interaction; we are going to talk about it in the same way, but here we are going to talk about the bacterial plaque and its effect on the hosts bone, and then the host tissue will go in a host response which is as we mentioned previously an inflammatory response. Now the problem we have in the periodontal diseases is that most of the problem or most of the destruction in the periodontal tissues is not from a direct effect of the bacterial plaque; but it is from a direct effect of the host response or immune response which is a result of the invasion of the bacteria into the tissues. The damage from the bacterial plaque against any other infection in the body generally will not lead to damage because of the bacteria or bacterial plaque or pathogens generally. So, let us ask this question: Does the immune response have a protective effect or a destructive effect? It is here a destructive
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effect. Why do we have this destructive effect of the immune system? Simply because the immune system is not aiming to protect the teeth or the periodontal tissues; but the general aim of the immune system is to protect the whole body. We are not comparing the health of a tooth with the health of for example cardiac tissues, both of them are important, but which one of them is more important? Of course cardiac tissues is more important; and because of this the immune system is always directed to protect the cardiac tissues, the whole body, the blood stream and the vital organs in the host tissues (generally), why the tooth and the periodontal tissues arent considered to be of a high priority to our body, it is just too bad .

*** Important points: There is a bacterial invasion of the tissues or bacterial plaque and an immune response, the destruction that happens due to the immune response is what causes the problem in the periodontal tissues. Accumulation of plaque on the gingival tissues and gingival margins and in the gingival pocket that will cause an inflammatory response. We have mentioned previously that it is only in the gingival tissues or in the periodontal tissues where I have the bone as close to the outer environment, while other bones in any other area of the body are well protected under layers of muscles, facial and skin. So, the presence of the dental pathogens alone is insufficient to cause the tissue destruction seen in periodontitis, and it is the body response to
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the periodontal pathogens that causes all the bone destruction seen in periodontitis.

Now I want to mention that we have two sides of our balance; dental plaque or bacterial plaque and immune response. As we mentioned in the previous lecture we have some factors that may affect this balance, so how can this balance get affected? 1. There are specific & non-specific theories of bacterial plaque accumulation that causes periodontitis. Specific is related to the number of specific bacterial type, the virulence of the bacteria; the mechanism that bacteria colonize and invade the periodontium regardless the amount of the bacteria. Non-specific is related to huge amount of bacterial plaque accumulation regardless the type of bacteria.

2. Immune response. We will start discussing the bacterial virulence factors and then through that we will reach the immune response. Bacteria virulence factor, mechanisms of bacteria to colonize and invade that area (periodontal tissue and periodontium). we have said before , why we have plaque accumulation in the oral cavity on teeth but we dont have this problem in anywhere else in the body. Why is that? Because there's shedding of soft tissues and epithelium generally but I don't have this shedding in enamel or dentine.
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Bacterial virulence factor is considered to be a minor cause of the periodontal destruction; it is a non-specific factor. So that the virulence factor of bacteria is not that important. Bacterial virulence factors can be divided into two parts; the characteristics of the bacteria itself and the products of the bacteria. When we talk about the characteristics of the bacteria itself we're talking about the pathogenicity which comes from the cell membrane or the cell wall of the bacteria, and the bacterial invasion factors; the ability of the bacteria to penetrate into the epithelium lining or the pocket core and the inter-radicular connective tissue; with peptides found on the bacterial cell membrane that will cause an immune response later on. Which types of cells are responsible of phagocytosis or presenting of these peptides or antigens for other types of immune system cells? Langerhans cells they are in the epithelium, the macrophages and monocytes generally. Talking about the bacterial products; one of the bacterial products is exotoxin, so what is exotoxin? It is harm proteins or potent toxins that produces by the bacteria and released from the bacteria itself; it is not connected or associated with the cell wall or cell membrane of the bacteria. Second thing is enzymes, bacteria always produce enzymes; proteins that catalyze the chemical reactions; like causing damage
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to the tissues and damage to the proteins proteinases is what we call it generally (type of enzymes), it opens the field for bacteria to invade the tissues. Now, let's go back and talk about the bacterial side or dental plaque side. Here we're talking about the bacterial colonization and plaque biofilm, weve talked in the previous lecture about the steps or the bases of the bacterial plaque maturation; it begins with a clean surface (after perform brushing, scaling, polishing), then there will be a thin coated of acquired pellicle, then building and ascending organism, and multiplication of these cells, growth of that area, and then mature biofilm within 28 days to complete the maturation of it; and then we will have mushroom shape like what we see in the bacterial plaque and fluid channels. Now, lets talk about the propagation of bacteria How does the propagation of bacteria differ from the colonization? Colonization is the whole procedure; from the attachment to the surface including the propagation of the bacteria, while propagation is cell-to-cell adherence of one colony of bacteria to one another. In propagation, we have a sequence, it is not a random procedure, each bacterial strain has a limited set of bacteria that it is able to adhere to it.

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Sometimes they talk about it like an alphabetical ordering, for example there are lines; line A, line B, line C, line D, and for example line C is not allowed to set unless line B is there. This point is very important in understanding the pathogenicity of the pathogens generally and periodontitis specifically. According to the propagation of bacteria we can also divide the whole process into three stages; early, intermediate and late: Firstly we have the pellicle on the tooth structure, and the first layer on the early phase of propagation of the bacteria which allows other layers to be accumulated in sequence and here some kinds of bacteria are obviously seen. In the early colonization the first bacteria to colonize is nonpathogenic, this non-pathogenic bacteria is what we have after tooth brushing, scaling or lets say after applying excellent oral hygiene measures on the tooth surface, we call this first layer acquired pellicle. Right after the acquired pellicle, there will be a single layer of bacteria, and this process takes around 24 hours; this allows us to remove it when we apply good oral hygiene measures, so we can prevent the accumulation of the pathogenic layer of the plaque that will be formed later on. Here we said that the tooth surface or the hard tissues in the oral cavity generally attaches firstly to a layer of non-pathogenic bacteria, and this layer of non-pathogenic bacteria allows the
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attachment of the pathogenic bacteria; because pathogenic bacteria are always free and floating in the oral cavity. Because they are floating, I can divide these types of bacteria according to the attachment into two types (attached or not attached): 1. Floating bacteria in oral cavity; and they cannot increase their number and they cannot secrete matrix or toxin basically until they are in the mature plaque or biofilm generally. 2. Attached bacteria; as what we have already said, the first bacteria to colonize are the non-pathogenic and the periodontal pathogens are unable to colonize the biofilm alone. The first thing we need is the acquired pellicle non-pathogenic bacteria pathogenic bacteria to be attached to all that system. So, this system or sequence of attachment allows me and the researchers to work against the plaque formation chemically such as using fluoroxidine or any other mouthwashes which is direct towards the attachment of one of these layers.

Colonization of acquired pellicle happens first, and then the gram-positive bacteria colonizes on it and attaches to it; which is mostly non-pathogenic, and later on the pathogenic bacteria get attached; such as streptococcus Sanguins the streptococcus bacteria causes other type of diseases; for example the caries are caused by streptococcus Mutans, so this indicates that all this
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species is attached directly to the tooth surface at the pellicle layer, and because of this these types of bacteria are responsible for caries. Also we have the streptococcus Sanguins or the Actinomycoses attached to the tongue area on the bony-rich proteins of saliva coated the tooth surface or what we call the pellicle. Early colonization as we said is the pellicle which consists of gram-positive; here were talking about the streptococcal species which takes place specifically in the early colonization; and there are many of the streptococcal species that have the ability to attach to the tooth pellicle, and also there're other non-streptococcus species which have the ability to attach to tooth pellicle, also other early colonizers co-aggregate with streptococcal species and held itself to it during colonization in the early phase.

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The importance of the early colonization is that the free-floating periodontal pathogens can't cause the disease; so I have always to keep the teeth surfaces free of these pathogenic bacteria, but how
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to do that? Every time the biofilm is disturbed, the process must start all over again with the early colonizers, so every time I apply the oral hygiene measures, I prevent the pathogenic bacteria from attaching to the biofilm layer presents there on the tooth surface. Now talking about the intermediate and late colonizers; like the early colonizers the intermediate and late bacterial colonizers must join the biofilm in the proper sequence. Also non-periodontal pathogens are late colonizers to the biofilm, the intermediate propagation then induce proliferation or increase in the number of the early colonizers; such as Streptococcus, also there will be an increase in the number of cells to provide the environment for the anaerobic and gram-negative bacteria that will come during the late stage. During the Late stage, the gram-negative organisms will come such as Prevotella intermedia which will attach to the 1st layer, and also the gram-negative bacteria of different late stages; such as Porphyromonas Gingivalis. Here we have seen this figure more than once; talking about the bacterial aggregation, a scientist came and divided the bacteria into complexes, he divided them into: 1) Red complex: The most pathogenic bacteria in the oral cavity in the sub-gingival group or the sub-gingival biofilm; such as P.gingivalis, T.forsythia and T.denticola. 2) Green complex: The least pathogenic compared to other bacteria generally regarding the periodontal diseases.
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** (these points are very important regarding the figure as Dr.Malik said).

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Now, I want to talk about the biochemical mediators, as we said last week there're innate and adaptive immune response, also we said that there're stages in periodontitis; initial, early, established and advanced, these stages start the processes to get gingivitis into periodontitis step by step. First there will be an invasion of the bacteria into the periodontal tissues which will stimulate Langerhans cell, phagocytes or any other cell that can present the antigen to the cells to start the immune response.

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The immune response in the body is generally carried out through the biochemical mediators. The biochemical mediators are active compounds which are secreted from the immune cells that activate the body inflammatory response, and they can be divided generally into cytokines, prostaglandins, and also (MMPs) MatrixMetalloProteinases. Note that biochemical mediators released by immune cells to activate, guide and remain the inflammatory response. Inflammatory mediators that are important in periodontal diseases are cytokines, prostaglandins and MMPs. Cytokines are considered to be cell-sickled protein molecules, last time we said there are cytokines and chemokines. We have chemotactic effect and cytokines, chemotactic effect come from the pathogens generally. Cytokines (cell signaling protein molecules) are considered to be powerful mediators produced by the immune cells generally, influence the behavior of other cells and give signals to the immune system to send more phagocytes to the site of infection, as we said it increases the epithelial permeability of the sub-epithelial plexus. Cytokines are produced by many different cells; especially the PMN (PolyMorphoNuclear) cells, Macrophages, B-Lymphocytes, epithelial cells, gingival fibroblasts and osteoblasts, and it produce the response to the tissue injury.

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Cytokines are important in the periodontal diseases and it includes IL-1, IL-6 and IL-8, TNF- (tumor necrosis factor-alpha), and their function is to recruit cells such as PMN cells and macrophages to the infection sites. So cytokines are produced by PMN cells and macrophages and they also recruit this type of cells to the site of infection, and they increase the vascular permeability that increases the movement of immune cells into the tissues, and last time weve talked about the macrophages and how it penetrate the epithelial cells towards the connective tissue where there is injury.

So, first of all is cytokines , then there will be a penetration between 2 epithelial cells. Later on it will initiate tissue destruction and bone loss in the site of infection such as periodontal diseases generally. About prostaglandins, it is also coated inflammatory mediators, we have several groups; D, E, F, G, H and I; but the one I care about usually is E, because most cells can produce prostaglandins, why? What are prostaglandins? Prostaglandins are arachidonic acid build the cell membrane itself. Macrophages and fibroblast produce prostaglandins, and this prostaglandins cause bone destruction, stimulate the osteoclast which will make bone resorption, it functions in increasing the permeability and dilatation of blood vessel to promote and increase the movement of immune cells and compliment to the infection site;
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(compliments are proteins approximately 30 in number that disturb the cell membrane of the bacteria), it triggers osteoclast bone consuming cells which cause resorption of the bone, it promotes the overproduction of destructive MMP's, enzymes and prostaglandins specially the E series to initiate most of the alveolar bone destruction in periodontitis.

Regarding the prostaglandins they care a lot about the non steroidal anti inflammatory drugs (NSAIDs); because generally they work against the prostaglandins, which may reduce the inflammation in the area, and reduce the destruction of the bone. But they still do researches regarding this information. About MatrixMetalloProteinases, it is a family of at least 12 different enzymes still under researches and they are produced by variant cells on the body; like PMN cells, macrophages, fibroblast, junctional epithelial cells, these enzymes acts together to breakdown the connective tissue matrix, which contains collagen, keratin and elastin. And when there is a destruction of the connective tissue this will cause edema, and there will be periodontal pocket, and recession, and these MMPs are present in the body in the healthy status before the diseased status, and they are present all over -not sure- the periodontal connective tissue matrix. In the chronic bacterial infection they are released and there is an overproduction of them, which will result in a breakdown of the
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connective tissue of periodontium, and the high MMP level in the area results in extensive collagen destruction of periodontal tissues, gingival rescission and pocket formation.

Getting back to our point host response in periodontal diseases, weve already discussed the stages of periodontitis and gingivitis; which are initial, early, established and advanced stages from the view of macrophages, neutrophils and the effect of them. Now we will discuss them in another way, in the initial stage we have the formation of the biofilm, look at this picture; here we can see the enamel, this is epithelium, this is cementum, junctional epithelium, sulcular epithelium, oral epithelium, this is the biofilm accumulated here, and there are polymorphinuclear cells released in the sulcus and also presents in some other areas. The bacteria colonizes the tooth near the gingival margin, within 24 hours, and it will be ready in 2-4 days ( 2 - 4 ) , bacteria initiates the host response, and there will be a dilation of the blood vessels, PMN cells pass the blood stream into the gingival connective tissue, PMN cells release cytokines and destroy the gingival connective tissue allowing PMN cells to move quickly towards the tissues, also there is releasing of it in the sulcus, PMN cells migrate into the sulcus and phagocytes the bacteria, and this is the initial stage.
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Now we will discuss the early stage gingivitis, here we are still talking about the subclinical gingivitis, in its early stage; there will be releasing of some cytokines, interleukin 1-BETA and also interleukin 6, Tumor Necrosis factor-alpha, prostaglandin E2. What will all of this do? It will stimulate the immune system generally. And the bacteria penetrate connective tissue cause releasing of interleukins 8 from the epithelial cells, and the PMN cells release cytokine causing more localized destruction of the connective tissue, and the macrophages release cytokines and prostaglandins E2 and MMPs in the area, and this will cause inflammation, redness .in the area, and there will be formation of rete-ridges in this area. There is penetration of bacteria into the epithelium or the soft tissue or the connective tissue beneath the epithelial layer, here it is the established stage (gingivitis) and then it will proceed to plaque biofilm extend subgingivally forming a pocket, and the biofilm goes more apically, releasing of cytokines and chemical mediators, interleukins 8, MMPs, TNF, and started to have plasma cells, lymphocytes, which we have in early and established stages, we have already talked about the age. Now the host cells produce more toxic chemicals or immune mediators and cytokines, there is an increase of the proportion of gram negative anaerobes; because the environment of apical region becomes completely anaerobic, that gives the best and excellent conditions to enhance the anaerobic bacteria growth and function. In advance stages (periodontitis), here there is start of destruction of the bone because of macrophages and prostaglandins E2, cytokines
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will destroy the connective tissue and periodontal ligament fibers, cytokines, prostaglandin E2 and MMPs destroy the connective tissue and bone, and prostaglandin E2 initiate the bone destruction.

(Name of 3 bacteria the red complex)this complex is considered to be responsible of the periodontitis generally, which is considered to be the most pathogenic bacteria in periodontitis, and this is what we have in the advanced stage. The mechanism of alveolar bone destruction, when there is a stimulation of macrophages, it releases interleukin 1-beta, TNFalpha to the fibroblast which will release prostaglandin E2 which will stimulate the osteoclasts, and cause stimulation of breakdown of collagen, elastin and extra cellular matrix generally and that is done by MMPs, and it stimulate the fibroblast to secrete MMPs to cause destruction of connective tissue, prostaglandins E2 stimulate osteoclasts to resorb the alveolar bone. Finally, the take home message is that, in order for the periodontium to remain healthy, the bacterial infection must be controlled so it does not trigger chronic exaggerated post immune response. How to control that? By excellent oral hygiene and mechanical brushing mechanical oral hygiene measures. Note that the bodys immune response to the bacteria causes most of the tissue destruction in the periodontal tissues. Edited By : Majd M. Hidmi
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