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Johns Hopkins Arthritis Center Grand RoundsPeriodontal Disease and Rheumatoid Arthritis: From Epidemiologic Associations to Shared Disease MechanismsClifton O. Bingham III, MDJune 8, 2009

Slide 1 (“Periodontal Disease and Rheumatoid Arthritis: From Epidemiologic Associations to Shared Disease Mechanisms”)

Slide 2 (“Disclosures”)Slide 3 (“Absolutely Complete Disclosure”)

…inhibitors of these proteins and cells. So that's my full disclosure, which Anthony encouraged all of us to do. So hopefully that gives you some better insight in terms of my conflicts of interest. So…but if we do everything that Ed Miller wants us to do according to the rules of the law, that's what we need to do now.

Slide 4 (“Outline”)

So the outline today. I'm going to be giving you sort of a primer on oral health and periodontal disease. We are going to talk about associations between periodontal disease and rheumatoid arthritis. And finally, we are going to discuss citrullination as a shared mechanism of disease.

Slide 5 (“Background”)

In terms of background, there have been multiple reports, and I'll go through some of these with you, that have described an association between periodontal disease and rheumatoid arthritis. And it's been recognized that periodontal disease is associated with other systemic conditions, including diabetes, atherosclerotic cardiovascular disease, low birth weight infants and preterm labor. And in fact, intervention by treating periodontal disease can actually improve diabetes control, can improve the weight of infants and decrease preterm labor. And there is an interventional study that has been ongoing looking at the effect in atherosclerotic cardiovascular disease. So it implies that there may be relationships between oral health and systemic diseases.

Slide 6 (“Periodontal Disease”)

Now periodontal disease is something that we need to learn about to understand its relevance to this talk – or to this topic. Periodontitis is an inflammatory disease affecting the gums and structures. While it initiates from an infectious trigger and involves gingival inflammation, which many of us – or most of us – have to some extent, the characterization of periodontal disease is when it goes beyond inflammation and becomes destructive, where there is separation of the tooth from its surrounding structure, destruction of the collagen, destruction of the matrix that begins to make the tooth loose.

The stages begin with gingivitis, or chronic inflammation, and again this if you look in your mouth, most of us will have some evidence of gingival inflammation. But it's beyond this when there is colonization of what are called the red complex organisms that we really look at the process of periodontitis beginning. And then again, this sequence that occurs with loss of connective tissue attachments, bone resorption and ultimately tooth loss.

Slide 7 (“Biofilm Development in PD”)

This is what a biofilm looks like on the tooth of a patient with periodontitis, and you see that there are multiple bacteria that are sort of piled on top of each other. And this little cartoon sort of outlines what happens in the stages of biofilm development in the mouth. There are certain bacteria that come along and colonize, but then there are bacteria that set up shop on top of this, the specific adhesion molecules between the two. And then there are further bacteria that come along with additional properties that build up on top of that.

Slide 8 (“Colonization in Periodontal Disease”)

And these are the late colonizers that really describe patients with periodontal disease. So we all have multiple organisms in our mouths. And we know that the mouth is very dirty and human bites are bad things that we have to treat with antibiotics because of all of the anaerobes.

Slide 9 (“Porphyromonas gingivalis”)

But patients with periodontitis have specific flora in their mouths, one of the most important of which is this bug called Porphyromonas gingivalis. Others that are also of importance, and they track along with it, are treponema denticola, certain species of Eubacterium, Providencia intermedia and actinomycetemcomitans organism here as well. But it's when these come along that the process goes from inflammation to one that has the potential for destruction.

Porphyromonas gingivalis is a gram negative anaerobic bacteria, and again it's one of these terminal complex organisms – these late colonizers. Several properties that are unique about P. gingivalis include its expression of LPS. Most of the other bugs that are down underneath there don't have LPs. Therefore, it can activate toll-like receptors. Also, Porphyromonas gingivalis has an awful lot of proteolytic enzymes. Many are involved in arginine, lysine and cysteine metabolism, and one of those which we'll talk about more is a peptidyl arginine deiminase. There are also many enzymes that are directly secreted by this bacteria that cause collagen degradation and other exotoxins and gingipains which make many proteins susceptible to further cleavage.

Recently it has been described that P. gingivalis has DNA for enolase, which has overlapping sequence with human alpha enolase, which is susceptible to citrullination. And finally, antibodies to P. gingivalis are a marker for periodontal disease. And one of the ways that people can be identified for having exposure to this organism by looking at serum antibody responses against it.

Slide 10 (“Periodontal Disease Epidemiology”)

The epidemiology of periodontal disease is something as well we need to learn about. Periodontal disease is the leading cause of tooth loss in the United States. Now the estimates of the prevalence of periodontal disease depend on the definition that you use, but using the NHANES III survey, about 35% of adults over age 35 have some degree of periodontal disease; 30% of these were classified as being moderate to severe; and a substantial proportion of these severe patients go on to progressive disease. So if we look at the expected prevalence using NHANES' definition, the expected prevalence of moderate to severe disease would be about 13% of the general population. Now the risk factors for the development of periodontal disease are cigarette smoking, certain medications, systemic diseases and what we have learned is that twin studies have shown us that about 50% of periodontal disease is

explained by some heritability, and many genetic associations have been reported, none of them well validated, but some of the ones that I will put up here that have at least one report include HLA-DR4 polymorphisms and TNF, and the TNF promoter that has been described for rheumatoid arthritis as well as in the IL1 promoter.

QUESTION: What is the magnitude of the effect of cigarette smoking?

Cigarette smoking is the major risk factor, so – for periodontal disease. It is the one that is absolutely very strongly associated. It's unusual, and Melanie, you can weigh in for me as well, that people who are not smokers will develop it although people do, as you will see from some of our data.

QUESTION: [Inaudible]

I don't know in terms of relative risk for cigarette smoking, but it is the major, major risk.

Other polymorphisms that have been described are in the toll-like receptor and CD14.

Slide 11 (“Photos: Healthy/Gingivitis/ Moderate Periodontitis/Severe Periodontitis”)

So the progression of gum disease is as follows. These are nice, healthy gums. These are the gums that most of us or many of us would have that have gingivitis. And what you can see is some hyperemia of the gingiva. You might also be able to appreciate a little bit of swelling here that is not present here. And this is a mild condition where you have colonization. You may have excessive plaque accumulation, and this is the type of thing that toothbrushing will help calm down, and good oral hygiene. But this is the progression to periodontitis. I hope you can appreciate some of the differences. Now you not only have the gum inflammation, but what you begin to see is recession of the gum line, and you begin to see parts of the tooth that weren't visible before. So you have had regression of the gingiva. You begin to expose parts of the tooth that weren't there. You begin to get these triangles and black spaces between teeth. And then this it the later stages of the disease where you see significant loss of the gingiva. You begin to see loss of the bone, and I'll show you what this looks like radiographically in just a second. And here obviously a lot of plaque accumulation as well. So this is a progressive disease and a progressive cycle. And what we're talking about is this type of lesion when we speak of periodontal disease.

Slide 12 (“Radiographic Changes in Periodontal Disease”)

Now there are many things that are similar between periodontal disease and rheumatoid arthritis, and one of those is the effect on bone. This is a normal bitewing x-ray that you might get when you go to the dentist with nice, healthy gums and here's a bone level demonstrated here around the bottom of the tooth. Here is that normal bone level again in a patient with periodontal disease. You see that the bone level has regressed significantly. And you begin to see even these spaces underneath the roots of the teeth, and I'll describe to you furcation later, which is putting a probe into the mouth and seeing if it will slip underneath this place in the tooth. In fact, that's something that I will show you some data on that we see very frequently in the RA patient population.

Slide 13 (“Pathogenesis of Periodontal Disease”)

So what do we know about the pathogenesis of this? Well, bacteria are necessary, but they are not sufficient. Eradication of the bacteria does not necessarily lead to resolution of the condition. And submicrobicidal concentrations of tetracyclines improve periodontal disease, explained for in part by matrix metalloproteinase activity. Antibody responses to certain bacteria predict progressive disease and bone loss, one of which is anti-P. gingivalis. There is a T-cell component of the disease that is seen, again, some suggestions of HLA associations, oligoclonal expansions of T-cells seen in the gingiva. There is cytokine-mediated effector damage driven by osteoclasts, RANK ligand and other pro-inflammatory cytokines, including TNF and IL1. And again, these genetic associations. Slide 14 (“Cartoon: Tooth”)

So if we look at a diagram – I think a diagram sort of will tell us a lot about what happens in disease – we've gotten used to seeing what this looks like in rheumatoid arthritis. I tried to set up a similar type of diagram for periodontal disease. Now I've shown you the accumulation of bacteria and plaque and biofilm formation that develops in this pocket. These bacteria begin to release substances such as LPS, activating neutrophils, macrophages, recruiting them into the site. But there are also T-cells that proliferate. There are plasma cells in the gingiva as well. These proliferate, and rheumatoid factors have also been described in patients with periodontal disease. There are also the antibodies that develop again to the bacteria. The release of multiple cytokines that are detectable both expressed in the gingival tissue as well as released into what's called the gingival crevicular fluid, which sits right here. Activation of fibroblasts that ends up leading to further connective tissue matrix degradation and osteoclast activation that leads to the degradation of bone. So many of the players that we are accustomed to in rheumatoid arthritis we see recapitulated in the periodontal microenvironment.

Slide 15 (“Previous Studies of RA and PD”)

So what about studies that have been done looking at RA and periodontal disease? One of the largest studies that was recently reported looked again back to the NHANES III study. It looked at subjects who were greater than 60 years of age, who had musculoskeletal disease – they looked at patients who were 60 years of age. Musculoskeletal examinations were performed, and there was a single quadrant dental examination performed on these patients as well. That means one-fourth of the teeth were looked at in patients to define oral health. From this study it was found that RA patients were more likely to be edentulous – an odds ratio of 2.27 – and have periodontal disease, with an odds ratio of 1.82 compared with non-RA subjects. This level of risk increase is similar to that seen for other conditions such as diabetes, preterm labor and low birth weight. So again, similar odds ratios to those conditions. And these data were adjusted for age, sex, race, ethnicity and smoking. But there really were no details whatsoever regarding the rheumatoid arthritis in this study.

Several other small case-control studies have reported similar results, and the odds ratios have ranged from 2.3 up to 8. And another study that I think is of interest is a study by a French group that looked at the association between bone loss on periodontal x-rays compared to bone loss on wrist radiographs of patients with rheumatoid arthritis and found that the RA-shared epitope is associated both with periodontal bone loss as well as with wrist damage. So sort of guilt by association from several different studies.

Slide 16 (“Recent RA-PD Case Control Study”)

Here is one of the recent case-control studies that has a little bit more information and better examinations than many of the others. It is in 57 RA subjects and 52 healthy controls. They did

measurements at four sites in each tooth looking at plaque index, gingival index, pocket depth, clinical attachment loss, and they defined patients who are periodontally diseased as having attachment loss greater than 4 mm. Now in this study that did step-wise logistic regression, and they found out that the odds were 8.05 for patients with RA for periodontitis compared with controls. When this was adjusted for the plaque and gingival index the odds ratio dropped, but it was still markedly significant. And it was also found that RA patients had higher indices of all of the different markers of periodontitis, including bleeding on probing, attachment loss, gingivitis and other measures of periodontal disease.

QUESTION: The definition of periodontitis there was….?

No, this was a standard – a single definition that was a mean attachment loss of 4 mm. This is not a standard definition, and that's one of the limitations of this study that is significant. There was no information on RA disease characteristics, and it was a non-standard definition of periodontal disease. So there are several definitions that I'll go through with you that have been established, but no studies have really fulfilled all of the obligations of having good information on RA disease activity on periodontal disease and using standard definitions of activity for both.

QUESTIONS: In the NHANES data on the prior slide, how is RA defined?

It's self-report by the participant.

Slide 17 (“RA, PD, and CVD”)

And here's another study that I just put up here because it's a little bit larger. But it begins to get at this issue of relationships between rheumatoid arthritis, periodontal disease and cardiovascular disease. As I said, periodontal disease has been described as a risk factor for atherosclerosis and intervention studies have been ongoing trying to look at this relationship a little bit better.

This is an Egyptian group who has been working on oral health for a while. They took 100 RA patients who had cardiovascular disease – I'm sorry – 100 RA patients, 50 of whom had cardiovascular disease, 50 of whom did not have cardiovascular disease and 50 controls without RA. Eighty-four percent of the RA patients with cardiovascular disease had periodontal disease compared to 60% of the patients without cardiovascular disease and 10% of the control subjects. So the conclusion of the group was that there was an additive effect of RA plus cardiovascular disease and the association with periodontal disease, periodontal disease further increasing the cardiovascular risk.

Now, it was not a terribly well-done study, but I think there was some interesting information that came out of it. They did have some information on RA disease activity in the study, and they found that the severity of periodontal disease was significantly correlated to RA disease duration, to the DAS28, to TNF levels and inflammatory markers. And they also found that again these periodontal indices were higher in RA patients compared to controls, and they were higher in RA patients with cardiovascular disease than those without cardiovascular disease.

Slide 18 (“TNF inhibition Improves Experimental Periodontitis”)

There have been several studies that have told us that cytokines that are involved in rheumatoid arthritis are also important in periodontal disease. Several studies have looked at models of

experimental periodontitis and shown decreased inflammatory cell infiltration and bone loss with TNF inhibition, and there was a very nice study that looked at P55 receptor knockout mice who had – that had less bone loss and less severe periodontitis with an induced model of bacteria.

Slide 19 (“TNF in Patients with Periodontal Disease”)

And TNF has been described increased in patients with periodontal disease, higher in patients who are smokers than are non-smokers. A TNF polymorphism has been reported and confirmed recently in Japanese patients with severe periodontal disease. And in a very recent study that came out at the end of last year, it looked at 19 patients with rheumatoid arthritis versus 30 healthy controls and found that the serum TNF level was the only independent variable to predict probing death, attachment loss of bleeding on probing. Again, a small study, not many controls, but again, since the suggestion of an association with TNF with the extent of periodontal disease.

Slide 20 (“Infliximab Treatment Does Not Improve Gingivitis”)

That was followed, however, with a study at the end of last year that showed that infliximab treatment did not improve gingivitis and this was a study from the Journal of Periodontology. It has a lot of weaknesses but some interesting data that came out of it. They took 40 RA subjects, 20 of whom had been treated with infliximab, 20 who had not been treated with infliximab. They looked at the periodontal status of these infliximab-treated patients every six weeks. Nine patients who had not been treated with infliximab began being treated with infliximab, and they looked at those patients over the next nine months. Now, there are many weaknesses in the study. In the whole mouth they only evaluated six index teeth. They recorded the maximum values for many parameters and calculated a mean to give a periodontal index. What they found, though, is that periodontal disease did not appear to be affected by infliximab in this group versus this group. And in these nine patients they had pre and post data with infliximab. There was a decrease in attachment loss, but the periodontal disease remained constant. In the gingivitis and bleeding index in contrast to what you might expect with TNF inhibition, actually were increased in these nine patients. So this is, you know, a study that would argue against there being a role of TNF significantly with periodontal disease, but I think there are many limitations to this study that make it far from conclusive in terms of this activity.

Slide 21 (“Shared Mechanisms of Disease in PD and RA”)

So turning back to the general paradigm of RA versus periodontal disease, we can really line up many of the mediators that we have seen in many of the cell types. And if you look histopathologically, this is a patient actually with periodontal disease here with an intense neutrophilic and mixed cellular infiltrate and even some small lymphoid follicles that appear to be developing in the gingiva, very similar to what we might see in a rheumatoid joint.

Slide 22 (“Rheumatoid Arthritis ? Periodontal Disease”)

Now, these relationships that have been described epidemiologically raise the question of which comes first, the RA or the periodontal disease. And the traditional paradigm was that RA preceded the development of periodontal disease. But I think that it's far from conclusive, and I think it's time for us to potentially begin thinking in the other direction.

Slide 23 (“Prior Studies of RA and PD”)

So problems with many of these other studies that have been case-control and otherwise – they have only looked at patients with established longstanding rheumatoid arthritis. There has been very poor characterization of RA disease activity. The influence of medications and other comorbid medical conditions has been unclear and uncontrolled. There has been poor characterization of any type of functional ability for the RA patients, especially hand function. There has been no control for Sjögren's, and they have been poorly controlled for smoking.

Slide 24 (“Old Hypothesis: RA is Risk Factor for Periodontal Disease”)

Again, this was the old hypothesis that RA came first. Patients with RA had poor oral hygiene because they couldn't grip their toothbrush and brush well or patients with RA had TMJ joints that were affected and couldn’t open their mouth enough to be able to brush their teeth. Or patients were immunosuppressed or received NSAIDs that could be risk factors for the development of periodontal disease or they had secondary Sjögren's. But none of these were proven in any way.

Slide 25 (“New Hypothesis: Periodontal Disease is a Risk Factor for Rheumatoid Arthritis”)

But I would like to take us back to what we've learned from the pathophysiology of the disease and say that perhaps periodontal disease is what comes first, before the rheumatoid arthritis. And we have seen that there are similar genetic susceptibilities, at least in terms of HLA-DR4 by two studies and some of these other polymorphisms that have been described. We see similar lesions in the inflammatory focus. And we also know that other bacterial infections can directly cause inflammatory arthritis, either by molecular mimicry or by other mechanisms, including rheumatic fever, lyme disease and reactive arthritis.

Slide 26 (“Questions”)

So as I started looking at this, and my entry into this area actually came from clinical concerns and clinical observations while I was in New York at Bellevue where we saw a lot of patients who had very low socioeconomic status, many patients very, very poor oral health, and it was at the time when TNF antagonists were just coming to the market. I had extreme concerns about whether or not these drugs would be safe in patients who had significant periodontal disease. I was worried about the risk potentially of these patients developing endocarditis or other abnormalities. But because of the extent of the arthritis in the patients, we had to really bite the bullet and treat their arthritis. And in fact we didn't see patients that were coming down with endocarditis or getting these overwhelming dental abscesses. And in fact, if anything, it appeared that some of the gingival inflammation was improving. So I started working with dental colleagues at NYU School of Dentistry, then I moved down here to Johns Hopkins and began collaborations with the group over at University of Maryland. And as I thought through this issue of periodontal disease and RA, these are the types of questions that started to come up. I told you that most of the studies that have been done were looking at patients with established rheumatoid arthritis. They were cross-sectional evaluations of patients which really tell you nothing about cause and effect. So it seemed to me that one of the most important things to study was a group of patients with incident rheumatoid arthritis to find out if periodontal disease was present at the beginning of the disease and then what happened to patients over time as their rheumatoid arthritis progressed. I also wondered if patients with rheumatoid arthritis and periodontal disease would have more severe rheumatoid arthritis and if patients with RA have laboratory evidence

of exposure to oral pathogens or did patients with periodontal disease have antibodies that are associated with RA. And sort of the ultimate clinical question is can careful attention to oral health improve the outcomes for patients with RA and potentially does periodontal disease contribute increased cardiovascular risk to patients with rheumatoid arthritis.

Slide 27 (“Do Patients with Early RA have Increased Periodontal Disease?”)

So if we go to the first question – this was do patients with early have increased periodontal disease.

Slide 28 (“Periodontal Assessment in RA Study (PARAS, R03 DE018094)”)

This takes me to an R03 that was funded that we called the periodontal assessment in RA study that continues to be ongoing. We looked at patients who are ages 18 to 75 who had definite or probable RA. RA was diagnosed within 12 months. One of our inclusion criteria was that patients have at least 20 teeth in their mouths that we could score, and this became a problem for us in terms of patient recruitment. Exclusions were other forms of inflammatory arthritis, other topical or systemic antibiotics, medications that may be associated with gingival hyperplasia, contraindications to probing such as coagulopathy, valvular heart disease, prosthesis and other conditions requiring antibiotic prophylaxis for dental procedures. We didn't exclude these patients unless they were unwilling to receive standard prophylaxis. So the patients could come into the study. We gave them antibiotics before we did any of the probing; even though the probing that we're doing should probably not cause bacteremia, we always wanted to be on the safe side.

Slide 29 (“RA Characteristics Explored”)

The RA characteristics that we explored included RA severity and extraarticular factors that have been looked at with the ESCAPE cohort, RA duration, rheumatoid factor, CCP status, nodules, damage. We also looked at Ro/La in this population with Antony and Livia and the group upstairs. We looked at Schirmer's test in patients. We measured measure of RA disease activity. We looked at treatment, and we also evaluated comorbidities, alcohol, smoking, aspirin use and other habits.

Slide 30 (“Dental Characteristics Explored”)

We also explored multiple measures related to dental health, including a dental history of tooth loss, oral hygiene, brushing, flossing habits, cigarette smoking and alcohol use and we designed several periodontal questions that we gave to patients looking at bleeding gums, swollen gums, halitosis, pockets and gaps between teeth.

We asked standard six-question Sjogren's questionnaires for oral dryness and ocular dryness and we conducted a dental examination looking at all of the teeth, six sites on every tooth with multiple measures, and we have begun to evaluate unstimulated salivary flow and collected saliva from patients as well.

Slide 31 (“Periodontal Examination”)

The periodontal examination that we're performing is an overall oral assessment. We are examining all teeth except the wisdom teeth. We're measuring at six sites on every tooth the following indices: plaque, gingivitis, pocket depth, distance from the edge of the gum down to where the bottom of the

pocket is, the amount of recession or attachment loss, bleeding on probing, tooth mobility and this issue of furcation where you stick the probe in and see if it can get underneath a root.

Slide 32 (“Periodontal Examination”)

This is what the probe looks like in the teeth. It's non-painful, and people really don't complain too much. But you go around six sites on every tooth. One, two, three on the front and one, two, three on the back. And then the probe had gradations in millimeters, and you can measure how deep the probe goes down into the pocket and get these different measurements. The exam itself takes about an hour to an hour and a half for patients to conduct. So it is an intensive experience for them. We have been doing this on a standard examination table, which in patients with RA can be quite uncomfortable, but nonetheless we have managed to do it. But it has presented an additional challenge to the study. So again you might have had this done by your own dentist, going around doing a periodontal examination on you. But this is the probe going into the pocket and showing you how with moderate periodontitis there is this attachment loss that begins where this probe goes deeper into a pocket and you begin to get exposed roots and exposed parts of the teeth that form.

Slide 33 (“Cartoons: Healthy/Gingivitis/ Moderate Periodontitis/Severe Periodontitis”)

Slide 34 (“Definitions”)

The definition that we used for the initial study was one that had been used for other studies of periodontal disease. There was no real well-established definition at the time we started with this for periodontal disease, but we looked at patients and called them diseased if they had at least 20 teeth and had at least 4 sites with pocket depths greater than 3 mm and 4 sites with attachment loss greater than 3 mm. And this definition approaches that of the NHANES III definition that was used in looking at periodontal disease. And we also looked at patients sequentially every six months for periodontal progression, and we set up a definition for this as well.

Slide 35 (“Oral Health Assessment in RA Patients”)

This is information on the first 26 patients that we have evaluated breaking down patients between those with early rheumatoid arthritis, and we also looked at a group of patients with established rheumatoid arthritis. And you can see that the difference in disease duration is shorter disease in the patients with early RA. The CRPs were a little bit lower in the group of patients with early RA, probably because there is a little bit more steroid use in these patients that we were just seeing. The tender joint counts were higher in the early rheumatoid arthritis patients. The Das values were not statistically significantly different, but the numbers of patients remained somewhat slow – low – with this preliminary data.

Slide 36 (“PD is Equally Present And Severe Early And Late In The Course of RA”)

We looked at the periodontal parameters. The numbers of teeth were similar. The number of patients who met our definition for periodontally diseased, 65.4% overall, 54.5% in the early and 73.3% in the established. And I remind you that based on the NHANES III definition, the expected value in the population would be about 35%.

Slide 37 (“Conclusions”)

Looking at a more stringent definition defined by the CDC in 2007 for moderate disease and severe disease, our numbers have increased to 38% with moderate disease and 26.9% with severe disease. And again, the expected levels for these would be somewhere around 10% and less than 5% in the general population. What you notice from here is that the parameters related to periodontal disease are all very similar between the two groups with no statistically significant difference except for bleeding on probing and number of sites. And I bring you back to this issue of furcation. Over 80% of patients we are able to probe down and get to the level of a root of a tooth, indicating pretty significant periodontal disease in most patients that participated in the study. And there was really no screening for patients. We asked patients as they came through the clinic, would you be willing to participate. Probably there is a bias because I actually look at people's teeth and think about this. But it's – nonetheless, I think that these data strongly suggest that this phenomenon takes place in early disease as well as with established disease. So I think the conclusions from the first part of the study are that periodontal disease is both prevalent and often severe in patients with RA. And the characteristics of the periodontal disease are also similar between patients in early and established disease.

Slide 38 (“Do Patients with RA and Periodontal Disease have More Severe RA?”)

So that brings us to question number two: Do patients with rheumatoid arthritis and periodontal disease have more severe RA? That is, does periodontal disease influence the level of rheumatoid arthritis?

Slide 39 (“Hypotheses/Objectives”)

Our hypothesis was that periodontal disease and oral symptoms would be common in RA patients, and we already – we had shown this in terms of the previous work. But what we wanted to do was to answer this question as quickly and easily as we could with a larger group of patients.

Slide 40 (“Methods”)

So we elected to move forward administering questionnaires, asking about periodontal disease to patients to try to get some idea concerning the relationships with rheumatoid arthritis. We had an available population of patients with the ESCAPE cohort, and we administered questionnaires regarding periodontal disease and oral health to these patients at their second visit.

Slide 41 (“Escape RA”)

So we took patients from the ESCAPE cohort, 183 patients took our questionnaires. We got all of the information that I showed you before that we were looking at in the patients who we were also doing examinations on. We analyzed RA disease characteristics and their oral health parameters, and we did a validation study in the 33 patients that we had evaluated with oral examinations and also given the same questionnaires to.

So ESCAPE we've heard about from Joan and John multiple times in the past, but just to remind you, these are patients aged 45 to 84. It was a cohort to evaluate the prevalence and progression of subclinical cardiovascular disease in men and women with RA.

Slide 42 (“Methods: Analysis”)

We looked at univariate associations for RA disease characteristics with periodontal characteristics. We calculated prevalence ratios and generated simple and multivariable models that were adjusted for multiple confounders between periodontal symptoms and oral symptoms.

Slide 43 (“Characteristics Explored”)

The characteristics explored were, again, were the same as what we had done with our early RA cohort.

Slide 44 (“RA Disease Characteristics (n=183)”)

And here were the characteristics of the ESCAPE group. Disease duration of 10.8 years, relatively low levels of disease activity with low swollen and low tender joint counts. HAQ scores also in the low range.

Slide 45 (“Oral Health Characteristics”)

But the oral health characterization from patients with self-reported questionnaires – periodontal symptoms – 78.7% of patients. Specifically, bleeding of gums with brushing – 67% of patients. Self-reported swelling of the gums in 18.7%. Bleeding or swollen gums in 52.5%. Oral dryness being reported by about 35.5%. Schirmer test positive in about 43% of these patients. Ro/La positive only in 7%.

Slide 46 (“Univariate Associations Between RA Characteristics and Gum Bleeding/Swelling”)

And we looked at univariate associations between the RA characteristics and whether or not patients had gum bleeding or gum swelling. Here are the patients with no gum symptoms. Here are those with – who reported frequent gum bleeding or swelling. And here are those that reported both frequent gum bleeding and gum swelling. And we looked and found that nodules, disease activity, swollen joint count, tender joint count, HAQ and physician global were all statistically significantly associated with patients' self-report of periodontal symptoms.

Slide 47 (“Associations of RA Disease Activity (DAS28) with Periodontal Symptoms”)

The only characteristics that remained associated after controls, though, were rheumatoid nodules in DAS28. When we looked further into the relationship between dAS28, here again with any periodontal symptoms, with swollen gums and with bleeding gums and we broke out DAS28 levels by patients with high disease activity, moderate disease activity and low disease activity, we found what looked like a dose-response relationship between the presence of periodontal symptoms and disease activity with RA. We adjusted for gender, race, age, education, smoking, alcohol, brushing, flossing and aspirin use. We also looked at all of the other variables that are listed here, and none of them were significant.

Slide 48 (“DAS28 Scores According to Combined Gum Bleeding, Swelling, and Oral Dryness”)

When we looked further into this relationship and threw oral dryness into the equation, it appeared that patient report of oral dryness further increased the disease activity for rheumatoid arthritis. So these are patients now reporting whether or not they had these periodontal symptoms of bleeding, swelling of their gums and now reporting whether or not they had dryness in their mouth, and there appeared to be an additive effect on top of the effect from the bleeding and swelling in terms of relationship with RA disease activity.

Slide 49 (“Periodontal Symptoms Account for 22% of variability in DAS28”)

In further modeling, John has shown that the periodontal symptoms account for about 22% of the variability that's associated with the DAS28, which is a pretty high level of association. And this was with multiple controls.

I'm not going to go into the data for that. I'm going to move on to some additional data that we have from the study. The data that I presented before that last one I presented is an oral presentation at ACR.

Slide 50 (“Validation of Periodontal Self-Report”)

Now these were self-reported data, and you would raise questions about how good is self-report in relating to the actual examination of patients. We didn't do the exams on the patients from the ESCAPE cohort, but we had been doing examinations on other patients with rheumatoid arthritis who completed the same questionnaires. We did a validation study initially based on 26 patients and then increased up to 33 patients. And we found that the odds of patients having periodontal disease in those reporting periodontal symptoms was 2.3 times higher than those without symptoms. Our simple questionnaire gave a specificity of 0.75 and a positive predictive value of about 0.6. So the questionnaires, although they did not detect all of the cases, they provided some indication of a true association with periodontal disease.

Slide 51 (“Self Reported Gum Bleeding with Examination”)

If we look at self-reported gum bleeding with the examination for bleeding on probing, we found that there was a significantly – statistically significant relationship between these. So patients can report – if the patient says their gums bleed, we stick a probe into their mouth, they bleed – they do a pretty good job.

Slide 52 (“Correlation of Gingival Bleeding with Periodontitis”)

And then we look at the correlation between gingival bleeding and periodontitis. We see a very nice relationship that is highly statistically significantly different. So even though in this group of 183 patients we haven't done periodontal examinations, we do have some evidence that the questionnaires that we have administered do have a relationship with the ultimate question of periodontal disease being there or not.

Slide 53 (“Limitations of Study”)

So the limitations of this part of the study, obviously, are that these are questionnaires and self-reports. The patients that I showed you had longstanding rheumatoid arthritis, and their disease activity was relatively well-controlled.

Slide 54 (“Conclusions of this Study”)

So the conclusions from this study – in a large cohort of patients with established RA there was a high prevalence of self-reported periodontal symptoms consistent with our earlier report based on oral examination. The questionnaires had a moderately high positive predictive value to detect periodontal disease. And periodontal symptoms were associated with increasing RA disease activity, even after adjustment for multiple confounders. And oral dryness appeared to be an additive factor associated with RA disease activity.

Slide 55 (“Untitled: Questions”)

So now we have early disease looking at actual examinations compared to later disease. We have a large cohort where we've tried to evaluate these questions related to periodontal disease and RA disease activity. And then the next questions that I told you about were trying to understand the mechanism between these two disease processes. The question is, do patients with RA have laboratory evidence of exposure to oral pathogens and do patients with periodontal disease have antibodies associated with RA?

Slide 56 (“Citrullination”)

And this comes back to the issue of citrullination. I told you that P. gingivalis was a unique organism in the mouth and that it has an endogenous peptidylarginine deiminase enzyme. And as we know, citrullination is one of the critical first steps in the development of rheumatoid arthritis where we have markers detected as antibodies against citrullinated peptides, a disease specific marker that also correlates with disease – with damage.

Slide 57 (“Citrullination”)

Citrullination is the conversion of a charged arginine residue to an uncharged citrulline residue, which is thought to lead to secondary and tertiary conformational changes in proteins, potentially forming neoepitopes. In rheumatoid arthritis numerous putative targets of citrullination have been identified, including fibrinogen, vimentin, enolase and even PAD4. And we know that there are five PAD4s in the human. We know that PAD2, PAD4 and PAD6 are expressed in RA synovium. We know that PAD4 polymorphisms have been described in some RA patients. We have also recently learned that PAD2 is upregulated with cigarette smoking. And I've told you that P. gingivalis also has an endogenous PAD. So why in the world would a bacteria had a peptidylarginine deiminase as one of the enzymes that it has? Well, it's felt that the reason that P. gingivalis has this is that one of the things that happens through this conversion of arginine to citrulline is the release of ammonia. And I remind you that in the mouth one of the NA immune defenses that we have is acidification of the environment of the mouth, which is typically inhospitable to bacteria. Now around the base of a tooth you think about the microenvironment there, it would be very advantageous for a bacteria to be able to neutralize acid. So if you can imagine a bacteria that is able to citrullinate proteins that are readily available, therefore generating ammonia, it may provide a more hospitable environment for it to evade host defense. So very interestingly, we have also learned that the process of citrullination at least

through human PADs can also inactivate certain chemokines that could lead to neutrophil recruitment into the area. So again, two potential mechanisms for host defense evasion from the bacteria through a PAD.


In the space around the tooth, the environment is normally acidic.

COMMENT: Okay, so my instinct is if saliva is alkaline and loss of saliva gives you rampant dental cares, that alkaline is good. I've always had that sense that alkaline is good, so if alkaline is good, then maybe be the ammonia from the [inaudible] isn't necessarily due to pH change but due to something else. I think the mechanism I would leave somewhat open as to [inaudible].

Yes. Again, this is one of the hypothesized mechanisms. I think that there are many other things that don't – that are a little bit atypical as well between Sjögren's and periodontal disease. Most of the studies that have actually been done looking at Sjögren's and periodontal disease at least with primary Sjögren's, not secondary Sjögren's, have shown that there is not really a relationship between the two, although there have been some suggestions in the RA population with secondary Sjögren's that there may be an effect. So – and I'm not quite sure how it all goes together. But at least from the dental literature, this has been the hypothesized mechanism for this issue of changing pH and making a more hospitable microenvironment.

Slide 58 (“Citrullinated Peptides in the Pathogenesis of RA”)

So when we put this all together in terms of the pathogenesis of rheumatoid arthritis, we have these PADs that are here. We have these human PAD mutations. We have innate immune responses and other things that can activate human PADs. The process of citrullination releases autoantigens that turn on T cells, autoantibody formation and other cells in the microenvironment.

Slide 59 (“Roles of Periodontitis and PADs in Citrullination”)

And then we throw in periodontitis into the mix. We think about smoking, which could be a trigger for periodontitis, potentially a trigger for turning on human PAD, such as PAD2. Periodontitis activating and releasing IL1 and TNF, which may also act to stimulate human PADs and periodontitis acting through LPS and toll-like receptors, which could potentially have an additional impact on human PAD regulation. P. gingivalis specifically having its own peptidylarginine deiminase. And P. gingivalis also having gingipains, which make many of these proteins potentially more susceptible to cleavage by other proteases. And then we have a whole panel of different putative citrullinated substrates that have been implicated for rheumatoid arthritis.

I point out here this bacterial enolase again. The group from London has shown that human enolase, alpha enolase, is a putative autoantigen for patients with rheumatoid arthritis. They found that the site that becomes citrullinated in this human enolase has shared sequence hemology with bacterial enolase that is made by P. gingivalis. They have also found that the response to and generation of autoantigens against these enolase products are very highly MHC restricted, not just within the shared epitope but with specific ileals. So it indicates that there may be a very fine specificity between what becomes citrullinated and what becomes neoantigenic in specific individuals. I think this is where I

would say based on this entire hypothesis, that periodontitis could play in. We really don't know if it's this mechanism, if it's this mechanism or if it's some other mechanism between the two.

Slide 60 (“Citrullinated Proteins are Detected in Saliva from Patients with Systemic Autoimmune Diseases”)

The question becomes do we see any evidence for citrullination in patients with periodontal disease and in patients with rheumatoid arthritis in the mouth.

Slide 61 (“Logistic Regression for Periodontal Disease with RA Parameters (n=26)”)

This is a blot from Felipe. I appreciate his letting me use it for the grant in terms of preliminary data. This is saliva just collected from patients with RA, psoriatic arthritis and Sjögren's syndrome run out on a blot and then probed with the AMC citrullination detection kit. And what you see is here is a positive control of neutrophils stimulated with calcium and ionomycin. And here is saliva from patients with various autoimmune conditions showing citrullinated substrates across the board. So certainly in saliva we detect citrullinated proteins, in some more than others, the identity of which are not clear. I'll also mention that we have recently done some additional experiments with Ginny VanIke's group that have shown that this method of just taking saliva and running it out on a gel, if you don't put in protease inhibitors immediately after the saliva is obtained, you will lose a lot of what used to be there. So we now have modified our procedure for saliva collection to include a cocktail of protease inhibitors to try to stabilize what we're looking at so we can evaluate it further in the future.

Slide 62 (“IgG Anti-CCP Antibodies are Associated with Antibody responses to P. gingivalis”)

Now, going back to the data that we had from our small group of patients in whom we had periodontal examinations and serum collected, we looked at the relationship between periodontal disease and various RA parameters. Now here are the three different definitions that we use for looking at periodontal disease. Our definition that we established – which is about the NHANES III definition, the CDC definition for moderate disease and the CDC definition for severe disease. Again, this is a small number of patients that this data is taken from. But what we see is that anti-CCP antibodies with our definition of any periodontal disease are extremely high. Fortunately, this approached significance, but with the small numbers it still was not statistically significant. But this level of effect was even stronger than that attributed by cigarette smoking, which is the major risk factor known to be associated with periodontal disease. So we really thought that this was something that was highly suggestive of a relationship between anti-CCP antibodies in the presence of periodontal disease.


Yeah, in the ESCAPE cohort the relationship didn't pan out, but again, based on symptoms.


The shared epitope and anti-CCP antibodies had no effect on the relationships. It was independent.

So we also looked at a random sample of RA patient serum to find out if we had evidence of exposure to periodontal bacteria. So we divided these patients between patients who were CCP positive and

CCP negative. There were an equal number in each group. Serum was batch-shipped up to NYU, where an antibody assay is available to look at antibodies against P. gingivalis using an ELISA test that was there. We found an odds of 2.67 for having anti-CCP antibodies along with anti-P. gingivalis antibodies. Now, recently Ted Mickels in the group from BRASS with Jim O'Dell showed a relationship as well between the presence of IgM and IgG2 subclass anti-CCP antibodies with antibodies against P. gingivalis. But this relationship didn't hold out with total IgG antibodies against P. gingivalis as we had detected here. But at least from our preliminary data as well as from this group there seems to be a consistent association that shows that antibody responses against periodontal bacteria and antibodies against citrullinated peptides appear to go together in patients with rheumatoid arthritis.


I'm sorry?


In terms of the….


And I think that's one of the limitations of the work that we've done at least in our original study on our patients, is we didn't look – we used whatever was available from the patient's medical records for CCPs, which were from various assays and from various labs because we didn't have funding to look at CCPs ourselves within the study. But I think it does get into this issue of some of how you are testing for these, but I did find it interesting that the level of association that they describe and the level that we saw were quite similar.

Slide 63 (“PAD-4 is Detected in Periodontal Tissue”)

We have also begun to look at periodontal tissue itself in collaboration with our colleagues over at the University of Maryland. Some of the very preliminary data that we have we have stained to see if PAD4 is expressed and upregulated in periodontal tissue. And I thank Livia for her work on this basically looking at staining for PAD4, here visualized by brown staining in the periodontium. And one of the things that Livia said is that PAD4 is not as abundantly expressed as it would be in the synovium or other places where it has been upregulated. So the question becomes are we dealing with a reason for citrullination in periodontal disease, is it due to endogenous upregulation of the PADs as we might hypothesize in rheumatoid arthritis or might it be due to one of these exogenous causes, such as the PAD obtained from the bacteria. I think we don't know. But it does imply that at least citrullination is taking place in the microenvironment of the periodontium, and we would expect to see citrullinated proteins that are present as well, and those studies are ongoing.

Slide 64 (“Summary”)


This was our preliminary data that was used for the grant, and that's one of my specific aims. So we also submitted an R21 that will be going in for reconsideration and one of the specific aims

specifically is to look with multiple methods in addition to immunohistochemistry at the range of PAD expression in the tissue and the range of citrullinated proteins.


That's a good question, and that's another future specific aim for another grant that would look at a well-characterized periodontal cohort of patients because nobody has looked at anti-CCP antibodies in overall patients with periodontal disease. Is that what you're –


Well, my reasoning would be is because we don't all have the appropriate HLA-DR4 shared epitope that would then make this something that becomes a true neoantigen.

Slide 65 (“Further Studies”)

In terms of further studies, comprehensive oral assessments of RA patients and match controls are underway to confirm the self-report findings that I showed you before. The grant that was funded by the Arthritis Foundation will allow us to go into the ESCAPE cohort now and perform periodontal examinations on all of those patients. Obviously evaluation of the mechanistic underpinnings of the disease relationships is also in progress. And I think this is the question that nobody knows – whether aggressive management of periodontal disease will result in improved RA outcomes.

Slide 66 (“Clinical Studies”)

The clinical studies that are ongoing include the continued longitudinal evaluation of the early RA patients with sequential exams to look at progression of periodontal disease, oral exams of the ESCAPE patients, determining the effect of RA related medications on PD parameters, further evaluation of some of these relationships between Sjögren's and periodontal disease. I have also given the questionnaire to Nancy Shaddock, who has administered it to over 1,000 RA patients in the BRASS cohort of RA patients cross-sectionally, so we'll be able to have a confirmation cohort for our data from the ESCAPE set. We have gingival tissue collection ongoing for patients undergoing periodontal surgery, hopefully identifying patients with RA that have severe enough periodontal disease that would be willing to have periodontal surgery done over at University of Maryland, where we can obtain their tissue. And ultimately, one would like to design interventional studies to determine the effect of treatment.

Slide 67 (“Current RA Patient Recruitment for Oral Health Assessment”)

So far with the study we've screened 97 patients. We have enrolled 56, and we've completed examinations on 38 and a second exam on 10. And this will be ongoing now with funding.

Slide 68 (“Treatment of PD Improves RA Disease Activity”)

I put up one recent study that came out actually just last week of a treatment study of periodontal disease in rheumatoid arthritis patients. It's a very strange study. It took 40 patients with RA, all of whom had generalized severe periodontitis. So how they found these patients, I don't – you know, they obviously must have a pretty large RA population. But I think it does speak again to the

prevalence of severe periodontal disease in RA patients if they could identify 40 patients over a year and a half to do the study. Twenty of the patients were on DMARDS. Twenty were on DMARDS plus TNF. And then in each group they were randomized either to get treatment for their periodontal disease or to wait six weeks, and the looked at the periodontal exam again after six weeks. There was a reduction in sed rate in DAS28, in periodontal parameters with the periodontal treatment over six weeks. An interestingly, there was a reduction in baseline – in that should not be baseline TNF levels – but a reduction from baseline TNF levels in 40% of the patients who received the periodontal treatment versus 20% of those who had not been treated, even in the patients receiving TNF antagonists. I think this is very intriguing data. Again, it's a small study, but it does hint at the possibility of doing an interventional study looking at RA with periodontal disease.

Slide 69 (“Triangulation of Inflammation”)

Now I finally come back to this diagram that looks at this what I call now a triad of inflammation where we have rheumatoid arthritis and periodontal disease that we have been working with where we have the opportunity to work with the RA cardiovascular disease link that Joan is working with and begin to put these three diseases together potentially for a future grant application.

Slide 70 (“Mechanistic Studies”)

The mechanistic studies from this REF grant – we're going to be looking at bacterial PAD expression and trying to understand how this differs from human PADs. For an R21 we're looking at gingival tissue, crevicular fluid and salivary expression of PADs and citrullinated proteins. And looking at antibodies to bacterial flora versus the colonization patterns which give a little bit better idea of exactly what's taking place in the mouth of an individual.

Slide 71 (“Untitled”)

So we go back to my original diagram, and now I think it can become a little bit more complicated, when we throw periodontal disease into the model. So we have cigarette smoking turning on PAD2. Cigarette smoking is a risk factor for periodontal disease. Periodontal disease through P. gingivalis potentially leading to upregulation of PADs and citrullination. Periodontal disease leading to an inflammatory disease state with TNF upregulation that may drive the upregulation of PADs. Periodontal disease itself activating innate immune responses and causing localized inflammation and immunologic activation.

Slide 72 (“Conclusions”)

And finally a system as we have with RA with the disease acting back upon itself with periodontal disease potentially acting back on itself as well. So potentially a way to put these two disease processes together.

Slide 73 (“Clinical Implications”)

The clinical implications of this, I think, are number one that oral health parameters needs to be monitored much more closely in patients with RA and with autoimmune disease. I think we'll find a lot if we start looking for it. And now we might have the opportunity to do that, not only for RA but

also for other diseases. And interventions to improve oral pathology may have direct and indirect systemic benefits.

Slide 74 (“Past (and Future?) Arthritis Treatment”)

I mentioned that until the 1930s one of the ways that rheumatism was treated was by pulling the teeth. And you wonder if people might have been onto something back then beyond the relationship with rheumatic fever.

Slide 75 (“Happy Teeth Make Happy Joints”)

So I put out for you maybe a future plug "Happy Teeth Make Happy Joints." And the control of your arthritis pain – brush more, hurt less.

Slide 76 (“Acknowledgements”)

So I'd like to acknowledge all the many people who have helped me up to this point, including everyone in the Arthritis Center, all the research coordinators, Antony, Livia, Felipe, Erika, my colleagues at University of Maryland, at NYU, including the late John Ship, who was really one of the great researches in Sjögren's syndrome who died last year, John Gunsolley – or Jack Gunsolley – who is now at Virginia Commonwealth, and then the multiple funding sources that have come into play for this work up to this point. Thank you.

Slide 77 (“Progression to Periodontitis”)

A couple of minutes of questions.

QUESTION: [Inaudible.]

It hasn't been – it hasn't been looked at well. But the associations when it has been looked at have certainly been stronger for RA than other diseases. I think we now have the opportunity as we get an infrastructure set up to do this obviously to expand beyond RA and look into other diseases, at least other autoimmune diseases, and obviously another inflammatory disease control would be ideal. One of the sets – the groups of patients that I wanted to use is sort of the ideal control that was in my original grant application that the NIH pooh-poohed a long time again and the second iteration of my R03 was to have a control group of patients with psoriatic arthritis who have inflammatory arthritis who should not have anti-CCP antibodies to see if the relationship holds or doesn't hold in that group of patients as would be expected.


I think some of the things that you can see again with aggressive periodontal treatment – you are not going to get bone growing back. That's never going to happen. But you can see an impact on some of the indices of gingival inflammation, for instance, that will go down, bleeding on probing that will go down again. So you're treating the inflammatory component. You can also, you know, some studies have shown improvement in some of the different pieces of periodontal disease with treatment. But it's more an issue of, I think, prevention of further progression because really with periodontal disease

once it enters to a certain state it does become really sort of an inevitable type of progressive response downward until the tooth is lost.


Right. So you may be able to, for instance, monitor radiographically over shorter periods of time and look for alveolar bone loss or not lost as one of the potential ways, as we do with Sharp scores in RA. It is one of the things that we have talked about and thought about that might be, you know, again sort of a way to judge a potential intervention.

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