How RA Begins: A Detective Story

What causes rheumatoid arthritis?

The search, by scientists around the world, has been exhaustive, and the prey surprisingly elusive – but in breakthrough Johns Hopkins research, scientists Maximilian Konig, M.D., and Felipe Andrade, M.D., Ph.D., believe they have pinpointed one culprit: a kind of bacteria that lives in the mouth and is present in gum disease.

This form of bacteria has an unpronounceable name, Aggregatibacter actinomycetemcomitans, so the scientists who have discovered the terrible and important role it might play in RA just call it by its initials: Aa. “We are very excited about it,” says Konig. If their hypothesis is verified by other scientists and in larger studies, the possibilities of preventing RA from developing, and of treating early RA and possibly reversing symptoms, are enormous. This work was presented at the Plenary Session of the American College of Rheumatology’s Annual Scientific Meeting in November 2016, and I recently wrote about it for Leap magazine, a publication of the Johns Hopkins Division of Rheumatology.

The idea itself – that RA, like tuberculosis, anthrax, or pneumonia, is caused by a bacterial infection – is not new. “For a long time, people thought that the disease did not start in the joints,” says Andrade, who is a rheumatologist and immunologist. Back in the days of Louis Pasteur, in the early days of microbiology, scientists “strongly suspected that RA was like rheumatic fever (caused by streptococcal bacteria), in that it was at least triggered by an infectious agent,” says Konig, who did a postdoctoral fellowship in Andrade’s lab and now is a resident physician at Massachusetts General Hospital.

But nobody could prove it. Unlike other diseases, in which the time between exposure to a certain pathogen and development of symptoms takes days or weeks – so it’s easier to make the link between cause and effect – the gap in RA between exposure to something bad and actual illness could be a matter of years. Also, scientists “didn’t know what they were looking for, because there was no understanding of what kind of disease RA is, and how it works immunologically.”

Within the last 20 years, scientists at Hopkins and elsewhere have made huge leaps in understanding what happens when the immune system attacks the body in RA. They found strange proteins in the joints of people with the disease. These proteins are modified –“as if they are wearing a costume or makeup,” says Andrade. The modifications are what scientists describe as citrullinated; they’re decorated at the molecular level. Imagine someone wearing a wig, a fake mustache and spectacles – in short, a suspicious-looking character. The immune system errs on the side of caution, decides these shady-looking proteins are up to no good, and attacks them.

“A lot of the field, including us, has focused on what it is that changes the structure of these proteins to let them be altered in this chemical way,” says Antony Rosen, M.D., Director of the Division of Rheumatology and Vice Dean of Research at Johns Hopkins. “The alteration is catalyzed by PADs,” enzymes found in inflammatory cells, “and when they’re activated, they change the structure of the molecules into something that is seen as a threat by the immune system.” The antibodies the body makes in response to this protein mutation are very specific. From long-term studies of military personnel, scientists learned that these citrullinated proteins, and antibodies to them, can show up in the blood as early as 14 years before someone develops the first symptom of RA. “We always believed that if we understood what drove it, we would understand what turns on and drives RA.”

So here’s the chain of events so far: the enzymes change the proteins, and the immune system makes antibodies against the interlopers. The antibodies cause inflammation, “which causes the damage that leads to this disabling and destructive joint disease,” says Konig.

The Link to Gum Disease

But what activates the enzymes? What starts the whole process?

This brings us to the mouth, and you may be wondering why. It’s because since the early 1900s, doctors have noticed that people with bad RA tend to have severe periodontal disease (gum disease). “That was something that was quite puzzling,” says Konig, “but doctors didn’t focus on this observation. They thought maybe patients were not brushing their teeth properly because of their disabling joint pain.”

Over time, scientists began to suspect that there was something in the gums that might trigger both the periodontal disease and the RA – here’s the caveat – in someone who has a genetic susceptibility, a faulty gene that’s involved in how the immune system recognizes enemies.

Clearly, not everyone who has poor oral hygiene gets gum disease, and not everyone who develops periodontal disease gets RA. “I think you need to have both,” says Konig: “to have the right genetic background to be susceptible and to be infected with a bacterium that can initiate the production of these abnormal proteins. And probably a dose of bad luck, too. Only then will you develop the antibodies that are involved in the sustained immune response.”

Over the last decade or so, scientists have been looking for answers in the microenvironment of the gums in people who have periodontal disease. Scientists in England were convinced that they had found a likely candidate, a bacterium called P. gingivalis. “People believed that this might contain a unique enzyme that could generate citrullination in the gums,” says Konig. In Andrade’s lab, he started investigating this. “Felipe and I didn’t have any preconceived ideas about it. But whatever we tried to do, whenever we tried to replicate the data that proposed to be the link between P.gingivalis and RA, all our experiments were negative. We could not replicate it. When we tried to understand more mechanistically whether this made sense, we came to the conclusion that even though this was a beautiful story, the biology tells us that this is likely not the way things work.”

“Max did elegant work,” says Rosen. “He purified the bug, made the enzyme, set up an assay to show whether it made RA antigens, and showed that it was not possible. The P.gingivalis PAD does not create RA antigens; it actually turns itself off and doesn’t work against self-proteins,” the proteins that are targeted by the antibodies. In fact, Konig and Andrade published a paper strongly suggesting that the P.gingivalis theory was incorrect.

Konig and Andrade were frustrated; in fact, Konig notes, “Felipe said, ‘That’s it, we’re dropping it. We are never going to work on gum disease and RA again.’” But Konig couldn’t let it go. “I was a little obsessed with the idea that we were missing something.” At that time, Felipe had already published some work about how RA in the joint behaves. He realized that probably one of the most important points in the story is an immune system cell called the neutrophil.”

Neutrophils are white blood cells. They rush to the site of an infection, eat microorganisms – think of Pac Man here – and release germ-killing enzymes. In the joints of people with RA, neutrophils turn on their PADs and generate a huge amount of the RA autoantigens (targets against which the immune system makes antibodies). In striking work, Andrade found the step that turns on the citrullination: in the joints of someone with RA, there are pathways that punch temporary holes in the membranes of the neutrophils, and this is what causes the many small changes in the molecules – which, in turn, stimulate the production of antibodies.

Think about crabs in the sand, scuttling around, making tiny holes that fill up again very quickly. These holes in the neutrophils are like little perforations that weaken paper – but unlike those in perforated paper, these holes may last only seconds. That tiny flash of time is enough for calcium to pour into the neutrophil. The calcium activates the enzymes that cause the citrullination. This overwhelms the immune cell processes, the neutrophil dies and spits out the “dressed up” proteins into the joint.

Konig believed that something in the mouth was punching the holes in the neutrophils, and must be the cause of the citrullination. “I was at the lab meeting when he came up with this proposal,” recalls Rosen. “I thought, that’s really clever, but what are the chances of finding it? But he went and searched for bugs in the mouth, for anything that would open a hole in a neutrophil membrane.”

In gum disease, Konig explains, “you have a very rich inflammation that is full of neutrophils and bacteria in the periodontal pocket,” the diseased area of gum around a tooth. “It is very important for the immune system to be able to fight off these chronic pathogens in gum disease. Maybe a form of bacteria in gum disease fights back by poking holes in the neutrophil.”

After months of hard work, and with support from the Jerome L. Greene Foundation, Konig found Aa. “It makes a specific toxin, called leukotoxin A,” he says. “This toxin is a bacterial pore-forming toxin.” In other words, it pokes holes. Perhaps “someone with chronic gum inflammation has this specific bacterium, Aa, that makes this toxin that binds to neutrophils and creates short-acting holes, and that creates these autoantigens.” Add chronic infection with this particular form of bacteria with a ready entry into the body – through the gums – to having the genetic susceptibility, and it is likely that “this initiates the disease.”

Says Andrade, “we developed a diagnostic assay to identify people who have been infected by Aa, and found that as many as 47 percent of RA patients have evidence of this infection. The association is extremely strong.”

Where Do We Go From Here?

What does this mean? First, says Andrade, “it means that if our work is confirmed by others, this bacteria could be the closest thing to the cause of RA, something people have been seeking for many years.”

Now, what does it mean beyond that? As excited as the scientists are by this work, by the brilliant idea and the elegant science, they live day and night with the knowledge that RA is a devastating disease, and beyond anything, they want to help the people who suffer from it. They also hope to prevent further suffering, and this may be the first place to start if these results hold true.

One thought, Konig says, is that “you might be able to specifically kill these bacteria in a patient who already has RA, and maybe if you take the bacterium away, you could either stop the disease or ameliorate the severity of it. We already have markers (the antibodies the body makes against the citrullinated proteins) that identify people at risk of developing the disease.” These antibodies may be circulating in the blood a decade or more before the onset of disease. “If we could reach people before they have the disease – but who already have a marker for it, because they’re starting to develop antibodies – we could specifically treat the inflammation in the gums by inhibiting the bacteria, or use specific drugs that inhibit the utility and function of the toxin, and maybe prevent people from getting RA in the first place,” continues Konig.

It may even be that one day, dentists will identify patients with periodontal disease, and a simple swab test might be able to determine the presence of the antibodies – and get these people to a rheumatologist for early treatment.

It may also be that treating the Aa bug may help prevent further damage and make life better for someone with established RA.

“We screen for heart disease, for some cancers,” says Konig. “Why not screen for RA?”

————————————————-

RA and Modern Life

In Europe, before the 1800s, RA was virtually unknown. It was not written about in the medical literature, and it does not seem to have been the widespread problem that it is now (an estimated 1.5 million Americans have RA today). “Somewhere between 1800 and 1900, it became this large epidemic,” says Max Konig, “where you see RA cases and descriptions popping up everywhere.”

Why? Konig speculates that the rise in RA may have had something to do with the diet. “Sugar consumption increased by about 100-fold. Also, people started to smoke more.” Undoubtedly, as travel increased between continents, some new bacterial species were introduced, as well. Whatever happened, it resulted in a “shift in the microbiome of the gums.”

©Janet Farrar Worthington