Dr. Steven R. Little
Photo courtesy of Joshua Franzos
November brought a number of exciting research findings in oral health. One of the most interesting is an article in the Proceedings of National Academy of Sciences titled “Prevention of inflammation-mediated bone loss in murine and canine periodontal disease via recruitment of regulatory lymphocytes.” The paper presents a fascinating first step in treating periodontal disease by inducing a more-balanced immune response. The Science Spotlight recently spoke in depth about the paper with Steven R. Little, Ph.D., senior author on the paper and an NIDCR grantee. Dr. Little is a scientist in the McGowan Institute for Regenerative Medicine at the University of Pittsburgh. If you would like get your hands on the article, it appeared in the November 12 issue of PNAS. The authors are: Andrew J. Glowacki, Sayuri Yoshizawa, Siddarth Jhunjhunwala, Andrela E. Vieira, Gustavo P. Garlet, Charles Sfeir, and Steven R. Little.
You’ve spent most of your career exploring issues related to chemical engineering and materials science. How did you go from pondering mathematical equations to learning how to say Actinobacillus actinomycetemcomitans, the oral bacterium used in your studies to model the periodontal inflammation?
It was just a case of the right research disciplines, or really the right people, talking to each other at the right time. What happened is I attended a seminar here at the University of Pittsburgh about three years ago given by Dr. Gustavo Garlet, a dentist, superb immunologist, and now a co-author on this paper. Gustavo showed that in chronic periodontal disease, there is less FOXp3 expression. FOXp3 is a hallmark marker of Tregs, or naturally occurring regulatory T cells.
Tregs formerly were called suppressor T cells. Is that right?
Image courtesy of Randal McKenzie
Yes. Tregs help to turn down an immune response and make inflammatory cells tolerant of self. Gustavo’s point was as periodontal disease worsens, the prevalence of these key immune cells decreases.
The two have an inverse relationship?
Exactly. I remember thinking that if the Tregs go away in a disease state, what would happen if we bring them back? I raised the issue with one of my collaborators Dr. Charles Sfeir, an outstanding oral biologist at Pitt and a practicing periodontist. Charles, Gustavo, and I talked about it and thought the idea might be worth pursuing, and we already had a strategy in mind.
As a graduate student, I read a paper in the journal Nature Medicine back in 2004 that really stuck with me. The researchers discovered that certain ovarian tumors release a protein called CCL22, and they apparently do so to fool the immune system into accepting them as self.
What is CCL22?
It’s a chemokine, which is a fancy word for a common type of immune signaling protein. Certain immune cells, i.e., macrophages and dendritic cells, release CCL22 to induce the migration of other types of immune cells.
Such as Tregs?
Exactly. The ovarian tumors secrete CCL22 to attract the Tregs and, in essence, disguise themselves from immune recognition. It’s as though the Tregs vouch for the tumor and tell the immune system not to pay attention to their irregularities. As a graduate student, what was so fascinating to me is the tumors hadn’t invented a mutant super molecule that subdues the immune system. The tumors take the opposite approach. They use CCL22 to tap directly into our own immune system to co-opt a key regulatory component in the network.
The tumors survive by stealth, not by might.
That’s right. The stealth is speaking the language of the immune system to encourage a desired outcome. And it works. The idea of using the same biological subterfuge seemed to be a very persuasive way forward to treat periodontitis. Right now, most periodontal treatments focus on clearing away the bacteria that infect the gum tissue and prompt the inflammation. But bacteria will re-colonize the tooth surfaces and the periodontium, leaving the larger problem of inflammation mostly unaddressed. Remember it’s the inflammation, not the bacteria, that causes the severe tissue damage, erodes bone, and can lead to tooth loss.
What’s fascinating is you wanted to recruit the Tregs not to block or suppress the inflammation. You wanted to promote inflammation. Why?
We have a lot of bacteria in our mouths. Probably more than we realize. Our immune system must be able to handle that kind of bacterial load, and blocking inflammation could be harmful if we’re not careful. What’s important here is that we are promoting a balanced inflammation, not just increasing the inflammation. Regulatory T cells are a part of that balance that allows the local environment to defend itself from microorganisms, but also keeps it from destroying its own tissue.
I noticed from the paper that to promote the needed balance, you sought to sustain the directionality of your signal, too.
And that was a major engineering challenge. If the CCL22 was present everywhere, it wouldn’t work at all. CCL22 is a chemokine that needs to induce cell migration in a directional-and-biologically precise fashion. That meant that we had to create and sustain a point-source directionality into our synthetically engineered controlled-release system.
When you say, “synthetically engineered controlled-release system,” are we talking about a high-tech, implantable microchip or complicated next-generation technology that might involve a steep learning curve for most practicing dentists?
Oh, no. Our group succeeded at encapsulating CCL22 into a simple degradable powder. So, in our study, the treatment consisted of a small, dry plug of powder that we placed directly into the periodontal pocket, the space between the tooth and gum. We think what happens is the saliva washes into the periodontal pocket, and the dry plug absorbs the liquid and swells, lodging it into place. So, administration would be straightforward in the hands of any dentist in a busy practice.
Fluorescent micrographs of degrading CCL22 microparticles.
So, if it’s not a brand-new material or device that is critical to programming the directionality, how did you fold the basic biology into the materials side and program a controlled-release gradient?
Although it doesn’t sound very sexy, the answer is better mathematics. My lab has gone back over the past four or five years and revisited the fundamentals of the controlled-release field using materials with an already existing track record of FDA approval, and it’s really paid off.
Why probe the mathematical equations?
It goes back to my days as a graduate student about 10 years ago. Researchers would ask me to make a controlled-release system for a new drug that had never been encapsulated. I soon discovered that all of the mathematical models that represented all of the underlying controlled-release behavior on a molecular scale were very ad hoc.
More art than science.
Precisely. My modus operandi was just to make the formulation. If it didn’t work, I’d tweak the formulation and make it again. And again. And again. I mean, it sometimes would take me years to complete a formulation that acts in a desired way. So, when I came to Pitt as an assistant professor, those long hours in the laboratory told me that I wanted my group to be in position that it could rapidly program the spatial and temporal information for unencapsulated compounds.
Such as CCL22?
It was at the top of my list. CCL22 had never been encapsulated and controllably released. I wanted to be able to release the exact amounts of CCL22 that I wanted at the exact rate and without any of the bursts that can occur in time release formulations. Long story short, I wanted to be able to describe controlled release mathematically in a way that was generalizable.
How’d you do it?
A talented student named Sam Rothstein, now chief scientific officer of the new spinout company, Qrono Inc. in Pittsburgh, PA. By doing molecular experiments and observing how these systems degrade and erode, Sam accounted for all of the possible phases of release. That allowed us to come up with a viable model construct. Now, I can specify any type of drug and basically program as input the release behavior I want, and the model construct spits out a recipe to make a formulation. That formulation almost always does exactly what we want it to do on the very first try.
It’s like physics.
That’s exactly right, and it raises another critical point. We used a nanogram of drug in our animal studies to treat periodontitis. That’s such a miniscule amount of drug by today’s standards. What this shows is when you’re talking the body’s language, you don’t need a lot of drug to elicit a response. When you don’t speak the body’s language, you must deliver milligrams or more of a drug to act locally. Think about how much drug you are taking systemically at the milligram level. It can be a real jolt to the system.
We’re doing some very early toxicity studies with CCL22 in mice. What’s really cool is we can ramp up the dosage to 40 times the amount of drug that we used in the paper. We inject it directly into the bloodstream, and the mice just smile at you. It’s such a miniscule amount of drug, even at 40 times our dosage.
Let’s go through the PNAS paper. Or better yet, the figures.
Sure. Figure 1 is here. Let’s look at B, C, and D first. This quantifies the amount of bone loss. As you can see, the mice treated with the CCL22-releasing formulation, or the Treg-recruiting formulation, have less bone loss versus the untreated mice. You also can see there are fewer inflammatory cells. What’s fascinating here is the bacterial load in the gum tissue of all of these animals is the same. So, as I’ve mentioned, the standard treatments for advanced periodontitis involve the attenuation of the bacterial load. What we’re showing is we can achieve these effects with the same bacterial load.
And, as we said, the inflammation causes the major damage.
Exactly right. And you can see in the picture here, which shows the qualitative difference, that the difference is striking in the amount of bone resorption that we can eliminate by treating with this Treg-recruiting formulation.
Figure 2 is the expression of genes, as measured by PCR. Panel one shows the amount of Foxp3, which is a transcription factor and the hallmark of regulatory T cells, goes up as we would expect. The next two markers that you see, IL-10 and TGF-β, those are known factors that are secreted from regulatory T cells. So we are not only showing the molecules that are transcription factors inside of regulatory T cells, we’re showing the things that they are known to secrete. And those go up as well.
Let’s go to Figure 3.
Yes, it’s is really interesting. If regulatory T cells are responsible for the effect, we should be able to treat the mouse so as to eliminate the regulatory T cells and the effect should go back. In other words, if we eliminate the Tregs, it doesn’t matter if we’re trying to recruit or not, it shouldn’t work. To do so, we used an antibody that binds to the GITR receptor, which is highly expressed on Tregs.
It puts them out of action. So, sure enough, even when we administered our Treg-recruiting formulation, the effects are reversed. You see even more alveolar bone loss, more inflammatory cells, less TGF-β, less IL-10. We can show that when we knock out the mechanism that we think is responsible, the effects are reversed. I thought that was pretty compelling.
What about Figure 4?
Figure 4 is one of the most interesting results. Because we’re seeing a pronounced treatment effect, we wanted to scan all of these different markers for hard and soft tissues. We wanted to see what these recruited Tregs do to the local environment. That’s what Figure 4 shows. What’s fascinating about this is you see upregulation of markers that are known in bone growth, such as BMP4, BMP7, TGF-β. You also see things like Runx2 and alkaline phosphatase go up. These are all markers that we would think are involved in bone regeneration. In the bottom column, you see some soft tissue markers. Collagen 1A1, collagen 1A2. They go up. Then you see some regulators, TIMP1 and TIMP3, go up. These are inhibitors of tissue-destroying factors. In some of these tissue-destroying factors, such as MMP2, MMP8, MMP9, do you see those go down? That may be because we’re increasing the TIMP1 and TIMP3. I think what we’re seeing here is the sign of a regenerative microenvironment.
But we’re not delivering any known pro-regenerative drugs. We’re recruiting a lymphocyte, which sets in motion all of the above. What’s so cool about this is we always thought when we recruited regulatory T cells, we would establish a pro-regulatory environment. That just seems obvious. But it appears these cells may – and I don’t know – promote a pro-regenerative environment. If that’s the case, that really could be paradigm shifting. Now, you’re not just using the immune system to stop tissue resorption, you might be using the immune system to promote tissue regeneration.
And none of this was expected when you went in?
No, this was completely unexpected.
Let’s skip ahead to Figure 6?
Those are our canine studies. This shows in the most recognized preclinical model for translation that the treatment effect works. Let me point out a couple of things. People look at clinical measures, such as probing depth of the periodontal pockets and bleeding upon probing. But to actually go in and measure the bone loss quantitatively is something new. Like the mice, we see decreases in bone loss in treated dogs. We would hope that would be the case because we saw it in mice. Look at the eight-week tissue with the CCL22 formulation in the photo. Then look at the blank in the untreated. Look at how much less plaque is visible. We’re not brushing those teeth, nor are we administering antibiotics. It’s not like we’re decreasing the amount of bacteria there by any direct means. What might be happening here is because of the healthy environment that we’re promoting and the possible pro-regenerative environment, i.e., less soft-and-hard tissue destroying factors, the bacteria have less of a chance to take hold and form plaques in the pockets. So the teeth look cleaner, even though we’re not cleaning them. I thought that was particularly striking.
Where do you go from here?
We’re in the process of doing the pharmacology and toxicology studies. At this point, we’re showing pretty easily that this treatment is super safe. That’s what we would expect. You’re not administering something that inherently would have a lot of side effects. You’re delivering very, very small quantities of drug that are locally presented with a spatial gradient. The translation potential for this drug and approach is tremendous.
Thanks for the time