In 1980, a then-National Institute of Dental Research grantee and colleagues reported in the New England Journal of Medicine about a five-year-old boy with an unusual history of chronic skin and ear infections. Their case study provided the scientific lead for what now is known in the medical literature as leukocyte adhesion deficiency type I (LAD-I), a rare primary immunodeficiency driven by a subtle-but-stymieing inherited change to prominent immune cells called neutrophils that impair their ability to fight off skin and mucosal infections, including in the mouth.
Four decades later, a new set of NIDCR scientists and grantees and their colleagues have made another major LAD-I discovery. This time, they explain why children with the condition are predisposed to an extremely aggressive, early-onset form of periodontitis that has puzzled patients, parents, and practitioners.
The scientists show that the problem is not as long suspected a case of bacteria opportunistically exploiting a hole in the immune system to infect cells in the subgingival space, the fluid-filled pocket between the tooth and gum. Bacteria, in fact, were only minimally present in gingival tissue.
Rather the scientists found the periodontitis stems from a communication breakdown among neutrophils and two other types of immune cells that normally coordinate their activities in the pocket. Instead of promptly turning off the immune response, the cells ping the same looping message to each other to stay in inflammatory mode. The glitch, like leaving the iron on, perpetuates the inflammation of the subgingival space longer than needed and, over time and multiple immune responses, eventually damages supportive bone and ligaments that hold a tooth in its socket.
The findings, published in the March issue of Science Translational Medicine, point the way to targeted treatments that specifically turn off the inflammation in the periodontal pocket and potentially save the teeth of kids and young adults with LAD-1.
The results also offer an interesting glimpse into a future in which today’s refractory or aggressive periodontitis — clinical cases that resist standard antibiotic and mechanical therapies — have been defined based on their precise microbial and inflammatory causes, diagnostically subclassified, and potentially treated more effectively with targeted therapies.
Niki Moutsopoulos, D.D.S., Ph.D.
NIDCR Assistant Clinical Investigator
Niki Moutsopoulos, D.D.S., Ph.D., a scientist at NIDCR and lead author on the study, said the results also should be of broad significance for immunologists. The communication breakdown that causes the periodontitis occurs in a closed signaling loop called the Neutrostat that was discovered first in mice. The Neutrostat was proposed as a regulatory circuit that controls the production of neutrophils in mammals, including humans.
"This is the first report in which a human disease has been linked to a breakdown in Neutrostat signaling," she said. "Our data offer strong support that the Neutrostat is a key regulatory circuit for controlling neutrophil production, and that’s certainly critical information to have not only for LAD-I but for understanding human immunity in general."
The human body operates two immune systems: innate and acquired. The latter is our learned immunity mediated by T and other memory cells, which store the hard-fought lessons of past infections to thwart their return.
Innate immunity is our non-specific defense system that protects us from birth onward against infections in general. Its workhorse is the neutrophil, a round, microbe-eating cell that flows by the tens of billions flattened out stingray-like sampling the interior walls of capillaries and venules for biochemical hints of invaders. Should neutrophils sense that microbes have strayed into tissues where they don’t belong, they will withstand the sheer unidirectional force of the bloodstream to descend to a firm landing.
Neutrophils do so by rolling along the blood vessel wall and forming a series of Velcro-like bind-and-quick-release bonds that progressively break their momentum. As the neutrophils decelerate into a final bumpy tumble, they unfold hook-like integrin proteins to adhere firmly to the blood vessel wall. Once docked and further primed for battle, the neutrophils can reorganize their cytoskeletons for motility and begin crawling. They typically find a gap in the blood-vessel wall, squeeze through, and pick up the scent of their target in the tissue.
For the estimated one in a million people born with LAD-I, an inherited gene mutation leaves a critical portion of the integrin hook either missing or malformed. Although their neutrophils roll normally, they adhere poorly to the blood vessel wall and never fully prime for motility.
As a result, the neutrophils amass inside the blood vessel unable to get out. They are like first responders at the scene of a one-alarm fire with no way to enter the building and investigate.
Just as an arsonist would prosper under these circumstances, bacteria seem to thrive in people with LAD-I. For those with extreme immunodeficiency who contend with chronic, life-threatening infections, bone-marrow transplantation can cure LAD-I, despite the inherent risks and ongoing challenges.
For children and young adults with less severe immunodeficiency, the case for bone marrow transplantation is less compelling. Most persevere without the transplantation and its risks, preferring to cope as best they can with various chronic inflammations, including severe periodontitis.
But coping with LAD-I periodontitis can be extremely difficult. Even with optimal oral care, including frequent dental visits and strict daily regimens of antibacterial therapy, the periodontitis tends to worsen. In some children, the inflammation is so severe that newly erupted permanent teeth sometimes loosen within a matter of years. By their 20s, most have lost several, if not all, of their teeth.
The million-dollar question is why?
In early 2008, Niki Moutsopoulos launched her research career. She had completed her specialty in periodontics and Ph.D. in Immunology and was accepted into NIDCR’s two-year Dental Clinical Research Fellows Training Program. The fellowship allowed her to interact with patients while pursuing her research interest in oral immunology.
That’s when Moutsopoulos was referred to Steven Holland, M.D., who heads the Laboratory of Clinical Infectious Diseases at NIH’s National Institute of Allergy and Infectious Diseases. Holland studies various human infectious and immunological conditions, including several rare primary immunodeficiencies.
Moutsopoulos consulted with Holland about spending part of her fellowship in his laboratory and contributing her expertise in oral immunology to his team. He agreed, and Moutsopoulos
attended Holland’s weekly lab meeting the very next day.
couldn’t have been happier with her choice.
“The enthusiasm for science that permeated the Holland lab was inspiring
to me,” she said. “Not only did I interact
clinically, I learned how to interrogate human immunity more systematically and
Moutsopoulos soon became interested in a small cohort of children and young adults that Holland had assembled with the less-severe form of LAD-I, meaning they had not received a bone-marrow transplant. She was perplexed by their rampant periodontitis and intrigued by the likely role of oral mucosal immunity in driving the disease process.
"I already knew from the scientific literature that LAD-I periodontitis had never been studied in depth and certainly not in a cohort of patients," she explained, noting that cohorts can be logistically challenging to assemble for rare conditions. "It was truly a unique research opportunity, and, as an oral immunologist, I couldn’t wait to examine the tissues of these patients and see what was driving the periodontitis."
For those examinations, Moutsopoulos said it became obvious that bacteria hadn’t overgrown their natural boundaries in the periodontal pocket, the space between the tooth and gum, as previously suspected. In fact, she found minimal evidence of bacteria infecting tissues. And yet, Moutsopoulos could see in patient x-rays that bone continued to erode in the tooth socket. It was as though any biochemical hint of the bacteria near the gingival cells produced the immunological equivalent of an avalanche.
That’s when Moutsopoulos made another critical discovery. She found in gingival samples extracted from the patients that all had extremely elevated levels of IL-17A. While the acronym may not ring a bell for many, it is meaningful for immunologists. IL-17A is an immune signaling compound, or cytokine, that immune T cells produce to promote inflammation. IL-17A does its pro-inflammatory trick by increasing G-CSF.
What does G-CSF do?
It ramps up neutrophil production in the bone marrow.
For Moutsopoulos, the implications were profound. The mouth, as a bacteria-rich environment, will unavoidably promote immune responses. They come with the territory. But in LAD-I periodontitis, immune signals were somehow getting crossed, and what should have been a brief immune flare up had become self-perpetuating and pathological.
George Hajishengallis, D.D.S., Ph.D.,
University of Pennsylvania School of Dental Medicine
For a second opinion on her data, Moutsopoulos sought out George Hajishengallis, D.D.S., Ph.D., at the University of Pennsylvania’s School of Dental Medicine. She viewed Hajishengallis as a perceptive oral immunologist with an admirable quality of thinking outside the box. He and his colleagues recently had published a paper that reported on a glycoprotein that has the unique ability to turn off activated neutrophils in the gingiva. The glycoprotein interacted with IL-17. In the back of her mind, she imagined Hajishengallis might be open to collaborating with her to better define the signaling pathways that drive LAD-I periodontitis.
But Moutsopoulos had a problem. The two had never met. After dashing off an introductory email to Hajishengallis that mustered a polite reply, Moutsopoulos had a better idea. She noticed in her inbox an announcement for an upcoming scientific presentation in Bethesda. The event’s featured speaker: Dr. George Hajishengallis.
"I crashed the meeting," she laughed. "During a break, I introduced myself to George holding a ream of paper and said, "I want to show you something. I have LAD-I patients, and my findings are almost opposite of yours.’ George almost immediately said, ‘Stop, I’ll tell you what you’re finding in the context of LAD-I is quite different.’"
As luck would have it, Hajishengallis had been working with a mouse model that mimics the error underlying LAD-I, in which neutrophils adhere poorly and accumulate inside blood vessels unable to exit. Hajishengallis already had generated data that were consistent with previously published work by Klaus Ley, M.D and colleagues then at the University of Virginia. The Ley group had proposed in 2005 what it called the Neutrostat, a closed signaling loop that regulates the production of neutrophils much like a sensor in a thermometer controls the temperature in a room.
According to Ley group’s model, the Neutrostat works like this:
- Immune dendritic cells and macrophages serve as sensors of infection. When microbial intruders are detected, they sound the alarm for help, in part by releasing large amounts of a cytokine called IL-23.
- The alarm induces nearby T cells to release IL-17, the cytokine that Moutsopoulos had detected in the gingival samples from her patients. IL-17 produces more G-CSF, and that leads to the release of neutrophils from the bone marrow. The neutrophils circulate to the hot spot, where they roll, crawl, and squeeze to enter the fray.
- After the neutrophils consume the intruders, they are programmed to die. Scavenging macrophages consume the spent neutrophils and, in the process, reduce their release of IL-23. That has the domino effect of lowering IL-17, G-CSF, and neutrophil production. The signaling loop closes, and the immune response subsides.
- That is, unless something goes wrong. Ley et al. proposed that dendritic cells and macrophages will release IL-23 as programmed, and T cells will follow suit by producing IL-17. But if poorly adhering neutrophils accumulate in blood vessels, there will be no quick way to shut of the Neutrostat. Inflammatory conditions will run far longer than needed.
Harkening back to the Neutrostat regulatory loop, Hajishengallis told Moutsopoulos that she also would see an elevation in IL-23, which he had seen in mice. He added more certain than hopeful, "I guess that’s what you’re seeing in your patients. I’ve been looking for somebody with access to people with this rare disease."
The Neutrostat remained somewhat controversial in immunology. As happens in science as a part of the discovery process, other research groups had produced conflicting data that raised questions about the signaling loop. But, more significantly, no human disease had been linked to a breakdown in the Neutrostat to confirm its existence in people. Hajishengallis, a co-senior author on the published manuscript, recognized that LAD-I periodontitis would be the first.
As the two talked through the implications of their work, a collaboration was born. "What’s amazing is George and I already had independently assembled about two-thirds of this research story, and the human and mouse data were consistent," said Moutsopoulos. "Now, we just had to combine the human and mouse data to tell the most complete and compelling story possible."
The two confirmed that IL-23 also was elevated in the gingival samples from the LAD-I patients. Interestingly, they detected little or no IL-23 and IL-17 in the bloodstream of these patients. Moutsopoulos said this shouldn’t be surprising, noting that IL-17 and IL-23 are produced locally within the gingiva and wouldn’t spill out into the circulation.
"If we had looked in the circulation for clues to the periodontitis, we would have never found anything," she said. "You really have to look at a disease where it occurs." Moutsopoulos said discussions are ongoing about where to go next with these findings, especially now that a clear target is in view to shut down the inflammation in the gingiva.