Identification of immune cells in healthy mouth lays groundwork for understanding disease

Maintaining the body’s barrier defenses at sites such as the skin and mucosal surfaces is critical for health and survival. These barriers are continually exposed to foreign substances and infectious agents, and are home to a diverse array of commensal microbes—microorganisms that cause no harm and are part of the normal flora. Immune cells at barrier sites performs the remarkable task of keeping​ peace with commensal microbes while fighting off dangerous pathogens.

To gain a better understanding of how the immune system achieves this delicate balance, researchers led by Niki M. Moutsopoulos, D.D.S., Ph.D., chief of the Oral Immunity and Infection Unit​ at NIH’s National Institute of Dental and Craniofacial Research (NIDCR), in collaboration with Joanne E. Konkel, Ph.D., of the University of Manchester​, set out to identify the types of immune cells present in the gingiva (gums), a key barrier site. Their findings appeared in the journal Mucosal Immunology.

The gingiva that lines the teeth is especially susceptible to infection because it lies close to the bacterial biofilms that coat the surface of the teeth. The gingiva is an important barrier site because it is continually exposed to large communities of bacteria called biofilms, which adhere to the teeth. In addition, the inner covering of the gingiva lining the teeth is especially thin, providing ready access to the underlying tissue.

By examining gingival biopsies from 50 young, healthy volunteers, the researchers observed a predominance of immune cells called T cells and neutrophils, as well as a sophisticated network of antigen-presenting cells and a small population of innate lymphoid cells, a type of cell not previously known to inhabit human gingiva.

Nicolas Dutzan, the study’s first author, was able to make these determinations thanks to advances in a technique called multicolor flow cytometry, which scientists use to identify cellular components. Current methodologies enabled Dutzan and colleagues to define as many as 15 different molecules per cell, which allowed them to identify definitively each cell’s type and to gain insight into how the cells may be functioning.

Moutsopoulos’ team also found evidence that many of the immune cells in healthy mouths, such as T cells and innate lymphoid cells, are tissue-resident, continually patrolling the area like a local police force. This finding is consistent with data from other studies, which have shown that barrier sites often rely heavily on local populations of immune cells rather than calling in the systemic immune system for protection.

To see how the pattern of immune cells changes with disease, the scientists next examined gingival biopsies from a small group of people with periodontitis, a serious inflammatory disease that, if left untreated, results in tissue damage and erosion of the bones that support the teeth. The diseased gingiva had elevated numbers of neutrophils, a type of immune cell that helps quell infections by engulfing bacteria. The researchers also saw increased levels of a molecule called IL-17, which recruits neutrophils to the site of infection.

Healthy and periodontal disease gingival tissue taken from biopsies of study participants. Large numbers of immune cells are visible in the tissue damaged by periodontal disease. Courtesy of Niki M. Moutsopoulos, NIDCR.

While IL-17 is part of the host’s defense, if it is overproduced it may contribute to damaging inflammation. IL-17 is known to be associated with arthritis and other types of bone loss such as that seen in periodontitis, and inhibitors of the IL-7 pathway are used to treat psoriatic arthritis, a form of the disease associated with the skin condition, psoriasis. In future work, Dr. Moutsopoulos’ team aims to test the effectiveness of IL-17-like inhibitors in alleviating aggressive forms of periodontitis.

While landmark classical clinical studies have surveyed the major immune cell populations in the gingiva in certain inflammatory states, this study is the first to make use of current methodologies to perform an in-depth characterization of the immune cell network in healthy gingiva in humans.

“Our work is important because it provides a detailed picture of what the immune cell landscape looks like at this important barrier site under normal circumstances—before aging, infection or other problems arise,” said Moutsopoulos. “Knowing how these cells keep you healthy under ideal conditions sets the stage for our understanding of oral immunity and for being able to track changes that occur as disease begins and progresses. Ultimately, we expect to be able to use this information to identify novel drug targets and to develop new therapies for periodontitis and other diseases.”

Characterization of the human immune cell network at the gingival barrier. Dutzan N, Konkel JE, Greenwell-Wild T, Moutsopoulos NM. Mucosal Immunol. 2016 Jan 6. doi: 10.1038/mi.2015.136. [Epub ahead of print] PMID: 26732676​​​​​​​​

Unique site-specific factors control oral immunity

The body’s barrier sites, such as the skin, mouth and intestines, are continually exposed to potentially harmful foreign substances and infectious agents, and they are also home to a diverse array of harmless microbes called commensal microorganisms. Oral immunity is dependent on the immune system performing a remarkable balancing act at these sites by fighting off dangerous pathogens while tolerating the presence of the normal flora.

Multiple cell types are involved in protecting the body’s barriers, including immune cells called Th17 cells. These cells have been shown to help assist oral immunity and combat infections in the oral cavity—people with genetic defects in Th17 cell function experience frequent fungal infections of the mouth. Conversely, exaggerated Th17 cell responses have been linked to periodontal disease, underscoring the importance of precise control of the cells’ function. Previous research has shown that certain microbes regulate Th17 cells in the skin and intestines, but the factors that control them in the mouth have been less clear.

Niki M. Moutsopoulos, D.D.S., Ph.D., chief of the Oral Immunity and Infection Unit at the NIH’s National Institute of Dental and Craniofacial Research (NIDCR), wondered if, similar to other barrier sites, microbes control Th17 cell accumulation in the mouth.

Dr. Moutsopoulos has a longstanding interest in how microorganisms and the immune system interact to preserve the health of the oral cavity. Her research team focuses on the molecular pathways that lead to periodontal disease, with the ultimate goal being to identify therapeutic targets for this common condition.

In the current study, which was published in the journal, Immunity, and was carried out in collaboration with Joanne E. Konkel, Ph.D., of the University of Manchester, the researchers observed that Th17 cells appear in the oral cavity with age. There were few Th17 cells in young, 8-week-old mice but by 24 weeks, middle age for mice, Th17 cell numbers had increased dramatically. Based upon what was known about Th17 cells in the skin and intestines, the researchers suspected that the cells’ surge in middle age might involve shifts in microbial populations.

To their surprise, they found that the composition and levels of microbes in the oral cavity of 8-week-old mice were comparable to those of the 24-week-old mice. In addition, mice raised in a germ-free environment had similar oral Th17 cell levels as mice raised under normal, non-sterile conditions. These findings revealed that, in contrast to other barrier sites, the presence of Th17 cells in the mouth does not depend on microbes. The researchers concluded that factors that regulate Th17 cells must differ depending on the unique characteristics of each site.

Th17 cells are key mediators of immunity at barrier sites such as the oral cavity. The factors that control them differ depending on cues unique to each site’s local environment. Credit: Niki M. Moutsopoulos, D.D.S., Ph.D., NIDCR.

A clue as to what these factors might be in the oral cavity came from experiments showing that a signaling molecule called IL-6 is needed for Th17 cell accumulation—mice engineered to lack IL-6 produced far fewer Th17 cells compared to control mice that have the molecule. Dr. Moutsopoulos’s team next focused on identifying the factors that induce IL-6 and drive Th17 cell accumulation at this barrier. They hypothesized that “damage signals” caused by chewing could stimulate the local immune response.

Subsequent experiments validated this idea. The researchers found that mice raised on a soft food diet requiring minimal chewing had fewer Th17 cells by 24 weeks than mice fed a normal diet. In addition, Th17 cell numbers rose in the mouths of young mice when the researchers hardened the diet or induced damage by mechanical abrasion. This rise in Th17 cell levels did not occur in mice lacking IL-6, confirming the key role of this signaling molecule in promoting Th17 cell accumulation.

Together, these findings have uncovered a role for mechanical damage in triggering local immunity in the oral cavity, and have linked induction of Th17 cells to the stimulation of protective immune responses. However, exaggeration of these signals could be pathogenic and contribute to inflammation and periodontal bone loss.

“We know that Th17 cells are important for preserving the health of the oral cavity, but they also may contribute to periodontal disease, which is why we want to understand the factors that regulate them in this unique environment,” said Dr. Moutsopoulos.

More generally, the findings highlight the importance of evaluating the local environment for novel factors that may control immune responses at barrier sites.

This study was funded in part by the intramural program of NIDCR, the Biotechnology and Biological Sciences Research Council, the Wellcome Trust and the Manchester Collaborative Centre for Inflammation Research.

On-going mechanical damage from mastication drives homeostatic Th17 responses at the oral barrier. Dutzan N, Abusleme L, Bridgeman H, Greenwell-Wild T, Zangerle-Murray T, Fife ME, Bouladoux N, Linley H, Brenchley L, Wemyss K, Calderon G, Hong B, Break TJ, Bowdish DME, Lionakis MS, Jones SA, Trinchieri G, Diaz PI, Belkaid Y, Konkel JE, and Moutsopoulos NM. Immunity. 2017 Jan 6. doi: 10.1016/j.immuni.2016.12.010. [Epub ahead of print] PMID: 28087239