Periodontitis is a common chronic inflammatory disease characterized by oral mucosal tissue destructive inflammation, which leads to periodontal ligament damage, loss of supporting bone, and ultimately, tooth loss (1, 2). Microbial dysbiosis is thought to be the trigger of the oral soft tissue inflammation and current standard of care largely rely on the removal of microbial biofilm by mechanical removal or antibiotics (3). Nevertheless, it is not curative. Therefore, there is an urgent need to understand the pathogenesis of the destructive tissue inflammation.

Innate lymphoid cells (ILCs) are a recently identified family of tissue-resident lymphocytes that lack adaptive antigen receptors. Based on the expression of signature cytokines and transcription factors, ILCs are divided into three major groups: T-bet⁺ ILC1s produce interferon-γ (IFN-γ); GATA3⁺ ILC2s secrete interleukin-5 (IL-5), IL-13, and amphiregulin; and RORγt⁺ ILC3s produce IL-17A and IL-22. ILC3s can be further divided into natural cytotoxicity receptor (NCR)⁺ ILC3s, NCR^-ILC3s and lymphoid tissue inducer (LTi) cells (4, 5). ILCs mainly reside at mucosal barrier surfaces that are able to provide an immediate immune response with rapid cytokines secretion in response to environmental stimuli (6). Thus, ILCs play important roles in regulating inflammation, immunity, tissue repair, and homeostasis.

Increasing numbers of studies have implicated that ILCs are critical regulators in multiple inflammatory diseases (7). ILC2s promote airway inflammation through secreting type 2 cytokines of IL-4, IL-5, and IL-13 and contribute to the pathogenesis of allergic asthma. Increased ILC2s are found in the blood and BALF of patients with asthma (8, 9). ILC3s and ILC1s are the abundant populations of ILCs in the human gut and have been extensively studied in inflammatory bowel disease (IBD) (10). The number of IFN-γ–producing ILC1s and IL-17A–producing NCR^- ILC3s are significantly increased in the inflamed intestinal mucosa of patients with Crohn’s disease (11). However, the increase of IFN-γ–producing ILC1s is accompanied by a large decrease in the number of NCR⁺ ILC3s, which is thought to be due to the plasticity between ILC3s and ILC1s (12, 13). Studies from both mice and human have demonstrated that NCR⁺ ILC3s can convert into IFN-γ–producing ILC1s (14, 15). ILC3s have also been shown to contribute to the skin inflammation of psoriasis (16).

As one of the important barrier sites, the oral mucosa is constantly exposed to a plethora of environmental stimuli, including microbes, food antigens, and airborne particles (17). Thus, immune responses in oral barrier need to be tightly controlled to maintain homeostatic balance between tolerance to commensals and immunity to pathogens (18). Oral mucosa harbors all ILC subsets (19–21). Two recent studies have shown that ILCs are involved in periodontitis (20, 21).One shows that all ILC subsets are increased in periodontitis patients, with a marked effect on ILC2s (20). Another study shows ILC1s are the predominant subset with RANKL expression in gingivitis and periodontitis (21). However, whether ILCs have any functional abnormalities in periodontitis remain largely unknown. In this study, we show ILCs in periodontal ligament are markedly increased in periodontitis patients compared with healthy controls. Among various ILCs, ILC1s and ILC3s exhibit increased numbers in periodontal ligament and enhanced production of inflammatory cytokines in periodontitis. Thus, our results highlight the role of ILCs in regulating oral immunity and periodontal ligament inflammation and provide insights into targeting ILCs for the treatment of periodontitis.

Innate lymphoid cells play critical roles in regulating immunity and inflammation, particularly at the mucosal barrier sites (6). Periodontitis is a common inflammatory disease in oral mucosal barrier that leads to immunopathology and destruction of supporting bone (1). However, the role of ILCs in periodontitis is poorly understood. Here, we have shown that both ILC1s and ILC3s are dramatically increased in periodontal ligament of periodontitis patients. Moreover, these cells exhibit an inflammatory phenotype indicated by enhanced production of IFN-γ and IL-17A in periodontitis. These data suggest ILC1s and ILC3s might promote the periodontal ligament inflammation and contribute to the pathogenesis of periodontitis.

Previous studies have shown that all ILCs subset are present in periodontal tissues from both gingivitis and periodontitis patients (20, 21). One study found that ILC1s are the major subset with ILC compartment in soft tissue from both gingivitis and periodontitis (21). While the other group showed that ILC2s are the predominant subset in periodontitis (20). The results from these two studies contradict each other. This might be because of the difference in samples collection. Soft tissues were used for the first study. The periodontal tissues were studied in the latter study, but they did not specify whether gingiva tissues or periodontal ligament tissues were used. In our study, we focused on the severe periodontitis patients who require surgery for tooth extraction. Periodontal ligament tissues were isolated and processed for subsequent cell analysis, which was thought to be more precise to reflect the local tissue-specific immune response during periodontitis. Periodontal ligament is the connective tissue that harbors both on tooth root and alveolar bone. When bacteria and LPS intrude the sulcular epithelium, the immune response is initiated in periodontal ligament that cause attachment loss, which forms periodontal pocket and alveolar bone loss. The immune response in periodontal ligament is the main cause of periodontal tissue destruction and alveolar bone loss.

The healthy individuals were included as control group. We chose a healthy third molar as a control. Although the mechanical load to the teeth maybe different from third molars, molars, or pre-molars. Biological mechanical load will do no harm to the periodontal tissue. Only occlusal trauma can lead periodontal tissue damage, which is excluded in our study. Furthermore, the construction and composition of periodontal ligament of molar and third molar is same, which can be used as healthy control group.

First, we found that ILC1s were the most abundant cell subset in periodontal ligament tissue from both healthy controls and periodontitis patients, accounting for about 50% of the total ILCs. By contrast, ILC2s were the least population of ILCs compartment account for less 10% of total ILCs. Then, we observed that total ILCs were dramatically increased in the periodontal ligament of periodontitis patients. Among them, the numbers of ILC1s and ILC3s were abnormally high in periodontal ligament tissues from periodontitis patients. These findings are consistent with the increased ILC1s and ILC3s in other inflammatory diseases, such as IBD and psoriasis.

Human ILC3s include NKp44⁺ILC3s and NKp44^-ILC3s. NKp44⁺ILC3s are able to produce IL-17A, IL-22 and IFN-γ, whereas NKp44^-ILC3s mainly produce IL-17A (30, 31). Previous reports have shown that NKp44⁺ILC3s are enriched in mucosal barrier tissues, such as intestinal tract, tonsils, and skin and are increased in inflammatory diseases, such as psoriasis and IBD (12, 30, 31). Despite NKp44⁺ILC3s are the largest population accounting for 70% of the total intestinal ILCs in the human gut (12, 14), they appeared to be a rare population in periodontal ligament accounting for less than 5% of the total ILCs. The difference in tissue distribution might reflect the influence of various tissue environment signals on the differentiation of ILC3 subsets. NKp44⁺ILC3s are considered as an inflammatory subset given their capability to produce IFN-γ and IL-17A. Increased NKp44⁺ILC3s have been shown in the skin lesions of psoriasis patients (16). Consistently, we observed that NKp44⁺ILC3s were increased as well as in inflamed periodontal ligament tissue. Together with the increased NKp44⁺ILC3s, the production of IFN-γ by ILC3s was enhanced as well. These data raised the possibility that increased IFN-γ might come from NKp44⁺ILC3s and contribute to the disease. These data indicate that ILC3s might play a pathogenic role in promoting the periodontal inflammation during periodontitis.

Previous reports have shown that inflammatory cytokines IL-17A and IFN-γ are implicated in driving the pathogenesis of periodontitis and consequent bone and tooth loss (32). IL-17A is increased in periodontitis and positively correlated with disease severity (33). IL-17A can promote osteoclastogenesis and bone resorption through activation osteoblasts to produce RANKL. In addition, IL-17A can synergize with other inflammatory cytokines to induce inflammatory responses, which result in destruction of periodontal tissues and alveolar bone (34). Similar to IL-17A, IFN-γ is also increased in periodontitis patients (35). IFN-γ has been shown not only to enhance the inflammation through induction of chemokines production by epithelial cells (36) but also to promote the destruction of periodontal tissues by inducing T cells to produce TNF-α and RANKL (37). Although Th17 cells are the major source of IL-17A, ILC3s are also capable of producing IL-17A (5). Indeed, we observed ILC3s produced more IL-17A in periodontal ligament from periodontitis patients. IFN-γ is another pathogenic cytokine and is thought to promote periodontal inflammation (19). As expected, the percentage of IFN-γ–producing ILC1 and ILC3 cells was markedly increased in periodontitis. Together, these data suggest that both ILC1s and ILC3s promote periodontal inflammation during periodontitis through secreting inflammatory cytokines IFN-γ and IL-17A. Thus, targeting ILCs might represent a promising approach for treating periodontitis.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by Ethical Review Board of Shanghai Stomatological Hospital, Fudan University. The patients/participants provided their written informed consent to participate in this study.

CL and JL collected the clinical samples, performed the experiments and analyzed the data. JP and YW collected the clinical samples. LS and YX conceived, designed and supervised the project. LS, YX, and JL wrote the manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by grant 202040495 (to YX) from Shanghai Municipal Health Commission, grant 81971487 (to LS) from the National Natural Science Foundation of China, grant 20ZR1430200 and 20142202300 (to LS) from Science and Technology Commission of Shanghai Municipality.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.