C-terminal citrullinated peptide alters antigen-specific APC:T cell interactions leading to breach of immune tolerance.

In rheumatoid arthritis, the emergence of anti-citrullinated autoimmunity is associated with HLA-antigen-T cell receptor complexes. The precise mechanisms underpinning this breach of tolerance are not well understood. Porphyromonas gingivalis expresses an enzyme capable of non-endogenous C-terminal citrullination with potential to generate citrullinated autoantigens. Here we document how C-terminal citrullination of ovalbumin peptide323-339 alters the interaction between antigen-presenting cells and OTII T cells to induce functional changes in responding T cells. These data reveal that C-terminal citrullination is sufficient to breach T cell peripheral tolerance in vivo and reveal the potential of C-terminal citrullination to lower the threshold for T cell activation. Finally, we demonstrate a role for the IL-2/STAT5/CD25 signalling axis in breach of tolerance. Together, our data identify a tractable mechanism and targetable pathways underpinning breach of tolerance in rheumatoid arthritis and provide new conceptual insight into the origins of anti-citrullinated autoimmunity.

Enrichment of Innate Lymphoid Cell Populations in Gingival Tissue.

Innate lymphoid cells (ILCs) are a population of lymphocytes that act as the first line of immunologic defense at mucosal surfaces. The ILC family in the skin, lungs, and gastrointestinal tissues has been investigated, and there are reports of individual subsets of ILCs in the oral tissues. We sought to investigate the whole ILC population (group 1, 2, and 3 subsets) in the murine gingivae and the lymph nodes draining the oral cavity. We show that ILCs made up a greater proportion of the whole CD45+ lymphocyte population in the murine gingivae (0.356% ± 0.039%) as compared with the proportion of ILCs in the draining lymph nodes (0.158% ± 0.005%). Cytokine profiling of the ILC populations demonstrated different proportions of ILC subsets in the murine gingivae versus the regional lymph nodes. The majority of ILCs in the draining lymph nodes expressed IL-5, whereas there were equal proportions of IFN-γ- and IL-5 expressing ILCs in the oral mucosa. The percentage of IL-17+ ILCs was comparable between the murine gingivae and the oral draining lymph nodes. These data suggest an enrichment of ILCs in the murine gingivae, and these ILCs reflect a cytokine profile discrepant to that of the local draining lymph nodes. These studies indicate diversity and enrichment of ILCs at the oral mucosal surface. The function of ILCs in the oral cavity remains to be determined; here, we provide a premise of ILC populations that merits future consideration in investigations of mouse models and human tissues.

Mast Cells Contribute to Porphyromonas gingivalis-induced Bone Loss.

Periodontitis is a chronic inflammatory and bone-destructive disease. Development of periodontitis is associated with dysbiosis of the microbial community, which may be caused by periodontal bacteria, such as Porphyromonas gingivalis Mast cells are sentinels at mucosal surfaces and are a potent source of inflammatory mediators, including tumor necrosis factors (TNF), although their role in the pathogenesis of periodontitis remains to be elucidated. This study sought to determine the contribution of mast cells to local bone destruction following oral infection with P. gingivalis Mast cell-deficient mice (Kit(W-sh/W-sh)) were protected from P. gingivalis-induced alveolar bone loss, with a reduction in anti-P. gingivalis serum antibody titers compared with wild-type infected controls. Furthermore, mast cell-deficient mice had reduced expression of Tnf, Il6, and Il1b mRNA in gingival tissues compared with wild-type mice. Mast cell-engrafted Kit(W-sh/W-sh) mice infected with P. gingivalis demonstrated alveolar bone loss and serum anti-P. gingivalis antibody titers equivalent to wild-type infected mice. The expression of Tnf mRNA in gingival tissues of Kit(W-sh/W-sh) mice was elevated following the engraftment of mast cells, indicating that mast cells contributed to the Tnf transcript in gingival tissues. In vitro, mast cells degranulated and released significant TNF in response to oral bacteria, and neutralizing TNF in vivo abrogated alveolar bone loss following P. gingivalis infection. These data indicate that mast cells and TNF contribute to the immunopathogenesis of periodontitis and may offer therapeutic targets.

IL-33 Exacerbates Periodontal Disease through Induction of RANKL.

Cytokines mediate the balance between protective and destructive immunity in periodontitis. We sought to investigate the role of IL-33 in periodontitis. The expression of IL-33 in gingival tissue from healthy controls (n = 10) and patients with chronic periodontitis (n = 17) was investigated. Based on a murine model of periodontal disease, the function of IL-33 was determined first by administration of exogenous IL-33 and second by inhibition of IL-33 signaling using mice deficient in the IL-33 receptor ST2. Alveolar bone level, serum antibody, and lymphocyte responses were assessed in the murine model. Expression of IL-33 and ST2 was elevated in gingival tissues from patients with chronic periodontitis as compared with healthy tissues (P < 0.05). Similarly, Il33 expression was higher in periodontal tissues of Porphyromonas gingivalis-infected mice as compared with sham-infected controls (P < 0.05). IL-33 treatment of P. gingivalis-infected mice significantly exacerbated alveolar bone loss when compared with infection or IL-33 treatment alone (P < 0.001). Conversely, P. gingivalis infection-induced alveolar bone loss was attenuated in mice lacking ST2. The percentages of T and B lymphocytes expressing nuclear factor κB ligand (RANKL) in the gingival tissues and T lymphocytes expressing RANKL in the cervical draining lymph nodes were higher in IL-33-treated P. gingivalis-infected mice versus phosphate buffered saline-treated P. gingivalis-infected controls (all P < 0.001). Targeting the RANKL pathway by osteoprotegerin administration abrogated periodontal bone destruction in P. gingivalis-infected, IL-33-treated mice. These data demonstrate a previously unrecognized role for IL-33 in exacerbating bone loss in a RANKL-dependent manner in the context of bacterial infection and suggest that this pathway may be amenable to manipulation as a novel therapeutic target in periodontitis.

Periodontitis in the absence of B cells and specific anti-bacterial antibody.

Periodontitis (PD) results from complex interactions between a dysbiotic oral microbiota and a dysregulated host immune response. The inflammatory infiltrate in the gingiva of PD patients includes an abundance of B cells, implicating these cells in the immunopathology. We sought to investigate the role of B cells in PD using a murine model. Wild-type or B-cell-deficient (µMT) mice were orally infected with Porphyromonas gingivalis. One or six weeks following infection, lymphocyte populations in the gingiva and cervical draining lymph nodes (dLN) were analysed by flow cytometry; serum anti-P. gingivalis IgG antibody titers were measured by enzyme-linked immunosorbent assay, and alveolar bone loss was determined. In wild-type mice, the percentage of gingival B cells expressing receptor activator of nuclear factor-κB ligand (RANKL) was significantly increased 1 week post-infection (5.36% control versus 11% PD, P < 0.01). The percentage of Fas(+) GL7(+) germinal centre B cells in the dLN was significantly increased at both 1 week (2.03% control versus 6.90% PD, P < 0.01) and 6 weeks (4.45% control versus 8.77% PD, P < 0.05) post-infection. B-cell-deficient mice were protected from P. gingivalis-induced alveolar bone loss, with a lack of B-cell proliferation and lack of CD4(+) CD44(+) CD62L(-) T-cell generation in the dLN, and absence of serum anti-P. gingivalis antibodies. Our data imply a pathological role for B cells in PD, and that selective targeting of this immune axis may have a role in treating severe periodontal disease.