IL‐22–expressing CD4⁺AhR⁺ T lymphocytes are associated with RANKL‐mediated alveolar bone resorption during experimental periodontitis

Abstract

Background and Objective Over the past few years, the importance of interleukin‐22 (IL‐22) and T‐helper (Th)22 lymphocytes in the pathogenesis of periodontitis has become apparent; however, there are still aspects that are not addressed yet. Cells expressing IL‐22 and aryl hydrocarbon receptor (AhR), transcription factor master switch gene implicated in the differentiation and function of Th22 lymphocytes, have been detected in periodontal tissues of periodontitis‐affected patients. In addition, IL‐22 has been associated with osteoclast differentiation and their bone resorptive activity in vitro. However, the destructive potential of IL‐22–expressing AhR+ Th22 lymphocytes over periodontal tissues during periodontitis has not been demonstrated in vivo yet. Therefore, this study aimed to analyze whether IL‐22–expressing CD4+AhR+ T lymphocytes detected in periodontal lesions are associated with alveolar bone resorption during experimental periodontitis. Material and Methods Using a murine model of periodontitis, the expression levels of IL‐22 and AhR, as well as the Th1‐, Th2‐, Th17‐ and T regulatory‐associated cytokines, were analyzed in periodontal lesions using qPCR. The detection of CD4+IL‐22+AhR+ T lymphocytes was analyzed in periodontal lesions and cervical lymph nodes that drain these periodontal lesions using flow cytometry. In addition, the expression of the osteoclastogenic mediator called receptor activator of nuclear factor‐κB ligand (RANKL) was analyzed by qPCR, western blot, and immunohistochemistry. Finally, alveolar bone resorption was analyzed using micro‐computed tomography and scanning electron microscopy, and the bone resorption levels were correlated with IL‐22 and RANKL expression. Results Higher levels of IL‐22, AhR, and RANKL, as well as IL‐1β, IL‐6, IL‐12, IL‐17, IL‐23, and TNF‐α, were expressed in periodontal lesions of infected mice compared with periodontal tissues of sham‐infected and non‐infected controls. Similarly, high RANKL immunoreaction was observed in periodontal tissues of infected mice; however, few or absent RANKL immunoreaction was observed in controls. This association between RANKL and periodontal infection was ratified by western blot. Furthermore, a higher detection of CD4+IL‐22+AhR+ T lymphocytes was found in periodontal lesions and cervical lymph nodes that drain these periodontal lesions in infected mice compared with non‐infected controls. Finally, the increased IL‐22 and RANKL expression showed positive correlation between them and with the augmented alveolar bone resorption observed in experimental periodontal lesions. Conclusion This study demonstrates the increase of IL‐22–expressing CD4+AhR+ T lymphocytes in periodontitis‐affected tissues and shows a positive correlation between IL‐22, RANKL expression, and alveolar bone resorption.

Humanized Mouse Models for the Study of Periodontitis: An Opportunity to Elucidate Unresolved Aspects of Its Immunopathogenesis and Analyze New Immunotherapeutic Strategies

“…The periodontal inoculation model comprises the localized microinjection of bacteria or some isolated bacterial component, such as lipopolysaccharide, directly into the palatal interproximal gingiva between the first, second, and third maxillary molar ( 18 , 35 ). This method promotes significant periodontal inflammation, characterized by an increased expression of pro-inflammatory cytokines, apical migration of the junctional epithelium, and activation of osteoclastogenesis, consistently resulting in alveolar bone resorption ( 36 , 40 ). Regarding the injection regimen, evidence commonly shows that the injections are performed two or three times per week under general anesthesia, generally using isoflurane ( 61 , 68 ).…”

“…This model has been used to evaluate different hypotheses regarding the mechanisms of periodontal inflammation and alveolar bone loss, due to the fact that it allows a reliable characterization of the immune response induced in the periodontal tissues and the cervical lymph nodes that drain the infected periodontium ( 6 , 40 ). Since the mono-infection with a known bacterium allows great experimental control over the pathogenic stimulus, this model has been shown to be useful for analyzing pathogenic differences between different periodontal bacteria, their different serotypes, or bacteria defective in a certain virulence factor ( 6 , 7 , 18 , 40 , 41 ).…”

O-Polysaccharide Plays a Major Role on the Virulence and Immunostimulatory Potential of Aggregatibacter actinomycetemcomitans During Periodontal Infection

“…To quantify the extent of alveolar bone loss, the maxillae were scanned using a micro-computed tomography (mCT) equipment (SkyScan 1272; Bruker, Belgium), as previously described (18,32). 3D-digitized images were generated using a modified conebeam algorithm in a reconstruction software (Nrecon software, Bruker, Belgium).…”

“…In order to obtain cells to perform the flow cytometric analysis, the palatal periodontal tissues and cervical lymph nodes that drain the periodontal tissues were processed, as previously described (32). For the recovery of palatal periodontal tissues, after accessing the oral cavity, whole maxillary blocks separated from the nasal cavity were dissected and immediately processed for enzymatic digestion.…”

“…The presence of Tbet + (Th1), RORgt + (Th17), and AhR + (Th22) lymphocytes within the periodontal lesions and cervical lymph nodes were analyzed by identifying the expression of their specific transcription factors using flow cytometry, as previously described (18,32). The identified transcription factors were T-bet for Th1 cells, RORgt for Th17 cells, and AhR for Th22 cells.…”