Roles Of CD4~+T Cells In Bone Resorption Of Smoking-associated Periodontitis

Periodontitis is one of the most common chronic oral infectious diseases inadults. However, the etiology of periodontitis is still not well understood.Recently, a number of studies have suggested that autoimmune, especially CD4~+Tcells,immune response, is critical to the development of periodontitis. In addition,tobacco smoking has been recognized as the main risk factor for the developmentand progression of periodontal diseases. In previous studies, we havedemonstrated that functional αlpha7subunit of nicotinic acetylcholine receptors(α7nAChR), which is not only a predominant subunit composition of nAChRsbut also a potent target of nicotine binding receptor, presents in the periodontaltissues of human and rats, the upregulation of α7nAChR level by administrationof nicotine could be partially suppressed by pretreatment with α-BTX, the α7nAChR antagonist.Previous studies of our research group have demonstrated that nicotinecould upregulate the expression of α7nAChR in rat periodontal tissues andhuman periodontal ligament cells, the upregulation of α7nAChR levels byadministration of nicotine could be partially suppressed by pretreatment withα-BTX, which is the specific antagonist of α7nAChR. Meanwhile, numerousdatas have demonstrated immune response, especially CD4~+T cells,immuneregulation, plays the key role of bone resorption in periodontal disease, indicating CD4~+T cells are closely related to the activity and development of periodontitis.However, whether nicotine activates α7nAChR via CD4~+T cells have adestructive effect on periodontal tissue? What is the immunological andmolecular mechanism? Currently, no related reports have been published.In-depth study of this question will help to reveal the role of nicotine onperiodontal tissue destruction, immunological and molecular mechanisms of thedevelopment of periodontitis, providing the relationship between experimentaland theoretical basis for prevention and treatment of smoking-associatedperiodontitis.PartⅠ: In vitro culture of human periodontal ligament cells andsorting CD4~+T cells1Materials and methodsTaking the first premolar from patients which are extracted because oforthodontic treatment. In sterile conditions scrape root mid-third periodontalligament tissue for primary culture. Extracting10ml peripheral venous bloodfrom healthy non-smoking volunteers, then separating peripheral bloodmononuclear cells by density gradient centrifugation. Stained with CD4fluorescein isothiocyanate antibody, incubation and sorting CD4~+T cells by flowcytometry.2ResultsHuman periodontal ligament cells were cultured in vitro successfully. CD4~+Tcells were sorted successfully. Prior to sorting, CD4~+T cells accounted for33.3%in the system; after sorting, accounted for98.2%in the system.3ConclusionsSuccessfully culturing hPDLCs in vitro and sorting the CD4~+T cells, establishing the experimental cell model for the further experiment.PartⅡ: Nicotine regulates the expression of osteoclastdifferentiation-associated factors from human periodontalligament cells cocultured with or without CD4~+T cells1Materials and methodsSelecting the fifth-passage hPDLCs for the further experiment, divided intotwo groups: coculture group and non-coculture group, treated with agonistnicotine (10-5M) and/or antagonists of alpha-BTX (10-8M); Then we usedreal-time quantitative PCR and Western blot to test M-CSF, OPG and RANKLmRNA and protein expression.2ResultsReal-time quantitative PCR result showed that:10–5M nicotine couldsignificantly (P<0.05) upregulate the gene expressions of RANKL and M-CSF innon-coculture group, and significantly (P<0.05) downregulate gene expression ofOPG. While pretreating with α-BTX could partially inhibit these effects.10–5Mnicotine could more significantly (P<0.05) upregulate the gene expression ofRANKL than non-coculture group, and M-CSF, OPG gene expression isapproximately the same with non-coculture group; Western blot presented thesimilar results to Real-time quantitative PCR.3ConclusionsPCR and Western blot results indicated that the nicotine upregulated RANKLand M-CSF gene and protein expressions, and downregulated OPG gene andprotein expressions, which were regulated by periodontal α7nAChR innon-coculture group. While in coculture group,10–5M nicotine could moresignificantly (P<0.05) upregulate the gene expression of RANKL than non-coculture group, and M-CSF, OPG gene expression is approximately thesame with non-coculture group, indicating nicotine may activates α7nAChR viaCD4~+T cells have a destructive effect on periodontal tissue.PartⅢ: Nicotine regulates the migration of CD4~+T cellscocultured with or without human periodontal ligament cells1Materials and methodsSelecting good conditioned CD4~+T cells for the further experiment, dividedinto two groups: coculture group and non-coculture group, treated with agonistnicotine (10–5M) and/or antagonists of alpha-BTX (10–8M); Then we usedtranswell migration chamber to observe the migration of CD4~+T cells.2ResultsTranswell migration showed that:10–5M nicotine could significantly inducethe CD4~+T cells migration in non-coculture group, while pretreating with α-BTXcould partially inhibit this effect. In coculture group,10–5M nicotine could inducemore significantly (P<0.05) the CD4~+T cells migration than non-coculture group.3ConclusionsTranswell migration indicated that nicotine significantly induced CD4~+T cellsmigration, and further improving the CD4~+T cells migration capability coculturewith human periodontal ligament cells; Indicating that after coculturing withhuman periodontal ligament cells, nicotine may regulate the effects betweenCD4~+T cells and human periodontal ligament cells, promoting the CD4~+T cellsmigration capability, and further increasing the local inflammatory responses.