o:25849 Ex vivo study of IL-6 expression and function in immune cell subsets from human periapical lesions en ABSTRACT Aim Even though IL-6 is a key inflammatory cytokine in periapical lesions (PLs), its function in stable periapical disease is unknown. Therefore, the aim of this study was to investigate the following: first, the ex vivo production of IL-6 by clinically different PLs; next, subsets of immune cells expressing IL-6 in PLs according to their inflammatory status and finally, modulatory effect of IL-6 on T-cell cytokine production in cell cultures. Methodology Inflammatory cells were isolated from a total of 95 human PLs. Detection of IL-6+ cells within the myeloid and lymphoid populations was performed by multicolour flow cytometry. ELISA and FlowCytomix Microbeads Assay were used to measure cytokine levels in culture supernatants. To study the role of IL-6 in PLs, mononuclear cells (MNC) from symptomatic (Sy) or asymptomatic (Asy) PLs were treated with IL-6 or Tocilizumab, an IL-6R blocking antibody. The differences between groups were tested by unpaired t-test, Mann–Whitney or Friedman tests. Results The levels of IL-6 in PL MNC culture supernatants were significantly higher compared with total PL cells and PL granulocytes (p < .001). MNC from Sy PLs produced significantly higher levels of IL-6 than MNC from Asy PLs (p < .001). Flow cytometry analysis showed that NKT cells, CD8+ T cells and M2 macrophages (MØ), were dominant IL-6+ cells, in contrast to CD4+ T cells. However, CD8+ and CD4+ T cells contributed the most to IL-6 production. IL-6hi producing MNC cultures had higher levels of Th1 (IFN-γ), Th17 (IL-17A), Tfh (IL-21) and RANKL, whereas Th2 (IL-4) and Tregs cytokines (IL-10, TGF-β) were lower compared with IL-6low-producing cultures. Exogenous IL-6 stimulated 17A, IL-21 and RANKL, independently of PL activation status, but decreased IFN-γ, IL-4 and IL-33 levels in IL-6hi-producing cultures. Tocilizumab increased IL-10 and TGF-β in IL-6low producing cultures. All differences were p < .05. Conclusions Most immune cells from Sy PLs expressed higher levels of IL-6 compared with Asy PLs, which correlated with IL-6 production in culture. Analysis of cytokines suggested a dominant pro- inflammatory T-cell response and osteodestructive function of IL-6 in PLs judging by Th17/Tfh cell activation, Tregs inhibition and increased RANKL/OPG ratio. Excessive IL-6 production decreased Th1/Th2 responses. cell cultures, cytokines, flow cytometry, periapical lesions 1552099 10.1111/iej.13704 2022-06-01T08:51:57.076Z 44 yes 46 Tanja Džopalić University of Niš, Medical Faculty, Serbia Miodrag Čolić University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia akademik person Mile Eraković Military Medical Academy, Clinic for Stomatology, Belgrade, Serbia person Dušan Mihajlović University of Defense in Belgrade, Medical Faculty of the Military Medical Academy, Serbia person Dragana Vučević University of Defense in Belgrade, Medical Faculty of the Military Medical Academy, Serbia person Marina Bekić University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia person 0000-0002-6945-4241 Sergej Tomić University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia doktor bioloških nauka person 0000-0003-2570-1295 application/pdf 79757 https://phaidrabg.bg.ac.rs/o:25849 no yes 1 70 1552260 11A29 11A2904 International Endodontic Journal 55 5 480 494 John Wiley & Sons Ltd 2022