Džopalić Tanja, Tomić Sergej, Bekić Marina, Vučević Dragana, Mihajlović Dušan, Eraković Mile, Čolić Miodrag
Medical Faculty, University of Niš, Niš, Serbia.
Medical Faculty of the Military Medical Academy, University of Defense in Belgrade, Belgrade, Serbia.
Int Endod J. 2022 May;55(5):480-494. doi: 10.1111/iej.13704. Epub 2022 Feb 25.
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.
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.
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-6 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-6 -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-6 -producing cultures. Tocilizumab increased IL-10 and TGF-β in IL-6 -producing cultures. All differences were p < .05.
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.
尽管白细胞介素-6（IL-6）是根尖周病变（PLs）中的关键炎症细胞因子，但其在稳定型根尖周病中的功能尚不清楚。因此，本研究的目的是调查以下内容：第一，临床上不同的PLs中IL-6的体外产生情况；第二，根据PLs的炎症状态，在PLs中表达IL-6的免疫细胞亚群；最后，IL-6对细胞培养中T细胞细胞因子产生的调节作用。
从总共95例人类PLs中分离炎症细胞。通过多色流式细胞术检测髓系和淋巴系群体中的IL-6细胞。采用酶联免疫吸附测定（ELISA）和流式细胞微珠分析来测量培养上清液中的细胞因子水平。为了研究IL-6在PLs中的作用，用IL-6或抗IL-6受体阻断抗体托珠单抗处理有症状（Sy）或无症状（Asy）PLs的单核细胞（MNC）。通过不成对t检验、曼-惠特尼检验或弗里德曼检验来检验组间差异。
与PLs总细胞和PLs粒细胞相比，PLs的MNC培养上清液中IL-6水平显著更高（p < 0.001）。Sy PLs的MNC产生的IL-6水平显著高于Asy PLs的MNC（p < 0.001）。流式细胞术分析表明，与CD4 T细胞相比，自然杀伤T细胞（NKT细胞）、CD8 T细胞和M2巨噬细胞（MØ）是主要的IL-6产生细胞。然而，CD8和CD4 T细胞对IL-6产生的贡献最大。产生IL-6的MNC培养物中Th1（干扰素-γ）、Th17（白细胞介素-17A）、滤泡辅助性T细胞（Tfh，白细胞介素-21）和核因子κB受体活化因子配体（RANKL）水平较高，而与不产生IL-6的培养物相比，Th2（白细胞介素-4）和调节性T细胞（Tregs）细胞因子（白细胞介素-10、转化生长因子-β）水平较低。外源性IL-6刺激产生白细胞介素-17A、白细胞介素-21和RANKL，与PLs的激活状态无关，但降低了产生IL-6的培养物中干扰素-γ、白细胞介素-4和白细胞介素-33的水平。托珠单抗增加了产生IL-6的培养物中白细胞介素-10和转化生长因子-β的水平。所有差异均为p < 0.05。
与Asy PLs相比，Sy PLs的大多数免疫细胞表达更高水平的IL-6，这与培养物中IL-6的产生相关。细胞因子分析表明从Th17/Tfh细胞活化、Tregs抑制以及RANKL/骨保护素（OPG）比值增加来看，IL-6在PLs中具有主要的促炎T细胞反应和骨破坏功能。过量的IL-6产生降低了Th1/Th2反应。
