Eken Ahmet, Okus Zehra, Erdem Serife, Azizoglu Zehra Busra, Haliloglu Yesim, Bicer Ayten, Gur Tugba Nur, Yilmaz Ebru, Karakukcu Musa, Altuntas Hamiyet Donmez, Canatan Halit
Department of Medical Biology, Erciyes University Faculty of Medicine, Betül-Ziya Eren Genome and Stem Cell Center (GENKOK), Kayseri, Turkey.
Department of Pediatric Hematology-Oncology, Erciyes University Faculty of Medicine, Kayseri, Turkey.
North Clin Istanb. 2019 Oct 24;6(4):379-387. doi: 10.14744/nci.2019.38802. eCollection 2019.
In this study, we aimed to assess the effects of long- and short-term IL-15 cytokine exposure of human monocyte-derived curdlan-matured dendritic cells (DCs) on the production of Th17 cell-polarizing cytokine IL-23 and subsequent Th17 cell activation.
Peripheral blood mononuclear cells (PBMCs) were purified using Ficoll-Paque from healthy donors. Monocytes were magnetically selected using CD14 Miltenyi beads and differentiated into DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for five days in the presence or absence of IL-15 (100ng/ml) for long-term exposure experiments. Then, DCs were matured with peptidoglycan (PGN), or curdlan for 24 hours. For short-term exposure experiments, IL-15 was added only during maturation of DCs. Then, DCs were characterized concerning the expression of MHC II and costimulatory molecules, production of cytokine subunits IL-23p19, IL-12p40, IL-12p35 and cytokine IL-23 via flow cytometry or real-time qPCR or ELISA. Finally, the phosphorylation of signaling molecules after curdlan stimulation was assessed using phospho-flow assays.
IL-15 exposure suppressed IL-23 production by DCs. As a result, IL-15-exposed DCs suppressed IL-17 production by allogeneic T cells. Importantly, we observed a reduction in the surface Dectin-1 receptor levels by IL-15-exposed DCs. In line with these observations, curdlan stimulation resulted in reduced phosphorylation of ERK1/2, NF-kB p65 and AKT by human DCs exposed to IL-15 compared with controls. These results may explain why IL-15-exposed DCs produce less IL-23 after maturation with curdlan, which is a ligand of Dectin-1.
Short- or long-term exposure to IL-15 of human DCs during their differentiation or maturation programs DCs against Th17 cell polarization, which suggests that IL-15 availability may affect CD4+ T cell-mediated protective immunity to fungal infections.
在本研究中，我们旨在评估人单核细胞来源的可溶葡聚糖成熟树突状细胞（DCs）长期和短期暴露于白细胞介素-15（IL-15）细胞因子对Th17细胞极化细胞因子IL-23产生及随后Th17细胞活化的影响。
使用Ficoll-Paque从健康供体中纯化外周血单核细胞（PBMCs）。使用CD14美天旎磁珠磁性分选单核细胞，并在存在或不存在IL-15（100ng/ml）的情况下，用粒细胞-巨噬细胞集落刺激因子（GM-CSF）和IL-4将其分化为DCs，用于长期暴露实验，持续5天。然后，用肽聚糖（PGN）或可溶葡聚糖使DCs成熟24小时。对于短期暴露实验，仅在DCs成熟期间添加IL-15。然后，通过流式细胞术、实时定量PCR或酶联免疫吸附测定法，对DCs的MHC II和共刺激分子表达、细胞因子亚基IL-23p19、IL-12p40、IL-12p35的产生以及细胞因子IL-23进行表征。最后，使用磷酸化流式细胞术评估可溶葡聚糖刺激后信号分子的磷酸化。
IL-15暴露抑制了DCs产生IL-23。结果，暴露于IL-15的DCs抑制了同种异体T细胞产生IL-17。重要的是，我们观察到暴露于IL-15的DCs表面脱噬素-1受体水平降低。与这些观察结果一致，与对照组相比，可溶葡聚糖刺激导致暴露于IL-15的人DCs的细胞外信号调节激酶1/2（ERK1/2）、核因子-κB p65（NF-κB p65）和蛋白激酶B（AKT）磷酸化减少。这些结果可能解释了为什么暴露于IL-15的DCs在用可溶葡聚糖成熟后产生较少的IL-23，可溶葡聚糖是脱噬素-1的配体。
人DCs在分化或成熟过程中短期或长期暴露于IL-15会使其针对Th17细胞极化进行编程，这表明IL-15的可获得性可能会影响CD4 + T细胞介导的针对真菌感染的保护性免疫。
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