BACKGROUND The aberrant recognition of self-nucleic acids by the innate immune system contributes to the pathology of several autoimmune diseases. Although microbial DNA and, in certain instances, self-DNA that is released from damaged cells are primarily recognized by Toll-like receptor 9 (TLR9), recent evidence suggests that other cytosolic sequence-nonspecific DNA sensors contribute to DNA recognition. In this study, we focused on the sensing of microbial and host DNA in type 1 diabetes (T1D) patients. METHODS Peripheral blood mononuclear cells (PBMCs) and monocytes from pediatric patients with T1D and from healthy donors were stimulated with microbial DNA (CpG) or with self-DNA (DNA contained within neutrophil extracellular traps, NETs). The production of cytokines was measured by flow cytometry and multiplex bead assays. The internalization of microbial DNA and its colocalization with STING was detected by image cytometry. Furthermore, the involvement of the TBK1 kinase was investigated by detecting its phosphorylation with phospho-flow cytometry or by using a TBK1 inhibition assay. RESULTS We observed a prominent proinflammatory response in T1D PBMCs, especially pDCs and monocytes, to microbial DNA in comparison to that in controls. We further confirmed that monocytes could bind and internalize DNA and respond by releasing proinflammatory cytokines in a more pronounced manner in T1D patients than those in controls. Surprisingly, this cytokine production was not affected by TLR9 blockade, suggesting the involvement of intracellular receptors in DNA recognition. We further identified TBK1 and STING as two crucial molecules in the DNA-sensing pathway that were involved in CpG-DNA sensing by T1D cells. A similar DNA-sensing pathway that was dependent on intracellular DNA sensors and the STING-TBK1 interaction was employed in response to NETs, which were used to model self-DNA. CONCLUSIONS Here, we show that there were significant differences in DNA sensing in T1D patients compared to that in controls. We demonstrate that monocytes from T1D patients are able to sense microbial- and self-DNA, leading to proinflammatory cytokine secretion through the adaptor protein STING and the TBK1 kinase.

中文翻译：

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在1型糖尿病患者中，单核细胞通过TBK1信号通路促进DNA传感。

背景技术先天免疫系统对自身核酸的异常识别导致了几种自身免疫疾病的病理。尽管微生物DNA以及在某些情况下从受损细胞释放的自身DNA主要被Toll样受体9（TLR9）识别，但最近的证据表明，其他胞质序列非特异性DNA传感器也有助于DNA识别。在这项研究中，我们专注于1型糖尿病（T1D）患者的微生物和宿主DNA的感测。方法用微生物DNA（CpG）或自身DNA（中性粒细胞外诱捕器中的DNA）刺激来自T1D患儿的小儿患者和健康供体的外周血单核细胞（PBMC）和单核细胞。细胞因子的产生通过流式细胞术和多重珠测定法测量。通过图像细胞术检测微生物DNA的内在化及其与STING的共定位。此外，通过用磷酸流式细胞仪检测其磷酸化或通过使用TBK1抑制试验研究了TBK1激酶的参与。结果与对照组相比，我们观察到T1D PBMC（尤其是pDC和单核细胞）对微生物DNA的明显促炎反应。我们进一步证实，在T1D患者中，单核细胞可以结合并内化DNA，并通过释放促炎性细胞因子而做出反应，而与对照组相比更为明显。出人意料的是，这种细胞因子的产生不受TLR9阻断的影响，表明细胞内受体参与了DNA识别。我们进一步确定了TBK1和STING是DNA传感途径中的两个关键分子，它们参与了T1D细胞CpG-DNA的传感。依赖于细胞内DNA传感器和STING-TBK1相互作用的类似DNA传感途径被用于响应NET，NET用于建模自我DNA。结论在这里，我们显示与对照组相比，T1D患者的DNA传感存在显着差异。我们证明，来自T1D患者的单核细胞能够感知微生物和自身DNA，从而通过衔接蛋白STING和TBK1激酶导致促炎性细胞因子分泌。结论在这里，我们显示与对照组相比，T1D患者的DNA传感存在显着差异。我们证明，来自T1D患者的单核细胞能够感知微生物和自身DNA，从而通过衔接蛋白STING和TBK1激酶导致促炎性细胞因子分泌。结论在这里，我们显示与对照组相比，T1D患者的DNA传感存在显着差异。我们证明，来自T1D患者的单核细胞能够感知微生物和自身DNA，从而通过衔接蛋白STING和TBK1激酶导致促炎性细胞因子分泌。