Visualization and modeling of inhibition of IL-1β and TNF-α mRNA transcription at the single-cell level.
Visualization and modeling of inhibition of IL-1β and TNF-α mRNA transcription at the single-cell level.
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DOI：
10.1038/s41598-021-92846-0
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发表时间：
2021-07-01
影响因子：
4.6
通讯作者：
Werner J
中科院分区：
文献类型：
--
作者：
Kalb D;Vo HD;Adikari S;Hong-Geller E;Munsky B;Werner J
IL-1β和TNF-α是典型的免疫反应介质，在慢性和急性疾病的广泛炎症反应中发挥关键的调节作用。在这里，我们采用自动化显微镜平台分析单细胞水平上IL-1β和TNF-α的信使RNA （mRNA）表达。将细胞暴露于革兰氏阴性菌的外膜成分脂多糖（LPS）后，利用单分子荧光原位杂交（smFISH）观察和计数人单核白血病细胞系（THP-1）中IL-1β和TNF-α mRNA的表达量。我们发现小分子抑制剂MG132（用于阻断NF-κB信号传导的26S蛋白酶体抑制剂）和U0126（用于阻断ccaat -增强子结合蛋白C/EBP的MAPK激酶抑制剂）成功阻断IL-1β和TNF-α mRNA的表达。基于这些单细胞mRNA表达数据，我们筛选了36种不同的基因表达数学模型，并发现了两种相似的模型，可以捕捉药物U0126和MG132对基因向高激活状态转变的速率的影响。当它们的参数分别由每种药物的作用告知时，两个模型都能够预测联合药物治疗的效果。根据我们的数据和模型，我们假设IL-1β同时被NF-κB和C/EBP激活，而TNF-α主要被NF-κB激活。我们的联合单细胞实验和建模工作显示了这两个基因之间的相互联系，并展示了单细胞反应，包括基因表达的分布形状，平均表达和动力学，如何随着抑制而变化。
IL-1β and TNF-α are canonical immune response mediators that play key regulatory roles in a wide range of inflammatory responses to both chronic and acute conditions. Here we employ an automated microscopy platform for the analysis of messenger RNA (mRNA) expression of IL-1β and TNF-α at the single-cell level. The amount of IL-1β and TNF-α mRNA expressed in a human monocytic leukemia cell line (THP-1) is visualized and counted using single-molecule fluorescent in-situ hybridization (smFISH) following exposure of the cells to lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria. We show that the small molecule inhibitors MG132 (a 26S proteasome inhibitor used to block NF-κB signaling) and U0126 (a MAPK Kinase inhibitor used to block CCAAT-enhancer-binding proteins C/EBP) successfully block IL-1β and TNF-α mRNA expression. Based upon this single-cell mRNA expression data, we screened 36 different mathematical models of gene expression, and found two similar models that capture the effects by which the drugs U0126 and MG132 affect the rates at which the genes transition into highly activated states. When their parameters were informed by the action of each drug independently, both models were able to predict the effects of the combined drug treatment. From our data and models, we postulate that IL-1β is activated by both NF-κB and C/EBP, while TNF-α is predominantly activated by NF-κB. Our combined single-cell experimental and modeling efforts show the interconnection between these two genes and demonstrates how the single-cell responses, including the distribution shapes, mean expression, and kinetics of gene expression, change with inhibition.
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影响因子：
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作者：
Chen L;Deng H;Cui H;Fang J;Zuo Z;Deng J;Li Y;Wang X;Zhao L
通讯作者：
Zhao L
影响因子：
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Jarvis, James N.
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作者：
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通讯作者：
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