Following TLR4 stimulation of macrophages, negative feedback mediated by the anti-inflammatory cytokine IL-10 limits the inflammatory response. However, extensive cell-to-cell variability in TLR4-stimulated cytokine secretion raises questions about how negative feedback is robustly implemented. To explore this, we characterized the TLR4-stimulated secretion program in primary murine macrophages using a single-cell microwell assay that enabled evaluation of functional autocrine IL-10 signaling. High-dimensional analysis of single-cell data revealed three distinct tiers of TLR4-induced proinflammatory activation based on levels of cytokine secretion. Surprisingly, while IL-10 inhibits TLR4-induced activation in the highest tier, it also contributes to the TLR4-induced activation threshold by regulating which cells transition from non-secreting to secreting states. This role for IL-10 in restraining TLR4 inflammatory activation is largely mediated by intermediate IFN-β signaling, while TNF-α likely mediates response resolution by IL-10. Thus, cell-to-cell variability in cytokine regulatory motifs provides a means to tailor the TLR4-induced inflammatory response.

中文翻译：

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单细胞分泌分析揭示了IL-10在抑制和解决TLR4诱导的炎症反应中的双重作用

在TLR4刺激巨噬细胞后，抗炎细胞因子IL-10介导的负反馈限制了炎症反应。但是，TLR4刺激的细胞因子分泌中广泛的细胞间变异性引发了有关如何可靠地实施负反馈的问题。为了探索这一点，我们使用单细胞微孔测定法对原代小鼠巨噬细胞中TLR4刺激的分泌程序进行了表征，从而能够评估功能性自分泌IL-10信号传导。单细胞数据的高维分析显示了基于细胞因子分泌水平的TLR4诱导的促炎激活的三个不同层次。令人惊讶的是，尽管IL-10在最高层抑制TLR4诱导的激活，它也通过调节哪些细胞从非分泌状态转变为分泌状态而有助于TLR4诱导的激活阈值。IL-10抑制TLR4炎症激活的这种作用很大程度上是由中间IFN-β信号传导介导的，而TNF-α可能介导了IL-10的应答消退。因此，细胞因子调节基序中的细胞间差异提供了一种调整TLR4诱导的炎症反应的方法。