IFN-? selectively suppresses a subset of TLR4-activated genes and enhancers to potentiate M1-like macrophage polarization [RNA-seq II]

Complete polarization of macrophages towards an M1-like proinflammatory and antimicrobial state requires combined action of IFN-? and LPS. Synergistic activation of canonical inflammatory NF-?B target genes by IFN-? and LPS is well appreciated, but less is known about whether IFN-? negatively regulates components of the LPS response, and how this affects polarization. A combined transcriptomic and epigenomic approach revealed that IFN-? selectively abrogates LPS-induced feedback and select metabolic pathways by suppressing TLR4-mediated activation of gene enhancers. In contrast to superinduction of inflammatory genes via enhancers that harbor IRF sequences and bind STAT1, IFN-?-mediated repression targeted enhancers with STAT sequences that bound STAT3. TLR4-activated IFN-?-suppressed enhancers comprised two subsets distinguished by differential regulation of histone acetylation and recruitment of STAT3, CDK8 and cohesin, and were functionally inactivated by IFN-?. These findings reveal that IFN-? suppresses feedback inhibitory and metabolic components of the TLR response to achieve full M1 polarization, and provide insights into mechanisms by which IFN-? selectively inhibits TLR4-induced transcription. Overall design: RNA-seq analysis of transcriptional changes in human macrophages (M-CSF/GM-CSF) that were cultured with or without IFN-? and then stimulated with LPS or vice versa

巨噬细胞向M1样促炎抗菌表型的完全极化，需要干扰素γ（IFN-γ）与脂多糖（LPS）的协同作用。干扰素γ与LPS对经典炎症核因子κB（NF-κB）靶基因的协同激活已得到广泛证实，但目前关于干扰素γ是否负调控LPS应答组分、以及该调控如何影响巨噬细胞极化的相关认知仍较为有限。本研究通过转录组与表观基因组联合分析策略发现，干扰素γ可通过抑制Toll样受体4（TLR4）介导的基因增强子激活，选择性阻断LPS诱导的反馈通路与部分代谢途径。与携带干扰素调节因子（IRF）结合序列并结合信号转导与转录激活因子1（STAT1）的增强子所介导的炎症基因超诱导不同，干扰素γ介导的基因沉默靶向的是结合信号转导与转录激活因子3（STAT3）的携带STAT结合序列的增强子。Toll样受体4（TLR4）激活且受干扰素γ抑制的增强子可分为两个亚群，二者在组蛋白乙酰化的差异调控以及STAT3、细胞周期蛋白依赖性激酶8（CDK8）和黏连蛋白（cohesin）的招募特征上存在差异，且可被干扰素γ功能性灭活。上述研究结果揭示，干扰素γ可通过抑制TLR应答的反馈抑制组分与代谢组分，以实现完全的M1极化，并为干扰素γ选择性抑制TLR4诱导的转录过程提供了机制层面的新见解。整体实验设计：对经巨噬细胞集落刺激因子（M-CSF）/粒细胞-巨噬细胞集落刺激因子（GM-CSF）培养的人源巨噬细胞，设置仅用干扰素γ处理、仅用脂多糖（LPS）刺激、干扰素γ预处理后再用LPS刺激，以及LPS刺激后再用干扰素γ处理的四组样本，随后通过RNA测序（RNA-seq）分析各组细胞的转录水平变化。