Super enhancer-mediated transcription of miR146a-5p drives M2 polarization during Leishmania donovani infection.
Super enhancer-mediated transcription of miR146a-5p drives M2 polarization during Leishmania donovani infection.
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超级增强子介导的miR146a-5p转录驱动多诺瓦利什曼原虫感染期间M2极化
DOI：
10.1371/journal.ppat.1009343
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发表时间：
2021-03
期刊：
影响因子：
6.7
通讯作者：
Ali N
中科院分区：
文献类型：
--
作者：
Das S;Mukherjee S;Ali N
杜氏利什曼原虫感染的结果取决于M1和M2巨噬细胞之间的动态交换。微RNA（miRNAs）和表观遗传修饰物参与调节L。杜氏感染仍然难以确定。极化调节miRNAs的差异表达分析显示，在杜氏利什曼原虫感染期间miR 146 a-5 p显著富集。在感染的骨髓衍生的巨噬细胞（BMDM）和BALB/c小鼠器官中观察到miR 146 a-5 p的持续富集。我们发现miR 146 a-5 p参与了寄生虫的吞噬作用和存活能力。此外，miR 146 a-5 pgot富含白细胞介素4刺激的BMDM，表明其可能参与M2极化。在用miRVANA抗146 a寡核苷酸、M2标志物（CCR 7、YM-1、FIZZ-1、β-内酰胺酶-1、IL 10和IL 4）和转录因子（p-STAT 6和c/EBPβ）随着M1极化转录因子的增加而减少（p-STAT 1、AP 1和IRF-1）、miR 146 a靶基因（TRAF 6和IRAK 1）、M1细胞因子（IL 12和TNFα）、iNOS、一氧化氮和磷酸化p-65亚基的核转位。感染的BALB/c小鼠中细胞内成熟miR 146 a-5 p库的中和降低了器官寄生虫负荷以及M2标志物和IL 10的表达，同时富集了M1标志物如iNOS和IL 12。此外，我们探索了超级增强子（SE）的新作用，一种顺式作用的调控成分，在感染过程中丰富miR 146 a-5 p的表达。在感染的BMDM中发现SE组分如BET溴结构域4（BRD 4）和p300的表达增强和核保留。在沉默BRD 4后，miR 146 a-5 p和M2标记物的表达下调，TRAF 6、IRAK 1和iNOS水平升高。基于STRING V.11的预测和免疫沉淀证实了BRD 4、p300和RNA pol II（RpbI）之间的强相互作用。染色质免疫沉淀研究表明，在感染过程中，BRD 4在miR 146 a-5 p基因的增强子位点上募集。总之，我们的研究结果揭示了BRD 4/p300依赖性超级增强子在L. Donovani感染，其又介导M2极化和免疫抑制。内脏利什曼病（VL），由原生动物寄生虫杜氏利什曼原虫引起，是最严重的形式的利什曼病，如果不治疗是高度致命的。黑热病后皮肤利什曼病（PKDL）患者在感染过程中引发的终身免疫抑制是VL成功治疗的主要障碍。识别这种免疫抑制背后的分子原理将增加VL治疗的成功。L. donovani劫持宿主巨噬细胞并将其从促炎性M1型转化为免疫抑制性M2型，这允许成功建立感染。在此，我们探讨了miRNA-146 a-5 p在感染过程中M1型向M2型转化中不可或缺的作用。体外和体内miRNA沉默均确立了miR 146 a-5 p作为M1极化的强制性负调控因子。计算分析和基于免疫沉淀的实验验证了L. donovani诱导超级增强子复合物介导的miR 146 a-5 p的转录上调。BET布罗莫结构域蛋白4（BRD 4）与p300组蛋白乙酰转移酶和RNA pol II一起形成该SE复合物沿着。BRD 4的沉默显著消除了miR 146 a-5 p介导的M2极化。简而言之，我们目前的发现确立了BRD 4依赖性超级增强子在协调巨噬细胞miR 146 a-5 p的持续转录中的先前未被认识的作用，所述miR 146 a-5 p反过来促进L. Donovani感染
The outcome of Leishmania donovani infection depends upon the dynamic interchanges between M1 and M2 macrophages. Information of the involvement of microRNAs (miRNAs) and epigenetic modifiers in regulating macrophage plasticity during L. donovani infection is still elusive. Differential expression analysis of polarization-regulating miRNAs, revealed significant enrichment of miR146a-5p during Leishmania donovani infection. A sustained enrichment of miR146a-5p was observed in both infected bone marrow derived macrophages (BMDMs) and BALB/c mice organs. We found involvement of miR146a-5p in phagocytosis and survivability of parasites. Moreover, miR146a-5pgot enriched in interleukin 4- stimulated BMDMs, indicating its possible involvement in M2 polarization. Upon transfecting BMDMs with miRVANA anti-146a oligos, M2 markers (CCR7, YM-1, FIZZ-1, arginase-1, IL10 and IL4) and transcription factors (p-STAT6 and c/EBPβ) got depleted with concomitant augmentation of M1-polarizing transcription factors (p-STAT1, AP1 and IRF-1), miR146a target genes (TRAF6 and IRAK1), M1 cytokines (IL12 and TNFα), iNOS, nitric oxide, and nuclear translocation of phospho p-65 subunit. Neutralization of intracellular mature miR146a-5p pool in infected BALB/c mice lower organ parasite burden and expressions of M2 markers and IL10 with enrichment of M1 markers like iNOS and IL12. Additionally, we explored the novel role of super enhancer (SE), a cis-acting regulatory component, to enrich miR146a-5p expression during infection. Enhanced expression and nuclear retention of SE components like BET bromodomain 4 (BRD4) and p300 were found in infected BMDMs. Upon silencing BRD4, expressions of miR146a-5p and M2 markers were down regulated and TRAF6, IRAK1 and iNOS levels increased. STRING V.11 based predication and immune precipitation confirmed the strong interaction amongst BRD4, p300 and RNA pol II (RpbI). Chromatin immune precipitation studies suggested the recruitment of BRD4 at the enhancer loci of miR146a-5p gene during infection. Altogether, our findings revealed a novel role of BRD4/p300-depdendent super-enhancer in regulating miR146a expression during L. donovani infection which in turn mediates M2 polarization and immune-suppression. Visceral leishmaniasis (VL), caused by protozoan parasites Leishmania donovani, is the most severe form of leishmaniasis and is highly lethal if left untreated. Major obstacle for successful therapy of VL originates from the life-long immune-suppression triggered in the post kala-azar dermal leishmaniasis (PKDL) patients during infection. Identification of molecular principles behind such immune-suppression will add success in VL therapeutics. L. donovani hijacks the host macrophages and converts them from pro-inflammatory M1 to immune-suppressive M2 type, which allows successful infection establishment. Herein, we explored the indispensable role of miRNA-146a-5p in conversion of M1 to M2 type during infection. Both in vitro and in vivo miRNA silencing established miR146a-5p as an imperative negative regulator ofM1 polarization. Computational analysis as well as immune precipitation based experiments authenticated that L. donovani induces super enhancer complex mediated transcriptional upregulation of miR146a-5p. BET bromodomain protein 4 (BRD4) forms this SE complex along with p300 histone acetyl transferase and RNA pol II. Silencing of BRD4 significantly abrogated miR146a-5p mediated M2 polarization. In short, our current findings established a previously unrecognized role of BRD4-depdendent super enhancers in orchestrating persistent transcription of macrophage miR146a-5p which in turn promotes M2 polarization during L. donovani infection.
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影响因子：
3.7
作者：
Jablonski KA;Amici SA;Webb LM;Ruiz-Rosado Jde D;Popovich PG;Partida-Sanchez S;Guerau-de-Arellano M
通讯作者：
Guerau-de-Arellano M
影响因子：
3.1
作者：
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DOI：
10.1038/mtna.2016.71
发表时间：
2016-09-27
期刊：
Molecular therapy. Nucleic acids
影响因子：
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作者：
通讯作者：
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影响因子：
39.3
作者：
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通讯作者：
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影响因子：
4.6
作者：
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通讯作者：
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