Department of Pulmonary and Critical Care Medicine, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China; Department of Pulmonary and Critical Care Medicine, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan, China.
Department of Pulmonary and Critical Care Medicine, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China; Department of Pulmonary and Critical Care Medicine, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan, China; Department of Critical Care Medicine, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China.
Int Immunopharmacol. 2021 Oct;99:108072. doi: 10.1016/j.intimp.2021.108072. Epub 2021 Aug 16.
BACKGROUND: Macrophage polarization has a causal role in the pathogenesis and resolution of various clinical diseases. DNA-binding transcription factors (TFs) have been identified as essential factors during gene transcription. Better insight into the TFs that regulate macrophage polarization could provide novel therapeutic targets. METHODS: IFN-γ (50 ng/mL) or IL4 (20 ng/mL) was utilized to stimulate bone marrow-derived macrophages from mice for 24 h for M1- and M2-polarized macrophage model construction, respectively. First, ATAC-seq (Assay for Targeting Accessible-Chromatin with high throughout sequencing) and motif analysis were conducted to identify potential transcription factors (TFs) involved in M1 and M2 macrophage polarization. Second, essential TFs were identified through RNA-seq, after which, their expression was compared between M0-polarized and M1/M2-polarized macrophages. Furthermore, a multiomic analysis of RNA-seq (siRNA knock down of the identified TFs), ChIP-seq and ATAC-seq was utilized to explore the TF-regulated molecular network. GO and KEGG analyses were used to expound the main functions of the TF-regulated molecular network. Finally, the top 5 TF-regulated genes were validated through flow cytometry, ELISA and qPCR. The cut-off values for high-throughput sequencing and qPCR were FDR < 0.05 and P < 0.05, respectively. RESULTS: Compared with M0 macrophages, 10,771 and 4,848 peaks were identified by ATAC-seq during M1 and M2 macrophage polarization, respectively (FDR < 0.05). Fifty and 62 TF binding motifs were identified for the TFs that participate in M1 and M2 macrophage polarization, respectively. The most significantly highly expressed TFs in M1 and M2 macrophages were identified by RNA-seq as Irf1 and Egr1, with LogFC values of 3.2 and 2.8, respectively. Multiomic analyses further found that Irf1 regulated the transcription of 90 genes and that Egr1 regulated the transcription of 116 genes. The Irf1-regulated molecular network played a key role in the inflammatory response and viral defence of M1 macrophages, and 116 Egr1-regulated genes included anti-inflammatory and cell proliferation genes. Validation experiments indicated that IFN-γ-induced Gbp5, Nos2, CD86, Cxcl10 and Cxcl5 expression was significantly downregulated in siIrf1-BMDMs, and IL4-induced Itgax, Nipal1, Bhlhe40, CD206 and Ffar4 expression was significantly downregulated in siEgr1-BMDMs (P < 0.05). CONCLUSIONS: Through multiomic analyses of epigenetic sequencing and RNA-seq with partial validation, the current study found that Irf1- and Egr1-induced transcription plays key roles in M1 and M2 macrophage polarization, respectively.
背景：巨噬细胞极化在各种临床疾病的发病机制和解决中起着因果作用。DNA 结合转录因子（TFs）已被确定为基因转录过程中的必需因子。更好地了解调节巨噬细胞极化的 TFs，可以为新的治疗靶点提供新的思路。
方法：利用 IFN-γ（50ng/mL）或 IL4（20ng/mL）分别刺激来自小鼠的骨髓来源的巨噬细胞 24 小时，以构建 M1 和 M2 极化的巨噬细胞模型。首先，通过 ATAC-seq（高吞吐量测序的靶向可及染色质分析）和 motif 分析鉴定参与 M1 和 M2 巨噬细胞极化的潜在 TFs。其次，通过 RNA-seq 鉴定必需的 TFs，然后比较它们在 M0 极化和 M1/M2 极化巨噬细胞中的表达。此外，还进行了 RNA-seq（鉴定的 TFs 的 siRNA 敲低）、ChIP-seq 和 ATAC-seq 的多组学分析，以探索 TF 调控的分子网络。GO 和 KEGG 分析用于阐述 TF 调控的分子网络的主要功能。最后，通过流式细胞术、ELISA 和 qPCR 验证前 5 个 TF 调控基因。高通量测序和 qPCR 的截止值分别为 FDR<0.05 和 P<0.05。
结果：与 M0 巨噬细胞相比，M1 和 M2 巨噬细胞极化过程中通过 ATAC-seq 分别鉴定出 10771 和 4848 个峰（FDR<0.05）。鉴定出 50 个和 62 个参与 M1 和 M2 巨噬细胞极化的 TF 结合基序。通过 RNA-seq 鉴定出在 M1 和 M2 巨噬细胞中表达水平最高的 TFs 分别为 Irf1 和 Egr1，LogFC 值分别为 3.2 和 2.8。多组学分析进一步发现，Irf1 调控 90 个基因的转录，Egr1 调控 116 个基因的转录。Irf1 调控的分子网络在 M1 巨噬细胞的炎症反应和病毒防御中起着关键作用，116 个 Egr1 调控的基因包括抗炎和细胞增殖基因。验证实验表明，IFN-γ诱导的 Gbp5、Nos2、CD86、Cxcl10 和 Cxcl5 在 siIrf1-BMDMs 中的表达明显下调，而 IL4 诱导的 Itgax、Nipal1、Bhlhe40、CD206 和 Ffar4 在 siEgr1-BMDMs 中的表达明显下调（P<0.05）。
结论：通过 RNA-seq 与表观遗传测序的多组学分析（部分验证），本研究发现 Irf1-和 Egr1 诱导的转录分别在 M1 和 M2 巨噬细胞极化中起关键作用。
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