IL-6 augments IL-4-induced polarization of primary human macrophages through synergy of STAT3, STAT6 and BATF transcription factors

Here we explored how cytokines of the tumor milieu, interleukin (IL)-6 and IL-4, interact to influence target gene expression in primary human monocyte-derived macrophages (hMDMs). We show that dual stimulation with IL-4 and IL-6 synergistically modified gene expression. Among the synergistically induced genes are several targets with known pro-tumorigenic properties, such as CC-chemokine ligand 18 (CCL18), transforming growth factor alpha (TGFA) or CD274 (programmed cell death 1 ligand 1 (PD-L1)). We found that transcription factors of the signal transducer and activator of transcription (STAT) family, STAT3 and STAT6 bind regulatory regions of synergistically induced genes in close vicinity. STAT3 and STAT6 co-binding further induces the basic leucine zipper ATF-like transcription factor (BATF), which participates in synergistic induction of target gene expression. Functional analyses revealed increased MCF-7 and MDA-MB 231 tumor cell motility in response to conditioned media from co-treated hMDMs compared to cells incubated with media from single cytokine-treated hMDMs. Flow cytometric analysis of T cell populations upon co-culture with hMDMs polarized by different cytokines indicated that dual stimulation promoted immunosuppressive properties of hMDMs in a PD-L1-dependent manner. Analysis of clinical data revealed increased expression of BATF together with TAM markers in tumor stroma of breast cancer patients as compared to normal breast tissue stroma. Collectively, our findings suggest that IL-4 and IL-6 cooperate to alter the human macrophage transcriptome, endowing hMDMs with pro-tumorigenic properties. Overall design: We compared synergistically induced genes in IL-4/IL-6 co-stimulation relative to IL-4, IL-6 and untreated monocyte derived human macrophages via RNA sequencing本研究探讨了肿瘤微环境中的细胞因子——白细胞介素（interleukin，IL）-6与IL-4——如何相互作用，以影响原代人单核细胞衍生巨噬细胞（human monocyte-derived macrophages，hMDMs）的靶基因表达。本研究证实，IL-4与IL-6联合刺激可协同调控基因表达。其中协同诱导的基因包含多个已被证实具有促肿瘤特性的靶点，例如CC趋化因子配体18（CC-chemokine ligand 18，CCL18）、转化生长因子α（transforming growth factor alpha，TGFA）以及CD274（程序性死亡蛋白1配体1，programmed cell death 1 ligand 1，PD-L1）。研究发现，信号转导与转录激活因子（signal transducer and activator of transcription，STAT）家族成员STAT3与STAT6可在协同诱导基因的调控区域附近结合。二者的共结合还可进一步激活碱性亮氨酸拉链ATF样转录因子（basic leucine zipper ATF-like transcription factor，BATF），而该因子参与靶基因的协同诱导过程。功能分析显示，与经单一细胞因子处理的hMDMs条件培养基孵育的肿瘤细胞相比，经联合处理的hMDMs条件培养基可增强MCF-7与MDA-MB 231肿瘤细胞的迁移能力。对经不同细胞因子极化解育的hMDMs与T细胞共培养后的T细胞群进行流式细胞术分析，结果显示联合刺激可通过PD-L1依赖的方式增强hMDMs的免疫抑制特性。临床数据分析显示，与正常乳腺组织间质相比，乳腺癌患者肿瘤间质中BATF与肿瘤相关巨噬细胞（tumor-associated macrophages，TAM）标志物的表达水平均显著升高。综上，本研究结果表明IL-4与IL-6可协同改变人巨噬细胞的转录组，使hMDMs获得促肿瘤特性。实验整体设计：通过RNA测序技术，对比IL-4/IL-6联合刺激组、单一IL-4刺激组、单一IL-6刺激组以及未处理的人单核细胞衍生巨噬细胞中协同诱导基因的表达差异。