MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBPβ regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection

For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 β (IL-1β) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPβ signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPβsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.

为高效清除结核分枝杆菌（Mycobacterium tuberculosis, Mtb），巨噬细胞会向M1极化，激活与抗菌效应分子（如一氧化氮（nitric oxide, NO））及促炎细胞因子（如白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α））生成相关的转录因子。与此同时，炎症消退与M2极化相关，此时精氨酸酶及白细胞介素10（IL-10）等细胞因子的表达水平升高。调控M1与M2极化平衡，以及细菌相关过程（如自噬、结核分枝杆菌向溶酶体的转运）的转录及转录后机制，目前尚未完全阐明。本研究首次报道，微小RNA-26a（miR-26a）可靶向调控转录因子Krüppel样因子4（KLF4）。在结核分枝杆菌感染过程中，miR-26a的表达下调（在离体及体内模型中均已观测到）可促进KLF4的表达上调，进而使精氨酸酶活性升高、诱导型一氧化氮合酶（iNOS）活性降低。本研究进一步证实，KLF4会阻碍结核分枝杆菌向溶酶体的转运。环腺苷酸应答元件结合蛋白-CCAAT增强子结合蛋白β（CREB-C/EBPβ）信号轴同样可促进M2极化。在感染的巨噬细胞及感染小鼠体内，均观测到miR-26a表达下调及C/ebpbeta表达上调。对C/ebpbeta进行基因敲低，可抑制感染巨噬细胞中选定的M2标志物（如Il10与Irf4）的表达。研究通过以下发现证实了上述通路的重要性：转染miR-26a模拟物，或对Klf4、Creb、C/ebpbeta进行基因敲低，均可减弱结核分枝杆菌在巨噬细胞内的存活能力。综上，本研究结果表明，miR-26a/KLF4及CREB-C/EBPβ信号通路在调控结核分枝杆菌在巨噬细胞内的存活过程中发挥关键作用。本研究加深了我们对结核分枝杆菌如何劫持宿主信号通路以在巨噬细胞内存活的认知，并为开发靶向宿主的干预手段提供了全新的探索方向。