BHLHE40 regulates myeloid cell polarization through IL-10-dependent and independent mechanisms

Better understanding of the host responses to Mycobacterium tuberculosis (Mtb) infections is required to prevent tuberculosis and develop new therapeutic interventions. The host transcription factor BHLHE40 is essential for controlling Mtb infection, in part by repressing Il10 expression, where excess IL-10 contributes to the early susceptibility of Bhlhe40-/- mice to Mtb infection. Deletion of Bhlhe40 in lung macrophages and dendritic cells is sufficient to increase the susceptibility of mice to Mtb infection, but how BHLHE40 impacts macrophage and dendritic cell responses to Mtb is unknown. Here we report that BHLHE40 is required in myeloid cells exposed to GM-CSF, an abundant cytokine in the lung, to promote the expression of genes associated with a pro-inflammatory state and better control of Mtb infection. Loss of Bhlhe40 expression in murine bone marrow derived myeloid cells cultured in the presence of GM-CSF results in lower levels of pro-inflammatory associated signaling molecules IL-1b, IL-6, IL-12, TNFa, iNOS, IL-2, KC, and RANTES, as well as higher levels of the anti-inflammatory associated molecules MCP-1 and IL-10 following exposure to heat-killed Mtb. Deletion of Il10 in Bhlhe40-/- myeloid cells restored some, but not all, pro-inflammatory signals, demonstrating that BHLHE40 promotes pro-inflammatory responses via both IL-10-dependent and independent mechanisms. In addition, we show that macrophages and neutrophils within the lungs of Mtb-infected Bhlhe40-/- mice exhibit defects in iNOS production compared to infected WT mice, supporting that BHLHE40 promotes pro-inflammatory responses in innate immune cells, which may contribute to the essential role for BHLHE40 during Mtb infection in vivo. Overall design: To investigate the role of BHLHE40 in inflammatory responses in GM-CSF-cultured cells and determine what effects were dependent on excess IL-10 in the absence of BHLHE40, we generated GM-CSF cultures from WT, Bhlhe40-/-, and Bhlhe40-/-Il10-/- GM-CSF mice and collected samples in the following conditions: naïve, mock-treated, and heat-killed Mycobacterium tuberculosis (HKTB)-treated.

为预防结核病并开发新型治疗干预手段，目前仍需更深入解析宿主对结核分枝杆菌（Mycobacterium tuberculosis, Mtb）感染的应答机制。
宿主转录因子BHLHE40是控制结核分枝杆菌感染的关键分子，其部分作用机制为抑制白细胞介素10（Interleukin-10, IL-10）的表达；过量的IL-10会导致Bhlhe40基因敲除（Bhlhe40-/-）小鼠在结核分枝杆菌感染早期更易受感染。
肺巨噬细胞与树突状细胞中Bhlhe40的特异性缺失足以提升小鼠对结核分枝杆菌感染的易感性，但BHLHE40如何调控巨噬细胞与树突状细胞对结核分枝杆菌的应答反应仍有待阐明。
本研究证实，在暴露于肺内高丰度细胞因子粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GM-CSF）的髓系细胞中，BHLHE40是促进促炎相关基因表达、更有效控制结核分枝杆菌感染所必需的分子。
在经GM-CSF培养的小鼠骨髓来源髓系细胞中，Bhlhe40表达缺失会导致经热灭活结核分枝杆菌刺激后，促炎相关信号分子IL-1β、IL-6、IL-12、TNF-α、诱导型一氧化氮合酶（iNOS）、IL-2、KC及RANTES的表达水平显著降低，而抗炎相关分子MCP-1与IL-10的表达水平显著升高。
在Bhlhe40-/-髓系细胞中敲除Il10可恢复部分（而非全部）促炎信号通路，这表明BHLHE40可通过IL-10依赖与非依赖两种途径介导促炎应答。
此外，本研究发现，相较于感染野生型（WT）小鼠的肺组织，结核分枝杆菌感染的Bhlhe40-/-小鼠肺内巨噬细胞与中性粒细胞的iNOS合成存在缺陷，这佐证了BHLHE40可在先天免疫细胞中促进促炎应答，这一效应可能是BHLHE40在体内结核分枝杆菌感染过程中发挥核心作用的关键机制之一。
实验整体设计：为探究BHLHE40在GM-CSF培养细胞炎症应答中的作用，并明确Bhlhe40缺失时过量IL-10所介导的效应，我们分别从野生型、Bhlhe40-/-及Bhlhe40-/-Il10-/-双基因敲除小鼠中构建GM-CSF培养体系，并在以下三种条件下收集样本：未致敏组、模拟处理组及热灭活结核分枝杆菌（heat-killed Mycobacterium tuberculosis, HKTB）处理组。