内质网应激调控的肺泡巨噬细胞M1/M2极化平衡在急性肺损伤中的作用

Acute lung injury (ALI) and its more severe manifestation, acute respiratory distress syndrome (ARDS), are characterised by acute inflammation that affects the function of the gas exchange surface of the lung. Dysregulated inflammation, inappropriate accumulation of inflammatory mediators, uncontrolled activation of inflammatory effector cells remain central pathophysiologic concepts in ALI and ARDS. Despite recent advances in diagnostic and therapeutic modalities, ALI/ARDS is still associated with significant morbidity and mortality. ..Alveolar macrophages (AMs) are highly heterogeneous and plastic cells that can rapidly change their function in response to local enviromental signals and display different polarization states, thus playing an important role in regulating the inflammatory response in ALI. Our preliminary data have demonstrated that AMs exhibit predominantly the inflammatory M1 phenotype in induction phase of ALI induced by LPS in mice, providing evidene for a potential role of alveolar macrophage polarization imbalance (M1 vs. M2) in LPS-induced ALI mouse model. Whereas, toll-like receptor 4 (TLR4) knockout or endoplasmic reticulum (ER) stress inhibitor salubrinal could promote the resolution of ALI induced by LPS. Based on those data we propose that ER stress signaling may serve as a novel mechanism that induces macrophage polarization toward M1 phenotype and M1/M2 imbalance in LPS-induced ALI, thus exaggerating the inflammatory process of ALI. ..In the present study, we firstly identify the macrophage pheotype by isolation of peripheral blood mononuclear cells and AMs from ALI/ARDS patients and LPS-induced ALI mouse model; we then investigate whether ER stress signaling is involved in regulation of macrophage phenotype selection toward M1 and the underlying molecular mechanism by using human PBMCs, bone marrow stem cells (BMSCs) from WT and TLR4, PERK, XBP1, ATF6 gene knockour mice and LPS-induced ALI mouse models.In addition, we explore whether inhibition of TLR4 and ER stress could shift alveolar macrophage from M1 to M2 and promote the resolution of ALI. Our study demonstrates the role of ER stress-regulated macrophage polarization in the process of ALI, providing evidence that therapeutic intervention to modulate the TLR4 and ER stress signaling may be an effective strategy to promote the resolution of ALI.

急性肺损伤/急性呼吸窘迫综合症（ALI/ARDS）的本质是肺内失控性炎症反应及组织损伤，其发病率及病死率较高。巨噬细胞极化的平衡失调能影响局部组织的微环境炎症状态。项目前期研究发现LPS诱导的小鼠ALI模型中，腹腔巨噬细胞（AMs）的M1/M2比例失调；TLR4受体敲除或者内质网应激抑制剂能够改善小鼠ALI症状。我们推测TLR4和内质网应激信号通路参与调节AMs的极化状态，导致M/M2比例失衡，促进ALI炎症反应过程。研究拟通过对ALI/ARDS患者AMs的表型进行分析，采用人外周血单核细胞，野生型和基因敲除小鼠骨髓基质干细胞及LPS诱导的小鼠ALI模型，探讨TLR4和内质网应激信号通路参与调节的AMs表型变化在LPS诱导ALI中的作用及可能机制。本研究从巨噬细胞极化平衡对局部微环境炎症影响方面揭示了内质网应激调节的AMs极化平衡在ALI疾病进程中的作用，可为临床开辟新的治疗策略提供指导。

急性肺损伤（ALI）的本质是肺内失控性炎症反应及组织损伤，其发病率及病死率较高。肺泡巨噬细胞（AM）能适应体内复杂的微环境变化，表现出不同的极化表型和功能，在始动ALI炎症反应、促进PMN浸润及肺组织损伤中发挥着重要作用。内质网应激作为多种应激反应的共同通路，广泛参与多种炎性疾病的发生和发展。但目前尚未见内质网应激调控的巨噬细胞极化状态在ALI发生发展中作用的研究报道。本项目主要研究结果显示：1）通过对临床样本和LPS诱导小鼠ALI模型的检测发现，ALI患者肺组织及LPS诱导ALI小鼠肺组织中存在内质网应激及M1/M2极化平衡失调；2）体外分离小鼠骨髓基质细胞（BM）并分化为M1和M2，检测发现M1型巨噬细胞中内质网应激及未折叠蛋白反应（UPR）信号通路明显激活；3）通过对体外诱导分化的M1和M2细胞给予内质网应激诱导剂或者抑制剂后发现，内质网应激促进M1，抑制M2，加重炎症反应；4）通过对体外诱导分化的M1和M2细胞给予UPR各信号通路抑制剂后发现，抑制IRE1α能够抑制内质网应激，促进M1向M2转化；5）从WT，MyD88 KO和TRIF KO肺泡灌洗液中分离AM后给予LPS刺激，研究发现抑制TLR4-TRIF信号通路能够抑制内质网应激，促进M1向M2转化；6）小鼠腹腔给予TUDCA预处理后再给予LPS，分离腹腔巨噬细胞，研究发现抑制UPR信号通路促进M1向M2转换，改善LPS诱导的ALI。本研究结果初步证实，LPS通过TLR4-TRIF-IRE1α信号通路诱导巨噬细胞向M1转化，进而参与LPS诱导的ALI发生，而抑制上述通路可以促进巨噬细胞由M1向M2转化，进而改善LPS诱导的ALI。本项目通过研究内质网应激对巨噬细胞极化平衡的影响，进而对肺组织局部微环境影响方面揭示了内质网应激调节的AM极化平衡在ALI疾病进程中的作用，为临床开辟新的治疗策略提供指导和帮助。