Toll-interacting protein inhibits transforming growth factor beta signaling in mouse lung fibroblasts.
Toll-interacting protein inhibits transforming growth factor beta signaling in mouse lung fibroblasts.
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DOI：
10.1096/fba.2023-00054
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发表时间：
2024-01
影响因子：
2.7
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中科院分区：
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在全基因组关联研究中已确定Toll相互作用蛋白（TOLLIP）基因的变异与特发性肺纤维化的疾病风险、死亡率和对N-乙酰半胱氨酸治疗的反应相关。尽管已知TOLLIP调节先天免疫应答，但其在器官纤维化中的相关性仍然未知。文献中的先前工作表明TOLLIP抑制人细胞系中的转化生长因子β（TGFβ）信号传导。在这项研究中，我们研究了TOLLIP在小鼠肺成纤维细胞（MLF）对TGFβ的反应中的作用，以及在使用Tollip−/−小鼠的实验性肺纤维化博莱霉素模型中的作用。我们假设，如果TOLLIP负调控TGFβ信号传导，那么Tollip−/−小鼠肺成纤维细胞（MLF）对TGFβ治疗的反应会增强，Tollip−/−小鼠在博来霉素激发后会发生纤维化增加。用TGFβ（1 ng/mL）刺激原代MLF 24 h。获得RNA以通过RNA-seq评估总体转录应答，并通过qPCR评估肌成纤维细胞转变的标志物。TGFβ刺激的MLF的功能评估包括通过划痕试验的细胞迁移、细胞增殖和通过Matrigel的基质侵袭。在肺纤维化的体内模型中，Tollip−/−小鼠和野生型（WT）同窝小鼠经气管内给予博来霉素，并评估纤维化。我们通过SMAD 2、ERK 1/2和TGFβR1蛋白质印迹进一步检测了博来霉素损伤后体内TGFβ信号转导。在对TGFβ处理的响应中，WT和Tollip−/− MLF均表现出与肌成纤维细胞分化一致的整体转录变化。然而，Tollip−/− MLF与WT MLF相比显示出更多的差异表达基因，qPCR显示出更大的Acta 2上调。在功能上，与WT相比，Tollip-/-MLF还表现出迁移和基质胶侵袭性增加。我们在体外和体内发现了Tollip−/−通过SMAD 2增强TGFβ信号传导的证据。Tollip−/−小鼠使用标准体重调整剂量时存活率较低，第21天无纤维化差异的证据。根据性别调整给药后，第21天未观察到纤维化差异。然而，与WT相比，Tollip−/−小鼠在第14天的早期消退期间体重减轻更大，支气管肺泡灌洗液总蛋白增加，而没有证据表明第7天的急性肺损伤存在差异，表明肺损伤消退受损。Toll相互作用蛋白（TOLLIP）缺乏增强了小鼠肺成纤维细胞和博莱霉素损伤肺中的TGFβ信号传导。尽管小鼠肺中成纤维细胞活化增强，TGFβ信号负调控受损，Tollip−/−小鼠体内肺纤维化并未恶化。然而，在Tollip−/−中观察到博莱霉素诱导的肺损伤后体重减轻和支气管肺泡灌洗液中总蛋白增加，表明肺修复和损伤消退中的其他因素可能受到TOLLIP缺乏的影响。
Variations in the Toll‐interacting protein (TOLLIP) gene have been identified in genome‐wide association studies to correlate with risk of disease, mortality, and response to N‐acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFβ) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFβ and in the bleomycin model of experimental lung fibrosis using Tollip−/− mice. We hypothesize that if TOLLIP negatively regulates TGFβ signaling, then Tollip−/− mouse lung fibroblasts (MLFs) would have enhanced response to TGFβ treatment, and Tollip−/− mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFβ (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA‐seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFβ‐stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the in vivo model of lung fibrosis, Tollip−/− mice and wild‐type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFβ signaling in vivo after bleomycin injury by SMAD2, ERK1/2, and TGFβR1 Western blot. In response to TGFβ treatment, both WT and Tollip−/− MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, Tollip−/− MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of Acta2 by qPCR. Functionally, Tollip−/− MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFβ signaling in Tollip−/− through SMAD2 in vitro and in vivo. Tollip−/− mice experienced lower survival using a standard weight‐adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, Tollip−/− mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury. Toll‐interacting protein (TOLLIP) deficiency enhances TGFβ signaling in mouse lung fibroblasts and lungs of bleomycin‐injured lung. Despite enhanced fibroblast activation and impaired negative regulation of TGFβ signaling in mouse lungs, Tollip−/− mice did not demonstrate worse lung fibrosis in vivo. However, increased weight loss and total protein in the bronchoalveolar lavage fluid were observed in Tollip−/− following bleomycin‐induced lung injury, suggesting other factors in lung repair and injury resolution may be affected by TOLLIP deficiency.