Identification and function of Tbx4 resident fibroblasts as a major source of fibrotic fibroblasts

Progressive tissue fibrosis is a major cause of morbidity, and idiopathic pulmonary fibrosis (IPF) is a terminal illness characterized by unremitting matrix deposition in the lung with very limited choice of therapies. The imcomplete understanding of the mechanisms of progressive fibrosis curbs the progress in therapeutics development. Of which, the origin of fibrotic fibroblasts has been poorly defined during the pathogenesis of tissue fibrosis. Here, we fate-mapped a early embryonic transcription factor T-box gene 4 (Tbx4)-derived mesenchymal progenitors in injured adult lung and found that Tbx4+ lineage cells are the major source of myofibroblasts. The ablation of Tbx4+ cells or disruption of Tbx4 signaling attenuated lung fibrosis in bleomycin injury model in mice in vivo. Furthermore, Tbx4+ fibroblasts are more invasive and the regulation of fibroblast invasiveness by Tbx4 is through mediating hyaluronan synthase 2 (HAS2). This study identified a major mesenchymal transcription factor driving the development of fibrotic fibroblasts during lung fibrosis. Understanding the origin, signaling, and functions of these fibroblasts would prove pivotal in the development of therapeutics for patients with progressive fibrotic diseases. We used microarrays to detail the gene expression of Tbx4 and non-Tbx4 cultured fibroblasts. Lung fibroblasts were cultured from Tbx4-Cre;RosaTm mice. Tbx4-Tdtomato positive and Tbx4-Tdtomato negative fibroblasts were sorted by flow cytometry. RNA were extracted and hybridized on Affymetrix microarrays.

进行性组织纤维化是引发患者发病的主要原因，特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）便是一种以肺部持续性基质沉积为特征的终末期疾病，可供选择的治疗手段极为有限。目前学界对进行性纤维化的发病机制认识不足，这制约了治疗药物的开发进程。其中，在组织纤维化的发病过程中，纤维化成纤维细胞的起源仍未得到明确阐明。本研究对损伤成年肺中的早期胚胎转录因子T-box基因4（T-box gene 4, Tbx4）阳性间充质祖细胞开展谱系示踪研究，发现Tbx4+谱系细胞是肌成纤维细胞的主要来源。在博莱霉素诱导的小鼠体内肺损伤模型中，清除Tbx4+细胞或阻断Tbx4信号通路，可有效减轻肺纤维化程度。进一步研究表明，Tbx4+成纤维细胞侵袭能力更强，且Tbx4通过介导透明质酸合酶2（hyaluronan synthase 2, HAS2）的表达，调控成纤维细胞的侵袭性。本研究明确了驱动肺纤维化进程中纤维化成纤维细胞生成的关键间充质转录因子。阐明此类成纤维细胞的起源、信号通路及功能，将为进行性纤维化疾病患者的治疗药物开发提供关键依据。本研究通过基因芯片技术详细分析了Tbx4+与Tbx4-体外培养成纤维细胞的基因表达谱。实验所用肺成纤维细胞源自Tbx4-Cre；RosaTm小鼠，经体外培养后，通过流式细胞术分选出Tbx4-Tdtomato阳性与Tbx4-Tdtomato阴性的成纤维细胞，提取RNA后在Affymetrix基因芯片上完成杂交实验。