Monoclonal Antibody Targeting Pear1 for Pulmonary Fibrosis Therapy [bulk RNA-seq]

Pulmonary fibrosis (PF) is a chronic interstitial lung disease that causes irreversible and progressive lung scarring and respiratory failure. Activation of fibroblasts (FBs) play a central role in progression of PF. Here we report that platelet endothelial aggregation receptor 1 (Pear1) in FBs is a new molecular target for PF therapy. Pear1 deficiency spontaneously caused respiratory function decline and alveolar collagens accumulation in old mice. The degree of PF and mortality induced by bleomycin were significantly enhanced in Pear1 deficient mice. FB Mesenchyme-specific Pear1 deficiency aggravated bleomycin-induced PF, confirming that Pear1 modulates PF progression probably byvia regulation of FBs function. Single cell RNA-seq analysis of pulmonary FB and functional enrichment analysis revealed drastic expansion of Aactivated- FB clusters and enrichment of activated FB marker genes in extracellular matrix (ECM) development and pulmonary fibrosis in Pear1-/- fibrotic lungs. CD140+ bulk tissue RNA-seq analysis further confirmed that multiple mesenchyme development pathways especially epithelial mesenchymal transition (EMT) are enriched with up-regulated genes involving FB mediated ECM organization and development in in Pear1-/- fibrotic lungs. We further found that Pear1 associated with Protein Phosphatase 1 to suppress fibrotic factors such as TGFß, FGF or PDGF-induced intracellular signalling and FB activation. Intratracheal aerosolization of monoclonal antibody activating Pear1 greatly ameliorates PF in both wild-type mice and Pear1-humanized mice, suggesting that targeting Pear1 may serve as a new therapeutic strategy for PFand significantly improves their survival rate. Fresh lung tissues were harvested from mouse bleomycin model. We used flow cytometry to sort out Cd140+ cells from total lung cells. Total RNA were isolated from the cells and used for RNA-seq assay.

肺纤维化（Pulmonary fibrosis, PF）是一类慢性间质性肺部疾病，可引发不可逆且进行性的肺部瘢痕形成与呼吸衰竭。成纤维细胞（fibroblasts, FBs）的活化在肺纤维化的疾病进展中发挥核心作用。本研究报道，成纤维细胞中的血小板内皮聚集受体1（platelet endothelial aggregation receptor 1, Pear1）是肺纤维化治疗的全新分子靶点。Pear1基因缺失可自发引发老年小鼠呼吸功能下降与肺泡胶原沉积。在Pear1缺失小鼠体内，博来霉素诱导的肺纤维化程度与死亡率均显著升高。成纤维细胞间质特异性Pear1缺失会加重博来霉素诱导的肺纤维化，证实Pear1或通过调控成纤维细胞功能来影响肺纤维化的进展。对肺部成纤维细胞开展的单细胞RNA测序（single cell RNA-seq）分析与功能富集分析结果显示，在Pear1基因敲除（Pear1-/-）的纤维化肺组织中，活化成纤维细胞簇出现显著扩增，且活化成纤维细胞的标志物基因富集于细胞外基质（extracellular matrix, ECM）发育与肺纤维化相关通路。CD140+ bulk组织RNA测序分析进一步证实，在Pear1-/-纤维化肺组织中，多条间质发育通路（尤其是上皮间质转化（epithelial mesenchymal transition, EMT））存在上调基因富集，这些基因涉及成纤维细胞介导的细胞外基质组织与发育过程。本研究还发现，Pear1可与蛋白磷酸酶1（Protein Phosphatase 1）结合，从而抑制转化生长因子β（transforming growth factor β, TGFβ）、成纤维细胞生长因子（fibroblast growth factor, FGF）与血小板衍生生长因子（platelet-derived growth factor, PDGF）等致纤维化因子所诱导的细胞内信号通路及成纤维细胞活化。通过气管内雾化递送靶向活化Pear1的单克隆抗体，可显著改善野生型小鼠与Pear1人源化小鼠的肺纤维化症状，提示靶向Pear1或可成为肺纤维化的全新治疗策略，并显著提升小鼠存活率。本研究从博来霉素诱导的小鼠肺纤维化模型中获取新鲜肺组织，采用流式细胞术（flow cytometry）从总肺细胞中分选得到CD140+细胞，提取细胞总RNA并开展RNA测序（RNA-seq）分析。