Novel Mechanisms of Pulmonary Fibrosis

肺纤维化的新机制

• 批准号: 10660225
• 负责人: Francis Xavier McCormack
• 金额: $ 64.31万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-20 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660225
• 关键词: Acceleration; Acids; Actins; Address; Alveolar; Alveolar Macrophages; Animals; Asbestos; Attenuated; Automobile Driving; Biochemical; Bone Marrow; Bone Matrix; Bone Resorption; Calcitonin Receptor; Calcium; Cell Differentiation process; Cell Lineage; Cell physiology; Cells; Clinical; Clinical Trials; Coculture Techniques; Complex; Defect; Development; Disease; Enzymes; Epithelium; Exposure to; Extracellular Matrix; FDA approved; Fibroblasts; Fibrosis; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Giant Cells; Goals; Histologic; Homeostasis; Host Defense; Human; Hydrochloric Acid; Hydroxyapatites; In Vitro; Inflammasome; Inhalation; Injury; Learning; Lesion; Liquid substance; Lung; Lung diseases; Macrophage; Macrophage Colony-Stimulating Factor; Measures; Mediating; Minerals; Molecular Target; Mus; Mutation; Mycobacterium tuberculosis; Myelogenous; Myeloid Cells; Occupational; Osteoclasts; PPAR gamma; Particulate; Pathogenesis; Pathway interactions; Patients; Pattern; Peptide Hydrolases; Phospholipid Metabolism; Physiological; Planet Earth; Pneumoconiosis; Predisposition; Prevalence; Process; Production; Proliferating; Proteins; Proton Pump; Pulmonary Alveolar Proteinosis; Pulmonary Challenge; Pulmonary Fibrosis; Pulmonary alveolar microlithiasis; Reporting; Role; Silicon Dioxide; Silicosis; Slice; Specific qualifier value; Stereotyping; Structure of parenchyma of lung; Syndrome; TNFSF11 gene; Testing; Tissues; Transforming Growth Factor beta; Up-Regulation; Zoledronic Acid; autosome; bisphosphonate; black lung; bone; calcium phosphate; cathepsin K; cell injury; cell transformation; cytokine; experimental study; fibrotic lung; fibrotic lung disease; gene repression; human RNA sequencing; in vivo; inorganic phosphate; insight; lipid metabolism; migration; monocyte; mouse model; novel; osteoclastogenesis; particle; pharmacologic; pre-clinical; prevent; programs; protein expression; recruit; response; single-cell RNA sequencing; sodium-phosphate cotransporter proteins; surfactant; tartrate-resistant acid phosphatase; theories

This proposal explores the role of a pulmonary osteoclast-like cell (POLC) in silicosis. We first discovered POLCs while studying pulmonary alveolar microlithiasis (PAM), a rare, autosomal recessive disorder caused by mutations in the epithelial sodium phosphate co-transporter, Npt2b. Phosphate accumulates in the alveolar lining fluid and complexes with calcium to form spherical hydroxyapatite microliths containing bone matrix proteins and surfactant components. Contact of microliths with tissue resident alveolar macrophages (TR-AM) and recruited monocytes (Mo-AM) induces osteoclastic transformation in the Npt2b-/- mouse, with expression of the full repertoire of osteoclast signature gene and protein expression in multinucleated giant cells (MNGC) including tartrate resistant acid phosphatase (TRAP), cathepsin K (CTSK), calcitonin receptor (CALCR) and the proton pump, ATP6V0D2. Single cell RNA sequencing (scRNAseq) of human PAM lung also confirmed a robust osteoclast signature in AM, and IHC confirmed the presence of TRAP and CTSK positive MNGC. Like humans, Npt2b-/- animals develop modest pulmonary fibrosis and a marked restrictive physiologic defect. We found that microliths induce alveolar expression of the requisite osteoclastogenic cytokines for POLC differentiation (i.e. M- CSF, RANKL) and expression of hydrochloric acid and CTSK that both dissolve stones and damage tissues. We were surprised to find that the Npt2b-/- mice also develop pulmonary alveolar proteinosis, which has not been reported in PAM, but is known to be associated with another particulate exposure syndrome, silicoproteinosis. This led us to consider that osteoclastic transformation of TR-AM and Mo-AM is a stereotypic response to inhaled particles, resulting in the concomitant loss of AM surfactant catabolic and host defense functions (in the form of susceptibility to Tb). Indeed, snRNAseq, rtPCR measures of gene expression and IHC assessments revealed that silica challenge is also associated with RANKL-dependent osteoclastic transformation of BAL and parenchymal cells, and TRAP, CTSK and hydrochloric acid production, culminating in destructive remodeling and pulmonary fibrosis, and associated with a decrease in proteins required for lipid metabolism. Anti- RANKL treatment of mice attenuates the silicoproteinosis, POLC formation, fibrosis and the restrictive physiologic defect that occurs in silica-challenged mice. The differential tissue responses to dissolvable vs. persistent particulates forms the basis for our hypothesis that persistent acid and matrix degrading enzymes produced by POLC may be primary drivers of particulate-induced fibrosis. To test this hypothesis in three aims, we will determine; 1) Cell lineages and alveolar factors that give rise to POLC; 2) The role of osteoclastic functions and products in silica- induced fibrosis; and 3) The role of POLC in the pathogenesis of pulmonary fibrosis, in vivo. This proposal directly addresses mechanisms relevant to the alarming rise in the prevalence of progressive massive fibrosis in black lung claimants, and successful completion of the aims will provide preclinical evidence supporting anti- osteoclastic strategies such as bisphosphonates and anti-RANKL in exposed subjects.

该提案探讨了肺破骨细胞样细胞(Polc)在矽肺中的作用。我们首先发现了POLC 在研究肺泡微石症(PAM)时，一种罕见的常染色体隐性遗传病由 上皮性磷酸钠共转运蛋白Npt2b的突变。磷酸盐在牙槽衬里中积聚 液体和与钙的络合物形成球形羟基磷灰石微石，含有骨基质蛋白和 表面活性成分。微石与组织常驻肺泡巨噬细胞的接触和招募 单核细胞(Mo-AM)诱导Npt2b-/-小鼠破骨细胞转化 破骨细胞特征基因和蛋白在多核巨细胞(MNGC)中的表达谱 抗酒石酸酸性磷酸酶(TRAP)、组织蛋白酶K(CTSK)、降钙素受体(CALCR)和质子 泵，ATP6V0D2。人PAM肺的单细胞RNA测序(ScRNAseq)也证实了 AM和IHC的破骨细胞特征证实存在TRAP和CTSK阳性的MNGC。像人类一样， Npt2b-/-动物出现轻度肺纤维化和明显的限制性生理缺陷。我们发现 微石诱导牙槽骨中破骨细胞分化所必需的细胞因子的表达(即M- CSF、RANKL)和既能溶解结石又能损伤组织的盐酸和CTSK的表达。我们 我们惊讶地发现，Npt2b-/-小鼠也患上了肺泡蛋白沉积症，这还没有 在PAM中报告，但已知与另一种颗粒物暴露综合征有关，即硅蛋白沉着症。 这使得我们认为，tr-AM和Mo-AM的破骨转化是对吸入的刻板印象反应 颗粒，导致AM表面活性物质分解代谢和宿主防御功能的伴随丧失(以 对结核病的易感性)。事实上，SnRNAseq、RTPCR基因表达的测量和IHC评估揭示了 这种二氧化硅挑战也与RANKL依赖的BAL破骨细胞转化有关， 实质细胞，以及TRAP、CTSK和盐酸的产生，最终导致破坏性的重建 和肺纤维化，并与脂肪代谢所需蛋白质的减少有关。反RANKL 治疗小鼠减轻硅蛋白沉着症、Polc形成、纤维化和限制性生理缺陷 这种情况发生在硅胶挑战的小鼠身上。组织对可溶解颗粒物和持久性颗粒物的不同反应 形成了我们假设的基础，即Polc产生的持久性酸和基质降解酶可能 是颗粒物引起的纤维化的主要驱动力。为了在三个目标上检验这一假设，我们将确定：1)细胞 导致Polc的谱系和牙槽因素；2)硅石中破骨细胞功能和产物的作用- 诱导纤维化；以及3)Polc在体内肺纤维化发病机制中的作用。这项建议直接 阐述了与进行性大规模纤维化患病率惊人上升相关的机制 肺索赔人，并成功完成AIMS将提供临床前证据支持反 暴露于受试者的破骨策略，如双磷酸盐和抗RANKL。