Role of Pulmonary Osteoclast-Like Cells in Lung Injury

该提案探讨了肺破骨细胞样细胞(Polc)在石棉诱导的肺纤维化中的作用。 我们在研究一种罕见的常染色体隐性遗传性肺泡微石症(PAM)时首次发现了POLC 由上皮性磷酸钠共转运蛋白Npt2b突变引起的疾病。磷酸盐的积累 在牙槽内衬液和钙的络合物中形成含骨的球形羟基磷灰石微石 基质蛋白和表面活性成分。微石粉与肺泡巨噬细胞的接触及募集 肺泡单核细胞(Alv-Mo)诱导破骨细胞转化 多核巨细胞(MNGC)中的破骨细胞特征基因和蛋白，包括抗酒石酸 磷酸酶(TRAP)、组织蛋白酶K(CTSK)和质子泵ATP6V0D2。单细胞核糖核酸测序 人PAM肺也证实了AM中有强烈的破骨细胞特征，IHC证实了TRAP的存在 CTSK阳性MNGC。像人类一样，Npt2b-/-动物也会出现适度的肺纤维化和明显的 限制性生理缺陷。我们发现，微石粉诱导牙槽骨中必需的 破骨细胞因子MCSF和RANKL对Polc分化及盐酸和 CTSK既能溶解结石，又能破坏组织。我们还发现，当微结石被采用时 转移到WT动物的肺内，附着在巨噬细胞上或被巨噬细胞降解，并被清除 28d内无残余炎症或纤维化，无肺损伤迹象。我们注意到高密度的浸润物 在我们的索引中，PAM患者在服用双膦酸类药物和抗RANKL时进展迅速 治疗减缓了Npt2b-/-小鼠体内微结石的清除。这些数据使我们得出结论，破骨细胞 AM和Alv-Mo的转化是肺对微结石的主要防御，它可能代表着一种 对包括石棉在内的其他颗粒的刻板反应。事实上，我们发现石棉的挑战也是 与髓系细胞中TRAP和CTSK的表达以及BAL和肺组织中MNGC的表达有关 最终导致破坏性重塑和肺纤维化。石棉未经生理盐水处理的BAL细胞 当小鼠被移植到牛骨片上时，小鼠会降解骨骼并从骨基质中释放出胶原碎片， 破骨细胞的标志性功能。完全愈合与纤维化对可溶性的不同组织反应 (微石)与持久性(石棉)颗粒形成了我们假设酸和基质降解的基础 当吸入的颗粒物是无敌的时，POLC产生的酶可能是纤维化的主要驱动因素。为了测试 这一假设有三个目的，我们将确定：1)肺性破骨细胞的基因程序 单核细胞对石棉反应的规范，2)单核细胞获得的破骨细胞功能 和巨噬细胞对石棉的攻击，3)多聚细胞在石棉诱导的发病机制中的作用 肺纤维化。该提案直接涉及与进行性肺纤维化相关的机制， 仍然发生在从20世纪30年代到20世纪30年代期间在军队所有部门接触石棉的数千名退伍军人中 越南、海湾和阿富汗战争，并寻求发展临床前证据支持反破骨 用于治疗石棉肺的策略，如Denosumab的双膦酸盐。

This proposal explores the role of a pulmonary osteoclast-like cell (POLC) in asbestos-induced pulmonary fibrosis. 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 alveolar macrophages (AM) and recruited alveolar monocytes (Alv-Mo) induces osteoclastic transformation, with expression of the full repertoire of osteoclast signature genes and proteins in multinucleated giant cells (MNGC) including tartrate resistant acid phosphatase (TRAP), cathepsin K (CTSK), and the proton pump ATP6V0D2. Single cell RNA sequencing 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, mCSF and RANKL, for POLC differentiation and expression of hydrochloric acid and CTSK that both dissolve stones and damage tissues. We also found that when microliths were adoptively transferred into the lungs of WT animals, they attached to or were degraded by macrophages, and were cleared within 28d without residual inflammation or fibrosis or evidence of lung injury. We noted that hyperdense infiltrates in our index PAM patient progressed rapidly when she was placed on bisphosphonates, and that anti-RANKL therapy slowed the clearance of microliths in Npt2b-/- mice. These data led us to the conclusion that osteoclastic transformation of AM and Alv-Mo is the lung's primary defense against microliths and that it may represent a stereotypic response to other particles, including asbestos. Indeed, we find that asbestos challenge is also associated with TRAP and CTSK expression in myeloid cells and MNGC in the BAL and lung tissue, and that it culminates in destructive remodeling and pulmonary fibrosis. The BAL cells from asbestos but not saline treated mice degrade bone and liberate collagen fragments from the bone matrix when plated on bovine bone slices, the signature function of osteoclasts. The differential tissue responses of complete healing vs. fibrosis to dissolvable (microliths) vs. persistent (asbestos) particulates forms the basis for our hypothesis that acid and matrix degrading enzymes produced by POLCs may be primary drivers of fibrosis when the inhaled particle is invincible. To test this hypothesis in three aims, we will determine; 1) the gene programs that underlie pulmonary osteoclastic specification of recruited monocytes in response to asbestos, 2) the osteoclast functions acquired by monocytes and macrophages upon asbestos challenge, 3) the role of POLCs in the pathogenesis of asbestos-induced pulmonary fibrosis. This proposal directly addresses mechanisms relevant to progressive pulmonary fibrosis that still occurs in the thousands of veterans exposed to asbestos in all branches of the military from the 1930s through the Vietnam, Gulf and Afghanistan Wars, and seeks to develop preclinical evidence supporting anti-osteoclastic strategies such as bisphosphonates of Denosumab for the treatment of asbestosis.