Role of Pulmonary Osteoclast-Like Cells in Lung Injury

本研究旨在探讨肺破骨细胞样细胞（POLC）在石棉诱导的肺纤维化中的作用。 我们在研究肺泡微石症（PAM）时首次发现了POLC， 由上皮磷酸钠协同转运蛋白Npt 2b突变引起的疾病。磷酸盐积累 并与钙络合形成含有骨的球形羟基磷灰石微石 基质蛋白和表面活性剂组分。微石与肺泡巨噬细胞（AM）接触并招募 肺泡单核细胞（Alv-Mo）诱导骨细胞转化，表达完整的 多核巨细胞（MNGC）中的破骨细胞标记基因和蛋白质，包括酒石酸抗性酸 磷酸酶（TRAP）、组织蛋白酶K（CTSK）和质子泵ATP 6V 0 D2。单细胞RNA测序 人PAM肺也证实了AM中强有力的破骨细胞特征，并且IHC证实了TRAP的存在 CTSK阳性MNGC。与人类一样，Npt 2b-/-动物发生中度肺纤维化和显著的肺纤维化。 限制性生理缺陷我们发现，微石诱导肺泡表达必需的 破骨细胞生成细胞因子mCSF和RANKL，用于POLC分化和盐酸表达， CTSK既溶解结石又损伤组织。我们还发现，当细石器被收养时， 转移到WT动物的肺中，它们附着于巨噬细胞或被巨噬细胞降解，并被清除 28天内无残留炎症或纤维化或肺损伤证据。我们注意到高密度浸润 在我们指数PAM患者中，当她被置于双膦酸盐时，她进展迅速，且抗RANKL 治疗减慢了Npt 2b-/-小鼠中微石的清除。这些数据使我们得出结论， AM和Alv-Mo的转化是肺对细石的主要防御，它可能代表了肺的一种免疫反应。 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的表达相关， 最终导致破坏性重塑和肺纤维化来自石棉但未经盐水处理的BAL细胞 当小鼠接种在牛骨切片上时，小鼠降解骨并从骨基质中释放胶原片段， 破骨细胞的标志性功能。完全愈合与纤维化对可溶性胶原的不同组织反应 （细石）与持久性（石棉）颗粒形成了我们假设酸和基质降解的基础 当吸入的颗粒不可抵抗时，POLC产生的酶可能是纤维化的主要驱动因素。测试 我们将从三个方面来研究这一假说：1）肺成骨细胞的基因程序 石棉对招募的单核细胞的特异性反应，2）单核细胞获得的破骨细胞功能 3）POLCs在石棉诱导的肺损伤发病机制中的作用 肺纤维化该提案直接针对与进行性肺纤维化相关的机制， 仍然发生在成千上万的退伍军人暴露于石棉在所有分支的军事从20世纪30年代通过 越南战争、海湾战争和阿富汗战争，并寻求开发支持抗骨质疏松的临床前证据。 例如地舒单抗的双膦酸盐用于治疗石棉肺的策略。

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.