Pulmonary osteoclast-like cells in silica induced pulmonary fibrosis.

The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored mechanisms of silica-induced pulmonary fibrosis in human lung samples collected from patients with occupational exposure to silica and in a longitudinal mouse model of silicosis using multiple modalities including whole-lung single-cell RNA sequencing and histological, biochemical, and physiologic assessments. In addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor κΒ ligand (RANKL) in pulmonary lymphocytes, and alveolar type II cells. Anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated pulmonary fibrosis. We conclude that silica induces differentiation of pulmonary osteoclast-like cells leading to progressive lung injury, likely due to sustained elaboration of bone-resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.

Insights into pulmonary phosphate homeostasis and osteoclastogenesis emerge from the study of pulmonary alveolar microlithiasis.

Pulmonary alveolar microlithiasis is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a pulmonary alveolar microlithiasis lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes and other proteins suggested a role for osteoclast-like cells in the host response to microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activation in a receptor activator of nuclear factor-κB ligand and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.

Pulmonary osteoclast-like cells in silica induced pulmonary fibrosis.

The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored the mechanisms of silica-induced pulmonary fibrosis in a mouse model using multiple modalities including whole-lung single-nucleus RNA sequencing. These analyses revealed that in addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor-κB ligand (RANKL) in pulmonary lymphocytes and alveolar type II cells. Furthermore, anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated silica-induced pulmonary fibrosis. We conclude that silica induces osteoclast-like differentiation of distinct recruited and tissue resident monocyte populations, leading to progressive lung injury, likely due to sustained elaboration of bone resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.

Mitogenic stimulation accelerates influenza-induced mortality by increasing susceptibility of alveolar type II cells to infection.

Development of pneumonia is the most lethal consequence of influenza, increasing mortality more than 50-fold compared with uncomplicated infection. The spread of viral infection from conducting airways to the alveolar epithelium is therefore a pivotal event in influenza pathogenesis. We found that mitogenic stimulation with keratinocyte growth factor (KGF) markedly accelerated mortality after infectious challenge with influenza A virus (IAV). Coadministration of KGF with IAV markedly accelerated the spread of viral infection from the airways to alveoli compared with challenge with IAV alone, based on spatial and temporal analyses of viral nucleoprotein staining of lung tissue sections and dissociated lung cells. To better define the temporal relationship between KGF administration and susceptibility to IAV infection in vivo, we administered KGF 120, 48, 24, and 0 h before intrapulmonary IAV challenge and assessed the percentages of proliferating and IAV-infected, alveolar type II (AECII) cells in dispersed lung cell populations. Peak AECII infectivity coincided with the timing of KGF administration that also induced peak AECII proliferation. AECII from mice that were given intrapulmonary KGF before isolation and then infected with IAV ex vivo exhibited the same temporal pattern of proliferation and infectious susceptibility. KGF-induced increases in mortality, AECII proliferation, and enhanced IAV susceptibility were all reversed by pretreatment of the animals with the mTOR inhibitor rapamycin before mitogenic stimulation. Taken together, these data suggest mTOR signaling-dependent, mitogenic conditioning of AECII is a determinant of host susceptibility to infection with IAV.

Modeling pulmonary alveolar microlithiasis by epithelial deletion of the Npt2b sodium phosphate cotransporter reveals putative biomarkers and strategies for treatment.

Pulmonary alveolar microlithiasis (PAM) is a rare, autosomal recessive lung disorder associated with progressive accumulation of calcium phosphate microliths. Inactivating mutations in SLC34A2, which encodes the NPT2b sodium-dependent phosphate cotransporter, has been proposed as a cause of PAM. We show that epithelial deletion of Npt2b in mice results in a progressive pulmonary process characterized by diffuse alveolar microlith accumulation, radiographic opacification, restrictive physiology, inflammation, fibrosis, and an unexpected alveolar phospholipidosis. Cytokine and surfactant protein elevations in the alveolar lavage and serum of PAM mice and confirmed in serum from PAM patients identify serum MCP-1 (monocyte chemotactic protein 1) and SP-D (surfactant protein D) as potential biomarkers. Microliths introduced by adoptive transfer into the lungs of wild-type mice produce marked macrophage-rich inflammation and elevation of serum MCP-1 that peaks at 1 week and resolves at 1 month, concomitant with clearance of stones. Microliths isolated by bronchoalveolar lavage readily dissolve in EDTA, and therapeutic whole-lung EDTA lavage reduces the burden of stones in the lungs. A low-phosphate diet prevents microlith formation in young animals and reduces lung injury on the basis of reduction in serum SP-D. The burden of pulmonary calcium deposits in established PAM is also diminished within 4 weeks by a low-phosphate diet challenge. These data support a causative role for Npt2b in the pathogenesis of PAM and the use of the PAM mouse model as a preclinical platform for the development of biomarkers and therapeutic strategies.