Epithelial cell dysfunction is postulated as an important component in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mutations in the surfactant protein C (SP-C) gene (SFTPC), an alveolar type II (AT2) cell–restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knockin mouse model capable of regulated expression of an IPF-associated isoleucine-to-threonine substitution at codon 73 (I73T) in Sftpc (SP-C^I73T). Tamoxifen-treated SP-C^I73T cohorts developed rapid increases in Sftpc^I73T mRNA and misprocessed proSP-C^I73T protein accompanied by increased early mortality (days 7–14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA content of select inflammatory cytokines. Resolution of alveolitis (2–4 weeks), commensurate with a rise in TGF-β1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, α–smooth muscle actin–positive (α-SMA–positive) cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP-C expression in vivo causes spontaneous lung fibrosis, strengthening the role of AT2 cell dysfunction as a key upstream driver of IPF pathogenesis.

Red blood cells detect and bind cell-free nucleic acids, contributing to anemia and immune cell activation during acute inflammation.

Alveolar type 2 (AT2) cell endoplasmic reticulum (ER) stress is a prominent feature in adult and pediatric interstitial lung disease (ILD and ChILD), but in vivo models linking AT2 cell ER stress to ILD have been elusive. Based on a clinical ChILD case, we identified a critical cysteine residue in the surfactant protein C gene ( SFTPC ) BRICHOS domain whose mutation induced ER stress in vitro. To model this in vivo, we generated a knockin mouse model expressing a cysteine-to-glycine substitution at codon 121 (C121G) in the Sftpc gene. Sftpc ^C121G expression during fetal development resulted in a toxic gain-of-function causing fatal postnatal respiratory failure from disrupted lung morphogenesis. Induced Sftpc ^C121G expression in adult mice resulted in an ER-retained pro-protein causing AT2 cell ER stress. Sftpc ^C121G AT2 cells were a source of cytokines expressed in concert with development of polycellular alveolitis. These cytokines were subsequently found in a high-dimensional proteomic screen of bronchoalveolar lavage fluid from ChILD patients with the same class of SFTPC mutations. Following alveolitis resolution, Sftpc ^C121G mice developed spontaneous pulmonary fibrosis and restrictive lung impairment. This model provides proof of concept linking AT2 cell ER stress to fibrotic lung disease coupled with translationally relevant biomarkers.

Nitrogen mustard (NM) is an alkylating agent known to cause extensive pulmonary injury progressing to fibrosis. This is accompanied by a persistent macrophage inflammatory response. In these studies, we characterized the phenotype of macrophages accumulating in the lung over time following NM exposure. Treatment of rats with NM (0.125 mg/kg, intratracheally) resulted in an increase in CD11b+ macrophages in histologic sections. These cells consisted of inducible nitric oxide synthase+ (iNOS) proinflammatory M1 macrophages, and CD68+, CD163+, CD206+, YM-1+, and arginase-II+antiinflammatory M2 macrophages. Although M1 macrophages were prominent 1–3 days after NM, M2 macrophages were most notable at 28 days. At this time, they were enlarged and vacuolated, consistent with a profibrotic phenotype. Flow cytometric analysis of isolated lung macrophages identified three phenotypically distinct subpopulations: mature CD11b−, CD43−, and CD68+ resident macrophages, which decreased in numbers after NM; and two infiltrating (CD11b+) macrophage subsets: immature CD43+ M1 macrophages and mature CD43− M2 macrophages, which increased sequentially. Time-related increases in M1 (iNOS, IL-12α, COX-2, TNF-α, matrix metalloproteinase-9, matrix metalloproteinase-10) and M2 (IL-10, pentraxin-2, connective tissue growth factor, ApoE) genes, as well as chemokines/chemokine receptors associated with trafficking of M1 (CCR2, CCR5, CCL2, CCL5) and M2 (CX3CR1, fractalkine) macrophages to sites of injury, were also noted in macrophages isolated from the lung after NM. The appearance of M1 and M2 macrophages in the lung correlated with NM-induced acute injury and the development of fibrosis, suggesting a potential role of these macrophage subpopulations in the pathogenic response to NM.

Nitrogen mustard (NM) is a vesicant that causes lung injury and fibrosis, accompanied by a persistent macrophage inflammatory response. In these studies we analyzed the spleen as a source of these cells. Splenectomized (SPX) and sham control rats were treated intratracheally with NM (0.125 mg/kg) or PBS control. Macrophage responses were analyzed 1–7 days later. Splenectomy resulted in an increase in lung macrophages expressing CCR2, but a decrease in ATR-1α⁺ cells, receptors important in bone marrow and spleen monocyte trafficking, respectively. Splenectomy was also associated with an increase in proinflammatory M1 (iNOS⁺, CD11b⁺CD43⁺) macrophages in lungs of NM-treated rats, as well as greater upregulation of iNOS and COX-2 mRNA expression. Conversely, a decrease in CD11b⁺CD43⁻ M2 macrophages was observed in SPX rats, with no changes in CD68⁺, CD163⁺, CD206⁺, or YM-1⁺ M2 macrophages, suggesting distinct origins of M2 subpopulations responding to NM. Macrophage expression of M2 genes including IL-10, ApoE, PTX-2, PTX-3, 5-HT2α, and 5-HT7 was also reduced in NM-treated SPX rats compared with shams, indicating impaired M2 activity. Changes in lung macrophages responding to NM as a consequence of splenectomy were correlated with exacerbated tissue injury and more rapid fibrogenesis. These data demonstrate that the spleen is a source of a subset of M2 macrophages with anti-inflammatory activity; moreover, in their absence, proinflammatory/cytotoxic M1 macrophages predominate in the lung, resulting in heightened pathology. Understanding the origin of macrophages and characterizing their phenotype after vesicant exposure may lead to more targeted therapeutics aimed at reducing toxicity and disease pathogenesis.

Patients with idiopathic pulmonary fibrosis (IPF) often experience precipitous deteriorations, termed “acute exacerbations” (AE), marked by diffuse alveolitis and altered gas exchange, resulting in a significant loss of lung function or mortality. The missense isoleucine to threonine substitution at position 73 (I73T) in the alveolar type 2 cell-restricted surfactant protein-C (SP-C) gene (SFTPC) has been linked to clinical IPF. To better understand the sequence of events that impact AE-IPF, we leveraged a murine model of inducible SP-C^I73T (SP-C^I73T/I73TFlp^+/−) expression. Following administration of tamoxifen to 8–12-wk-old mice, an upregulation of Sftpc^I73T initiated a diffuse lung injury marked by increases in bronchoalveolar lavage fluid (BALF) protein and histochemical evidence of CD45⁺ and CD11b⁺ cell infiltrates. Flow cytometry of collagenase-digested lung cells revealed a transient, early reduction in SiglecF^hiCD11b^lowCD64^hiCD11c^hi macrophages, countered by the sequential accumulation of SiglecF^loCD11b⁺CD64⁻CD11c⁻CCR2⁺Ly6C⁺ immature macrophages (3 d), Ly6G⁺ neutrophils (7 d), and SiglecF^hiCD11b^hiCD11c^lo eosinophils (2 wk). By mRNA analysis, BALF cells demonstrated a time-dependent phenotypic shift from a proinflammatory (3 d) to an anti-inflammatory/profibrotic activation state, along with serial elaboration of monocyte and eosinophil recruitment factors. The i.v. administration of clodronate effectively reduced total BALF cell numbers, CCR2⁺ immature macrophages, and eosinophil influx while improving survival. In contrast, resident macrophage depletion from the intratracheal delivery of clodronate liposomes enhanced Sftpc^I73T-induced mortality. These results using Sftpc^I73T mice provide a detailed ontogeny for AE-IPF driven by alveolar epithelial dysfunction that induces a polycellular inflammation initiated by the early influx of proinflammatory CCR2⁺Ly6C^hi immature macrophages. Key Points In a murine model of lung fibrosis, an ontogeny of inflammatory events was defined. Mutant Sftpc^I73T expression caused early lung injury marked by a polycellular alveolitis. Depletion of peripheral monocytes was protective from Sftpc^I73T injury.