Durable alveolar engraftment of PSC-derived lung epithelial cells into immunocompetent mice.

Durable reconstitution of the distal lung epithelium with pluripotent stem cell (PSC) derivatives, if realized, would represent a promising therapy for diseases that result from alveolar damage. Here, we differentiate murine PSCs into self-renewing lung epithelial progenitors able to engraft into the injured distal lung epithelium of immunocompetent, syngeneic mouse recipients. After transplantation, these progenitors mature in the distal lung, assuming the molecular phenotypes of alveolar type 2 (AT2) and type 1 (AT1) cells. After months in vivo, donor-derived cells retain their mature phenotypes, as characterized by single-cell RNA sequencing (scRNA-seq), histologic profiling, and functional assessment that demonstrates continued capacity of the engrafted cells to proliferate and differentiate. These results indicate durable reconstitution of the distal lung's facultative progenitor and differentiated epithelial cell compartments with PSC-derived cells, thus establishing a novel model for pulmonary cell therapy that can be utilized to better understand the mechanisms and utility of engraftment.

PRDM3/16 Regulate Chromatin Accessibility Required for NKX2-1 Mediated Alveolar Epithelial Differentiation and Function.

Differential chromatin accessibility accompanies and mediates transcriptional control of diverse cell fates and their differentiation during embryogenesis. While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Here, we demonstrate that the paired domain zinc finger transcriptional regulators PRDM3 and PRDM16 regulate chromatin accessibility to mediate cell differentiation decisions during lung morphogenesis. Combined deletion of Prdm3 and Prdm16 in early lung endoderm caused perinatal lethality due to respiratory failure from loss of AT2 cell function. Prdm3/16 deletion led to the accumulation of partially differentiated AT1 cells and loss of AT2 cells. Combination of single cell RNA-seq, bulk ATAC-seq, and CUT&RUN demonstrated that PRDM3 and PRDM16 enhanced chromatin accessibility at NKX2-1 transcriptional targets in peripheral epithelial cells, all three factors binding together at a multitude of cell-type specific cis-active DNA elements. Network analysis demonstrated that PRDM3/16 regulated genes critical for perinatal AT2 cell differentiation, surfactant homeostasis, and innate host defense. Lineage specific deletion of PRDM3/16 in AT2 cells led to lineage infidelity, with PRDM3/16 null cells acquiring partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16.

Surfactant protein C mutation links postnatal type 2 cell dysfunction to adult disease.

Mutations in the gene SFTPC, encoding surfactant protein C (SP-C), are associated with interstitial lung disease in children and adults. To assess the natural history of disease, we knocked in a familial, disease-associated SFTPC mutation, L188Q (L184Q [LQ] in mice), into the mouse Sftpc locus. Translation of the mutant proprotein, proSP-CLQ, exceeded that of proSP-CWT in neonatal alveolar type 2 epithelial cells (AT2 cells) and was associated with transient activation of oxidative stress and apoptosis, leading to impaired expansion of AT2 cells during postnatal alveolarization. Differentiation of AT2 to AT1 cells was also inhibited in ex vivo organoid culture of AT2 cells isolated from LQ mice; importantly, treatment with antioxidant promoted alveolar differentiation. Upon completion of alveolarization, SftpcLQ expression was downregulated, leading to resolution of chronic stress responses; however, the failure to restore AT2 cell numbers resulted in a permanent loss of AT2 cells that was linked to decreased regenerative capacity in the adult lung. Collectively, these data support the hypothesis that susceptibility to disease in adult LQ mice is established during postnatal lung development, and they provide a potential explanation for the delayed onset of disease in patients with familial pulmonary fibrosis.

IGF1R controls mechanosignaling in myofibroblasts required for pulmonary alveologenesis.

Ventilation throughout life is dependent on the formation of pulmonary alveoli, which create an extensive surface area in which the close apposition of respiratory epithelium and endothelial cells of the pulmonary microvascular enables efficient gas exchange. Morphogenesis of the alveoli initiates at late gestation in humans and the early postnatal period in the mouse. Alveolar septation is directed by complex signaling interactions among multiple cell types. Here, we demonstrate that IGF1 receptor gene (Igf1r) expression by a subset of pulmonary fibroblasts is required for normal alveologenesis in mice. Postnatal deletion of Igf1r caused alveolar simplification, disrupting alveolar elastin networks and extracellular matrix without altering myofibroblast differentiation or proliferation. Moreover, loss of Igf1r impaired contractile properties of lung myofibroblasts and inhibited myosin light chain (MLC) phosphorylation and mechanotransductive nuclear YAP activity. Activation of p-AKT, p-MLC, and nuclear YAP in myofibroblasts was dependent on Igf1r. Pharmacologic activation of AKT enhanced MLC phosphorylation, increased YAP activation, and ameliorated alveolar simplification in vivo. IGF1R controls mechanosignaling in myofibroblasts required for lung alveologenesis.

In Vivo Generation of Lung and Thyroid Tissues from Embryonic Stem Cells Using Blastocyst Complementation.

Rationale: The regeneration and replacement of lung cells or tissues from induced pluripotent stem cell- or embryonic stem cell-derived cells represent future therapies for life-threatening pulmonary disorders but are limited by technical challenges to produce highly differentiated cells able to maintain lung function. Functional lung tissue-containing airways, alveoli, vasculature, and stroma have never been produced via directed differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells. We sought to produce all tissue components of the lung from bronchi to alveoli by embryo complementation.Objectives: To determine whether ESCs are capable of generating lung tissue in Nkx2-1-/- mouse embryos with lung agenesis.Methods: Blastocyst complementation was used to produce chimeras from normal mouse ESCs and Nkx2-1-/- embryos, which lack pulmonary tissues. Nkx2-1-/- chimeras were examined using immunostaining, transmission electronic microscopy, fluorescence-activated cell sorter analysis, and single-cell RNA sequencing.Measurements and Main Results: Although peripheral pulmonary and thyroid tissues are entirely lacking in Nkx2-1 gene-deleted embryos, pulmonary and thyroid structures in Nkx2-1-/- chimeras were restored after ESC complementation. Respiratory epithelial cell lineages in restored lungs of Nkx2-1-/- chimeras were derived almost entirely from ESCs, whereas endothelial, immune, and stromal cells were mosaic. ESC-derived cells from multiple respiratory cell lineages were highly differentiated and indistinguishable from endogenous cells based on morphology, ultrastructure, gene expression signatures, and cell surface proteins used to identify cell types by fluorescence-activated cell sorter.Conclusions: Lung and thyroid tissues were generated in vivo from ESCs by blastocyst complementation. Nkx2-1-/- chimeras can be used as "bioreactors" for in vivo differentiation and functional studies of ESC-derived progenitor cells.

Proteasome dysfunction in alveolar type 2 epithelial cells is associated with acute respiratory distress syndrome.

Proteasomes are a critical component of quality control that regulate turnover of short-lived, unfolded, and misfolded proteins. Proteasome activity has been therapeutically targeted and considered as a treatment option for several chronic lung disorders including pulmonary fibrosis. Although pharmacologic inhibition of proteasome activity effectively prevents the transformation of fibroblasts to myofibroblasts, the effect on alveolar type 2 (AT2) epithelial cells is not clear. To address this knowledge gap, we generated a genetic model in which a proteasome subunit, RPT3, which promotes assembly of active 26S proteasome, was conditionally deleted in AT2 cells of mice. Partial deletion of RPT3 resulted in 26S proteasome dysfunction, leading to augmented cell stress and cell death. Acute loss of AT2 cells resulted in depletion of alveolar surfactant, disruption of the alveolar epithelial barrier and, ultimately, lethal acute respiratory distress syndrome (ARDS). This study underscores importance of proteasome function in maintenance of AT2 cell homeostasis and supports the need to further investigate the role of proteasome dysfunction in ARDS pathogenesis.

EMC3 coordinates surfactant protein and lipid homeostasis required for respiration.

Adaptation to respiration at birth depends upon the synthesis of pulmonary surfactant, a lipid-protein complex that reduces surface tension at the air-liquid interface in the alveoli and prevents lung collapse during the ventilatory cycle. Herein, we demonstrated that the gene encoding a subunit of the endoplasmic reticulum membrane complex, EMC3, also known as TMEM111 (Emc3/Tmem111), was required for murine pulmonary surfactant synthesis and lung function at birth. Conditional deletion of Emc3 in murine embryonic lung epithelial cells disrupted the synthesis and packaging of surfactant lipids and proteins, impaired the formation of lamellar bodies, and induced the unfolded protein response in alveolar type 2 (AT2) cells. EMC3 was essential for the processing and routing of surfactant proteins, SP-B and SP-C, and the biogenesis of the phospholipid transport protein ABCA3. Transcriptomic, lipidomic, and proteomic analyses demonstrated that EMC3 coordinates the assembly of lipids and proteins in AT2 cells that is necessary for surfactant synthesis and function at birth.

Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells.

Lung alveoli, which are unique to air-breathing organisms, have been challenging to generate from pluripotent stem cells (PSCs) in part because there are limited model systems available to provide the necessary developmental roadmaps for in vitro differentiation. Here we report the generation of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, from human PSCs. Using multicolored fluorescent reporter lines, we track and purify human SFTPC+ alveolar progenitors as they emerge from endodermal precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells form monolayered epithelial "alveolospheres" in 3D cultures without the need for mesenchymal support, exhibit self-renewal capacity, and display additional AEC2 functional capacities. Footprint-free CRISPR-based gene correction of PSCs derived from patients carrying a homozygous surfactant mutation (SFTPB121ins2) restores surfactant processing in AEC2s. Thus, PSC-derived AEC2s provide a platform for disease modeling and future functional regeneration of the distal lung.

The Phosphatidylcholine Transfer Protein Stard7 is Required for Mitochondrial and Epithelial Cell Homeostasis.

Mitochondria synthesize select phospholipids but lack the machinery for synthesis of the most abundant mitochondrial phospholipid, phosphatidylcholine (PC). Although the phospholipid transfer protein Stard7 promotes uptake of PC by mitochondria, the importance of this pathway for mitochondrial and cellular homeostasis represents a significant knowledge gap. Haploinsufficiency for Stard7 is associated with significant exacerbation of allergic airway disease in mice, including an increase in epithelial barrier permeability. To test the hypothesis that Stard7 deficiency leads to altered barrier structure/function downstream of mitochondrial dysfunction, Stard7 expression was knocked down in a bronchiolar epithelial cell line (BEAS-2B) and specifically deleted in lung epithelial cells of mice (Stard7epi∆/∆). Stard7 deficiency was associated with altered mitochondrial size and membrane organization both in vitro and in vivo. Altered mitochondrial structure was accompanied by disruption of mitochondrial homeostasis, including decreased aerobic respiration, increased oxidant stress, and mitochondrial DNA damage that, in turn, was linked to altered barrier integrity and function. Both mitochondrial and barrier defects were largely corrected by targeting Stard7 to mitochondria or treating epithelial cells with a mitochondrial-targeted antioxidant. These studies suggest that Stard7-mediated transfer of PC is crucial for mitochondrial homeostasis and that mitochondrial dysfunction contributes to altered barrier permeability in Stard7-deficient mice.

Airway Epithelial KIF3A Regulates Th2 Responses to Aeroallergens.

KIF3A, the gene encoding kinesin family member 3A, is a susceptibility gene locus associated with asthma; however, mechanisms by which KIF3A might influence the pathogenesis of the disorder are unknown. In this study, we deleted the mouse Kif3a gene in airway epithelial cells. Both homozygous and heterozygous Kif3a gene-deleted mice were highly susceptible to aeroallergens from Aspergillus fumigatus and the house dust mite, resulting in an asthma-like pathology characterized by increased goblet cell metaplasia, airway hyperresponsiveness, and Th2-mediated inflammation. Deletion of the Kif3a gene increased the severity of pulmonary eosinophilic inflammation and expression of cytokines (Il-4, Il-13, and Il-17a) and chemokine (Ccl11) RNAs following pulmonary exposure to Aspergillus extract. Inhibition of Kif3a disrupted the structure of motile cilia and impaired mucociliary clearance, barrier function, and epithelial repair, demonstrating additional mechanisms by which deficiency of KIF3A in respiratory epithelial cells contributes to pulmonary pathology. Airway epithelial KIF3A suppresses Th2 pulmonary inflammation and airway hyperresponsiveness following aeroallergen exposure, implicating epithelial microtubular functions in the pathogenesis of Th2-mediated lung pathology.

Reserve autophagic capacity in alveolar epithelia provides a replicative niche for influenza A virus.

Autophagy contributes to cellular homeostasis through metabolite recycling and degradation of cytotoxic protein aggregates and damaged organelles. Although recent studies have established that the requirement for basal autophagy is largely tissue specific, the importance of autophagy for alveolar epithelial cell homeostasis remains an important knowledge gap. In the present study we generated two mouse models, with > 90% or > 50% recombination at the Atg5 locus in the distal respiratory epithelium, to assess the effect of dose-dependent decreases in autophagy on alveolar homeostasis. A 90% decrease in autophagy was well tolerated in young adult mice but resulted in alveolar septal thickening and altered lung mechanics in aged animals, consistent with accumulation of damage over time. By comparison, a 50% decrease in autophagy had no effect on alveolar structure or function throughout the murine life span, indicating that basal autophagy in this compartment exceeds that required for homeostasis. A 50% decrease in autophagy in the bronchoalveolar epithelium significantly attenuated influenza A/H3N2 viral replication, leading to improved lung structure and function and reduced morbidity and mortality after infection. The reserve of autophagic capacity in the alveolar epithelium may provide a niche for replication of influenza A virus.

Deficiency of the BiP cochaperone ERdj4 causes constitutive endoplasmic reticulum stress and metabolic defects.

Endoplasmic reticulum-localized DnaJ 4 (ERdj4) is an immunoglobulin-binding protein (BiP) cochaperone and component of the endoplasmic reticulum-associated degradation (ERAD) pathway that functions to remove unfolded/misfolded substrates from the ER lumen under conditions of ER stress. To elucidate the function of ERdj4 in vivo, we disrupted the ERdj4 locus using gene trap (GT) mutagenesis, leading to hypomorphic expression of ERdj4 in mice homozygous for the trapped allele (ERdj4(GT/GT)). Approximately half of ERdj4(GT/GT) mice died perinatally associated with fetal growth restriction, reduced hepatic glycogen stores, and hypoglycemia. Surviving adult mice exhibited evidence of constitutive ER stress in multiple cells/tissues, including fibroblasts, lung, kidney, salivary gland, and pancreas. Elevated ER stress in pancreatic ß cells of ERdj4(GT/GT) mice was associated with ß cell loss, hypoinsulinemia, and glucose intolerance. Collectively these results suggest an important role for ERdj4 in maintaining ER homeostasis during normal fetal growth and postnatal adaptation to metabolic stress.

4-Phenylbutyric acid treatment rescues trafficking and processing of a mutant surfactant protein-C.

Mutations in the SFTPC gene, encoding surfactant protein-C (SP-C), are associated with interstitial lung disease (ILD). Knowledge of the intracellular fate of mutant SP-C is essential in the design of therapies to correct trafficking/processing of the proprotein, and to prevent the formation of cytotoxic aggregates. We assessed the potential of a chemical chaperone to correct the trafficking and processing of three disease-associated mutant SP-C proteins. HEK293 cells were stably transfected with wild-type (SP-C(WT)) or mutant (SP-C(L188Q), SP-C(Δexon4), or SP-C(I73T)) SP-C, and cell lines with a similar expression of SP-C mRNA were identified. The effects of the chemical chaperone 4-phenylbutyric acid (PBA) and lysosomotropic drugs on intracellular trafficking to the endolysosomal pathway and the subsequent conversion of SP-C proprotein to mature peptide were assessed. Despite comparable SP-C mRNA expression, proprotein concentrations varied greatly: SP-C(I73T) was more abundant than SP-C(WT) and was localized to the cell surface, whereas SP-C(Δexon4) was barely detectable. In contrast, SP-C(L188Q) and SP-C(WT) proprotein concentrations were comparable, and a small amount of SP-C(L188Q) was localized to the endolysosomal pathway. PBA treatment restored the trafficking and processing of SP-C(L188Q) to SP-C(WT) concentrations, but did not correct the mistrafficking of SP-C(I73T) or rescue SP-C(Δexon4). PBA treatment also promoted the aggregation of SP-C proproteins, including SP-C(L188Q). This study provides proof of the principle that a chemical chaperone can correct the mistrafficking and processing of a disease-associated mutant SP-C proprotein.

Comparative proteomic analysis of lung lamellar bodies and lysosome-related organelles.

Pulmonary surfactant is a complex mixture of lipids and proteins that is essential for postnatal function. Surfactant is synthesized in alveolar type II cells and stored as multi-bilayer membranes in a specialized secretory lysosome-related organelle (LRO), known as the lamellar body (LB), prior to secretion into the alveolar airspaces. Few LB proteins have been identified and the mechanisms regulating formation and trafficking of this organelle are poorly understood. Lamellar bodies were isolated from rat lungs, separated into limiting membrane and core populations, fractionated by SDS-PAGE and proteins identified by nanoLC-tandem mass spectrometry. In total 562 proteins were identified, significantly extending a previous study that identified 44 proteins in rat lung LB. The lung LB proteome reflects the dynamic interaction of this organelle with the biosynthetic, secretory and endocytic pathways of the type II epithelial cell. Comparison with other LRO proteomes indicated that 60% of LB proteins were detected in one or more of 8 other proteomes, confirming classification of the LB as a LRO. Remarkably the LB shared 37.8% of its proteins with the melanosome but only 9.9% with lamellar bodies from the skin. Of the 229 proteins not detected in other LRO proteomes, a subset of 34 proteins was enriched in lung relative to other tissues. Proteins with lipid-related functions comprised a significant proportion of the LB unique subset, consistent with the major function of this organelle in the organization, storage and secretion of surfactant lipid. The lung LB proteome will facilitate identification of molecular pathways involved in LB biogenesis, surfactant homeostasis and disease pathogenesis.

Nedd4-2-mediated ubiquitination facilitates processing of surfactant protein-C.

We previously proposed a model of surfactant protein (SP)-C biosynthesis in which internalization of the proprotein from the limiting membrane of the multivesicular body to internal vesicles represents a key step in the processing and secretion of SP-C. To test this hypothesis, alanine mutagenesis of the N-terminal propeptide of SP-C was performed. Adenoviruses encoding mutant proproteins were infected into type II cells isolated from Sftpc(-/-) mice, and media analyzed for secreted SP-C 24 hours after infection. Mutation of S(12)PPDYS(17) completely blocked secretion of SP-C. PPDY (PY motif) has previously been shown to bind WW domains of neural precursor cell-expressed developmentally down-regulated (Nedd) 4-like E3 ubiquitin ligases. Purified recombinant glutathione S-transferase-SP-C propeptide (residues 1-35) bound recombinant Nedd4-2 strongly, and Nedd4 weakly; the S(12)PPDYS(17)mutation abrogated binding of SP-C to Nedd4-2. Immobilized recombinant Nedd4-2 WW domain captured SP-C proprotein from mouse type II cell lysates; in the reverse pulldown, endogenous SP-C in type II cells was captured by recombinant Nedd4-2. To determine if the interaction of Nedd4-2 and SP-C resulted in ubiquitination, the SP-C proprotein was immunoprecipitated from transiently transfected human embryonic kidney 293 cells, and analyzed by SDS-PAGE/Western blotting with ubiquitin antibody. Two ubiquitinated forms of SP-C were detected; ubiquitination was blocked by mutation of K6, but not K34, in the SP-C propeptide. Mutation of K6 also inhibited processing of SP-C proprotein to the mature peptide in human embryonic kidney 293 cells. Nedd4-2-mediated ubiquitination regulates lumenal relocation of SP-C, leading to processing and, ultimately, secretion of SP-C.

Meckel-Gruber syndrome protein MKS3 is required for endoplasmic reticulum-associated degradation of surfactant protein C.

Autosomal dominant mutations in the SFTPC gene are associated with idiopathic pulmonary fibrosis, a progressive lethal interstitial lung disease. Mutations that cause misfolding of the encoded proprotein surfactant protein C (SP-C) trigger endoplasmic reticulum (ER)-associated degradation, a pathway that segregates terminally misfolded substrate for retrotranslocation to the cytosol and degradation by proteasome. Microarray screens for genes involved in SP-C ER-associated degradation identified MKS3/TMEM67, a locus previously linked to the ciliopathy Meckel-Gruber syndrome. In this study, MKS3 was identified as a membrane glycoprotein predominantly localized to the ER. Expression of MKS3 was up-regulated by genetic or pharmacological inducers of ER stress. The ER lumenal domain of MKS3 interacted with a complex that included mutant SP-C and associated chaperones, whereas the region predicted to encode the transmembrane domains of MKS3 interacted with cytosolic p97. Deletion of the transmembrane and cytosolic domains abrogated interaction of MKS3 with p97 and resulted in accumulation of mutant SP-C proprotein; knockdown of MKS3 also inhibited degradation of mutant SP-C. These results support a model in which MKS3 links the ER lumenal quality control machinery with the cytosolic degradation apparatus.

Adaptation and increased susceptibility to infection associated with constitutive expression of misfolded SP-C.

Mutations in the gene encoding SP-C (surfactant protein C; SFTPC) have been linked to interstitial lung disease (ILD) in children and adults. Expression of the index mutation, SP-C(Deltaexon4), in transiently transfected cells and type II cells of transgenic mice resulted in misfolding of the proprotein, activation of endoplasmic reticulum (ER) stress pathways, and cytotoxicity. In this study, we show that stably transfected cells adapted to chronic ER stress imposed by the constitutive expression of SP-C(Deltaexon4) via an NF-kappaB-dependent pathway. However, the infection of cells expressing SP-C(Deltaexon4) with respiratory syncytial virus resulted in significantly enhanced cytotoxicity associated with accumulation of the mutant proprotein, pronounced activation of the unfolded protein response, and cell death. Adaptation to chronic ER stress imposed by misfolded SP-C was associated with increased susceptibility to viral-induced cell death. The wide variability in the age of onset of ILD in patients with SFTPC mutations may be related to environmental insults that ultimately overwhelm the homeostatic cytoprotective response.

Partial SP-B deficiency perturbs lung function and causes air space abnormalities.

Surfactant protein B (SP-B) is required for function of newborn and adult lung, and partial deficiency has been associated with susceptibility to lung injury. In the present study, transgenic mice were produced in which expression of SP-B in type II epithelial cells was conditionally regulated. Concentrations of SP-B were maintained at 60-70% of that normally present in control. Immunostaining for SP-B demonstrated cellular heterogeneity in expression of the protein. In subsets of type II cells in which SP-B staining was decreased, immunostaining for pro-SP-C was increased and lamellar body ultrastructure was disrupted, consistent with focal SP-B deficiency. Fluorescence-activated cell sorting analyses of freshly isolated type II cells identified a population of cells with low SP-B content and a smaller population with increased SP-B content, confirming nonuniform expression of the SP-B transgene. Focal air space enlargement, without cellular infiltration or inflammation, was observed. Pressure-volume curves indicated that maximal tidal volume was unchanged; however, hysteresis was modestly altered and residual volumes were significantly decreased in the SP-B-deficient mice. Chronic, nonuniform SP-B deficiency perturbed pulmonary function and caused air space enlargement.

Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells.

Surfactant protein D (SP-D) is a member of the collectin family of the innate host defense proteins. In the lung, SP-D is expressed primarily by type II cells. Gene-targeted SP-D-deficient [SP-D(-/-)] mice have three- to fivefold higher surfactant lipid pool sizes. However, surfactant synthesis and secretion by type II cells and catabolism by alveolar macrophages are normal in SP-D(-/-) mice. Therefore, we hypothesized that SP-D might regulate surfactant homeostasis by influencing surfactant structure, thereby altering its uptake by type II cells. Large (LA) and small aggregate (SA) surfactant were isolated from bronchoalveolar lavage fluid (BALF) from SP-D(-/-), wild-type [SP-D(+/+)], and transgenic mice in which SP-D was expressed under conditional control of doxycycline in alveolar type II cells. Uptake of both LA and SA isolated from SP-D(-/-) mice by normal type II cells was decreased. Abnormally dense lipid forms were observed by electron microscopy of LA from SP-D(-/-) mice. SA from SP-D(-/-) mice consisted of atypical multilamellated small vesicles. Abnormalities in surfactant uptake by type II cells and in surfactant ultrastructure were corrected by conditional expression of SP-D in vivo. Preincubation of BALF from SP-D(-/-) mice with SP-D changed surfactant ultrastructure to be similar to that of SP-D(+/+) mice in vitro. The rapid changes in surfactant structure, increased uptake by type II cells, and decreased pool sizes normally occurring in the postnatal period were not seen in SP-D(-/-) mice. SP-D regulates uptake and catabolism by type II cells and influences the ultrastructure of surfactant in the alveolus.

N-terminally extended surfactant protein (SP) C isolated from SP-B-deficient children has reduced surface activity and inhibited lipopolysaccharide binding.

In both humans and mice, a deficiency of surfactant protein B (SP-B) is associated with a decreased concentration of mature SP-C and accumulation of a larger SP-C peptide, denoted SP-C(i), which is not observed under normal conditions. Isolation of hydrophobic polypeptides from the lungs of children who died with two different SP-B mutations yielded pure SP-C(i) and showed only trace amounts of mature SP-C. Determination of the SP-C(i) covalent structure revealed a 12-residue N-terminal peptide segment, followed by a 35-residue segment that is identical to mature SP-C. The SP-C(i) structure determined herein is similar to that of a proposed late intermediate in the processing of proSP-C, suggesting that SP-C(i) is the immediate precursor of SP-C. In bronchoalveolar lavage fluid from transgenic mice with a focal deficiency of SP-B, SP-C(i) was detected in the biophysically active, large aggregate fraction and was associated with membrane structures that are typical for a large aggregate surfactant. However, unlike SP-C, SP-C(i) exhibited a very poor ability to promote phospholipid adsorption, gave high surface tension during cyclic film compression, and did not bind lipopolysaccharide in vitro. SP-C(i) is thus capable of associating with surfactant lipids, but its N-terminal dodecapeptide segment must be proteolytically removed to generate a biologically functional peptide. The results of this study indicate that the early postnatal fatal respiratory distress seen in SP-B-deficient children is combined with the near absence of active variants of SP-C.