Synergistic Action of Antimicrobial Lung Proteins against Klebsiella pneumoniae.

As key components of innate immunity, lung antimicrobial proteins play a critical role in warding off invading respiratory pathogens. Lung surfactant protein A (SP-A) exerts synergistic antimicrobial activity with the N-terminal segment of the SP-B proprotein (SP-BN) against Klebsiella pneumoniae K2 in vivo. However, the factors that govern SP-A/SP-BN antimicrobial activity are still unclear. The aim of this study was to identify the mechanisms by which SP-A and SP-BN act synergistically against K. pneumoniae, which is resistant to either protein alone. The effect of these proteins on K. pneumoniae was studied by membrane permeabilization and depolarization assays and transmission electron microscopy. Their effects on model membranes of the outer and inner bacterial membranes were analyzed by differential scanning calorimetry and membrane leakage assays. Our results indicate that the SP-A/SP-BN complex alters the ultrastructure of K. pneumoniae by binding to lipopolysaccharide molecules present in the outer membrane, forming packing defects in the membrane that may favor the translocation of both proteins to the periplasmic space. The SP-A/SP-BN complex depolarized and permeabilized the inner membrane, perhaps through the induction of toroidal pores. We conclude that the synergistic antimicrobial activity of SP-A/SP-BN is based on the capability of this complex, but not either protein alone, to alter the integrity of bacterial membranes.

Preclinical MRI to Quantify Pulmonary Disease Severity and Trajectories in Poorly Characterized Mouse Models: A Pedagogical Example Using Data from Novel Transgenic Models of Lung Fibrosis.

Structural remodeling in lung disease is progressive and heterogeneous, making temporally and spatially explicit information necessary to understand disease initiation and progression. While mouse models are essential to elucidate mechanistic pathways underlying disease, the experimental tools commonly available to quantify lung disease burden are typically invasive (e.g., histology). This necessitates large cross-sectional studies with terminal endpoints, which increases experimental complexity and expense. Alternatively, magnetic resonance imaging (MRI) provides information noninvasively, thus permitting robust, repeated-measures statistics. Although lung MRI is challenging due to low tissue density and rapid apparent transverse relaxation (T2* <1 ms), various imaging methods have been proposed to quantify disease burden. However, there are no widely accepted strategies for preclinical lung MRI. As such, it can be difficult for researchers who lack lung imaging expertise to design experimental protocols-particularly for novel mouse models. Here, we build upon prior work from several research groups to describe a widely applicable acquisition and analysis pipeline that can be implemented without prior preclinical pulmonary MRI experience. Our approach utilizes 3D radial ultrashort echo time (UTE) MRI with retrospective gating and lung segmentation is facilitated with a deep-learning algorithm. This pipeline was deployed to assess disease dynamics over 255 days in novel, transgenic mouse models of lung fibrosis based on disease-associated, loss-of-function mutations in Surfactant Protein-C. Previously identified imaging biomarkers (tidal volume, signal coefficient of variation, etc.) were calculated semi-automatically from these data, with an objectively-defined high signal volume identified as the most robust metric. Beyond quantifying disease dynamics, we discuss common pitfalls encountered in preclinical lung MRI and present systematic approaches to identify and mitigate these challenges. While the experimental results and specific pedagogical examples are confined to lung fibrosis, the tools and approaches presented should be broadly useful to quantify structural lung disease in a wide range of mouse models.

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.

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.

Air Space Distension Precedes Spontaneous Fibrotic Remodeling and Impaired Cholesterol Metabolism in the Absence of Surfactant Protein C.

Surfactant protein (SP)-C deficiency is found in samples from patients with idiopathic pulmonary fibrosis, especially in familial forms of this disease. We hypothesized that SP-C may contribute to fibrotic remodeling in aging mice and alveolar lipid homeostasis. For this purpose, we analyzed lung function, alveolar dynamics, lung structure, collagen content, and expression of genes related to lipid and cholesterol metabolism of aging SP-C knockout mice. In addition, in vitro experiments with an alveolar macrophage cell line exposed to lipid vesicles with or without cholesterol and/or SP-C were performed. Alveolar dynamics showed progressive alveolar derecruitment with age and impaired oxygen saturation. Lung structure revealed that decreasing volume density of alveolar spaces was accompanied by increasing of the ductal counterparts. Simultaneously, septal wall thickness steadily increased, and fibrotic wounds appeared in lungs from the age of 50 weeks. This remarkable phenotype is unique to the 129Sv strain, which has an increased absorption of cholesterol, linking the accumulation of cholesterol and the absence of SP-C to a fibrotic remodeling process. The findings of this study suggest that overall loss of SP-C results in an age-dependent, complex, heterogeneous phenotype characterized by a combination of overdistended air spaces and fibrotic wounds that resembles combined emphysema and pulmonary fibrosis in patients with idiopathic pulmonary fibrosis. Addition of SP-C to cholesterol-laden lipid vesicles enhanced the expression of cholesterol metabolism and transport genes in an alveolar macrophage cell line, identifying a potential new lipid-protein axis involved in lung remodeling.

Surfactant Protein B Deficiency Induced High Surface Tension: Relationship between Alveolar Micromechanics, Alveolar Fluid Properties and Alveolar Epithelial Cell Injury.

High surface tension at the alveolar air-liquid interface is a typical feature of acute and chronic lung injury. However, the manner in which high surface tension contributes to lung injury is not well understood. This study investigated the relationship between abnormal alveolar micromechanics, alveolar epithelial injury, intra-alveolar fluid properties and remodeling in the conditional surfactant protein B (SP-B) knockout mouse model. Measurements of pulmonary mechanics, broncho-alveolar lavage fluid (BAL), and design-based stereology were performed as a function of time of SP-B deficiency. After one day of SP-B deficiency the volume of alveolar fluid V(alvfluid,par) as well as BAL protein and albumin levels were normal while the surface area of injured alveolar epithelium S(AEinjure,sep) was significantly increased. Alveoli and alveolar surface area could be recruited by increasing the air inflation pressure. Quasi-static pressure-volume loops were characterized by an increased hysteresis while the inspiratory capacity was reduced. After 3 days, an increase in V(alvfluid,par) as well as BAL protein and albumin levels were linked with a failure of both alveolar recruitment and airway pressure-dependent redistribution of alveolar fluid. Over time, V(alvfluid,par) increased exponentially with S(AEinjure,sep). In conclusion, high surface tension induces alveolar epithelial injury prior to edema formation. After passing a threshold, epithelial injury results in vascular leakage and exponential accumulation of alveolar fluid critically hampering alveolar recruitability.

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.

Natural Anti-Infective Pulmonary Proteins: In Vivo Cooperative Action of Surfactant Protein SP-A and the Lung Antimicrobial Peptide SP-BN.

The anionic antimicrobial peptide SP-B(N), derived from the N-terminal saposin-like domain of the surfactant protein (SP)-B proprotein, and SP-A are lung anti-infective proteins. SP-A-deficient mice are more susceptible than wild-type mice to lung infections, and bacterial killing is enhanced in transgenic mice overexpressing SP-B(N). Despite their potential anti-infective action, in vitro studies indicate that several microorganisms are resistant to SP-A and SP-B(N). In this study, we test the hypothesis that these proteins act synergistically or cooperatively to strengthen each other's microbicidal activity. The results indicate that the proteins acted synergistically in vitro against SP-A- and SP-B(N)-resistant capsulated Klebsiella pneumoniae (serotype K2) at neutral pH. SP-A and SP-B(N) were able to interact in solution (Kd = 0.4 µM), which enabled their binding to bacteria with which SP-A or SP-B(N) alone could not interact. In vivo, we found that treatment of K. pneumoniae-infected mice with SP-A and SP-B(N) conferred more protection against K. pneumoniae infection than each protein individually. SP-A/SP-B(N)-treated infected mice showed significant reduction of bacterial burden, enhanced neutrophil recruitment, and ameliorated lung histopathology with respect to untreated infected mice. In addition, the concentrations of inflammatory mediators in lung homogenates increased early in infection in contrast with the weak inflammatory response of untreated K. pneumoniae-infected mice. Finally, we found that therapeutic treatment with SP-A and SP-B(N) 6 or 24 h after bacterial challenge conferred significant protection against K. pneumoniae infection. These studies show novel anti-infective pathways that could drive development of new strategies against pulmonary infections.

Alveolar development and disease.

Gas exchange after birth is entirely dependent on the remarkable architecture of the alveolus, its formation and function being mediated by the interactions of numerous cell types whose precise positions and activities are controlled by a diversity of signaling and transcriptional networks. In the later stages of gestation, alveolar epithelial cells lining the peripheral lung saccules produce increasing amounts of surfactant lipids and proteins that are secreted into the airspaces at birth. The lack of lung maturation and the associated lack of pulmonary surfactant in preterm infants causes respiratory distress syndrome, a common cause of morbidity and mortality associated with premature birth. At the time of birth, surfactant homeostasis begins to be established by balanced processes involved in surfactant production, storage, secretion, recycling, and catabolism. Insights from physiology and engineering made in the 20th century enabled survival of newborn infants requiring mechanical ventilation for the first time. Thereafter, advances in biochemistry, biophysics, and molecular biology led to an understanding of the pulmonary surfactant system that made possible exogenous surfactant replacement for the treatment of preterm infants. Identification of surfactant proteins, cloning of the genes encoding them, and elucidation of their roles in the regulation of surfactant synthesis, structure, and function have provided increasing understanding of alveolar homeostasis in health and disease. This Perspective seeks to consider developmental aspects of the pulmonary surfactant system and its importance in the pathogenesis of acute and chronic lung diseases related to alveolar homeostasis.

Establishment of LC-MS methods for the analysis of palmitoylated surfactant proteins.

The surfactant proteins (SPs), SP-B and SP-C, are important components of pulmonary surfactant involved in the reduction of alveolar surface tension. Quantification of SP-B and SP-C in surfactant drugs is informative for their quality control and the evaluation of their biological activity. Western blot analysis enabled the quantification of SP-B, but not SP-C, in surfactant drugs. Here, we report a new procedure involving chemical treatments and LC-MS to analyze SP-C peptides. The procedure enabled qualitative analysis of SP-C from different species with discrimination of the palmitoylation status and the artificial modifications that occur during handling and/or storage. In addition, the method can be used to estimate the total amount of SP-C in pulmonary surfactant drugs. The strategy described here might serve as a prototype to establish analytical methods for peptides that are extremely hydrophobic and behave like lipids. The new method provides an easy measurement of SP-C from various biological samples, which will help the characterization of various experimental animal models and the quality control of surfactant drugs, as well as diagnostics of human samples.

Haploinsufficiency for Stard7 is associated with enhanced allergic responses in lung and skin.

Allergic asthma is a chronic inflammatory disorder that affects ∼20% of the population worldwide. Microarray analyses of nasal epithelial cells from acute asthmatic patients detected a 50% decrease in expression of Stard7, an intracellular phosphatidylcholine transport protein. To determine whether loss of Stard7 expression promotes allergic responses, mice were generated in which one allele of the Stard7 locus was globally disrupted (Stard7 (+/-) mice). OVA sensitization and challenge of Stard7(+/-) mice resulted in a significant increase in pulmonary inflammation, mucous cell metaplasia, airway hyperresponsiveness, and OVA-specific IgE compared with OVA-sensitized/challenged wild-type (WT) mice. This exacerbation was largely Th2-mediated with a significant increase in CD4(+)IL-13(+) T cells and IL-4, IL-5, and IL-13 cytokines. The loss of Stard7 was also associated with increased lung epithelial permeability and activation of proinflammatory dendritic cells in sensitized and/or challenged Stard7 (+/-) mice. Notably, OVA-pulsed dendritic cells from Stard7(+/-) mice were sufficient to confer an exaggerated allergic response in OVA-challenged WT mice, although airway hyperresponsiveness was greater in Stard7(+/-) recipients compared with WT recipients. Enhanced allergic responses in the lung were accompanied by age-dependent development of spontaneous atopic dermatitis. Overall, these data suggest that Stard7 is an important component of a novel protective pathway in tissues exposed to the extracellular environment.

Diseases of pulmonary surfactant homeostasis.

Advances in physiology and biochemistry have provided fundamental insights into the role of pulmonary surfactant in the pathogenesis and treatment of preterm infants with respiratory distress syndrome. Identification of the surfactant proteins, lipid transporters, and transcriptional networks regulating their expression has provided the tools and insights needed to discern the molecular and cellular processes regulating the production and function of pulmonary surfactant prior to and after birth. Mutations in genes regulating surfactant homeostasis have been associated with severe lung disease in neonates and older infants. Biophysical and transgenic mouse models have provided insight into the mechanisms underlying surfactant protein and alveolar homeostasis. These studies have provided the framework for understanding the structure and function of pulmonary surfactant, which has informed understanding of the pathogenesis of diverse pulmonary disorders previously considered idiopathic. This review considers the pulmonary surfactant system and the genetic causes of acute and chronic lung disease caused by disruption of alveolar homeostasis.

The BiP cochaperone ERdj4 is required for B cell development and function.

ERdj4 is a BiP cochaperone regulated by the unfolded protein response to facilitate degradation of unfolded and/or misfolded proteins in the endoplasmic reticulum. As the unfolded protein response plays a critical role in B cell maturation and antibody production, ERdj4 gene trap mice were generated to determine if this chaperone was required for B cell homeostasis. Homozygosity for the trapped allele resulted in hypomorphic expression of ERdj4 in bone marrow cells and abnormal development of hematopoietic lineages in the bone marrow. The number of myeloid cells was increased, while the number of erythroid and B lymphoid cells was reduced in ERdj4 gene trap mice compared to controls. An intrinsic B cell defect was identified that decreased survival of B cell precursors including large and small pre-B, and immature B cells. Consistent with impaired B lymphopoiesis, the number of mature follicular B cells was reduced in both the bone marrow and spleen of ERdj4 gene trap mice. Paradoxically, unchallenged ERdj4 gene trap mice showed non-specific hypergammaglobulinemia and gene trap B cells exhibited increased proliferation, survival and isotype switching in response to LPS stimulation. Although ERdj4 gene trap mice responded normally to T cell-independent antigen, they failed to mount a specific antibody response to T cell-dependent antigen in vivo. Collectively, these findings demonstrate that the chaperone activity of ERdj4 is required for survival of B cell progenitors and normal antibody production.

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.