The expression of the surfactant proteins SP-A and SP-B during postnatal alveolarization of the rat lung.

OBJECTIVE: Surfactant-specific proteins (SP) are responsible for the functional and structural integrity as well as for the stabilization of the intra-alveolar surfactant. Morphological lung maturation starts in rat lungs after birth. The aim of this study was to investigate whether the expression of the hydrophilic SP-A and the hydrophobic SP-B is associated with characteristic postnatal changes characterizing morphological lung maturation. METHODS: Stereological methods were performed on the light microscope. Using immunohistochemical and molecular biological methods (Western Blot, RT-qPCR), the SP-A and SP-B of adult rat lungs and of those with different postnatal developmental stages (3, 7, 14 and 21 days after birth) were characterized. RESULTS: As signs of alveolarization the total septal surface and volume increased and the septal thickness decreased. The significantly highest relative surface fraction of SP-A labeled alveolar epithelial cells type II (AEII) was found together with the highest relative SP-A gene expression before the alveolarization (3th postnatal day). With the downregulation of SP-A gene expression during and after alveolarization (between postnatal days 7 and 14), the surface fraction of the SP-A labeled AEII also decreased, so they are lowest in adult animals. The surface fraction of SP-B labeled AEII and the SP-B gene expression showed the significantly highest levels in adults, the protein expression increased also significantly at the end of morphological lung maturation. There were no alterations in the SP-B expression before and during alveolarization until postnatal day 14. The protein expression as well as the gene expression of SP-A and SP-B correlated very well with the total surface of alveolar septa independent of the postnatal age. CONCLUSION: The expression of SP-A and SP-B is differentially associated with morphological lung maturation and correlates with increased septation of alveoli as indirect clue for alveolarization.

Lung Surfactant Protein B Peptide Mimics Interact with the Human ACE2 Receptor.

Lung surfactant is a complex mixture of phospholipids and surfactant proteins that is produced in alveolar type 2 cells. It prevents lung collapse by reducing surface tension and is involved in innate immunity. Exogenous animal-derived and, more recently, synthetic lung surfactant has shown clinical efficacy in surfactant-deficient premature infants and in critically ill patients with acute respiratory distress syndrome (ARDS), such as those with severe COVID-19 disease. COVID-19 pneumonia is initiated by the binding of the viral receptor-binding domain (RBD) of SARS-CoV-2 to the cellular receptor angiotensin-converting enzyme 2 (ACE2). Inflammation and tissue damage then lead to loss and dysfunction of surface activity that can be relieved by treatment with an exogenous lung surfactant. Surfactant protein B (SP-B) is pivotal for surfactant activity and has anti-inflammatory effects. Here, we study the binding of two synthetic SP-B peptide mimics, Super Mini-B (SMB) and B-YL, to a recombinant human ACE2 receptor protein construct using molecular docking and surface plasmon resonance (SPR) to evaluate their potential as antiviral drugs. The SPR measurements confirmed that both the SMB and B-YL peptides bind to the rhACE2 receptor with affinities like that of the viral RBD-ACE2 complex. These findings suggest that synthetic lung surfactant peptide mimics can act as competitive inhibitors of the binding of viral RBD to the ACE2 receptor.

Alveolar-Capillary Barrier Protection In Vitro: Lung Cell Type-Specific Effects and Molecular Mechanisms Induced by 1α, 25-Dihydroxyvitamin D3.

Low serum levels of 1α, 25-dihydroxyvitamin D3 (VD3) are associated with a higher mortality in trauma patients with sepsis or ARDS. However, the molecular mechanisms behind this observation are not yet understood. VD3 is known to stimulate lung maturity, alveolar type II cell differentiation, or pulmonary surfactant synthesis and guides epithelial defense during infection. In this study, we investigated the impact of VD3 on the alveolar-capillary barrier in a co-culture model of alveolar epithelial cells and microvascular endothelial cells respectively in the individual cell types. After stimulation with bacterial LPS (lipopolysaccharide), gene expression of inflammatory cytokines, surfactant proteins, transport proteins, antimicrobial peptide, and doublecortin-like kinase 1 (DCLK1) were analyzed by real-time PCR, while corresponding proteins were evaluated by ELISA, immune-fluorescence, or Western blot. The effect of VD3 on the intracellular protein composition in H441 cells was analyzed by quantitative liquid chromatography-mass spectrometry-based proteomics. VD3 effectively protected the alveolar-capillary barrier against LPS treatment, as indicated by TEER measurement and morphological assessment. VD3 did not inhibit the IL-6 secretion by H441 and OEC but restricted the diffusion of IL-6 to the epithelial compartment. Further, VD3 could significantly suppress the surfactant protein A expression induced in the co-culture system by LPS treatment. VD3 induced high levels of the antimicrobial peptide LL-37, which counteracted effects by LPS and strengthened the barrier. Quantitative proteomics identified VD3-dependent protein abundance changes ranging from constitutional extracellular matrix components and surfactant-associated proteins to immune-regulatory molecules. DCLK1, as a newly described target molecule for VD3, was prominently stimulated by VD3 (10 nM) and seems to influence the alveolar-epithelial cell barrier and regeneration.

Synchrotron radiation circular dichroism spectroscopy reveals that gold and silver nanoparticles modify the secondary structure of a lung surfactant protein B analogue.

Inhaled nanoparticles (NPs) depositing in the alveolar region of the lung interact initially with a surfactant layer and in vitro studies have demonstrated that NPs can adversely affect the biophysical function of model pulmonary surfactants (PS), of which surfactant protein B (SP-B) is a key component. Other studies have demonstrated the potential for NPs to modify the structure and function of proteins. It was therefore hypothesised that NPs may affect the biophysical function of PS by modifying the structure of SP-B. Synchrotron radiation circular dichroism (SRCD) spectroscopy was used to explore the effect of various concentrations of gold nanoparticles (AuNPs) (5, 10, 20 nm), silver nanoparticles (AgNPs) (10 nm) and silver citrate on the secondary structure of surfactant protein B analogue, SP-B1-25, in a TFE/PB dispersion. For Au and Ag NPs the SRCD spectra indicated a concentration dependent reduction in the α-helical structure of SP-B1-25 (5 nm AuNP ≈ 10 nm AgNP ≫ 10 nm AuNP > 20 nm AuNP). For AuNPs the effect was greater for the 5 nm size, which was not fully explained by consideration of surface area. The impact of the 10 nm AgNPs was greater than that of the 10 nm AuNPs and the effect of AgNPs was greater than that of silver citrate at equivalent Ag mass concentrations. For 10 nm AuNPs, SRCD spectra for dispersions in, the more physiologically relevant, DPPC showed a similar concentration dependent pattern. The results demonstrate the potential for inhaled NPs to modify SP-B1-25 structure and thus potentially adversely impact the physiological function of the lung, however, further studies are necessary to confirm this.

Mass Spectrometry and Machine Learning Reveal Determinants of Client Recognition by Antiamyloid Chaperones.

The assembly of proteins and peptides into amyloid fibrils is causally linked to serious disorders such as Alzheimer's disease. Multiple proteins have been shown to prevent amyloid formation in vitro and in vivo, ranging from highly specific chaperone-client pairs to completely nonspecific binding of aggregation-prone peptides. The underlying interactions remain elusive. Here, we turn to the machine learning-based structure prediction algorithm AlphaFold2 to obtain models for the nonspecific interactions of ß-lactoglobulin, transthyretin, or thioredoxin 80 with the model amyloid peptide amyloid ß and the highly specific complex between the BRICHOS chaperone domain of C-terminal region of lung surfactant protein C and its polyvaline target. Using a combination of native mass spectrometry (MS) and ion mobility MS, we show that nonspecific chaperoning is driven predominantly by hydrophobic interactions of amyloid ß with hydrophobic surfaces in ß-lactoglobulin, transthyretin, and thioredoxin 80, and in part regulated by oligomer stability. For C-terminal region of lung surfactant protein C, native MS and hydrogen-deuterium exchange MS reveal that a disordered region recognizes the polyvaline target by forming a complementary ß-strand. Hence, we show that AlphaFold2 and MS can yield atomistic models of hard-to-capture protein interactions that reveal different chaperoning mechanisms based on separate ligand properties and may provide possible clues for specific therapeutic intervention.

Dimerization of the pulmonary surfactant protein C in a membrane environment.

Surfactant protein C (SP-C) has several functions in pulmonary surfactant. These include the transfer of lipids between different membrane structures, a role in surfactant recycling and homeostasis, and involvement in modulation of the innate defense system. Despite these important functions, the structures of functional SP-C complexes have remained unclear. SP-C is known to exist as a primarily α-helical structure with an apparently unstructured N-terminal region, yet there is recent evidence that the functions of SP-C could be associated with the formation of SP-C dimers and higher oligomers. In this work, we used molecular dynamics simulations, two-dimensional umbrella sampling, and well-tempered metadynamics to study the details of SP-C dimerization. The results suggest that SP-C dimerizes in pulmonary surfactant membranes, forming dimers of different topologies. The simulations identified a dimerization motif region V21xxxVxxxGxxxM33 that is much larger than the putative A30xxxG34 motif that is commonly assumed to control the dimerization of some α-helical transmembrane domains. The results provide a stronger basis for elucidating how SP-C functions in concert with other surfactant proteins.

Umbilical Cord-derived Mesenchymal Stem Cells with Surfactant Protein B Alleviates Inflammatory Response in Acute Respiratory Distress Syndrome by Regulating Macrophage Polarization.

Background: Acute respiratory distress syndrome (ARDS) is a severe disorder that is related to a high mortality. Mesenchymal stem cells (MSCs) have shown strong effects in relieving lung injury. Aims: To determine the role of umbilical cord-derived MSCs (UC-MSCs) together with surfactant protein B (SP-B) in ARDS. Study Design: Animal experimentation. Methods: Immunophenotypic characteristics of UC-MSCs were identified. BALB/c mice were intratracheally administrated with lipopolysaccharide (LPS) and received UC-MSCs or UC-MSCs transfected with SP-B (UC-MSCs-SP-B). Pathological changes and lung injury degrees after transplantation were assessed by histological and biochemical analyses. Inflammatory chemokine and cytokine production in the bronchoalveolar lavage fluid (BALF) was measured using enzyme-linked immunoassay. Flow cytometry was used to examine macrophage phenotypes and differentiation of T-helper 17 (Th17) and T-regulatory (Treg) in the BALF. Results: Our results showed that isolated UC-MSCs possessed multilineage differentiation potential. SP-B transfection into UC-MSCs strengthened the effects of UC-MSCs on lung function repair in LPS-induced ARDS. UC-MSCs and UC-MSCs-SP-B attenuated cellular infiltration. Additionally, UC-MSCs and UC-MSCs-SP-B inhibited the inflammatory response by promoting M2-like polarization, as well as reduced Th17 differentiation and promoted Treg differentiation. Conclusion: UC-MSCs in combination with SP-B, alleviates inflammatory reaction in ARDS by regulating macrophage polarization.

A dominant negative variant of RAB5B disrupts maturation of surfactant protein B and surfactant protein C.

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.

Differential LysoTracker Uptake Defines Two Populations of Distal Epithelial Cells in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal degenerative lung disease of unknown etiology. Although in its final stages it implicates, in a reactive manner, all lung cell types, the initial damage involves the alveolar epithelial compartment, in particular the alveolar epithelial type 2 cells (AEC2s). AEC2s serve dual progenitor and surfactant secreting functions, both of which are deeply impacted in IPF. Thus, we hypothesize that the size of the surfactant processing compartment, as measured by LysoTracker incorporation, allows the identification of different epithelial states in the IPF lung. Flow cytometry analysis of epithelial LysoTracker incorporation delineates two populations (Lysohigh and Lysolow) of AEC2s that behave in a compensatory manner during bleomycin injury and in the donor/IPF lung. Employing flow cytometry and transcriptomic analysis of cells isolated from donor and IPF lungs, we demonstrate that the Lysohigh population expresses all classical AEC2 markers and is drastically diminished in IPF. The Lysolow population, which is increased in proportion in IPF, co-expressed AEC2 and basal cell markers, resembling the phenotype of the previously identified intermediate AEC2 population in the IPF lung. In that regard, we provide an in-depth flow-cytometry characterization of LysoTracker uptake, HTII-280, proSP-C, mature SP-B, NGFR, KRT5, and CD24 expression in human lung epithelial cells. Combining functional analysis with extracellular and intracellular marker expression and transcriptomic analysis, we advance the current understanding of epithelial cell behavior and fate in lung fibrosis.

Protective effects of Surfacen® in allergen-induced asthma mice model.

Airway obstruction with increased airway resistance in asthma, commonly caused by smooth muscle constriction, mucosal edema and fluid secretion into the airway lumen, may partly be due to a poor function of pulmonary surfactant. Surfacen®, a clinical pulmonary surfactant, has anti-inflammatory action, but its effect on asthma has not been studied. This work aimed to evaluate the effect of Surfacen® in a murine allergen-induced acute asthma model, using house dust mite allergens. In a therapeutic experimental setting, mice were first sensitized by being administered with two doses (sc) of Dermatophagoides siboney allergen in aluminum hydroxide followed by one intranasal administration of the allergen. Then, sensitized mice were administered with aerosol of hypertonic 3% NaCl, Salbutamol 0.15 mg/kg, or Surfacen® 16 mg in a whole-body chamber on days 22, 23, and 24. Further, mice were subjected to aerosol allergen challenge on day 25. Surfacen® showed bronchial dilation and inhibition of Th2 inflammation (lower levels of IL-5 and IL-13 in broncoalveolar lavage) which increased IFN-γ and unchanged IL-10 in BAL. Moreover, Sufacen® administration was associated with a marked inhibition of the serum specific IgE burst upon allergen exposure, as well as, IgG2a antibody increase, suggesting potential anti-allergy effects with inclination towards Th1. These results support also the effectiveness of the aerosol administration method to deliver the drug into lungs. Surfacen® induced a favorable pharmacological effect, with a bronchodilator outcome comparable to Salbutamol, consistent with its action as a lung surfactant, and with an advantageous anti-inflammatory and anti-allergic immunomodulatory effect.

Proteinosis alveolar pulmonar: reporte de caso en paciente con linfoma no hodgkin mesentérico / Pulmonary alveolar proteinosis: report of a case in patient with history of mesenteric non-hodgkin lymphoma

Objetivos: Exponer en base a un caso clínico una revisión de literatura reciente sobre Proteinosis alveolar pulmonar (PAP). Presentación del caso: Revisión de ficha clínica electrónica de paciente de sexo masculino de 76 años con antecedente de linfoma no Hodgkin (LNH) mesentérico, estirpe B de tipo folicular, quien acude en forma reiterada a servicios de urgencia por cuadro de dos meses de evolución de fiebre, compromiso del estado general y tos. Al examen físico destaca crépitos en hemitórax derecho. Se realizó Tomografía computada (TC) de tórax que mostró opacidades pulmonares en vidrio esmerilado periféricas, con engrosamiento septal liso y algunas bandas retráctiles subpleurales. Se manejó ambulatoriamente con Azitromicina por una semana. Sin respuesta, evoluciona con baja de peso y diaforesis nocturna. Nueva TC de tórax en enero 2021, muestra nuevos focos de "empedrado" periféricos extensos, descrito como "crazy paving", focos de vidrio esmerilado difusos extensos, sin condensación y con resolución de bandas retráctiles. Estudio infeccioso negativo. Se realiza lavado broncoalveolar (LBA) con estudio histológico de líquido que muestra proceso inflamatorio crónico con abundantes macrófagos y material proteináceo. Discusión: Tras el descarte de patología infecciosa, se orientó el estudio hacia otras causas de enfermedad parenquimatosa pulmonar. Así, resulta fundamental la descripción correcta del patrón imagenológico tomográfico y el LBA que resultaron compatibles con PAP. Conclusión: La PAP es una patología infrecuente, pero una historia clínica adecuada, el planteamiento de diagnósticos diferenciales de neumonía de lenta resolución, asociado el reconocimiento del patrón radiológico característico y el estudio histológico con LBA permiten realizar un diagnóstico certero, con gran implicancia terapéutica.

Objective: To present a review of recent literature on pulmonary alveolar proteinosis (PAP) based on a clinical case. Presentation of the case: Review of electronic clinical record of a 76 years-old masculine patient with history of mesenteric Non-Hodgkin Lymphoma (NHL) follicular-type lineage B, who repeatedly attended the emergency services due to a two-month history of symptoms of fever, compromised general condition and cough. Physical examination revealed crepitus in the right hemithorax. Chest computed tomography (CT) was performed, which showed peripheral ground-glass pulmonary opacities, with smooth septal thickening and some subpleural retractile bands. He was managed on an outpatient basis with Azithromycin for one week. No response; evolves with weight loss and night diaphoresis. New chest CT in January 2021, shows new extensive peripheral "cobblestone" foci, described as "crazy paving", extensive diffuse ground glass foci, without condensation and with resolution of retractile bands. Negative infectious study. Bronchoalveolar lavage (BAL) was performed with a histological study of the fluid showing a chronic inflammatory process with abundant macrophages and proteinaceous material. Discussion: After ruling out infectious pathology, the study was oriented towards other causes of pulmonary parenchymal disease. Thus, the correct description of the tomographic imaging pattern and the BAL that were compatible with PAP are essential. Conclusion: PAP is an infrequent pathology, but an adequate clinical history, the approach to differential diagnoses of slowly resolving pneumonia, associated with the recognition of the characteristic radiological pattern and the histological study with BAL allow an accurate diagnosis to be made, with great therapeutic implications.

Alveolar type II cells and pulmonary surfactant in COVID-19 era.

In this review, we discuss the role of pulmonary surfactant in the host defense against respiratory pathogens, including novel coronavirus SARS-CoV-2. In the lower respiratory system, the virus uses angiotensin-converting enzyme 2 (ACE2) receptor in conjunction with serine protease TMPRSS2, expressed by alveolar type II (ATII) cells as one of the SARS-CoV-2 target cells, to enter. ATII cells are the main source of surfactant. After their infection and the resulting damage, the consequences may be severe and may include injury to the alveolar-capillary barrier, lung edema, inflammation, ineffective gas exchange, impaired lung mechanics and reduced oxygenation, which resembles acute respiratory distress syndrome (ARDS) of other etiology. The aim of this review is to highlight the key role of ATII cells and reduced surfactant in the pathogenesis of the respiratory form of COVID-19 and to emphasize the rational basis for exogenous surfactant therapy in COVID-19 ARDS patients.

Surfactant Proteins A/D-CD14 on Alveolar Macrophages Is a Common Pathway Associated With Phagocytosis of Nanomaterials and Cytokine Production.

Alveolar macrophages are responsible for clearance of airborne dust and pathogens. How they recognize and phagocytose a variety of engineered nanomaterials (ENMs) with different properties is an important issue for safety assessment of ENMs. Surfactant-associated proteins, specifically existing in the pulmonary surfactant, are important opsonins for phagocytosis of airborne microorganisms. The purposes of the current study are to understand whether opsonization of ENMs by surfactant-associated proteins promotes phagocytosis of ENMs and cytokine production, and to determine whether a common pathway for phagocytosis of ENMs with different properties exists. For these purposes, four ENMs, MWCNT-7, TiO2, SiO2, and fullerene C60, with different shapes, sizes, chemical compositions, and surface reactivities, were chosen for this study. Short-term pulmonary exposure to MWCNT-7, TiO2, SiO2, and C60 induced inflammation in the rat lung, and most of the administered ENMs were phagocytosed by alveolar macrophages. The ENMs were phagocytosed by isolated primary alveolar macrophages (PAMs) in vitro, and phagocytosis was enhanced by rat bronchioalveolar lavage fluid (BALF), suggesting that proteins in the BALF were associated with phagocytosis. Analysis of proteins bound to the 4 ENMs by LC/MS indicated that surfactant-associated proteins A and D (SP-A, SP-D) were common binding proteins for all the 4 ENMs. Both BALF and SP-A, but not SP-D, enhanced TNF-α production by MWCNT-7 treated PAMs; BALF, SP-A, and SP-D increased IL-1ß production in TiO2 and SiO2 treated PAMs; and BALF, SP-A, and SP-D enhanced IL-6 production in C60 treated PAMs. Knockdown of CD14, a receptor for SP-A/D, significantly reduced phagocytosis of ENMs and SP-A-enhanced cytokine production by PAMs. These results indicate that SP-A/D can opsonize all the test ENMs and enhance phagocytosis of the ENMs by alveolar macrophages through CD14, suggesting that SP-A/D-CD14 is a common pathway mediating phagocytosis of ENMs. Cytokine production induced by ENMs, however, is dependent on the type of ENM that is phagocytosed. Our results demonstrate a dual role for surfactant proteins as opsonins for both microbes and for inhaled dusts and fibers, including ENMs, allowing macrophages to recognize and remove the vast majority of these particles, thereby, greatly lessening their toxicity in the lung.

Immunohistochemical findings in the lungs of COVID-19 subjects: evidence of surfactant dysregulation.

OBJECTIVE: Acute respiratory distress syndrome (ARDS) is characterized by quantitative and qualitative changes in surfactant composition, leading to surfactant dysregulation with alveolar collapse and acute respiratory hypoxic failure. Recently, surfactant has been hypothesized to play a relevant role in COVID-19, representing a strong defender against SARS-CoV-2 infection. The aim of our work was the study of immunohistochemical surfactant expression in the lungs of patients died following SARS-CoV-2 ARDS, in order to shed light on a possible therapeutic surfactant administration. PATIENTS AND METHODS: We investigated four patients who died due to ARDS following SARS-COV-2 infection and four patients submitted to lung biopsy, in the absence of SARS-CoV-2 infection. In all 8 cases, lung specimens were immunostained with anti-surfactant protein A (SP-A) and B (SP-B). RESULTS: In control subjects, reactivity for SP-B was restricted to type II alveolar cells. Immunostaining for SP-A was observed on the surface of alveolar spaces. In the COVID-19 positive lungs, immunoreactivity for SP-B was similar to that observed in control lungs; SP-A was strongly expressed along the alveolar wall. Moreover, dense aggregates of SP-A positive material were observed in the alveolar spaces. CONCLUSIONS: Our immunohistochemical data show the dysregulation of surfactant production in COVID-19 patients, particularly regarding SP-A expression. The increased presence of SP-A in condensed masses inside alveolar spaces could invalidate the therapeutic efficacy of the treatment with exogenous surfactant.

Heterogeneity in Human Induced Pluripotent Stem Cell-derived Alveolar Epithelial Type II Cells Revealed with ABCA3/SFTPC Reporters.

Alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, are typically identified through the use of the canonical markers, SFTPC and ABCA3. Self-renewing AEC2-like cells have been generated from human induced pluripotent stem cells (iPSCs) through the use of knock-in SFTPC fluorochrome reporters. However, developmentally, SFTPC expression onset begins in the fetal distal lung bud tip and thus is not specific to mature AEC2s. Furthermore, SFTPC reporters appear to identify only those iPSC-derived AEC2s (iAEC2s) expressing the highest SFTPC levels. Here, we generate an ABCA3 knock-in GFP fusion reporter (ABCA3:GFP) that enables the purification of iAEC2s while allowing visualization of lamellar bodies, organelles associated with AEC2 maturation. Using an SFTPCtdTomato and ABCA3:GFP bifluorescent line for in vitro distal lung-directed differentiation, we observe later onset of ABCA3:GFP expression and broader identification of the subsequently emerging iAEC2 population based on ABCA3:GFP expression compared with SFTPCtdTomato expression. Comparing ABCA3:GFP/SFTPCtdTomato double-positive with ABCA3:GFP single-positive (SP) cells by RNA sequencing and functional studies reveals iAEC2 cellular heterogeneity with both populations functionally processing surfactant proteins but the SP cells exhibiting faster growth kinetics, increased clonogenicity, increased expression of progenitor markers, lower levels of SFTPC expression, and lower levels of AEC2 maturation markers. Over time, we observe that each population (double-positive and SP) gives rise to the other and each can serve as the parents of indefinitely self-renewing iAEC2 progeny. Our results indicate that iAEC2s are a heterogeneous population of cells with differing proliferation versus maturation properties, the majority of which can be tracked and purified using the ABCA3:GFP reporter or surrogate cell surface proteins, such as SLC34A2 and CPM.

Familial Pulmonary Fibrosis: Genetic Features and Clinical Implications.

Pulmonary fibrosis comprises a wide range of fibrotic lung diseases with unknown pathogenesis and poor prognosis. Familial pulmonary fibrosis (FPF) represents a unique subgroup of patients in which at least one other relative is also affected. Patients with FPF exhibit a wide range of pulmonary fibrosis phenotypes, although idiopathic pulmonary fibrosis is the most common subtype. Despite variable disease manifestations, patients with FPF experience worse survival compared with their counterparts with the sporadic disease form. Therefore, ascertaining a positive family history not only provides prognostic value but should also raise suspicion for the inheritance of an underlying causative genetic variant within kindreds. By focusing on FPF kindreds, rare variants within surfactant metabolism and telomere maintenance genes have been discovered. However, such genetic variation is not solely restricted to FPF, as similar rare variants are found in patients with seemingly sporadic pulmonary fibrosis, further supporting the idea of genetic susceptibility underlying pulmonary fibrosis as a whole. Researchers are beginning to show how the presence of rare variants may inform clinical management, such as informing predisposition risk for yet unaffected relatives as well as informing prognosis and therapeutic strategy for those already affected. Despite these advances, rare variants in surfactant and telomere-related genes only explain the genetic basis in about one-quarter of FPF kindreds. Therefore, research is needed to identify the missing genetic contributors of pulmonary fibrosis, which would not only improve our understanding of disease pathobiology but may offer additional opportunities to improve the health of patients.

The History and Mystery of Alveolar Epithelial Type II Cells: Focus on Their Physiologic and Pathologic Role in Lung.

Alveolar type II (ATII) cells are a key structure of the distal lung epithelium, where they exert their innate immune response and serve as progenitors of alveolar type I (ATI) cells, contributing to alveolar epithelial repair and regeneration. In the healthy lung, ATII cells coordinate the host defense mechanisms, not only generating a restrictive alveolar epithelial barrier, but also orchestrating host defense mechanisms and secreting surfactant proteins, which are important in lung protection against pathogen exposure. Moreover, surfactant proteins help to maintain homeostasis in the distal lung and reduce surface tension at the pulmonary air-liquid interface, thereby preventing atelectasis and reducing the work of breathing. ATII cells may also contribute to the fibroproliferative reaction by secreting growth factors and proinflammatory molecules after damage. Indeed, various acute and chronic diseases are associated with intensive inflammation. These include oedema, acute respiratory distress syndrome, fibrosis and numerous interstitial lung diseases, and are characterized by hyperplastic ATII cells which are considered an essential part of the epithelialization process and, consequently, wound healing. The aim of this review is that of revising the physiologic and pathologic role ATII cells play in pulmonary diseases, as, despite what has been learnt in the last few decades of research, the origin, phenotypic regulation and crosstalk of these cells still remain, in part, a mystery.

Pulmonary surfactant-associated protein B regulates prostaglandin-endoperoxide synthase-2 and inflammation in chronic obstructive pulmonary disease.

NEW FINDINGS: What is the central question of this study? It is reported that polymorphism of the gene for pulmonary surfactant-associated protein B (SFTPB) is associated with chronic obstructive pulmonary disease (COPD): what are the function and mechanism of action of SFTPB in COPD? What is the main finding and its importance? Under stimulation of the risk factors of COPD, SFTPB expression is decreased, which may be involved in the formation of COPD. The progress of COPD induces an inflammatory response and reduces SFTPB expression. Levels of prostaglandin-endoperoxide synthase-2 (PTGS2) and inflammatory responses are changed by SFTPB, which indicates that SFTPB promotes the progression of COPD by PTGS2 and inflammation. ABSTRACT: Pulmonary surfactant-associated protein B (SFTPB) is a critical protein for lung homeostasis, and polymorphism of its gene is associated with chronic obstructive pulmonary disease (COPD). However, few studies have so far confirmed the functional involvement of SFTPB in COPD. Serum SFTPB and inflammatory cytokine levels were measured in 54 patients with acute exacerbation of COPD and 29 healthy controls. A549 cells were induced using 10% cigarette smoke extract (CSE) and treated with dexamethasone to investigate the effect of inflammation on SFTPB expression, and the effect of SFTPB overexpression and silencing on inflammatory cytokines was measured using real-time PCR and enzyme-linked immunosorbent assay. SFTPB expression was assessed in mouse lung tissues using immunofluorescence. Serum levels of SFTPB were significantly lower in COPD patients than in controls (P = 0.009). Conversely, levels of interleukin (IL)-6 and prostaglandin-endoperoxide synthase-2 (PTGS2) were increased in COPD patients (IL-6: P = 0.006; PTGS2: P = 0.043). After CSE treatment, SFTPB mRNA and protein levels were significantly decreased compared to controls (mRNA: P = 0.002; protein: P = 0.011), while IL-6, IL-8 and PTGS2 were elevated. Dexamethasone treatment increased SFTPB levels. Following overexpression of SFTPB in A549 cells, mRNA and protein levels of IL-6, IL-8 and PTGS2 were significantly reduced, while gene silencing induced the opposite effect. SFTPB levels were significantly reduced in the lung tissue of a mouse model of COPD compared to controls. Reduced SFTPB levels may induce PTGS2 and inflammatory responses in COPD and SFTPB could be a key protein for evaluation of COPD progression.

A primate-specific RNA-binding protein (RBMXL3) is involved in glucocorticoid regulation of human pulmonary surfactant protein B (SP-B) mRNA stability.

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate levels of surfactant protein B (SP-B). Dexamethasone (DEX) increases human SP-B expression, in part, through increased SP-B mRNA stability. A 30-nt-long hairpin element (RBE) in the 3'-untranslated region of human SP-B mRNA mediates both DEX-induced and intrinsic mRNA stabilities, but the mechanism is unknown. Proteomic analysis of RBE-interacting proteins identified a primate-specific protein, RNA-binding motif X-linked-like-3 (RBMXL3). siRNA directed against RBMXL3 reduces DEX-induced SP-B mRNA expression in human bronchoalveolar cells. Human SP-B mRNA stability, measured by our dual cistronic plasmid assay, is unaffected by DEX in mouse lung epithelial cells lacking RBMXL3, but DEX increases human SP-B mRNA stability when RBMXL3 is expressed and requires the RBE. In the absence of DEX, RBE interacts with cellular proteins, reducing intrinsic SP-B mRNA stability in human and mouse lung epithelial cells. RBMXL3 specifically binds the RBE in vitro, whereas RNA immunoprecipitation and affinity chromatography analyses indicate that binding is enhanced in the presence of DEX. These results describe a model where intrinsic stability of human SP-B mRNA is reduced through binding of cellular mRNA decay factors to RBE, which is then relieved through DEX-enhanced binding of primate-specific RBMXL3.