Surfactant Metabolism Research Articles

Glutamine metabolism is required for alveolar macrophage proliferation

Abstract Alveolar macrophages (AMs) are the most abundant immune cells in the lung during homeostasis. AMs are critical for normal lung development, surfactant metabolism and host defense against various respiratory pathogens. Despite being terminally differentiated cells, AMs are able to proliferate and self-renew to maintain their compartment without the input of the hematopoietic system in the adulthood during homeostasis. However, the molecular and metabolic mechanisms modulating AM proliferative responses are still incompletely understood. Here we have investigated the metabolic regulation of AM proliferation and self-renewal. We found that mitochondrial metabolism is critical for AM proliferation in vitro and in vivo. Interestingly, inhibition of glucose uptake or fatty acid oxidation did not significantly impact AM proliferation. Rather, inhibition of the glutamine metabolic process abolished AM proliferation in vitro and in vivo following their partial depletion during influenza infection. We further demonstrated that glutamine metabolism was required for AM mitochondrial respiration. Our data indicate that glutamine is a critical substrate fueling mitochondrial metabolism required for AM proliferation. Our study is expected to shed light on the metabolic regulation of AM maintenance and repopulation during homeostasis and following acute respiratory viral infection.

Impact of high tidal volume ventilation on surfactant metabolism and lung injury in infant rats - Supplemental material

Impact of high tidal volume ventilation on surfactant metabolism and lung injury in infant rats - Supplemental material

Impact of high tidal volume ventilation on surfactant metabolism and lung injury in infant rats - Supplemental material

Impact of high tidal volume ventilation on surfactant metabolism and lung injury in infant rats - Supplemental material

Metabolic labelling of choline phospholipids probes ABCA3 transport in lamellar bodies

In the metabolism of pulmonary surfactant, the ATP-binding cassette sub-family A member 3 (ABCA3) is a crucial protein in the formation of the storage compartment for surfactant, the lamellar body (LB), and the transport of phospholipids in it. Mutations in ABCA3 not only disturb surfactant metabolism but also cause chronic interstitial lung diseases. Assays for ABCA3 transport function are needed to investigate pathophysiology of the mutations and treatment options for the patients.We metabolically labeled choline (Cho) head phospholipids with the Cho analogue, propargyl-Cho. The universal incorporation of propargyl-Cho was confirmed by mass spectrometry and labeled lipids were visualized in confocal microscopy by click reaction with an azide fluorophore. After pulse-labeling propargyl-Cho labeled lipids accumulated in ABCA3+ vesicles in a time and concentration dependent manner. When treated with the choline kinase inhibitor MN58b during the first 12 h, the lipids intensity inside ABCA3+ vesicles decreased, whereas intensity was unchanged when treated after 12 h. Miltefosine, a substrate of ABCA3, decreased the incorporation of labeled lipids in ABCA3+ vesicles at all time points. The lipids intensity inside the mutated (p.N568D or p.L1580P) ABCA3+ vesicles was decreased compared to wild type, while the intensity outside of vesicles showed no difference.Propargyl-Cho can metabolically pulse-label Cho phospholipids. Visualization and quantification of fluorescence intensity of the labeled lipids inside ABCA3+ vesicles at equilibrium can specifically assess the transport function of ABCA3.

Surfactant Deficiency: Late Presentation in a Term 2-Year-Old Male

Introduction Neonatal reactive airways disease (RDS) and childhood interstitial lung disease (chILD) include a heterogeneous spectrum of disorders. With recent advances, specific genetic mutations have been identified in disease pathogenesis: chief among these are inborn errors of surfactant metabolism. Research over the past decade has demonstrated that carrier genotypes and certain compound heterozygotes may have mild phenotypes, which remain undetected until childhood. The case herein corroborates recent data and demonstrates obstacles to final diagnosis. Case Report A 2-year-old male was admitted for persistent hypoxia following a sedated MRI brain. The patient was a term AGA male, with history of RDS, …

Major Effect of Oxidative Stress on the Male, but Not Female, SP-A1 Type II Cell miRNome.

Pulmonary surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. In humans there are two proteins, SP-A1 and SP-A2, encoded by SFTPA1 and SFTPA2, respectively, which are produced by the alveolar type II cells (T2C). We sought to investigate the differential influence of SP-A1 and SP-A2 in T2C miRNome under oxidative stress (OxS). SP-A knock out (KO) and hTG male and female mice expressing SP-A1 or SP-A2 as well as gonadectomized (Gx) mice were exposed to O3-induced oxidative stress (OxS) or filtered air (FA). Expression of miRNAs and mRNAs was measured in the T2C of experimental animals. (a) In SP-A1 males after normalizing to KO males, significant changes were observed in the miRNome in terms of sex-OxS effects, with 24 miRNAs being differentially expressed under OxS. (b) The mRNA targets of the dysregulated miRNAs included Ago2, Ddx20, Plcg2, Irs1, Elf2, Jak2, Map2k4, Bcl2, Ccnd1, and Vhl. We validated the expression levels of these transcripts, and observed that the mRNA levels of all of these targets were unaffected in SP-A1 T2C but six of these were significantly upregulated in the KO (except Bcl2 that was downregulated). (c) Gondadectomy had a major effect on the expression of miRNAs and in three of the mRNA targets (Irs1, Bcl2, and Vhl). Ccnd1 was upregulated in KO regardless of Gx. (d) The targets of the significantly changed miRNAs are involved in several pathways including MAPK signaling pathway, cell cycle, anti-apoptosis, and other. In conclusion, in response to OxS, SP-A1 and male hormones appear to have a major effect in the T2C miRNome.

Grouping of Poorly Soluble Low (Cyto)Toxic Particles: Example with 15 Selected Nanoparticles and A549 Human Lung Cells.

Poorly soluble, low (cyto)toxic particles (PSLTs) are often regarded as one group, but it is important that these particles can be further differentiated based on their bioactivity. Currently, there are no biological endpoint based groupings for inhaled nanoparticles (NPs) that would allow us to subgroup PSLTs based on their mode of action. The aim of this study was to group NPs based on their cytotoxicity and by using the in vitro response of the endo-lysosomal system as a biological endpoint. The endo-lysosomal system is a main cellular loading site for NPs. An impaired endo-lysosomal system in alveolar type II cells may have serious adverse effects on the maintenance of pulmonary surfactant homeostasis. The 15 different NPs were tested with human lung adenocarcinoma (A549) cells. The highly soluble NPs were most cytotoxic. With respect to PSLTs, only three NPs increased the cellular load of acid and phospholipid rich organelles indicating particle biopersistence. All the rest PSLTs could be regarded as low hazardous. The presented in vitro test system could serve as a fast screening tool to group particles according to their ability to interfere with lung surfactant metabolism. We discuss the applicability of the suggested test system for bringing together substances with similar modes-of-action on lung epithelium. In addition, we discuss this approach as a benchmark test for the comparative assessment of biopersistence of PSLTs.

Full-Term Neonatal Respiratory Distress and Chronic Lung Disease.

Respiratory distress occurs in 5% to 7% of live births at term gestation. Most cases are mild and transient and can be attributed to transient tachypnea of the newborn or "wet lung." Severe respiratory distress is often due to nonpulmonary causes such as sepsis or congenital heart disease. Occasionally, term neonatal respiratory distress is associated with an inherited primary lung disease such as primary ciliary dyskinesia or surfactant metabolism defects. These lung diseases have characteristic presentations in the neonatal period and are important to recognize, as they necessitate different management approaches and have lifelong implications. Suspicion for these diseases should prompt referral to a pediatric pulmonologist. [Pediatr Ann. 2019;48(4):e175-e181.].

TREATMENT OF RESPIRATORY DISTRESS-SYNDROME IN PRETERM NEWBORNS WITH THE USE OF EXOGENOUS SURFACTANT (PORACTANT ALFA): CLINLCAL GUIDLINES

Respiratory distress-syndrome (RDS) is an acute lung disease that occurs mainly in pretermnewborns due to deficiency of pulmonary surfactant and immaturity of the respiratory system. Its symptoms appear shortly after birth, and the clinical course in the absence of treatment is characterized by a worsening condition during the first 2 days of life.Despite the improvement and effectiveness of the prevention and treatment of RDS, this disease remains an important cause of death in newborns. According to European data, mortality associated with RDS can reach 10%, and according to the latest official statistical data from the United States, this disease ranks 8thamong the most important causes of death in infants.According to official statistical data from obstetrical hospitals, the incidence of RDS in Ukraine in 2016 was 194 cases per 1000 premature babies. The majority of patients in modern Ukrainianneonatal intensive care units die from the complications associated with this disease, especially intraventricular hemorrhages.The clinical recommendations contain sections on the etiology, pathogenesis, clinical presentation, antenatal prevention of RDS with recommendations for stabilizationafter birth and further,at the neonatal intensive care unit. Separately, the metabolism of surfactant, the importance of drug selection and the correct dose of surfactant use, practical recommendations on its use, indications, preparation for use and administration techniques are presented taking into account the international evidence-based data.Clinical guideline was elaborated for neonatologists, pediatric anesthesiologists and residents to improve the quality of medical care for preterm newborns at risk for developing or having RDS; the optimization of methods of medical care for preterm newborns to decrease the incidence of the disease, development of complications and mortality of premature babies at risk for or developing RDS.

Differential Effects of Human SP-A1 and SP-A2 on the BAL Proteome and Signaling Pathways in Response to Klebsiella pneumoniae and Ozone Exposure

Surfactant protein A (SP-A) plays critical roles in host defense, regulation of inflammation and surfactant metabolism in the lung. The human SP-A locus consists of two functional genes, SFTPA1 and SFTPA2 encoding surfactant proteins SP-A1 and SP-A2, respectively. Structural and functional differences exist between SP-A1 and SP-A2 in vitro and in vivo. Ozone is a major air pollutant with a negative impact on many biological processes. In this study we used humanized transgenic (hTG) SP-A1 and SP-A2 mice, and SP-A KO mice to study in vivo effects of SP-A1 and SP-A2 on the bronchoalveolar lavage (BAL) proteomic profile and associated signaling pathways in response to ozone or filtered air (FA) exposure and Klebsiella pneumoniae infection. The BAL samples were harvested 24 h after ozone (2 ppm for 3 h) or FA exposure and infection and analyzed by two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-ToF/ToF. We found: that (1) Ozone exposure, but not infection, is a major factor for increases in total BAL protein content. (2) A total of 36 proteins were identified, accounting for 89.62% of the BAL proteins resolved by the 2D-DIGE system. (3) The number of proteins in which levels were altered more than 25% following infection and FA exposure was: SP-A2 > SP-A1 > KO for male mice, and SP-A2 ≈ SP-A1 > KO for female mice. (4) The number of proteins with more than 25% increase/decrease after ozone exposure and infection was: SP-A2 > SP-A1 ≈ KO, with the majority being increases in male mice and decreases in female mice. (5) Eleven out of the 36 proteins, including annexin A5, glutathione S-transferase A4, SP-A1/SP-A2, and 14-3-3 zeta protein, exhibited significant differences among SP-A genotypes. The acute phase response (APR) that includes the NF-kB signaling pathway plays a critical role, followed by Nrf2-mediated oxidative response, and others. These associated with SP-A genotype, sex, and ozone-induced oxidative stress in response to infection. We concluded that human SP-A2 and SP-A1 exhibit differential genotype-and sex-dependent innate immune responses to microbial pathogens and/or ozone-induced oxidative stress by modulating proteomic patterns and signaling pathways in the lung.

Gene correlation network analysis to identify regulatory factors in idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a severe lung disease characterised by extensive pathological changes. The objective for this study was to identify the gene network and regulators underlying disease pathology...

CT findings in pulmonary alveolar proteinosis: serial changes and prognostic implications.

Pulmonary alveolar proteinosis (PAP) is a rare disease of unknown origin, characterized by impaired surfactant metabolism. In some patients, residual disease is observed after treatment; notably, this may progress to end-stage pulmonary fibrosis. This study was performed to evaluate changes in lung abnormalities on serial CT scans performed on the lungs of patients diagnosed with PAP, as well as to identify factors that can be used to predict clinical improvement in PAP. Twenty-five patients (16 men, nine women) were diagnosed with PAP at a single tertiary hospital. The extent and distribution of PAP were assessed on baseline and follow-up CT scans (median, 38 months; range, 2-96 months). Serial CT scans and clinical findings were analyzed to identify the predictive factors for clinical improvement in PAP. Baseline CT scans of patients diagnosed with PAP revealed that ground-glass opacity was the most common abnormality (100%); the second most common abnormality was interlobular/intralobular septal thickening (88%). Importantly, the final follow-up CT scans showed that the extent of lung abnormalities had decreased (n=13), including complete resolution (n=5), unchanged (n=9), and increased (n=3). Traction bronchiectasis and architectural distortion were detected in two patients (8%). On univariate and multivariate analyses, the change in the overall extent of lung abnormalities was a predictive factor for clinical improvement in PAP (odds ratio: 55.780; P=0.038). Most patients with PAP exhibited residual disease; however, progression to pulmonary fibrosis was rare. Analyses of overall changes, with respect to lung abnormalities on serial CT scans, may be predictive of the extent of clinical improvement in given patient.

Serum and sputum surfactants -A and -D in multidrug-resistant and drugsensitive tuberculosis patients

Abnormal production and function of surfactants are associated with pulmonary diseases. Also, pulmonary infections alter surfactant metabolism. Due to lack of information on the levels of surfactants A (SP-A) and D (SP-D) in Nigerian tuberculosis (TB) patients, this study assessed these surfactants in both sputum and serum of drug sensitive- and multidrug resistant- TB patients using ELISA. The aim is to explore the diagnostic or therapeutic potential of SP-A and SP-D in TB patients. Also, to find out appropriate sample for the analyses of SP-A and SP-D in TB patients. The mean serum levels of SP-A and SP-D were not significantly reduced in MDR-TB (n=30) and DS-TB patients (n=30) compared with non-TB apparently healthy controls (n=30) (p > 0.05). Mean sputum levels of SP-A and SP-D were significantly reduced in DS-TB patients compared with the levels in MDR-TB patients (p < 0.05). These findings suggest that the sputum SP-D and SP-A levels but not serum SP-D and SP-A levels are useful indicators of the disease activity in pulmonary TB patients. Keywords: Tuberculosis, Surfactants, Diagnosis, Nigeria

OP-348 - Preoperative Incentive Spirometry Exercise Reduces the Risk of Atelectasis in Obese Cabg Patients

Objective: General anesthesia, accumulation of secretions, impaired ciliary activity, alterations of surfactant metabolism and suspended ventilation during perfusion result in atelectasis development in cardiac surgery. Obese patients are particularly at high risk for the development of atelectasis. We sought to evaluate the influence of preoperative precautions for atelectasis, such as incentive spirometers, on postoperative pulmonary outcome.

Time-resolved proteome profiling of normal lung development.

Biochemical networks mediating normal lung morphogenesis and function have important implications for ameliorating morbidity and mortality in premature infants. Although several transcript-level studies have examined normal lung development, corresponding protein-level analyses are lacking. Here we performed proteomics analysis of murine lungs from embryonic to early adult ages to identify the molecular networks mediating normal lung development. We identified 8,932 proteins, providing a deep and comprehensive view of the lung proteome. Analysis of the proteomics data revealed discrete modules and the underlying regulatory and signaling network modulating their expression during development. Our data support the cell proliferation that characterizes early lung development and highlight responses of the lung to exposure to a nonsterile oxygen-rich ambient environment and the important role of lipid (surfactant) metabolism in lung development. Comparison of dynamic regulation of proteomic and recent transcriptomic analyses identified biological processes under posttranscriptional control. Our study provides a unique proteomic resource for understanding normal lung formation and function and can be freely accessed at Lungmap.net.

Pulmonary Alveolar Proteinosis- A Case Report

ABSTRACT Background: Pulmonary Alveolar Proteinosis (PAP) is a rare pulmonary disorder caused by a congregation of excessive lipoproteinaceous material in the alveolar spaces due to impaired surfactant metabolism. The congregation of the protein in the alveolar space leads to difficulty in breathing, impaired pulmonary immunity, and susceptibility to both opportunistic and acquired pulmonary infections. Although Pulmonary Alveolar Proteinosis is rare, there are potential treatments. Whole-lung lavage is the most widely accepted therapy and course of treatment. An additional form of therapy, GM-CSF stimulating therapy, uses recombinant deoxyribonucleic acid (DNA) technology to increase white cell production. Purpose: The purpose of this case report is to follow a patient through whole-lung lavage therapy to determine outcome and clinical improvement. Case Description: This case report follows a 55-year-old female patient diagnosed with secondary, idiopathic Pulmonary Alveolar Proteinosis through eight normal saline whole-lung lavages. The patient’s treatment was led by a pulmonologist with previous Pulmonary Alveolar Proteinosis experience. Outcome: By the end of the first four lavages, the patient showed clinical improvement, but during a two-month break from therapy, symptoms returned. Following the break, the patient underwent four additional lung lavage sessions and experienced similar relief as in previous courses of treatment. Discussion: The patient did complete an additional eight lavages to wash out excess lipoproteinaceous material to provide a longer period of symptom relief. The additional form of therapy, GM-CSF, is not a therapy option for this patient as the disease was idiopathic in nature. The last available treatment option for this patient is a lung transplant.

Surfactant proteins gene variants in premature newborn infants with severe respiratory distress syndrome

ObjectiveGenetic surfactant dysfunction causes respiratory failure in term and near-term newborn infants, but little is known of such condition in prematures. We evaluated genetic surfactant dysfunction in premature newborn infants with severe RDS.Patients and methodsA total of 68 preterm newborn infants with gestational age ≤32 weeks affected by unusually severe RDS were analysed for mutations in SFTPB, SFTPC and ABCA3. Therapies included oxygen supplementation, nasal CPAP, different modalities of ventilatory support, administration of exogenous surfactant, inhaled nitric oxide and steroids. Molecular analyses were performed on genomic DNA extracted from peripheral blood and Sanger sequencing of whole gene coding regions and intron junctions. In one case histology and electron microscopy on lung tissue was performed.ResultsHeterozygous previously described rare or novel variants in surfactant proteins genes ABCA3, SFTPB and SFTPC were identified in 24 newborn infants. In total, 11 infants died at age of 2 to 6 months. Ultrastructural analysis of lung tissue of one infant showed features suggesting ABCA3 dysfunction.DiscussionRare or novel genetic variants in genes encoding surfactant proteins were identified in a large proportion (35%) of premature newborn infants with particularly severe RDS. We speculate that interaction of developmental immaturity of surfactant production in association with abnormalities of surfactant metabolism of genetic origin may have a synergic worsening phenotypic effect.

1284P - HOXA-related long non-coding RNAs impact prognosis in early stage NSCLC patients

Background: HOX genes, grouped in four clusters in humans (HOXA-D), encode for transcription factors that are master regulators of embryonic development and oncogenesis. HOXA cluster, located in chromosome 7, play a critical role in the patterning of tissues with mesodermal components such as lungs. Long non-coding RNAs (lncRNAs) are especially abundant in the HOXA cluster (HOXA10-AS, HOXAS2, HOXAS3, HOXA11-AS, HOTAIRM1 and HOTTIP). We aimed to evaluate the prognostic role of HOXA-related lncRNAs in early stage non-small cell lung cancer (NSCLC). Methods: 100 early stage NSCLC patients resected in our center from June 2007 to November 2013 were studied. Patient characteristics: median age, 65 (32-84); 78% males; 74% stage I; 56% ADK; 23% received adjuvant treatment. With a mean follow-up of 28.7 months, 31% relapsed. As validation data set 200 NSCLC patients from TCGA Research Network were used (RNAseq data). Only stage I-II TCGA samples without prior malignancy or synchronous cancer that not received neoadjuvant treatment were included. Statistical analysis was performed using R and TANRIC. Results: HOTTIP and HOXA11AS impacted prognosis in our cohort of patients. HOTTIP was expressed in all samples and patients with high levels of HOTTIP had shorter TTR (78.3 vs 58 months; p = 0.048) and shorter OS (81.2 vs 61 months; p = 0.023). HOTTIP was overexpressed in SCC (p = 0.007) and in smokers (p = 0.018). HOXA11AS was expressed only in 9% of patients but the patients expressing HOXA11AS had shorter TTR (73.5 vs 32 months; p = 0.002). In the multivariate analysis HOXA11AS emerged as an independent prognostic marker for TTR (OR: 3.13, 95%CI: 1.3-7.3; p = 0.009), while HOTTIP (OR: 2.357, 95%CI: 1.1-5.2; p = 0.036) and age>65 (p = 0.022) for OS. In the validation data set HOXA11AS was validated as prognostic marker (p = 0.019). HOXA11AS expression correlated positively with development genes HOXA11, HOXA13, HOXA10, HOXA9, HOXA3, FOXD1, ZIC5 and miR-196b (HOXA cluster miRNA) and negatively with surfactant metabolism genes SFTPB and NAPSA or let-7a(p < 0.001). Interestingly, the high expression was associated to patients harboring RTN1 mutations (p < 0.0001). Conclusions: HOXA11AS expression in early stage NSCLC patients is a high-risk relapse marker. Legal entity responsible for the study: University of Barcelona, Barcelona, Spain Funding: None Disclosure: All authors have declared no conflicts of interest. Keywords: HOTTIP, NSCLC, lncRNA, HOXA11AS

Congenital Pulmonary Alveolar Proteinosis: From Birth to Ten-years of Age.

Pulmonary alveolar proteinosis is a rare lung disease in which lipoproteinaceous material accumulates within the alveoli, interfering with gas exchange. The disease is classified into congenital, secondary, and acquired. The congenital form includes inborn errors of surfactant metabolism, lysinuric protein intolerance and mutations in the components of granulocyte-macrophage colony-stimulating factor receptor. The main symptoms are non-specific. The radiologic appearance of pulmonary alveolar proteinosis is bilateral, symmetric and perihilar airspace consolidation. Bronchoalveolar lavage is crucial for diagnosis of the disease. There is only one ten-year-old patient with diagnosed congenital form in Croatia. What makes him different from other children in the world is that since the ninth month of his life he has been mechanically ventilated. Diagnosis of postnatal alveolar proteinosis should be considered in every infant with respiratory distress with diffuse alveolar and interstitial infiltrate.

ABCG1 regulates pulmonary surfactant metabolism in mice and men

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of surfactant. Surfactant synthesis and secretion are restricted to epithelial type 2 (T2) pneumocytes (also called T2 cells). Clearance of surfactant is dependent upon T2 cells and macrophages. ABCG1 is highly expressed in both T2 cells and macrophages. ABCG1-deficient mice accumulate surfactant, lamellar body-loaded T2 cells, lipid-loaded macrophages, B-1 lymphocytes, and immunoglobulins, clearly demonstrating that ABCG1 has a critical role in pulmonary homeostasis. We identify a variant in the ABCG1 promoter in patients with PAP that results in impaired activation of ABCG1 by the liver X receptor α, suggesting that ABCG1 basal expression and/or induction in response to sterol/lipid loading is essential for normal lung function. We generated mice lacking ABCG1 specifically in either T2 cells or macrophages to determine the relative contribution of these cell types on surfactant lipid homeostasis. These results establish a critical role for T2 cell ABCG1 in controlling surfactant and overall lipid homeostasis in the lung and in the pathogenesis of human lung disease.