Surfactant Protein Research Articles

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.

Hypomorphic pathogenic variant in SFTPB leads to adult pulmonary fibrosis.

Biallelic pathogenic variants in the surfactant protein (SP)-B gene (SFTPB) have been associated with fatal forms of interstitial lung diseases (ILD) in newborns and exceptional survival in young children. We herein report the cases of two related adults with pulmonary fibrosis due to a new homozygous SFTPB pathogenic variant, c.582G>A p.(Gln194=). In vitro transcript studies showed that this SFTPB synonymous pathogenic variant induces aberrant splicing leading to three abnormal transcripts with the preservation of the expression of a small proportion of normal SFTPB transcripts. Immunostainings on lung biopsies of the proband showed an almost complete loss of SP-B expression. This hypomorphic splice variant has thus probably allowed the patients' survival to adulthood while inducing an epithelial cell dysfunction leading to ILD. Altogether, this report shows that SFTPB pathogenic variants should be considered in atypical presentations and/or early-onset forms of ILD particularly when a family history is identified.

GAA deficiency disrupts distal airway cells in Pompe disease.

Pompe disease is an autosomal recessive glycogen storage disease caused by mutations in the gene that encodes acid alpha-glucosidase (GAA)-an enzyme responsible for hydrolyzing lysosomal glycogen. GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption. Glycogen accumulation in skeletal muscles, motor neurons, and airway smooth muscle cells is known to contribute to respiratory insufficiency in Pompe disease. However, the impact of GAA deficiency on the distal alveolar type 1 and type 2 cells (AT1 and AT2) has not been evaluated. AT1 cells rely on lysosomes for cellular homeostasis so that they can maintain a thin barrier for gas exchange, whereas AT2 cells depend on lysosome-like structures (lamellar bodies) for surfactant production. Using a mouse model of Pompe disease, the Gaa-/- mouse, we investigated the consequences of GAA deficiency on AT1 and AT2 cells using histology, pulmonary function and mechanics, and transcriptional analysis. Histological analysis revealed increased accumulation of lysosomal-associated membrane protein 1 (LAMP1) in the Gaa-/- mice lungs. Furthermore, ultrastructural examination showed extensive intracytoplasmic vacuoles enlargement and lamellar body engorgement. Respiratory dysfunction was confirmed using whole body plethysmography and forced oscillometry. Finally, transcriptomic analysis demonstrated dysregulation of surfactant proteins in AT2 cells, specifically reduced levels of surfactant protein D in the Gaa-/- mice. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the distal airway cells that disrupts surfactant homeostasis and contributes to respiratory impairments in Pompe disease.NEW & NOTEWORTHY This research highlights the impact of Pompe disease on distal airway cells. Prior to this work, respiratory insufficiency in Pompe disease was classically attributed to pathology in respiratory muscles and motor neurons. Using the Pompe mouse model, we note significant pathology in alveolar type 1 and 2 cells with reductions in surfactant protein D and disrupted surfactant homeostasis. These novel findings highlight the potential contributions of alveolar pathology to respiratory insufficiency in Pompe disease.

SP-A and SP-D surfactant proteins and conventional risk factors for chronic non-infectious human diseases

Surfactant proteins SP-A and SP-D, which belong to the family of collagen-containing type C lectins, are used as diagnostic and prognostic markers for many acute and chronic respiratory diseases. The aim of the study is to assess the impact of conventional risk factors for chronic non-infectious diseases on SP-A and SP-D protein levels by means of systemic and structural analysis on the basis of relevant publications from international databases and official WHO reports. This analytical review concludes that widespread expression of SP-A and SP-D is documented in numerous studies, and, although the lungs remain the main site of synthesis of surfactant proteins, one can expect its significant impact on the immune and inflammatory response in many organs and tissues. The authors note that there are several known extrapulmonary effects of these proteins. However, many mechanisms of additional cellular effects of SP-A and SP-D outside the bronchopulmonary system still remain unstudied, which indicates the prospects for further research in this area.

Long-term clinical outcomes of aspirin-exacerbated respiratory disease: Real-world data from an adult asthma cohort.

Aspirin-exacerbated respiratory disease (AERD) is a phenotype of severe asthma, but its disease course has not been well documented compared with that of aspirin-tolerant asthma (ATA). This study aimed to investigate the long-term clinical outcomes between AERD and ATA. AERD patients were identified by the diagnostic code and positive bronchoprovocation test in a real-world database. Longitudinal changes in lung function, blood eosinophil/neutrophil counts, and annual numbers of severe asthma exacerbations (AEx) were compared between the AERD and the ATA groups. Within a year after baseline, two or more severe AEx events indicated severe AERD, whereas less than two AEx events indicated nonsevere AERD. Among asthmatics, 353 had AERD in which 166 and 187 patients had severe and nonsevere AERD, respectively, and 717 had ATA. AERD patients had significantly lower FEV1%, higher blood neutrophil counts, and higher sputum eosinophils (%) (all p < .05) as well as higher levels of urinary LTE4 and serum periostin, and lower levels of serum myeloperoxidase and surfactant protein D (all p < .01) than those with ATA. In a 10-year follow-up, the severe AERD group maintained lower FEV1% with more severe AEs than the nonsevere AERD group. We demonstrated that AERD patients presented poorer long-term clinical outcomes than ATA patients in real-world data analyses.

The role of surfactant system dysfunction in the formation of dyspnea in patients with COVID-19-associated pneumonia

OBJECTIVE. To determine the contribution of surfactant system dysfunction (by level surfactant protein A (SP-A) of serum) to the formation of dyspnea in the subacute post-COVID (coronavirus disease) period in patients with COVID-19-associated pneumonia.&#x0D; MATERIALS AND METHODS. We screened 102 patients with dyspnea who had COVID-19-associated pneumonia in April – November 2021. The study was conducted in the subacute period of COVID-19 on day 47 (38; 62) after the first symptoms of coronavirus infectious. We excluded 25 patients whose dyspnea was associated with bronchial obstructive syndrome, signs of heart failure or anemia, and formed the main group of 77 patients with dyspnea due to lung parenchyma damage: mean age – 56 (49; 65) years, men – 37 (48.1 %), women – 40 (51.9 %). The control group consisted of 15 practically healthy individuals: mean age – 51 (45; 60) years, men – 6 (40.0 %), women – 9 (60.0 %). The main group was divided into three subgroups depending on the severity of COVID-19 in the acute period: subgroup 1 – 26 patients with dyspnea (mean age – 57 (53; 64) years; men – 12 (46.2 %), women – 14 (53.8 %)) who had a moderate course of the acute period of the disease; subgroup 2 – 36 patients (mean age – 55 (49; 61) years, men – 18 (50.0 %), women – 18 (50.0 %)) who had a severe course of the acute period of COVID-19; subgroup 3 – 15 patients (mean age – 55 (52; 63) years, men – 7 (46.7 %), women – 8 (53.3 %)) who had a critical course of the acute period of COVID-19. The examination of patients had consist general clinical methods, determination of level SpO2, assessment of dyspnea severity by the modified Borg scale, the 6-minute walk test (6MWD), and measurement levels SP-A of serum.&#x0D; RESULTS AND DISCUSSION. Patients of subgroups 3 of COVID-19 in the subacute period had quite severe dyspnea both before 6MWD (4 (3; 6) points on the Borg scale) and after it (7 (5; 9) points on the Borg scale), whereas patients of subgroups 1 and 2 had less severe dyspnea both before 6MWD (1 (0.5; 2) and 2 (1; 4) points, respectively) and after it (2 (1; 3) and 5 (4; 7) points, respectively). The level of SP-A in the subacute period was significantly higher in patients of subgroups 2 and 3 compared to patients of subgroup 1 (73.8 (59.0; 87.2), 88.1 (51.1; 100.8) ng/ml and 46.6 (21.1; 77.1) ng/ml, respectively) (p&lt;0.05). There was no significant difference between patients in subgroup 1 and the control group (46.6 (21.1; 77.1) vs 29.6 (14.0; 53.1) ng/ml) (p&gt;0.05). Analysis of the area under the ROC curve between patients in subgroup 1 and subgroups 2 and 3 showed a high diagnostic ability of the test: AUC 0.866 (95 % confidence interval 0.766-0.935; p&lt;0.0001) with an optimal cut-off point of 59 ng/ml (sensitivity – 78.4 %, specificity – 95.5 %). At a serum SP-A level &gt;59 ng/ml, the risk of lung surfactant system dysfunction in severe or critical patients in the subacute period of the disease increases 6-fold (odds ratio 6.1; 95 % confidence interval 2.2-17.3; p=0.0006).&#x0D; CONCLUSIONS. The severity of dyspnea in the subacute period of COVID-19 due to lung parenchymal damage depends on the severity of the acute course of the disease. Elevated SP-A levels (&gt;59 ng/ml) in the subacute period of COVID-19 are observed in patients with severe or critical acute disease and reflect the presence of signs of long-term surfactant system dysfunction.

Circulating Biomarkers and Disease Activity in Systemic Sclerosis-Associated Interstitial Lung Disease

Background: Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is a major cause of death in SSc patients. Several studies have reported that serum anti-Scl-70, Krebs von Lungren-6 (KL-6), and surfactant protein D (SP-D) levels are associated with the presence and progression of ILD in SSc patients. Objective: To examine the correlation between levels of these serum biomarkers and disease severity determined by baseline dyspnea index (BDI), high-resolution computed tomography (HRCT) score, and pulmonary function tests. Materials and Methods: The present study was a single-center, cross-sectional study. Serum anti-Scl-70, KL-6, and SP-D from 20 SSc-ILD patients and five non-ILD subjects were measured. The BDI, HRCT score, and pulmonary function tests were used to assess the severity of ILD in SSc-ILD patients. HRCT abnormalities, including ground-glass opacity (GGO), fibrosis, and honeycombing, were scored by using the semi-quantitative scoring system. Results: Serum anti-Scl-70, KL-6, and SP-D in SSc-ILD patients were significantly higher than those in non-ILD subjects. There was a moderate correlation between diffusing capacity for carbon monoxide (DLCO) and serum KL-6 levels (r=–0.551, p=0.022), while the pulmonary fibrosis (PF) score exhibited a strong correlation with serum KL-6 levels (r=0.630, p=0.003). The PF score had a moderate negative correlation with forced vital capacity (FVC) (r=–0.515, p=0.034) and a strong negative correlation with total lung capacity (TLC) and DLCO (r=–0.625, p=0.007, and r=–0.762, p&lt;0.001, respectively). Conclusion: The levels of serum KL-6, and SP-D are elevated in SSc-ILD patients. Serum KL-6 may be a useful non-invasive biomarker for the disease severity, as determined by DLCO and the extent of fibrosis on HRCT, in patients with SSc-ILD. Trial registration: Thai Clinical Trials Registry, TCTR20200314001, registered 13 March 2020, retrospectively registered at http://www.thaiclinicaltrials.org/show/TCTR20200314001 Keywords: Scleroderma; Systemic sclerosis; Interstitial pneumonia; Interstitial lung disease; Fibrosis; Biomarker

Differentiation of human induced pluripotent stem cells into functional lung alveolar epithelial cells in 3D dynamic culture.

Introduction: Understanding lung epithelium cell development from human induced pluripotent stem cells (IPSCs) in vitro can lead to an individualized model for lung engineering, therapy, and drug testing. Method: We developed a protocol to produce lung mature type I pneumocytes using encapsulation of human IPSCs in 1.1% (w/v) alginate solution within a rotating wall bioreactor system in only 20days without using feeder cells. The aim was to reduce exposure to animal products and laborious interventions in the future. Results: The three-dimensional (3D) bioprocess allowed cell derivation into endoderm, and subsequently into type II alveolar epithelial cells within a very short period. Cells successfully expressed surfactant proteins C and B associated with type II alveolar epithelial cells, and the key structure of lamellar bodies and microvilli was shown by transmission electron microscopy. The survival rate was the highest under dynamic conditions, which reveal the possibility of adapting this integration for large-scale cell production of alveolar epithelial cells from human IPSCs. Discussion: We were able to develop a strategy for the culture and differentiation of human IPSCs into alveolar type II cells using an in vitro system that mimics the in vivo environment. Hydrogel beads would offer a suitable matrix for 3D cultures and that the high-aspect-ratio vessel bioreactor can be used to increase the differentiation of human IPSCs relative to the results obtained with traditional monolayer cultures.

Glycosylation of H4 influenza strains with pandemic potential and susceptibilities to lung surfactant SP-D.

We recently reported that members of group 1 influenza A virus (IAV) containing H2, H5, H6, and H11 hemagglutinins (HAs) are resistant to lung surfactant protein D (SP-D). H3 viruses, members of group 2 IAV, have high affinity for SP-D, which depends on the presence of high-mannose glycans at glycosite N165 on the head of HA. The low affinity of SP-D for the group 1 viruses is due to the presence of complex glycans at an analogous glycosite on the head of HA, and replacement with high-mannose glycan at this site evoked strong interaction with SP-D. Thus, if members of group 1 IAV were to make the zoonotic leap to humans, the pathogenicity of such strains could be problematic since SP-D, as a first-line innate immunity factor in respiratory tissues, could be ineffective as demonstrated in vitro. Here, we extend these studies to group 2 H4 viruses that are representative of those with specificity for avian or swine sialyl receptors, i.e., those with receptor-binding sites with either Q226 and G228 for avian or recent Q226L and G228S mutations that facilitate swine receptor specificity. The latter have increased pathogenicity potential in humans due to a switch from avian sialylα2,3 to sialylα2,6 glycan receptor preference. A better understanding of the potential action of SP-D against these strains will provide important information regarding the pandemic risk of such strains. Our glycomics and in vitro analyses of four H4 HAs reveal SP-D-favorable glycosylation patterns. Therefore, susceptibilities to this first-line innate immunity defense respiratory surfactant against such H4 viruses are high and align with H3 HA glycosylation.

Macrophage-derived exosomal TNF-α promotes pulmonary surfactant protein expression in PM2.5-induced acute lung injury

Short-term high-concentration exposure to airborne fine particulate matter (PM2.5) is strongly associated with the risk of acute lung injury (ALI). It has been recently reported that exosomes (Exos) involve in the progression of respiratory diseases. However, the molecular mechanisms by which exosome-mediated intercellular signaling exacerbate PM2.5-induced ALI remains largely unaddressed. In the present study, we firstly investigated the effect of macrophage-derived exosomal tumor necrosis factor α (TNF-α) on pulmonary surfactant proteins (SPs) expression in epithelial MLE-12 cells after PM2.5 exposure. The higher levels of exosomes in the bronchoalveolar lavage fluid (BALF) of PM2.5-induced ALI mice were found. BALF-exosomes significantly up-regulated SPs expression in MLE-12 cells. Moreover, we found that remarkably high expression of TNF-α in exosomes secreted by PM2.5-treated RAW264.7 cells. Exosomal TNF-α promoted thyroid transcription factor-1 (TTF-1) activation and SPs expression in MLE-12 cells. Furthermore, intratracheal instillation of macrophage-derived TNF-α-containing exosomes increased epithelial cell SPs expression in the lungs of mice. Taken together, these results suggest that macrophages-secreted exosomal TNF-α can trigger epithelial cell SPs expression, which provides new insight and potential target in the mechanism of epithelial cell dysfunction in PM2.5-induced ALI.

Engineered Extracellular Vesicles Derived from Dermal Fibroblasts Attenuate Inflammation in a Murine Model of Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) represents a significant burden to the healthcare system, with ≈200000 cases diagnosed annually in the USA. ARDS patients suffer from severe refractory hypoxemia, alveolar-capillary barrier dysfunction, impaired surfactant function, and abnormal upregulation of inflammatory pathways that lead to intensive care unit admission, prolonged hospitalization, and increased disability-adjusted life years. Currently, there is no cure or FDA-approved therapy for ARDS. This work describes the implementation of engineered extracellular vesicle (eEV)-based nanocarriers for targeted nonviral delivery of anti-inflammatory payloads to the inflamed/injured lung. The results show the ability of surfactant protein A (SPA)-functionalized IL-4- and IL-10-loaded eEVs to promote intrapulmonary retention and reduce inflammation, both in vitro and in vivo. Significant attenuation is observed in tissue damage, proinflammatory cytokine secretion, macrophage activation, influx of protein-rich fluid, and neutrophil infiltration into the alveolar space as early as 6 h post-eEVs treatment. Additionally, metabolomics analyses show that eEV treatment causes significant changes in the metabolic profile of inflamed lungs, driving the secretion of key anti-inflammatory metabolites. Altogether, these results establish the potential of eEVs derived from dermal fibroblasts to reduce inflammation, tissue damage, and the prevalence/progression of injury during ARDS via nonviral delivery of anti-inflammatory genes/transcripts.

Study of Serum Surfactant Protein D Levels in Chronic Obstructive Pulmonary Disease Patients

ABSTRACT Background: An increasing prevalence of chronic obstructive pulmonary disease (COPD) worldwide is one of the major reasons for morbidity and mortality. Surfactant protein D (SP-D) has been identified to have a role in the host defence system of the lungs and regulation of surfactant homeostasis. SP-D is considered as a biomarker of ascertaining the severity of COPD and risk of future exacerbations. Objectives: This study aimed to investigate changes in lung functions and SP-D levels in COPD patients for early diagnosis and timely intervention to prevent progression of the disease. Methods: One hundred stable COPD patients were enrolled for the study and were compared to 100 normal healthy individuals of the same age. Those fulfilling the inclusion and exclusion criteria were enrolled for the study. Pulmonary function tests were performed via spirometry. Blood samples were collected and estimation of SP-D was done using enzyme-linked immunosorbent assay (ELISA). Results: The observation revealed a significant decline in ventilatory functions with increasing severity of COPD, as reflected by the decrease in FEV1% and FEV1-to-FVC ratio compared to controls (P &lt; 0.001). Serum SP-D levels were significantly increased in COPD patients (P &lt; 0.001) compared to controls. Conclusion: SP-D estimation in COPD patients helps in monitoring the progression and prognosis of the diseases; formulate strategies for early therapeutic intervention to prevent risk of exacerbations to improve quality of life.

Cellular proteins act as surfactants to control the interfacial behavior and function of biological condensates.

Interfacial tension governs the behaviors and physiological functions of multiple biological condensates during diverse biological processes. Little is known about whether there are cellular surfactant factors that regulate the interfacial tension and functions of biological condensates within physiological environments. TFEB, a master transcription factor that controls expression of autophagic-lysosomal genes, assembles into transcriptional condensates to control the autophagy-lysosome pathway (ALP). Here, we show that interfacial tension modulates the transcriptional activity of TFEB condensates. MLX, MYC, and IPMK act as synergistic surfactants to decrease the interfacial tension and consequent DNA affinity of TFEB condensates. The interfacial tension of TFEB condensates is quantitatively correlated to their DNA affinity and subsequent ALP activity. The interfacial tension and DNA affinity of condensates formed by TAZ-TEAD4 are also regulated by the synergistic surfactant proteins RUNX3 and HOXA4. Our results indicate that the interfacial tension and functions of biological condensates can be controlled by cellular surfactant proteins in human cells.

Epithelial folding of alveolar cells derived from human induced pluripotent stem cells on artificial basement membrane.

Epithelial folding depends on mechanical properties of both epithelial cells and underlying basement membrane (BM). While folding is essential for tissue morphogenesis and functions, it is difficult to recapitulate features of a growing epithelial monolayer for in vitro modeling due to lack of in vivo like BM. Herein, we report a method to overcome this difficulty by culturing on an artificial basement membrane (ABM) the primordial lung progenitors (PLPs) from human induced pluripotent stem cells (hiPSCs). The ABM was achieved by self-assembling collagen IV and laminin, the two principal natural BM proteins, in the pores of a monolayer of crosslinked gelatin nanofibers deposited on a honeycomb micro-frame. The hiPSC-PLPs were seeded on the ABM for alveolar differentiation under submerged and air-liquid interface culture conditions. As results, the forces generated by the growing epithelial monolayer led to a geometry-dependent folding. Analysis of strain distribution in a clamped membrane provided instrumental insights into some of the observed phenomena. Moreover, the forces generated by the growing epithelial layer led to a high-level expression of surfactant protein C and a high percentage of aquaporin 5 positive cells compared with the results obtained with a nanofiber-covered bulk substrate. Thus, this work demonstrated the importance of recapitulating natural BM for advanced epithelial modeling. STATEMENT OF SIGNIFICANCE: The effort to develop in vitro epithelial models has not been entirely successful to date, due to lack of in vivo like basement membrane (BM). This lack has been overcome by using a microfabricated dense thin and pliable sheet like structure made of natural BM proteins. With such an artificial BM, alveolar epithelial deformation and folding could be studied and date could be correlated to numerical analyses of a plate theory. This method is simple and effective, enabling further developments in epithelial tissue modeling.

High-frequency chest wall oscillation multiple times daily can better reduce the loss of pulmonary surfactant and improve lung compliance in mechanically ventilated patients

BackgroundHigh-frequency chest wall oscillation (HFCWO) has been widely recognized for its airway secretion clearance effectiveness in critically ill ICU patients. ObjectivesThe purpose of this randomized controlled trial is to validate and compare the effects of different frequencies of HFCWO on oxygenation, lung compliance, and pulmonary surfactant proteins (SPs) in critically ill patients admitted to the intensive care unit (ICU). MethodsSixty patients with severe craniocerebral injury treated with a tracheostomy and mechanical ventilation were randomized into three groups (20 patients in each group): a single group (treated with 30 minutes of HFCWO once daily) and a double group (treated with 30 minutes of HFCWO twice daily), and a blank group (treated without HFCWO). Primary outcome measures included results on several specific proteins (SP-A, SP-B, SP-C, and SP-D) in serum and alveolar lavage fluid. Secondary outcome measures were lung static compliance test and oxygenation. ResultsPatients in both the single and double groups exhibited significant oxygenation and static compliance improvement. Similar results were observed in changes in SPs concentrations in the alveolar lavage fluid. However, a significant reduction of SPs levels was observed in the serum. In the group comparison analysis for the same variables between the single and double group, twice daily HFCWO treatments showed a significantly better result. ConclusionCompared with HFCWO once daily, HFCWO twice daily is advantageous in patients with tracheostomy and prolonged ventilation, which demonstrated significantly greater effectiveness in improving oxygenation and lung static compliance linked to the increase of and SPs contents in the airways as well as a reduction of SPs shift from airways to the blood.

Severe Neonatal Interstitial Lung Disease Caused by a Rare Surfactant Protein C Mutation.

Childhood interstitial lung disease (chILD) is a collective term for a group of rare lung disorders of heterogeneous origin. Surfactant dysfunction disorders are a cause of chILD with onset during the neonatal period and infancy. Clinical signs of tachypnea and hypoxemia are nonspecific and usually caused by common conditions like lower respiratory tract infections. We report on a full-term male newborn who was readmitted to the hospital at 7 days of age with marked tachypnea and poor feeding during the respiratory syncytial virus season. After exclusion of infection and other, more common congenital disorders, chILD was diagnosed using chest computed tomography and genetic analysis. A likely pathogenic heterozygous variant of SFTPC (c.163C>T, L55F) was detected by whole exome sequencing. The patient received supplemental oxygen and noninvasive respiratory support and was treated with intravenous methylprednisolone pulses and hydroxychloroquine. Despite the treatment, his respiratory situation deteriorated continuously, leading to several hospitalizations and continuous escalation of noninvasive ventilatory support. At 6 months of age, the patient was listed for lung transplant and transplanted successfully aged 7 months.

Increased Alveolar Epithelial Damage Markers and Inflammasome-Regulated Cytokines Are Associated with Pulmonary Superinfection in ARDS.

Acute respiratory distress syndrome (ARDS) is a life-threatening form of respiratory failure defined by dysregulated immune homeostasis and alveolar epithelial and endothelial damage. Up to 40% of ARDS patients develop pulmonary superinfections, contributing to poor prognosis and increasing mortality. Understanding what renders ARDS patients highly susceptible to pulmonary superinfections is therefore essential. We hypothesized that ARDS patients who develop pulmonary superinfections display a distinct pulmonary injury and pro-inflammatory response pattern. Serum and BALF samples from 52 patients were collected simultaneously within 24 h of ARDS onset. The incidence of pulmonary superinfections was determined retrospectively, and the patients were classified accordingly. Serum concentrations of the epithelial markers soluble receptor for advanced glycation end-products (sRAGE) and surfactant protein D (SP-D) and the endothelial markers vascular endothelial growth factor (VEGF) and angiopoetin-2 (Ang-2) as well as bronchoalveolar lavage fluid concentrations of the pro-inflammatory cytokines interleukin 1ß (IL-1ß), interleukin 18 (IL-18), interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-a) were analyzed via multiplex immunoassay. Inflammasome-regulated cytokine IL-18 and the epithelial damage markers SP-D and sRAGE were significantly increased in ARDS patients who developed pulmonary superinfections. In contrast, endothelial markers and inflammasome-independent cytokines did not differ between the groups. The current findings reveal a distinct biomarker pattern that indicates inflammasome activation and alveolar epithelial injury. This pattern may potentially be used in future studies to identify high-risk patients, enabling targeted preventive strategies and personalized treatment approaches.

Association of Surfactant Protein D Single Nucleotide Polymorphisms rs721917, rs2243639, rs3088308 with Recurrent Aphthous Stomatitis in Pakistani Population.

Recurrent aphthous stomatitis (RAS) is a benign ulcerative condition, defined by the recurrent formation of non-contagious mucosal ulcers. Surfactant protein D (SP-D) is secreted frequently at surfaces exposed directly to body fluids. This study aims to investigate the association of SP-D single nucleotide polymorphisms (SNPs) with the onset of RAS. Blood samples from 212 subjects (106 cases/controls each) were collected during 2019 and genotyped for SP-D SNPs (rs721917, rs2243639, rs3088308) by polymerase chain reaction and restriction fragment length polymorphism followed by 12% polyacrylamide gel electrophoresis. Minor aphthous (75.5%) was the commonly observed ulcer type as compared to herpetiform (21.7%) and major aphthous ulcers (2.8%). A family history of RAS was reported in 70% of cases. RAS was found significantly associated with rs3088308 genotypes T/A (95% (Cl): 1.57-5.03, p = 0.0005), A/A (95% (Cl): 1.8-6.7, p = 0.0002), T-allele (95% (Cl): 1.09-2.36, p = 0.01), A-allele (95% (Cl): 1.42-3.91, p = 0.01), rs721917 genotype T/T (95% (Cl): 1.15-25.35, p = 0.03), and T-allele (95% (Cl): 1.28-3.10, p = 0.002). Female gender and obese body mass index (BMI) were significantly associated with rs3088308 genotypes T/A (95% (CI): 1.89-15.7, p = 0.001), T/T (95% (Cl): 1.52-11.9, p = 0.005), A-allele (95% (Cl): 1.65-7.58, p < 0.001), and T-allele (95% (Cl): 1.4-10.1, p <0.001) and rs721917 genotype T/T (95% (CI) = 1.3-33, p = 0.02), respectively. This study describes the association of SP-D SNPs (rs721917, rs3088308) with RAS in the Pakistani population.

Activation of Wnt/β‑Catenin‑p130/E2F4 promotes the differentiation of bone marrow‑derived mesenchymal stem cells into typeII alveolar epithelial cells through cell cycle arrest.

The results of our previous study demonstrated that activation of the Wnt/β-catenin pathway increased the differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells; however, the specific mechanisms remain unclear. The present study aimed to evaluate the role of Wnt/β-catenin-p130/E2F transcription factor 4 (E2F4) in regulating the differentiation of mouse MSCs (mMSCs) into AT II cells, and to determine the specific mechanisms. mMSCs with p130 or E2F4 overexpression were constructed using lentiviral vectors. Differentiation of mMSCs into AT II cells was promoted using a modified coculture system with murine lung epithelial-12 cells incubated in small airway growth medium for 7-14 days. The differentiation efficiency was detected using immunofluorescence, western blot analysis and transmission electron microscopy. To detect the association between the canonical Wnt pathway and p130/E2F4, 4 mmol/l lithium chloride (LiCl) or 200 ng/ml Dickkopf-related protein 1 (DKK-1) was also added to the coculture system. Following differentiation, the cell cycle of mMSCs was evaluated using flow cytometry. The results of the present study demonstrated that surfactant protein C (SP-C) protein expression was higher in the p130 overexpression (MSC-p130) and E2F4 overexpression (MSC-E2F4) groups compared with the normal control mMSCs group following differentiation into AT II cells. Similar results for SP-C protein expression and lamellar body-like structures were also observed using immunofluorescence analysis and electron microscopy. Following the addition of LiCl into the coculture system for activation of the Wnt/β-catenin signaling pathway, phosphorylated (p)-p130/p130 was slightly decreased at 7 days and E2F4 was increased both at 7 and 14 days in mMSCs. Furthermore, the p-p130/p130 ratio was significantly increased at 14 days and E2F4 was decreased both at 7 and 14 days following DKK-1-mediated inhibition of the Wnt pathway. The results of the present study demonstrated that the numbers of cells in G1 and S phases were increased following activation of the Wnt pathway and decreased following Wnt pathway inhibition. However, the number of cells in G1 phase was increased following the differentiation of mMSCs overexpressing p130 or E2F4. Therefore, the results of the present study revealed that the canonical Wnt signaling pathway may affect the differentiation of MSCs into AT II cells via regulation of downstream p130/E2F4. The specific mechanisms may be associated with G1 phase extension in the cell cycle of MSCs.

Cytokine signaling converging on IL11 in ILD fibroblasts provokes aberrant epithelial differentiation signatures.

Interstitial lung disease (ILD) is a heterogenous group of lung disorders where destruction and incomplete regeneration of the lung parenchyma often results in persistent architectural distortion of the pulmonary scaffold. Continuous mesenchyme-centered, disease-relevant signaling likely initiates and perpetuates the fibrotic remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. With the aim of identifying functional mediators of the lung mesenchymal-epithelial crosstalk with potential as new targets for therapeutic strategies, we developed a 3D organoid co-culture model based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells that form alveolar organoids in presence of lung fibroblasts from fibrotic-ILD patients, in our study referring to cases of pulmonary fibrosis, as well as control cell line (IMR-90). While organoid formation capacity and size was comparable in the presence of fibrotic-ILD or control lung fibroblasts, metabolic activity was significantly increased in fibrotic-ILD co-cultures. Alveolar organoids cultured with fibrotic-ILD fibroblasts further demonstrated reduced stem cell function as reflected by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. To screen for key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used mass spectrometry and characterized the secretome of end stage fibrotic-ILD lung fibroblasts in comparison to non-chronic lung disease (CLD) patient fibroblasts. Out of the over 2000 proteins detected by this experimental approach, 47 proteins were differentially abundant comparing fibrotic-ILD and non-CLD fibroblast secretome. The fibrotic-ILD secretome profile was dominated by chemokines, including CXCL1, CXCL3, and CXCL8, interfering with growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating alveolar organoids with IL11, we recapitulated the co-culture results obtained with primary fibrotic-ILD fibroblasts including changes in metabolic activity. We identified mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD. In our alveolar organoid co-cultures, we were able to highlight the importance of fibroblast-initiated aberrant epithelial differentiation and confirmed IL11 as a key player in fibrotic-ILD pathogenesis by unbiased fibroblast secretome analysis.