Sodium butyrate ameliorates sepsis-associated lung injury by enhancing gut and lung barrier function in combination with modulation of CD4+Foxp3+ regulatory T cells.

Sepsis-associated lung injury often coexists with intestinal dysfunction. Butyrate, an essential gut microbiota metabolite, participates in gut-lung crosstalk and has immunoregulatory effects. This study aims to investigate the effect and mechanism of sodium butyrate (NaB) on lung injury. Sepsis-associated lung injury was established in mice by cecal ligation and puncture (CLP). Mice in treatment groups received NaB gavage after surgery. The survival rate, the oxygenation index and the lung wet-to-dry weight (W/D) ratio were calculated respectively. Pulmonary and intestinal histologic changes were observed. The total protein concentration in bronchoalveolar lavage fluid (BALF) was measured, and inflammatory factors in serum and BALF were examined. Diamine oxidase (DAO), lipopolysaccharide (LPS), and surfactant-associated protein D (SP-D) levels in serum and amphiregulin in lung tissue were assessed. Intercellular junction protein expression in the lung and intestinal tissues were examined. Changes in immune cells were analyzed. NaB treatment improved the survival rate, the oxygenation index and the histologic changes. NaB decreased the W/D ratio, total protein concentration, and the levels of proinflammatory cytokines, as well as SP-D, DAO and LPS, while increased the levels of anti-inflammatory cytokines and amphiregulin. The intercellular junction protein expression were improved by NaB. Furthermore, the CD4+/CD8+ T-cell ratio and the proportion of CD4+Foxp3+ regulatory T cells (Tregs) were increased by NaB. Our data suggested that NaB gavage effectively improved the survival rate and mitigated lung injury in CLP mice. The possible mechanism was that NaB augmented CD4+Foxp3+ Tregs and enhanced the barrier function of the gut and the lung.

Exogenous Apelin-13 Administration Ameliorates Cyclophosphamide- Induced Oxidative Stress, Inflammation, and Apoptosis in Rat Lungs.

BACKGROUND: Apelin-13 is an endogenous adipocytokine known for its antioxidant, antiinflammatory, and antiapoptotic properties. OBJECTIVE: We aimed to investigate the possible protective effects of exogenous Apelin-13 administration on oxidative stress, inflammation, and apoptosis induced by the cytotoxic agent cyclophosphamide (CP) in the lungs. METHODS: Twenty-four male Wistar albino rats were divided into four groups: Control (saline), CP (200 mg/kg), Apelin-13 (10 µg/kg/day), and CP+Apelin-13. CP was administered as a single dose on the fifth day, and apelin-13 was administered intraperitoneally for five days. Total oxidant status (TOS), total antioxidant status (TAS), and lipid peroxidation were determined with spectrophotometry, TNFα and IL1ß were determined with ELISA, APJ, Sirt1, NF-κB, and p53 mRNA expressions were determined with qRT-PCR, cytochrome (Cyt) C and caspase-3 protein expressions were studied with western blotting in lung tissues. The oxidative stress index (OSI) was also calculated. Furthermore, serum surfactant protein-D (SP-D) and Krebs von den Lungen-6 (KL-6) levels were measured with ELISA. RESULTS: Compared to the control group, TOS, OSI, lipid peroxidation, TNFα, IL1ß, cyt C, caspase-3, APJ, NF-κB, and p53 were higher, and Sirt1 was lower in the lung tissue of rats in the CP group. Serum KL-6 and SP-D levels were higher in the CP group. Co-administration of CP with Apelin-13 completely reversed the changes induced by CP administration. CONCLUSION: Exogenous Apelin-13 treatment protected lung tissue against injury by inhibiting cyclophosphamide-induced oxidative stress, inflammation, and apoptosis. This protective effect of apelin-13 was accompanied by upregulation of the Sirt1 and downregulation of NF-κB/p53 in the lungs.

Respiratory Effects of Traffic-Related Air Pollution: A Randomized, Crossover Analysis of Lung Function, Airway Metabolome, and Biomarkers of Airway Injury.

BACKGROUND: Exposure to traffic-related air pollution (TRAP) has been associated with increased risks of respiratory diseases, but the biological mechanisms are not yet fully elucidated. OBJECTIVES: Our aim was to evaluate the respiratory responses and explore potential biological mechanisms of TRAP exposure in a randomized crossover trial. METHODS: We conducted a randomized crossover trial in 56 healthy adults. Each participant was exposed to high- and low-TRAP exposure sessions by walking in a park and down a road with high traffic volume for 4 h in random order. Respiratory symptoms and lung function, including forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), the ratio of FEV1 to FVC, and maximal mid-expiratory flow (MMEF), were measured before and after each exposure session. Markers of 8-isoprostane, tumor necrosis factor-α (TNF-α), and ezrin in exhaled breath condensate (EBC), and surfactant proteins D (SP-D) in serum were also measured. We used linear mixed-effects models to estimate the associations, adjusted for age, sex, body mass index, meteorological condition, and batch (only for biomarkers). Liquid chromatography-mass spectrometry was used to profile the EBC metabolome. Untargeted metabolome-wide association study (MWAS) analysis and pathway enrichment analysis using mummichog were performed to identify critical metabolomic features and pathways associated with TRAP exposure. RESULTS: Participants had two to three times higher exposure to traffic-related air pollutants except for fine particulate matter while walking along the road compared with in the park. Compared with the low-TRAP exposure at the park, high-TRAP exposure at the road was associated with a higher score of respiratory symptoms [2.615 (95% CI: 0.605, 4.626), p=1.2×10-2] and relatively lower lung function indicators [-0.075L (95% CI: -0.138, -0.012), p=2.1×10-2] for FEV1 and -0.190L/s (95% CI: -0.351, -0.029; p=2.4×10-2) for MMEF]. Exposure to TRAP was significantly associated with changes in some, but not all, biomarkers, particularly with a 0.494-ng/mL (95% CI: 0.297, 0.691; p=9.5×10-6) increase for serum SP-D and a 0.123-ng/mL (95% CI: -0.208, -0.037; p=7.2×10-3) decrease for EBC ezrin. Untargeted MWAS analysis revealed that elevated TRAP exposure was significantly associated with perturbations in 23 and 32 metabolic pathways under positive- and negative-ion modes, respectively. These pathways were most related to inflammatory response, oxidative stress, and energy use metabolism. CONCLUSIONS: This study suggests that TRAP exposure might lead to lung function impairment and respiratory symptoms. Possible underlying mechanisms include lung epithelial injury, inflammation, oxidative stress, and energy metabolism disorders. https://doi.org/10.1289/EHP11139.

Intravenous surfactant protein D inhibits lipopolysaccharide-induced systemic inflammation.

BACKGROUND: Surfactant protein D (SP-D) is an innate host defense protein that clears infectious pathogens from the lung and regulates pulmonary host defense cells. SP-D is also detected in lower concentrations in plasma and many other non-pulmonary tissues. Plasma levels of SP-D increase during infection and other proinflammatory states; however, the source and functions of SP-D in the systemic circulation are largely unknown. We hypothesized that systemic SP-D may clear infectious pathogens and regulate host defense cells in extrapulmonary systems. METHODS: To determine if SP-D inhibited inflammation induced by systemic lipopolysaccharide (LPS), E.coli LPS was administered to mice via tail vein injection with and without SP-D and the inflammatory response was measured. RESULTS: Systemic SP-D has a circulating half-life of 6 h. Systemic IL-6 levels in mice lacking the SP-D gene were similar to wild type mice at baseline but were significantly higher than wild type mice following LPS treatment (38,000 vs 29,900 ng/ml for 20 mg/kg LPS and 100,700 vs 73,700 ng/ml for 40 mg/kg LPS). In addition, treating wild type mice with purified intravenous SP-D inhibited LPS induced secretion of IL-6 and TNFα in a concentration dependent manner. Inhibition of LPS induced inflammation by SP-D correlated with SP-D LPS binding suggesting SP-D mediated inhibition of systemic LPS requires direct SP-D LPS interactions. CONCLUSIONS: Taken together, the above results suggest that circulating SP-D decreases systemic inflammation and raise the possibility that a physiological purpose of increasing systemic SP-D levels during infection is to scavenge systemic infectious pathogens and limit inflammation-induced tissue injury.

Malondialdehyde acetaldehyde adduction of surfactant protein D attenuates SARS-CoV-2 spike protein binding and virus neutralization.

BACKGROUND: Over 43% of the world's population regularly consumes alcohol. Although not commonly known, alcohol can have a significant impact on the respiratory environment. Living in the time of the COVID-19 pandemic, alcohol misuse can have a particularly deleterious effect on SARS-CoV-2-infected individuals and, in turn, the overall healthcare system. Patients with alcohol use disorders have higher odds of COVID-19-associated hospitalization and mortality. Even though the detrimental role of alcohol on COVID-19 outcomes has been established, the underlying mechanisms are yet to be fully understood. Alcohol misuse has been shown to induce oxidative damage in the lungs through the production of reactive aldehydes such as malondialdehyde and acetaldehyde (MAA). MAA can then form adducts with proteins, altering their structure and function. One such protein is surfactant protein D (SPD), which plays an important role in innate immunity against pathogens. METHODS AND RESULTS: In this study, we examined whether MAA adduction of SPD (SPD-MAA) attenuates the ability of SPD to bind SARS-CoV-2 spike protein, reversing SPD-mediated virus neutralization. Using ELISA, we show that SPD-MAA is unable to competitively bind spike protein and prevent ACE2 receptor binding. Similarly, SPD-MAA fails to inhibit entry of wild-type SARS-CoV-2 virus into Calu-3 cells, a lung epithelial cell line, as well as ciliated primary human bronchial epithelial cells isolated from healthy individuals. CONCLUSIONS: Overall, MAA adduction of SPD, a consequence of alcohol overconsumption, represents one mechanism of compromised lung innate defense against SARS-CoV-2, highlighting a possible mechanism underlying COVID-19 severity and related mortality in patients who misuse alcohol.

Surfactant protein D inhibits lipid-laden foamy macrophages and lung inflammation in chronic obstructive pulmonary disease.

Increased levels of surfactant protein D (SP-D) and lipid-laden foamy macrophages (FMs) are frequently found under oxidative stress conditions and/or in patients with chronic obstructive pulmonary disease (COPD) who are also chronically exposed to cigarette smoke (CS). However, the roles and molecular mechanisms of SP-D and FMs in COPD have not yet been determined. In this study, increased levels of SP-D were found in the bronchoalveolar lavage fluid (BALF) and sera of ozone- and CS-exposed mice. Furthermore, SP-D-knockout mice showed increased lipid-laden FMs and airway inflammation caused by ozone and CS exposure, similar to that exhibited by our study cohort of chronic smokers and COPD patients. We also showed that an exogenous recombinant fragment of human SP-D (rfhSP-D) prevented the formation of oxidized low-density lipoprotein (oxLDL)-induced FMs in vitro and reversed the airway inflammation and emphysematous changes caused by oxidative stress and CS exposure in vivo. SP-D upregulated bone marrow-derived macrophage (BMDM) expression of genes involved in countering the oxidative stress and lipid metabolism perturbations induced by CS and oxLDL. Our study demonstrates the crucial roles of SP-D in the lipid homeostasis of dysfunctional alveolar macrophages caused by ozone and CS exposure in experimental mouse emphysema, which may provide a novel opportunity for the clinical application of SP-D in patients with COPD.

Dimethyl itaconate inhibits LPS-induced inflammatory release and apoptosis in alveolar type II epithelial and bronchial epithelial cells by activating pulmonary surfactant proteins A and D.

BACKGROUND: Injury to the lung is a common, clinically serious inflammatory disease. However, its pathogenesis remains unclear, and the existing treatments, including cytokine therapy, stem cell therapy, and hormone therapy, are not completely effective in treating this disease. Dimethyl itaconate (DMI) is a surfactant with important anti-inflammatory effects. OBJECTIVE: The present study used alveolar type II (AT II) and bronchial epithelial cells as models to determine the role of DMI in lung injury. MATERIAL AND METHODS: First, the effects of DMI were established on the survival, inflammatory release, and apoptosis in lipopolysaccharide (LPS)-induced AT II and bronchial epithelial cells. The association between DMI and Sirtuin1 (SIRT1) was assessed using molecular docking. Next, by constructing interference plasmids to inhibit surfactant protein (SP)-A and SP-D expressions, the effect of DMI was observed on inflammatory release and apoptosis. RESULTS: The results revealed that DMI increased the survival rate and expression levels of SP-A, SP-D, and SIRT1, and inhibited inflammatory factors as well as apoptosis in LPS-induced cells. Furthermore, DMI could bind to SIRT1 to regulate SP-A and SP-D expressions. After SP-A and SP-D expressions were inhibited, the inhibitory effect of DMI was reversed on inflammatory release and apoptosis. CONCLUSION: The findings of the present study revealed that DMI inhibited LPS-induced inflammatory release and apoptosis in cells by targeting SIRT1 and then activating SP-A and SP-D. This novel insight into the pharmacological mechanism of DMI lays the foundation for its later use for alleviating lung injury.

Innate/Inflammatory Bioregulation of Surfactant Protein D Alleviates Rat Osteoarthritis by Inhibiting Toll-Like Receptor 4 Signaling.

Osteoarthritis (OA) is a deteriorating disease of cartilage tissues mainly characterized as low-grade inflammation of the joint. Innate immune molecule surfactant protein D (SP-D) is a member of collectin family of collagenous Ca2+-dependent defense lectins and plays a vital role in the inflammatory and innate immune responses. The present study investigated the SP-D-mediated innate/inflammatory bioregulation in OA and explored the underlying molecular mechanism. Transcriptome analysis revealed that SP-D regulated genes were strongly enriched in the inflammatory response, immune response, cellular response to lipopolysaccharide (LPS), PI3K-Akt signaling, Toll-like receptor (TLR) signaling, and extracellular matrix (ECM)-receptor interaction pathways. Knockdown of the SP-D gene by the recombinant adeno-associated virus promoted the macrophage specific markers of CD68, F4/80 and TLR4 in the articular cartilage in vivo. SP-D alleviated the infiltration of synovial macrophages and neutrophils, and inhibited TLR4, TNF-α and the phosphorylation of PI3K, Akt and NF-κB p65 in cartilage. SP-D suppressed cartilage degeneration, inflammatory and immune responses in the rat OA model, whilst TAK-242 strengthened this improvement. In in vitro conditions, SP-D pre-treatment inhibited LPS-induced overproduction of inflammation-correlated cytokines such as IL-1ß and TNF-α, and suppressed the overexpression of TLR4, MD-2 and NLRP3. SP-D prevented the LPS-induced degradation of ECM by down-regulating MMP-13 and up-regulating collagen II. Blocking of TLR4 by TAK-242 further enhanced these manifestations. We also demonstrated that SP-D binds to the TLR4/MD-2 complex to suppress TLR4-mediated PI3K/Akt and NF-κB signaling activation in chondrocytes. Taken together, these findings indicate that SP-D has chondroprotective properties dependent on TLR4-mediated PI3K/Akt and NF-κB signaling and that SP-D has an optimal bioregulatory effect on the inflammatory and innate responses in OA.

Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function.

Alcohol consumption with concurrent cigarette smoking produces malondialdehyde acetaldehyde (MAA)-adducted lung proteins. Lung surfactant protein D (SPD) supports innate immunity via bacterial aggregation and lysis, as well as by enhancing macrophage-binding and phagocytosis. MAA-adducted SPD (SPD-MAA) has negative effects on lung cilia beating, macrophage function, and epithelial cell injury repair. Because changes in SPD multimer structure are known to impact SPD function, we hypothesized that MAA-adduction changes both SPD structure and function. Purified human SPD and SPD-MAA (1 mg/mL) were resolved by gel filtration using Sephadex G-200 and protein concentration of each fraction determined by Bradford assay. Fractions were immobilized onto nitrocellulose by slot blot and assayed by Western blot using antibodies to SPD and to MAA. Binding of SPD and SPD-MAA was determined fluorometrically using GFP-labeled Streptococcus pneumoniae (GFP-SP). Anti-bacterial aggregation of GFP-SP and macrophage bacterial phagocytosis were assayed by microscopy and permeability determined by bacterial phosphatase release. Viral injury was measured as LDH release in RSV-treated airway epithelial cells. Three sizes of SPD were resolved by gel chromatography as monomeric, trimeric, and multimeric forms. SPD multimer was the most prevalent, while the majority of SPD-MAA eluted as trimer and monomer. SPD dose-dependently bound to GFP-SP, but SPD-MAA binding to bacteria was significantly reduced. SPD enhanced, but MAA adduction of SPD prevented, both aggregation and macrophage phagocytosis of GFP-SP. Likewise, SPD increased bacterial permeability while SPD-MAA did not. In the presence of RSV, BEAS-2B cell viability was enhanced by SPD, but not protected by SPD-MAA. Our results demonstrate that MAA adduction changes the quaternary structure of SPD from multimer to trimer and monomer leading to a decrease in the native anti-microbial function of SPD. These findings suggest one mechanism for increased pneumonia observed in alcohol use disorders.

Ammonia exposure by intratracheal instillation causes severe and deteriorating lung injury and vascular effects in mice.

OBJECTIVE: Ammonia (NH3) is a corrosive alkaline gas that can cause life-threatening injuries by inhalation. The aim was to establish a disease model for NH3-induced injuries similar to acute lung injury (ALI) described in exposed humans and investigate the progression of lung damage, respiratory dysfunction and evaluate biomarkers for ALI and inflammation over time. METHODS: Female BALB/c mice were exposed to an NH3 dose of 91.0 mg/kg·bw using intratracheal instillation and the pathological changes were followed for up to 7 days. RESULTS: NH3 instillation resulted in the loss of body weight along with a significant increase in pro-inflammatory mediators in both bronchoalveolar lavage fluid (e.g. IL-1ß, IL-6, KC, MMP-9, SP-D) and blood (e.g. IL-6, Fibrinogen, PAI-1, PF4/CXCL4, SP-D), neutrophilic lung inflammation, alveolar damage, increased peripheral airway resistance and methacholine-induced airway hyperresponsiveness compared to controls at 20 h. On day 7 after exposure, deteriorating pathological changes such as increased macrophage lung infiltration, heart weights, lung hemorrhages and coagulation abnormalities (elevated plasma levels of PAI-1, fibrinogen, endothelin and thrombomodulin) were observed but no increase in lung collagen. Some of the analyzed blood biomarkers (e.g. RAGE, IL-1ß) were unaffected despite severe ALI and may not be significant for NH3-induced damages. CONCLUSIONS: NH3 induces severe acute lung injuries that deteriorate over time and biomarkers in lungs and blood that are similar to those found in humans. Therefore, this model has potential use for developing diagnostic tools for NH3-induced ALI and for finding new therapeutic treatments, since no specific antidote has been identified yet.

Diagnosis of Chronic Obstructive Pulmonary Disease and Regulatory Mechanism of miR-149-3p on Alveolar Inflammatory Factors and Expression of Surfactant Proteins A (SP-A) and D (SP-D) on Lung Surface Mediated by Wnt Pathway.

Objective: To study the mechanism of chronic obstructive pulmonary disease (COPD) in diagnosing alveolar factors and analyze the effect of miR-149-3p on alveolar inflammatory factors and the expression of surfactant protein D (SP-D) and SP-A on the lung surface mediated by Wnt pathway. Methods: Patients with stable COPD were taken as the research subjects, and healthy volunteers as the control group. Cardiac color Doppler ultrasound was adopted to measure the ventricular structure of patients. The ultrasound simulation method was introduced in the ultrasound imaging. The ultrasound image was processed based on the intelligent ultrasound simulation algorithm. The changes in the structure of the left and right ventricles were analyzed and compared in the two groups. The expression changes of miR-149-3p, Wnt1, ß-catenin, RhoA, and Wnt5a in lung tissues of mice in three groups were detected, as well as the content of tumor necrosis factor- (TNF-) α, IL-1ß, interleukin (IL-6), nuclear factor kB (NF-kB), and other inflammatory factors in bronchoalveolar tissues of mice in three groups. Results: The position where the attenuation ratio was less than 0.92 in the experiment under the ultrasonic simulation algorithm had a gray value of 50. Compared with the control group, the right ventricular mass index of patients with stable COPD was statistically considerable (P < 0.05). In patients with stable COPD, the overall right ventricular longitudinal strain, right ventricular diastolic longitudinal strain rate (RV DLSR), right ventricular diastolic circumferential strain rate, and right ventricular longitudinal displacement were significantly impaired (P < 0.05). The content of miR-149-3p in the lung tissue of the model group was dramatically inferior to that of the control group and the interference group (P < 0.05). The contents of Wnt1, ß-catenin, RhoA, and Wnt5a in the lung tissue of the model group were dramatically superior to those of the control group (P < 0.05). In addition, the expressions of TNF-α, IL-1ß, IL-6, and NF-kB in the alveolar lavage fluid of the model group were statistically different from those of control group (P < 0.05). The expression levels of SP-D and surfactant protein A (SP-A) in the COPD group were also statistically different from those of control group (P < 0.05). Conclusion: miR-149-3p regulated the expression of Wnt1, ß-catenin, RhoA, and Wnt5a, which also affected the signal transmission of the Wnt pathway, causing changes in the expression of alveolar inflammatory factors. Eventually, it affected the development of COPD.

Lower Oligomeric Form of Surfactant Protein D in Murine Acute Lung Injury Induces M1 Subtype Macrophages Through Calreticulin/p38 MAPK Signaling Pathway.

Surfactant protein D (SP-D) plays an important role in innate and adaptive immune responses. In this study, we found that the expression of total and de-oligomerized SP-D was significantly elevated in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI). To investigate the role of the lower oligomeric form of SP-D in the pathogenesis of ALI, we treated bone marrow-derived macrophages (BMDMs) with ALI-derived bronchoalveolar lavage (BAL) and found that SP-D in ALI BAL predominantly bound to calreticulin (CALR) on macrophages, subsequently increasing the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and expression of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, IL-10, and CD80. However, anti-SP-D (aSP-D) and anti-calreticulin (aCALR) pretreatment reversed the SP-D binding and activation of macrophages induced by ALI BAL or de-oligomerized recombinant murine SP-D (rSP-D). Lack of signal transducer and activator of transcription (STAT)6 in STAT6-/- macrophages resulted in resistance to suppression by aCALR. Further studies in an ALI mouse model showed that blockade of pulmonary SP-D by intratracheal (i.t.), but not intraperitoneal (i.p.), administration of aSP-D attenuated the severity of ALI, accompanied by lower neutrophil infiltrates and expression of IL-1beta and IL-6. Furthermore, i.t. administration of de-oligomerized rSP-D exacerbated the severity of ALI in association with more pro-inflammatory CD45+Siglec-F(-) M1 subtype macrophages and production of IL-6, TNF-alpha, IL-1beta, and IL-18. The results indicated that SP-D in the lungs of murine ALI was de-oligomerized and participated in the pathogenesis of ALI by predominantly binding to CALR on macrophages and subsequently activating the pro-inflammatory downstream signaling pathway. Targeting de-oligomerized SP-D is a promising therapeutic strategy for the treatment of ALI and acute respiratory distress syndrome (ARDS).

Research article expression of surfactant protein-A and D, and CD9 in lungs of 1 and 30 day old foals.

BACKGROUND: Respiratory diseases are a major cause of morbidity and mortality in the horses of all ages including foals. There is limited understanding of the expression of immune molecules such as tetraspanins and surfactant proteins (SP) and the regulation of the immune responses in the lungs of the foals. Therefore, the expression of CD9, SP-A and SP-D in foal lungs was examined. RESULTS: Lungs from one day old (n = 6) and 30 days old (n = 5) foals were examined for the expression of CD9, SP-A, and SP-D with immunohistology and Western blots. Western blot data showed significant increase in the amount of CD9 protein (p = 0.0397) but not of SP-A and SP-D at 30 days of age compared to one day. Immunohistology detected CD9 in the alveolar septa and vascular endothelium but not the bronchiolar epithelium in the lungs of the foals in both age groups. SP-A and SP-D expression was localized throughout the alveolar septa including type II alveolar epithelial cells and the vascular endothelium of the lungs in all the foals. Compared to one day old foals, the expression of SP-A and SP-D appeared to be increased in the bronchiolar epithelium of 30 day old foals. Pulmonary intravascular macrophages were also positive for SP-A and SP-D in 30 days old foals and these cells are not developed in the day old foals. CONCLUSIONS: This is the first data on the expression of CD9, SP-A and SP-D in the lungs of foals.

The role of angiopoietin-2 and surfactant protein-D levels in SARS-CoV-2-related lung injury: A prospective, observational, cohort study.

INTRODUCTION: Coronavirus disease-2019 (COVID-19) is a respiratory disease whose clinical manifestation ranges from asymptomatic to severe respiratory failure. The purpose of this study was to investigate the place of serum surfactant-D (SP-D) and angiopoetin-2 (Ang-2) levels in predicting severity of disease in patients diagnosed with COVID-19. METHODS: Sixty-four patients diagnosed with COVID-19 between September 2020 and February 2021, 50 patients diagnosed with community-acquired pneumonia and a 50-member healthy control group were included in the study. Plasma samples and clinical data were collected within 72 h after admission, during hospital stay. Serum SP-D and Ang-2 concentrations were measured using the enzyme-linked immunosorbent assay. RESULTS: SP-D and Ang-2 levels were significantly higher in the mild-moderate pneumonia and severe/critical patient groups compared to the asymptomatic and noncomplicated COVID-19 patients (p < 0.001 for all groups). Serum SP-D and Ang-2 levels of severe-critical COVID-19 patients were significantly higher than CAP patients (p < 0.001). Powerful correlation was present between clinical severity of COVID-19 and SP-D and Ang-2 levels (r = 0.885 p < 0.001 and r = 0.913 p < 0.001, respectively). Cut-off values of 37.7 ng/ml (AUC = 0.763, p < 0.001, 95% confidence interval [CI] = 0.667-0.860) for SP-D and 4208.3 pg/ml (AUC = 0.659, p = 0.004, 95% CI = 0.554-0.763) for Ang-2 were identified as predictors of COVID-19 disease at receiver operating characteristic curve analysis. CONCLUSION: SP-D and Ang-2 are predictive factors in differentiating COVID-19 patients and determining severity of disease. These data may be important for the initiation of treatment in the early stage of the disease in patients with COVID-19.

Light and electron-microscopic localization of CD9 and surfactant protein A and D in normal lungs of the horse.

The lung is a complex organ, and its physiology and immunology are regulated by various immune molecules and cells. Lung surfactant, a mixture of phospholipids and proteins produced by the bronchiolar and type II alveolar epithelial cells, is one such important player in lung physiology. Compared to knowledge about the biology of the surfactant in rodents and humans, only limited data are available on the surfactant in the horse. Although there are data linking levels of surfactant proteins with respiratory disease in the horse, there are no data on the cellular localization of surfactant protein A (SP-A) and surfactant protein D (SP-D). A member of the tetraspanin family of proteins, CD9 is a cell-signaling and adhesion protein and its expression has been detected in both normal and cancer cells, including those in the lung. Because there are no immunolocalization data on SP-A, SP-D, and CD9 in the normal lungs of the horse, our objective was to conduct a light and electron microscopic immunocytochemical study on normal lungs of the horse. The data showed SP-A and SP-D in bronchiolar epithelial and type II alveolar epithelial cells. These proteins were also localized in type I alveolar epithelial cells, pulmonary intravascular macrophages, and neutrophils, which is likely an outcome of endocytosis of the proteins by these cells. CD9 was present in the airway and vascular smooth muscle cells, endothelium, and blood cells, but not in the airway epithelium. These new data provide a baseline to further examine the expression and functions of SP-A, SP-D, and CD9 proteins in inflammation associated with respiratory diseases in the horse.

Le poumon est un organe complexe, et sa physiologie et son immunologie sont régulées par diverses molécules et cellules immunitaires. Le surfactant pulmonaire, un mélange de phospholipides et de protéines produits par les cellules épithéliales bronchiolaires et alvéolaires de type II, est un acteur important de la physiologie pulmonaire. Par rapport aux connaissances sur la biologie du surfactant chez les rongeurs et les humains, seules des données limitées sont disponibles sur le surfactant chez le cheval. Bien qu'il existe des données reliant les niveaux de protéines du surfactant à une maladie respiratoire chez le cheval, il n'y a pas de données sur la localisation cellulaire de la protéine de surfactant A (SP-A) et de la protéine de surfactant D (SP-D). Membre de la famille des protéines tétraspanines, CD9 est une protéine de signalisation et d'adhésion cellulaire et son expression a été détectée dans les cellules normales et cancéreuses, y compris celles du poumon. Comme il n'y a pas de données d'immunolocalisation pour SP-A, SP-D et CD9 dans les poumons normaux du cheval, notre objectif était de mener une étude immunocytochimique au microscope optique et électronique sur les poumons normaux du cheval. Les données ont montré la présence de SP-A et SP-D dans les cellules épithéliales bronchiolaires et alvéolaires de type II. Ces protéines étaient également localisées dans les cellules épithéliales alvéolaires de type I, les macrophages intravasculaires pulmonaires et les neutrophiles, ce qui est probablement le résultat de l'endocytose des protéines par ces cellules. Le CD9 était présent dans les cellules des voies respiratoires et des muscles lisses vasculaires, l'endothélium et les cellules sanguines, mais pas dans l'épithélium des voies respiratoires. Ces nouvelles données fournissent une base de référence pour examiner plus à fond l'expression et les fonctions des protéines SP-A, SP-D et CD9 dans l'inflammation associée aux maladies respiratoires chez le cheval.(Traduit par Docteur Serge Messier).

Surfactant protein D and bronchopulmonary dysplasia: a new way to approach an old problem.

Surfactant protein D (SP-D) is a collectin protein synthesized by alveolar type II cells in the lungs. SP-D participates in the innate immune defense of the lungs by helping to clear infectious pathogens and modulating the immune response. SP-D has shown an anti-inflammatory role by down-regulating the release of pro-inflammatory mediators in different signaling pathways such as the TLR4, decreasing the recruitment of inflammatory cells to the lung, and modulating the oxidative metabolism in the lungs. Recombinant human SP-D (rhSP-D) has been successfully produced mimicking the structure and functions of native SP-D. Several in vitro and in vivo experiments using different animal models have shown that treatment with rhSP-D reduces the lung inflammation originated by different insults, and that rhSP-D could be a potential treatment for bronchopulmonary dysplasia (BPD), a rare disease for which there is no effective therapy up to date. BPD is a complex disease in preterm infants whose incidence increases with decreasing gestational age at birth. Lung inflammation, which is caused by different prenatal and postnatal factors like infections, lung hyperoxia and mechanical ventilation, among others, is the key player in BPD. Exacerbated inflammation causes lung tissue injury that results in a deficient gas exchange in the lungs of preterm infants and frequently leads to long-term chronic lung dysfunction during childhood and adulthood. In addition, low SP-D levels and activity in the first days of life in preterm infants have been correlated with a worse pulmonary outcome in BPD. Thus, SP-D mediated functions in the innate immune response could be critical aspects of the pathogenesis in BPD and SP-D could inhibit lung tissue injury in this preterm population. Therefore, administration of rhSP-D has been proposed as promising therapy that could prevent BPD.

Neutrophils and IL-1α Regulate Surfactant Homeostasis during Cigarette Smoking.

Cigarette smoke exposure induces inflammation marked by rapid and sustained neutrophil infiltration, IL-1α, release and altered surfactant homeostasis. However, the extent to which neutrophils and IL-1α contribute to the maintenance of pulmonary surfactant homeostasis is not well understood. We sought to investigate whether neutrophils play a role in surfactant clearance as well as the effect of neutrophil depletion and IL-1α blockade on the response to cigarette smoke exposure. In vitro and in vivo administration of fluorescently labeled surfactant phosphatidylcholine was used to assess internalization of surfactant by lung neutrophils and macrophages during or following cigarette smoke exposure in mice. We also depleted neutrophils using anti-Ly-6G or anti-Gr-1 Abs, or we neutralized IL-1α using a blocking Ab to determine their respective roles in regulating surfactant homeostasis during cigarette smoke exposure. We observed that neutrophils actively internalize labeled surfactant both in vitro and in vivo and that IL-1α is required for smoke-induced elevation of surfactant protein (SP)-A and SP-D levels. Neutrophil depletion during cigarette smoke exposure led to a further increase in SP-A levels in the bronchoalveolar lavage and increased IL-1α, CCL2, GM-CSF, and G-CSF release. Finally, macrophage expression of Mmp12, a protease linked to emphysema, was increased in neutrophil-depleted groups and decreased following IL-1α blockade. Taken together, our results indicate that neutrophils and IL-1α signaling are actively involved in surfactant homeostasis and that the absence of neutrophils in the lungs during cigarette smoke exposure leads to an IL-1α-dependent exacerbation of the inflammatory response.

Hydroxypropyl Cyclodextrin Improves Amiodarone-induced Aberrant Lipid Homeostasis of Alveolar Cells.

Alveolar epithelial type II (AT2) cells secrete pulmonary surfactant via lamellar bodies (LBs). Abnormalities in LBs are associated with pulmonary disorders, including fibrosis. However, high-content screening (HCS) for LB abnormalities is limited by the lack of understanding of AT2 cell functions. In the present study, we have developed LB cells harboring LB-like organelles that secrete surfactant proteins. These cells were more similar to AT2 cells than to parental A549 cells. LB cells recapitulated amiodarone (AMD)-induced LB enlargement, similar to AT2 cells of patients exposed to AMD. To reverse AMD-induced LB abnormalities, we performed HCS of approved drugs and identified 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a cyclic oligosaccharide, as a potential therapeutic agent. A transcriptome analysis revealed that HPßCD modulates lipid homeostasis. In addition, HPßCD inhibited AMD-induced LB abnormalities in human induced pluripotent stem cell-derived AT2 cells. Our results demonstrate that LB cells are useful for HCS and suggest that HPßCD is a candidate therapeutic agent for AMD-induced interstitial pneumonia.

MEG3 Alleviated LPS-Induced Intestinal Injury in Sepsis by Modulating miR-129-5p and Surfactant Protein D.

Sepsis and intestinal injury triggered by sepsis are common in intensive care units, which can contribute to a high mortality. lncRNAs can modulate gene expression, and they are closely involved in multiple diseases, including sepsis. In our present study, we investigated the biological function of MEG3 in sepsis, especially during the intestinal injury. Currently, we observed that in LPS-induced sepsis mouse models, the intestinal injury was triggered. Meanwhile, we reported that MEG3 was greatly decreased in vivo, with an increase of miR-129-5p and inhibition of SP-D. Then, MEG3 was overexpressed, and we found that its overexpression repressed the intestinal injury via downregulating miR-129-5p in sepsis mice. Moreover, TNF-α and IL-6 expression was elevated in intestinal tissues compared to the control groups. MEG3 restrained the activation of TNF-α and IL-6, in sepsis models. Subsequently, to induce the inflammatory injury of sepsis, human colorectal Caco2 cells were treated with 10 ng/ml LPS. 10 ng/ml LPS significantly inhibited Caco2 cell proliferation and increased the apoptosis. Additionally, MEG3 was decreased whereas miR-129-5p was obviously increased in Caco2 cells incubated with LPS. Interestingly, we showed that MEG3 repressed cell apoptosis partly and enhanced Caco2 cell proliferation. miR-129-5p overexpression could reverse the effect of MEG3 in vitro. Previously, we proved SP-D was reduced in sepsis and it depressed the intestinal injury in vivo. Finally, the correlation among MEG3, miR-129-5p, and SP-D was predicted and confirmed in our investigation. These findings indicated that MEG3 might be a potential target for intestinal damage caused by sepsis via regulating miR-129-5p and SP-D.

miR-182-5p contributes to intestinal injury in a murine model of Staphylococcus aureus pneumonia-induced sepsis via targeting surfactant protein D.

Sepsis is a severe clinical disease, which is resulted from the excessive host inflammation response to the infection. Growing evidence indicates that Staphylococcus aureus pneumonia is a significant cause of sepsis, which can lead to intestinal injury, inflammation, and apoptosis. Studies have shown that miR-182-5p can serve as a tumor oncogene or a tumor suppressive microRNA in various cancers, however, its biological role in sepsis is still uninvestigated. Here, we reported that miR-182-5p was obviously increased in S. aureus pneumonia mice models. Loss of miR-182-5p inhibited intestinal damage and intestinal apoptosis as indicated by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, we observed the lack of miR-182-5p altered the local inflammatory response to pneumonia in the intestine. Elevated tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were observed in intestinal tissue of pneumonia groups compared with the shams. Furthermore, miR-182-5p knockout (KO) pneumonia group demonstrated decreased levels of intestinal TNF-α and IL-6. Primary murine intestinal epithelial cells were isolated and cultured in our investigation. We exhibited downregulation of miR-182-5p repressed intestinal epithelial cells apoptosis and rescued the cell viability. Meanwhile, miR-182-5p caused elevated cell apoptosis and reduced cell proliferation. Moreover, the surfactant protein D (SP-D) binds with the bacterial pathogens and remove the pathogens and apoptotic bodies, which exhibits important roles in modulating immune responses. It was displayed in our study that SP-D was greatly decreased in pneumonia mice models. SP-D was predicted as a downstream target of miR-182-5p. These data concluded that miR-182-5p promoted intestinal injury in S. aureus pneumonia-induced sepsis via targeting SP-D.