Surfactant Protein B Reduces Respiratory Viral Infectivity in Human Lungs

Purpose of Study: COVID pneumonia caused by SARS-CoV-2 can result in a depletion of surfactant & lung injury, which resembles neonatal respiratory distress syndrome. Exogenous surfactant has shown promise as a therapeutic option in intubated hospitalized patients. Our preliminary data in human lung organoids (LOs) with a deficiency of surfactant protein B (SP-B) showed an increased viral load compared to normal LOs. Single cell RNA sequencing (scRNAseq) revealed that SP-B-deficient cells showed increased viral entry genes (ACE2 receptor) & dysregulated inflammatory markers emanating from the lung cells themselves. Our objective was to determine: (1) cell-specific transcriptional differences between normal & SP-B deficient human lung cells after infection with SARS-CoV-2 and (2) a therapeutic role of SP-B protein & surfactant in COVID-19 pneumonia. Methods Used: We used normal and SP-B mutant (homozygous, frameshift, loss of function mutation p.Pro133GlnfsTer95, previously known as 121ins2) human induced pluripotent stem cells (hiPSC) and differentiated them into 3D proximal lung organoids. The organoids were infected with the delta variant of SARS-CoV-2 for 24 hours at an MOI of 1. Infected and uninfected organoids were fixed in trizol in triplicate and underwent processing for bulk RNA sequencing. We tested for differentially expressed genes using the program DEseq. We also plated normal iPSC derived lung organoids as a monolayer and pre-treated them with 1mg/ml of Poractant alfa or 5 uM of recombinant SP-B protein. The delta strain of SARS-CoV-2 was added to the 96 wells at an MOI of 0.1 for one hour with shaking, then an overlay with DMEM/CMC/FBS was added and left on for 23 hours. The plate was fixed and stained for nucleocapsid (NC) protein. Summary of Results: Bioinformatic analysis of the bulk RNA sequencing data showed an increase in the multiple cytokines and chemokines in the SP-B mutant LOs compared to control. We also saw differential gene expression patterns in the SP-B mutant LOs including a reduction in SFTPC, FOXA2, and NKX2-1 and an increase in IL1A, VEGFA, PPARG and SMAD3. In the exogenous surfactant experiments, there was a decrease in total expression of viral NC in the Poractant alfa & rSP-B-treated cells compared to SARS-CoV-2 infection alone (p<0.001). Conclusion(s): Surfactant modulates the viral load of SARS-CoV-2 infection in the human lung. Deficiency in SP-B results in the dysregulation of the lung epithelial inflammatory signaling pathways resulting in worsening infections.

Elucidating the enhanced binding affinity of a double mutant SP-D with trimannose on the influenza A virus using molecular dynamics.

Surfactant protein D (SP-D) is an essential component of the human pulmonary surfactant system, which is crucial in the innate immune response against glycan-containing pathogens, including Influenza A viruses (IAV) and SARS-CoV-2. Previous studies have shown that wild-type (WT) SP-D can bind IAV but exhibits poor antiviral activities. However, a double mutant (DM) SP-D consisting of two point mutations (Asp325Ala and Arg343Val) inhibits IAV more potently. Presently, the structural mechanisms behind the point mutations' effects on SP-D's binding affinity with viral surface glycans are not fully understood. Here we use microsecond-scale, full-atomistic molecular dynamics (MD) simulations to understand the molecular mechanism of mutation-induced SP-D's higher antiviral activity. We find that the Asp325Ala mutation promotes a trimannose conformational change to a more stable state. Arg343Val increases the binding with trimannose by increasing the hydrogen bonding interaction with Glu333. Free energy perturbation (FEP) binding free energy calculations indicate that the Arg343Val mutation contributes more to the increase of SP-D's binding affinity with trimannose than Asp325Ala. This study provides a molecular-level exploration of how the two mutations increase SP-D binding affinity with trimannose, which is vital for further developing preventative strategies for related diseases.

Type 2 alveolar epithelial cell-derived circulating extracellular vesicle-encapsulated surfactant protein C as a mediator of cardiac inflammation in COVID-19.

Among the countless endeavours made at elucidating the pathogenesis of COVID-19, those aimed at the histopathological alterations of type 2 alveolar epithelial cells (AT2) are of outstanding relevance to the field of lung physiology, as they are the building blocks of the pulmonary alveoli. A merit of high regenerative and proliferative capacity, exocytotic activity resulting in the release of extracellular vesicles (EVs) is particularly high in AT2 cells, especially in those infected with SARS-CoV-2. These AT2 cell-derived EVs, containing the genetic material of the virus, might enter the bloodstream and make their way into the cardiovascular system, where they may infect cardiomyocytes and bring about a series of events leading to heart failure. As surfactant protein C, a marker of AT2 cell activity and a constituent of the lung surfactant complex, occurs abundantly inside the AT2-derived EVs released during the inflammatory stage of COVID-19, it could potentially be used as a biomarker for predicting impending heart failure in those patients with a history of cardiovascular disease.

Plasma and Airway Levels of Surfactant Protein D Degradation Reflect Progression of ARDS in Ventilated Patients with SARS-CoV-2

Background: Patients with COVID-19 present severe respiratory symptoms progressing to acute respiratory distress syndrome (ARDS). Upon infection, SARS-CoV-2 destroys cells expressing the ACE2 receptor including alveolar type II cells (AT2). These cells are found in the alveolar-capillary barrier which normally secrete pulmonary surfactant, a complex of lipid and surfactant proteins (SPA, SP-B, SP-C, SP-D). Exogenous surfactant therapy (mainly composed of phospholipids, SP-B, and SP-C) has been successful in treating neonatal respiratory distress syndrome (nRDS) caused by surfactant deficiency in preterm babies.Plasma SP-D has been proposed as a marker of lung injury in COVID-19 but so far, no reports have evaluated sequential SP-D levels in both airway and plasma. As part of a clinical trial repurposing surfactant therapy to treat adult ventilated COVID-19 patients, we hypothesized that plasma SP-D levels may reflect decreased lung integrity and that SP-D degradation in plasma and airway samples from COVID-19 patients may reflect disease progression and severity. Methods: Enzyme-linked immunosorbent assay (ELISA) was used to quantify SP-D concentration in patient plasma and tracheal aspirate samples. Western Blotting was used to identify any protein degradation. Sequential daily plasma and airway samples were analysed. Results: SP-D concentration in serum was 10-20 times higher in patients ventilated for COVID-19 than in healthy volunteers. Additionally, the concentration of SP-D in plasma has shown to be 10-100-fold higher than in tracheal aspirates. Furthermore, degraded fragments of SP-D were detected at a higher ratio than intact SP-D in plasma of ventilated patients. This ratio decreased with administration of surfactant therapy (containing phospholipids and SP-B and SP-C but no SPA or SP-D). Conclusions: Increased serum SP-D and decreased tracheal aspirate SP-D from ventilated COVID-19 patients suggested leakage of pulmonary surfactant into the bloodstream caused by damage to the alveolar-capillary barrier in diseased lungs. The ratio of degraded vs. intact SP-D found in the plasma was compared before and after therapeutic surfactant administration. The results indicated that levels of SP-D in plasma and tracheal aspirates together with the ratio of degraded and intact SP-D in the plasma may be useful indicators of the severity of COVID-19 lung disease progression.

Highly Expandable Human Alveolar Organoids to Study Respiratory Diseases

Organoids are emerging to be an excellent tool for studying human development and disease. The COVID-19 pandemic has highlighted the importance of physiologically relevant alveolar infection models that include both alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. To address the need for an alveolar organoid culture system for respiratory research, we developed the PneumaCult™ Alveolar Organoid Expansion and Differentiation Media for the highly efficient expansion of isolated primary human AT2 cells and subsequent differentiation into AT1 cells. Alveolar organoids were established from a panel of various donors (n=5) by culturing purified human AT2 cells in Corning® Matrigel® domes with serum-free PneumaCult™ Alveolar Organoid Expansion Medium. Typically by day 10-14 the organoids are fully established and display a spherical morphology. Alveolar organoids can then be either expanded long-term by passaging cultures as single cells in Expansion Medium or differentiated into AT1 cells using the PneumaCult™ Alveolar Organoid Differentiation Medium. Organoids in PneumaCult™ Alveolar Organoid Expansion Medium contain self-renewing AT2 cells marked by the expression of HT2-280 in 89.9 +/- 14.5 (mean +/- SD;n=5 donors) of cells and the presence of Pro-SPC, demonstrate a great expansion potential of > 10,000-fold with more than 13 population doublings within 10 passages (n=5 donors). Alveolar organoids differentiated for 10 days in the PneumaCult™ Alveolar Organoid Differentiation Medium downregulate AT2 markers HT2-280 and Pro-SPC and start expressing AT1 markers HT1-56 in 93.8 +/- 7.2 (mean +/- SD;n=5 donors) of cells and are positive for RAGE and GPRC5a. Furthermore, we assessed the expression of SARS-CoV-2 entry receptor ACE2, which is present in both undifferentiated and differentiated alveolar organoids.To investigate the use of these alveolar organoids for infectious disease modeling, AT2-containing alveolar organoids were transduced with a GFP-labelled Respiratory Syncytial Virus (RSV). Alveolar organoids were susceptible to viral infection and replication was confirmed by fluorescence microscopy and quantitative PCR. In summary, the PneumaCult™ Alveolar Organoid Expansion and Differentiation Media are highly efficient and reproducible tools for the feeder-free expansion of AT2 cells and robust differentiation into AT1 cells, which can be used for infectious disease modeling.