SP-A and SP-D: Dual Functioning Immune Molecules With Antiviral and Immunomodulatory Properties.

Surfactant proteins A (SP-A) and D (SP-D) are soluble innate immune molecules which maintain lung homeostasis through their dual roles as anti-infectious and immunomodulatory agents. SP-A and SP-D bind numerous viruses including influenza A virus, respiratory syncytial virus (RSV) and human immunodeficiency virus (HIV), enhancing their clearance from mucosal points of entry and modulating the inflammatory response. They also have diverse roles in mediating innate and adaptive cell functions and in clearing apoptotic cells, allergens and other noxious particles. Here, we review how the properties of these first line defense molecules modulate inflammatory responses, as well as host-mediated immunopathology in response to viral infections. Since SP-A and SP-D are known to offer protection from viral and other infections, if their levels are decreased in some disease states as they are in severe asthma and chronic obstructive pulmonary disease (COPD), this may confer an increased risk of viral infection and exacerbations of disease. Recombinant molecules of SP-A and SP-D could be useful in both blocking respiratory viral infection while also modulating the immune system to prevent excessive inflammatory responses seen in, for example, RSV or coronavirus disease 2019 (COVID-19). Recombinant SP-A and SP-D could have therapeutic potential in neutralizing both current and future strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus as well as modulating the inflammation-mediated pathology associated with COVID-19. A recombinant fragment of human (rfh)SP-D has recently been shown to neutralize SARS-CoV-2. Further work investigating the potential therapeutic role of SP-A and SP-D in COVID-19 and other infectious and inflammatory diseases is indicated.

A small key unlocks a heavy door: The essential function of the small hydrophobic proteins SP-B and SP-C to trigger adsorption of pulmonary surfactant lamellar bodies.

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air-liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air-liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air-liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air-liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air-liquid interface.

Critical structural and functional roles for the N-terminal insertion sequence in surfactant protein B analogs.

BACKGROUND: Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity. METHODOLOGY/RESULTS: FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B. CONCLUSION: Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.

Pulmonary surfactant surface tension influences alveolar capillary shape and oxygenation.

Alveolar capillaries are located in close proximity to the alveolar epithelium and beneath the surfactant film. We hypothesized that the shape of alveolar capillaries and accompanying oxygenation are influenced by surfactant surface tension in the alveolus. To prove our hypothesis, surfactant surface tension was regulated by conditional expression of surfactant protein (SP)-B in Sftpb(-/-) mice, thereby inhibiting surface tension-lowering properties of surfactant in vivo within 24 hours after depletion of Sftpb. Minimum surface tension of isolated surfactant was increased and oxygen saturation was significantly reduced after 2 days of SP-B deficiency in association with deformation of alveolar capillaries. Intravascularly injected 3.2-mum-diameter microbeads through jugular vein were retained within narrowed pulmonary capillaries after reduction of SP-B. Ultrastructure studies demonstrated that the capillary protrusion typical of the normal alveolar-capillary unit was reduced in size, consistent with altered pulmonary blood flow. Pulmonary hypertension and intrapulmonary shunting are commonly associated with surfactant deficiency and dysfunction in neonates and adults with respiratory distress syndromes. Increased surfactant surface tension caused by reduction in SP-B induced narrowing of alveolar capillaries and oxygen desaturation, demonstrating an important role of surface tension-lowering properties of surfactant in the regulation of pulmonary vascular perfusion.

Pulmonary surfactant protein B in the peripheral circulation and functional impairment in patients with chronic heart failure.

INTRODUCTION AND OBJECTIVES: Surfactant protein B (SP-B) is a marker of damage to the alveolar-capillary barrier that could be useful for monitoring functional impairment in patients with chronic heart failure (HF). METHODS: Dyspnea-limited cardiopulmonary exercise testing was carried out in 43 outpatients with chronic HF (age 51+/-10 years, 77% male, left ventricular ejection fraction [LVEF] 33+/-11%). Peripheral blood serum samples were obtained at rest and during the first minute of peak exercise. The presence and concentration of SP-B in the serum samples were determined by Western blot analysis. RESULTS: At rest, SP-B was detected in 35 (82%) patients compared with only six (23%) healthy volunteers in a control group (n=26, age 51+/-10 years, 77% male). The median circulating SP-B level was higher in HF patients, at 174 [interquartile range, 70-283] vs. 77 [41-152] (P< .001) in the control group. In HF patients, the presence of circulating SP-B was associated with a lower LVEF (31.4+/-9.6% vs. 41.8+/-15%; P=.01). Multivariate analysis showed that the resting SP-B level correlated with a greater VE/VCO2 slope (beta=1.45; P=.02). The peak-exercise SP-B level correlated almost perfectly with the resting level (r=0.980; P< .001), but there was no significant increase with exercise (P=.164). Nor was there a correlation with any other exercise parameter. CONCLUSIONS: In patients with chronic HF, the level of pulmonary surfactant protein B in the peripheral circulation is increased and is correlated with ventilatory inefficiency during exercise, as indicated by the VE/VCO2 slope.

Surfactant proteins B and C are both necessary for alveolar stability at end expiration in premature rabbits with respiratory distress syndrome.

Modified natural surfactant preparations, used for treatment of respiratory distress syndrome in premature infants, contain phospholipids and the hydrophobic surfactant protein (SP)-B and SP-C. Herein, the individual and combined effects of SP-B and SP-C were evaluated in premature rabbit fetuses treated with airway instillation of surfactant and ventilated without positive end-expiratory pressure. Artificial surfactant preparations composed of synthetic phospholipids mixed with either 2% (wt/wt) of porcine SP-B, SP-C, or a synthetic poly-Leu analog of SP-C (SP-C33) did not stabilize the alveoli at the end of expiration, as measured by low lung gas volumes of approximately 5 ml/kg after 30 min of ventilation. However, treatment with phospholipids containing both SP-B and SP-C/SP-C33 approximately doubled lung gas volumes. Doubling the SP-C33 content did not affect lung gas volumes. The tidal volumes were similar in all groups receiving surfactant. This shows that SP-B and SP-C exert different physiological effects, since both proteins are needed to establish alveolar stability at end expiration in this animal model of respiratory distress syndrome, and that an optimal synthetic surfactant probably requires the presence of mimics of both SP-B and SP-C.

Hydrophobic surfactant proteins and their analogues.

Lung surfactant is a complex mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C and SP-D). Its major function in the lung alveolus is to reduce surface tension at the air-water interface in the terminal airways by the formation of a surface-active film enriched in surfactant lipids, hence preventing cellular collapse during respiration. Surfactant therapy using bovine or porcine lung surfactant extracts, which contain only polar lipids and native SP-B and SP-C, has dramatically improved the therapeutic outcomes of preterm infants with respiratory distress syndrome (RDS). One important goal of surfactant researchers is to replace animal-derived therapies with fully synthetic preparations based on SP-B and SP-C, produced by recombinant technology or peptide synthesis, and reconstituted with selected synthetic lipids. Here, we review recent research developments with peptide analogues of SP-B and SP-C, designed using either the known primary sequence and three-dimensional (3D) structure of the native proteins or, alternatively, the known 3D structures of closely homologous proteins. Such SP-B and SP-C mimics offer the possibility of studying the mechanisms of action of the respective native proteins, and may allow the design of optimized surfactant formulations for specific pulmonary diseases (e.g., acute lung injury (ALI) or acute respiratory distress syndrome (ARDS)). These synthetic surfactant preparations may also be a cost-saving therapeutic approach, with better quality control than may be obtained with animal-based treatments.

No association between coding polymorphism within Exon 4 of the human surfactant protein B gene and pulmonary function in healthy men.

The coding polymorphism (rs1130866) within the surfactant protein B gene is known to associate with certain respiratory abnormalities. We investigated, using spirometry and fluorescence-based PCR, whether this variant influenced pulmonary function in healthy, nonsmoking men. We found no association of pulmonary function with genotype at the rs1130866 locus.

[Surfactant protein B expression and its role in the development of human fetal lung epithelial cells].

OBJECTIVE: To investigate the expression patterns of surfactant protein B (SP-B) and its role in the development of human fatal lung epithelial cells. METHODS: Human fetal lung tissues were obtained from 37 fetuses of 10-34 weeks at abortion with parental consent and from two newborn infants who died of non-pulmonary causes. SP-B expression in the lung tissues was examined by immunohistochemistry. RESULTS: SP-B was detected in the cytoplasm of nonciliated columnar epithelial cells of the human fetal lung in as early as the 16th week of gestation. The positive reaction of SP-B was enhanced during canalicular stages and was more intense in the distal than in the proximal airway epithelium. From the 25th week to the prenatal stage, SP-B expression underwent no significant changes in the primitive alveolar stage, but increased remarkably after birth. CONCLUSION: The expression and secretion of SP-B reflects the maturation of the epithelial cells in human fatal lungs, and may closely associate with the survival ability of the newborn infants.

[Pseudomonas aeruginosa and Surfactant-associated Proteins A and D]. / Pseudomonas aeruginosa et surfactant rôle de SP-A et SP-D.

Surfactant-associated proteins A and D (SP-A and SP-D) are two pulmonary collectins that bind to bacterial, fungal and viral pathogens and have multiples classes of receptors on pneumocyte and macrophage membrane. They are chemoattractant for phagocytes, enhance uptake and killing of bacteria by macrophages and neutrophils. These molecules also act as activation ligand on macrophages and neutrophils to enhance phagocytosis, resulting in an increased bacterial clearance. Depending on activation of cells by stimuli, SP-A and SP-D modulate production of antimicrobial free radicals by phagocytes and secretion of cytokines. In vivo, SP-A deficient mice infected with Pseudomonas aeruginosa (P. aeruginosa) have decreased bacterial clearance and exacerbated inflammatory response in the lungs. Serious alterations in macrophages and increased production of reactive oxygen species were found in non-infected SP-D deficient mice. Patients with cystic fibrosis are frequently colonized by P. aeruginosa. Decreased levels of SP-A and SP-D have been measured in bronchoalveolar lavage fluid of these patients, as well as patients with acute pneumonia but no chronic lung disease. P. aeruginosa secretes various proteases, among them, elastase and protease IV have been found to degrade SP-A and SP-D and abrogate their immune function. However, further investigations are necessary to examine whether these deficiencies facilitate P. aeruginosa infections or stand as consequences.

Surfactant protein B 1580 polymorphism is associated with susceptibility to chronic obstructive pulmonary disease in Chinese Han population.

Whether surfactant protein B (SP-B)-18A/C and 1580C/T polymorphism were associated with susceptibility to chronic obstructive pulmonary disease (COPD) in Chinese Han population was investigated. After genomic DNA was isolated from blood of COPD smokers and control smokers, the genotypes of SP-B-18A/C and SP-B1580C/T polymorphism loci were determined by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) respectively. The results showed that there was significant difference in genotypes distribution frequency of SP-B1580C/T polymorphism locus between COPD smokers and control smokers. C-->T mutation rate (including TT homozygote and CT heterozygote) in COPD smokers was higher than in control smokers (57.9% vs 41.7%, chi2 = 4.93, P<0.05), whereas there was no significant difference in genotypes distribution frequency of SP-B1580-18A/C locus between COPD smokers and control smokers. The allele frequency (29.1%) of SP-B1580-18A/C locus is lower than T allele (70.9%) in Chinese Han Population, and the distribution was different from that in Mexican, in which, the A and T allele frequencies were 85% and 15% respectively. It was concluded that SP-B1580 T allele was probably associated with increased susceptibility to COPD in Chinese Han population; The polymorphism of SP-B-18A/C locus maybe varied with race.

[Surfactant-associated proteins B and C: molecular biology and physiologic properties]. / Surfactantproteine SP-B und SP-C: Molekularbiologie und Physiologie.

Treatment of neonatal RDS in premature infants with intratracheal administration of natural surfactant has become gold standard therapy. Natural surfactant preparations mainly contain, apart from phospholipids, the surfactant associated proteins B and C (SP-B and SP-C). Both proteins are synthesized mainly in alveolar type-II cells and Clara-cells, SP-B, also in the gastrointestinal tract and the auditive tube. SP-B is encoded on chromosome 2 over a region with 11 exons, whereas the SP-C gene is localized on chromosome 8 in a region containing 6 exons. Transcription of both SP-B and SP-C is induced by TTF-1. Furthermore SP-1 and SP-3 are known as transcription factors for SP-B. The main function of SP-B and SP-C is to maintain physiologic surface properties enabeling adequate lung mechanics. A complete SP-B deficiency following homozygous mutations in the SP-B gene (e. g. 121-ins 2-mutation) therefore leads to severe respiratory failure postnatally, due to the lack of functional surfactant. On the other hand complete deficiency of SP-C causes chronic interstitial pneumonitis as well in infants as in adults depending on disease-modifiers yet unknown. Besides the surface tension lowering property, SP-B reveals immunological functions regarding its interaction with different proinflammatory cellular systems as well as other inflammatory mediators, e. g. following hyperoxia. For SP-C first studies have described modulation of inflammatory reactions in macrophages, suggesting similar immune-modulatory effects. Whereas basic effects on lung mechanisms of both lipophilic surfactant proteins seem to be well understood, their immunologic local pulmonary functions and effects on surfactant metabolism require further investigations.

Surfactant protein B: structure and function.

Pulmonary surfactant is a mixture of phospholipids, neutral lipids, and associated proteins. A specific phospholipid, dipalmitoylphosphatidylcholine, is predominantly responsible for the modulation of surface tension at the alveolar air-liquid interface, but other surfactant lipid and protein components play important roles in surfactant function and metabolism. This review will focus on just one of the apoproteins, surfactant protein B, with a description of protein structure and the actions of surfactant protein B on surfactant lipid membranes.

Association between surfactant protein B + 1580 polymorphism and the risk of respiratory failure in adults with community-acquired pneumonia.

OBJECTIVE: Pulmonary surfactant protein (SP)-B plays a vital role in the formation and function of surfactant in the lung. A genetic polymorphism (SP-B + 1580) is postulated to result in diminished activity of SP-B. The objective was to determine whether the SP-B + 1580 CC genotype is associated with an increased risk of respiratory failure and ARDS in adults with community-acquired pneumonia. DESIGN: Prospective cohort of adults diagnosed with community-acquired pneumonia. SETTING: Hospital system. PATIENTS: We enrolled 402 adults > or = 18 yrs of age with community-acquired pneumonia; 158 were white, 243 were African American, and one was Asian. INTERVENTIONS: Genotypic analysis was performed on DNA isolated from whole blood using polymerase chain reaction amplification and DdeI restriction enzyme digestion. MEASUREMENTS AND MAIN RESULTS: We recorded the requirement for mechanical ventilation, the presence of acute respiratory distress syndrome (ARDS) or septic shock, and mortality. Sixty-three patients required mechanical ventilation, 12 patients developed ARDS, and 35 patients developed septic shock. Genotypic frequencies at the SP-B + 1580 site were T/T 183 of 402 (0.45), T/C 160 of 402 (0.40), and C/C 59 of 402 (0.15). Of the 59 patients who were C/C at the SP-B + 1580 site, 21 (0.356) required mechanical ventilation, compared with 26 of 160 patients (0.163) who were T/C and 16 of 183 (0.087) patients who were T/T (p < .001). ARDS developed in five of 59 (0.085) patients with the C/C genotype, compared with six of 160 (.038) patients with T/C and one of 183 patients with T/T (0.005, p < .009). Septic shock occurred in 12 of 59 (0.203) patients with the C/C genotype, compared with 13 of 160 (0.081) patients with T/C and ten of 183 (0.055) patients with T/T (p < .001). Mortality rate was not different between the three genotypes. CONCLUSION: Carriage of the C allele at the SP-B + 1580 site is associated with ARDS, septic shock, and the need for mechanical ventilation in adults with community-acquired pneumonia.

Nitric oxide synthase-2 down-regulates surfactant protein-B expression and enhances endotoxin-induced lung injury in mice.

Acute respiratory distress syndrome (ARDS) is a life-threatening ailment characterized by severe lung injury involving inflammatory cell recruitment to the lung, cytokine production, surfactant dysfunction, and up-regulation of nitric oxide synthase 2 (NOS2) resulting in nitric oxide (NO) production. We hypothesized that NO production from NOS2 expressed in lung parenchymal cells in a murine model of ARDS would correlate with abnormal surfactant function and reduced surfactant protein-B (SP-B) expression. Pulmonary responses to nebulized endotoxin (lipopolysaccharide, LPS) were evaluated in wild-type (WT) mice, NOS2 null (-/-) mice, and NOS2-chimeric animals derived from bone marrow transplantation. NOS2-/- animals exhibited significantly less physiologic lung dysfunction and loss of SP-B expression than did WT animals. However, lung neutrophil recruitment and bronchoalveolar lavage cytokine levels did not significantly differ between NOS2-/- and WT animals. Chimeric animals for NOS2 exhibited the phenotype of the recipient and therefore demonstrated that parenchymal production of NOS2 is critical for the development of LPS-induced lung injury. Furthermore, administration of NO donors, independent of cytokine stimulation, decreased SP-B promoter activity and mRNA expression in mouse lung epithelial cells. This study demonstrates that expression of NOS2 in lung epithelial cells is critical for the development of lung injury and mediates surfactant dysfunction independent of NOS2 inflammatory cell expression and cytokine production.

Stat-3 is required for pulmonary homeostasis during hyperoxia.

Acute lung injury syndromes remain common causes of morbidity and mortality in adults and children. Cellular and physiologic mechanisms maintaining pulmonary homeostasis during lung injury remain poorly understood. In the present study, the Stat-3 gene was selectively deleted in respiratory epithelial cells by conditional expression of Cre-recombinase under control of the surfactant protein C gene promoter. Cell-selective deletion of Stat-3 in respiratory epithelial cells did not alter prenatal lung morphogenesis or postnatal lung function. However, exposure of adult Stat-3-deleted mice to 95% oxygen caused a more rapidly progressive lung injury associated with alveolar capillary leak and acute respiratory distress. Epithelial cell injury and inflammatory responses were increased in the Stat-3-deleted mice. Surfactant proteins and lipids were decreased or absent in alveolar lavage material. Intratracheal treatment with exogenous surfactant protein B improved survival and lung histology in Stat-3-deleted mice during hyperoxia. Expression of Stat-3 in respiratory epithelial cells is not required for lung formation, but plays a critical role in maintenance of surfactant homeostasis and lung function during oxygen injury.

Surfactant protein B 1580 polymorphism is associated with susceptibility to chronic obstructive pulmonary disease in Chinese Han population / 华中科技大学学报（医学）（英德文版）

Whether surfactant protein B (SP-B)-18A/C and 1580C/T polymorphism were associated with susceptibility to chronic obstructive pulmonary disease (COPD) in Chinese Han population was investigated. After genomic DNA was isolated from blood of COPD smokers and control smokers, the genotypes of SP-B-18A/C and SP-B1580C/T polymorphism loci were determined by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) respectively. The results showed that there was significant difference in genotypes distribution frequency of SP-B1580C/T polymorphism locus between COPD smokers and control smokers. C-->T mutation rate (including TT homozygote and CT heterozygote) in COPD smokers was higher than in control smokers (57.9% vs 41.7%, chi2 = 4.93, P<0.05), whereas there was no significant difference in genotypes distribution frequency of SP-B1580-18A/C locus between COPD smokers and control smokers. The allele frequency (29.1%) of SP-B1580-18A/C locus is lower than T allele (70.9%) in Chinese Han Population, and the distribution was different from that in Mexican, in which, the A and T allele frequencies were 85% and 15% respectively. It was concluded that SP-B1580 T allele was probably associated with increased susceptibility to COPD in Chinese Han population; The polymorphism of SP-B-18A/C locus maybe varied with race.