Could SP-A and SP-D Serum Levels Predict COVID-19 Severity?

Given the various clinical manifestations that characterize Coronavirus Disease 2019 (COVID-19), the scientific community is constantly searching for biomarkers with prognostic value. Surfactant proteins A (SP-A) and D (SP-D) are collectins that play a crucial role in ensuring proper alveolar function and an alteration of their serum levels was reported in several pulmonary diseases characterized by Acute Respiratory Distress Syndrome (ARDS) and pulmonary fibrosis. Considering that such clinical manifestations can also occur during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, we wondered if these collectins could act as prognostic markers. In this regard, serum levels of SP-A and SP-D were measured by enzyme immunoassay in patients with SARS-CoV-2 infection (n = 51) at admission (T0) and after seven days (T1) and compared with healthy donors (n = 11). SP-D increased in COVID-19 patients compared to healthy controls during the early phases of infection, while a significant reduction was observed at T1. Stratifying SARS-CoV-2 patients according to disease severity, increased serum SP-D levels were observed in severe compared to mild patients. In light of these results, SP-D, but not SP-A, seems to be an eligible marker of COVID-19 pneumonia, and the early detection of SP-D serum levels could be crucial for preventive clinical management.

Cooperation of immune regulators Tollip and surfactant protein A inhibits influenza A virus infection in mice.

BACKGROUND: Influenza A virus (IAV) infection is a significant risk factor for respiratory diseases, but the host defense mechanisms against IAV remain to be defined. Immune regulators such as surfactant protein A (SP-A) and Toll-interacting protein (Tollip) have been shown to be involved in IAV infection, but whether SP-A and Tollip cooperate in more effective host defense against IAV infection has not been investigated. METHODS: Wild-type (WT), Tollip knockout (KO), SP-A KO, and Tollip/SP-A double KO (dKO) mice were infected with IAV for four days. Lung macrophages were isolated for bulk RNA sequencing. Precision-cut lung slices (PCLS) from WT and dKO mice were pre-treated with SP-A and then infected with IAV for 48 h. RESULTS: Viral load was significantly increased in bronchoalveolar lavage (BAL) fluid of dKO mice compared to all other strains of mice. dKO mice had significantly less recruitment of neutrophils into the lung compared to Tollip KO mice. SP-A treatment of PCLS enhanced expression of TNF and reduced viral load in dKO mouse lung tissue. Pathway analysis of bulk RNA sequencing data suggests that macrophages from IAV-infected dKO mice reduced expression of genes involved in neutrophil recruitment, IL-17 signaling, and Toll-like receptor signaling. CONCLUSIONS: Our data suggests that both Tollip and SP-A are essential for the lung to exert more effective innate defense against IAV infection.

Human surfactant protein A inhibits SARS-CoV-2 infectivity and alleviates lung injury in a mouse infection model.

Introduction: SARS coronavirus 2 (SARS-CoV-2) infects human angiotensin-converting enzyme 2 (hACE2)-expressing lung epithelial cells through its spike (S) protein. The S protein is highly glycosylated and could be a target for lectins. Surfactant protein A (SP-A) is a collagen-containing C-type lectin, expressed by mucosal epithelial cells and mediates its antiviral activities by binding to viral glycoproteins. Objective: This study examined the mechanistic role of human SP-A in SARS-CoV-2 infectivity and lung injury in vitro and in vivo. Results: Human SP-A can bind both SARS-CoV-2 S protein and hACE2 in a dose-dependent manner (p<0.01). Pre-incubation of SARS-CoV-2 (Delta) with human SP-A inhibited virus binding and entry and reduced viral load in human lung epithelial cells, evidenced by the dose-dependent decrease in viral RNA, nucleocapsid protein (NP), and titer (p<0.01). We observed significant weight loss, increased viral burden, and mortality rate, and more severe lung injury in SARS-CoV-2 infected hACE2/SP-A KO mice (SP-A deficient mice with hACE2 transgene) compared to infected hACE2/mSP-A (K18) and hACE2/hSP-A1 (6A2) mice (with both hACE2 and human SP-A1 transgenes) 6 Days Post-infection (DPI). Furthermore, increased SP-A level was observed in the saliva of COVID-19 patients compared to healthy controls (p<0.05), but severe COVID-19 patients had relatively lower SP-A levels than moderate COVID-19 patients (p<0.05). Discussion: Collectively, human SP-A attenuates SARS-CoV-2-induced acute lung injury (ALI) by directly binding to the S protein and hACE2, and inhibiting its infectivity; and SP-A level in the saliva of COVID-19 patients might serve as a biomarker for COVID-19 severity.

High risk of lung cancer in surfactant-related gene variant carriers.

BACKGROUND: Several rare surfactant-related gene (SRG) variants associated with interstitial lung disease are suspected to be associated with lung cancer, but data are missing. We aimed to study the epidemiology and phenotype of lung cancer in an international cohort of SRG variant carriers. METHODS: We conducted a cross-sectional study of all adults with SRG variants in the OrphaLung network and compared lung cancer risk with telomere-related gene (TRG) variant carriers. RESULTS: We identified 99 SRG adult variant carriers (SFTPA1 (n=18), SFTPA2 (n=31), SFTPC (n=24), ABCA3 (n=14) and NKX2-1 (n=12)), including 20 (20.2%) with lung cancer (SFTPA1 (n=7), SFTPA2 (n=8), SFTPC (n=3), NKX2-1 (n=2) and ABCA3 (n=0)). Among SRG variant carriers, the odds of lung cancer was associated with age (OR 1.04, 95% CI 1.01-1.08), smoking (OR 20.7, 95% CI 6.60-76.2) and SFTPA1/SFTPA2 variants (OR 3.97, 95% CI 1.39-13.2). Adenocarcinoma was the only histological type reported, with programmed death ligand-1 expression ≥1% in tumour cells in three samples. Cancer staging was localised (I/II) in eight (40%) individuals, locally advanced (III) in two (10%) and metastatic (IV) in 10 (50%). We found no somatic variant eligible for targeted therapy. Seven cancers were surgically removed, 10 received systemic therapy, and three received the best supportive care according to their stage and performance status. The median overall survival was 24 months, with stage I/II cancers showing better survival. We identified 233 TRG variant carriers. The comparative risk (subdistribution hazard ratio) for lung cancer in SRG patients versus TRG patients was 18.1 (95% CI 7.1-44.7). CONCLUSIONS: The high risk of lung cancer among SRG variant carriers suggests specific screening and diagnostic and therapeutic challenges. The benefit of regular computed tomography scan follow-up should be evaluated.

Surfactant protein A promotes western diet-induced hepatic steatosis and fibrosis in mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) remains the most common cause of liver disease in the United States due to the increased incidence of metabolic dysfunction and obesity. Surfactant protein A (SPA) regulates macrophage function, strongly binds to lipids, and is implicated in renal and idiopathic pulmonary fibrosis (IPF). However, the role of SPA in lipid accumulation, inflammation, and hepatic fibrosis that characterize MASLD remains unknown. SPA deficient (SPA-/-) and age-matched wild-type (WT) control mice were fed a Western diet for 8 weeks to induce MASLD. Blood and liver samples were collected and used to analyze pathological features associated with MASLD. SPA expression was significantly upregulated in livers of mice with MASLD. SPA deficiency attenuated lipid accumulation along with downregulation of genes involved in fatty acid uptake and reduction of hepatic inflammation as evidenced by the diminished macrophage activation, decreased monocyte infiltration, and reduced production of inflammatory cytokines. Moreover, SPA-/- inhibited stellate cell activation, collagen deposit, and liver fibrosis. These results highlight the novel role of SPA in promoting fatty acid uptake into hepatocytes, causing excessive lipid accumulation, inflammation, and fibrosis implicated in the pathogenesis of MASLD.

Decreased LPS-induced lung injury in pigs treated with a lung surfactant protein A-derived nonapeptide that inhibits peroxiredoxin 6 activity and subsequent NOX1,2 activation.

This study addressed the efficacy of a liposome-encapsulated nine amino acid peptide [peroxiredoxin 6 PLA2 inhibitory peptide-2 (PIP-2)] for the prevention or treatment of acute lung injury (ALI) +/- sepsis. PIP-2 inhibits the PLA2 activity of peroxiredoxin 6 (Prdx6), thereby preventing rac release and activation of NADPH oxidases (NOXes), types 1 and 2. Female Yorkshire pigs were infused intravenously with lipopolysaccharide (LPS) + liposomes (untreated) or LPS + PIP-2 encapsulated in liposomes (treated). Pigs were mechanically ventilated and continuously monitored; they were euthanized after 8 h or earlier if preestablished humane endpoints were reached. Control pigs (mechanical ventilation, no LPS) were essentially unchanged over the 8 h study. LPS administration resulted in systemic inflammation with manifestations of clinical sepsis-like syndrome, decreased lung compliance, and a marked decrease in the arterial Po2 with vascular instability leading to early euthanasia of 50% of untreated animals. PIP-2 treatment significantly reduced the requirement for supportive vasopressors and the manifestations of lung injury so that only 25% of animals required early euthanasia. Bronchoalveolar lavage fluid from PIP-2-treated versus untreated pigs showed markedly lower levels of total protein, cytokines (TNF-α, IL-6, IL-1ß), and myeloperoxidase. Thus, the porcine LPS-induced sepsis-like model was associated with moderate to severe lung pathophysiology compatible with ALI, whereas treatment with PIP-2 markedly decreased lung injury, cardiovascular instability, and early euthanasia. These results indicate that inhibition of reactive oxygen species (ROS) production via NOX1/2 has a beneficial effect in treating pigs with LPS-induced ALI plus or minus a sepsis-like syndrome, suggesting a potential role for PIP-2 in the treatment of ALI and/or sepsis in humans.NEW & NOTEWORTHY Currently available treatments that can alter lung inflammation have failed to significantly alter mortality of acute lung injury (ALI). Peroxiredoxin 6 PLA2 inhibitory peptide-2 (PIP-2) targets the liberation of reactive O2 species (ROS) that is associated with adverse cell signaling events, thereby decreasing the tissue oxidative injury that occurs early in the ALI syndrome. We propose that treatment with PIP-2 may be effective in preventing progression of early disease into its later stages with irreversible lung damage and relatively high mortality.

Multivalent, calcium-independent binding of surfactant protein A and D to sulfated glycosaminoglycans of the alveolar epithelial glycocalyx.

Lung surfactant collectins, surfactant protein A (SP-A) and D (SP-D), are oligomeric C-type lectins involved in lung immunity. Through their carbohydrate recognition domain, they recognize carbohydrates at pathogen surfaces and initiate lung innate immune response. Here, we propose that they may also be able to bind to other carbohydrates present in typical cell surfaces, such as the alveolar epithelial glycocalyx. To test this hypothesis, we analyzed and quantified the binding affinity of SP-A and SP-D to different sugars and glycosaminoglycans (GAGs) by microscale thermophoresis (MST). In addition, by changing the calcium concentration, we aimed to characterize any consequences on the binding behavior. Our results show that both oligomeric proteins bind with high affinity (in nanomolar range) to GAGs, such as hyaluronan (HA), heparan sulfate (HS) and chondroitin sulfate (CS). Binding to HS and CS was calcium-independent, as it was not affected by changing calcium concentration in the buffer. Quantification of GAGs in bronchoalveolar lavage (BAL) fluid from animals deficient in either SP-A or SP-D showed changes in GAG composition, and electron micrographs showed differences in alveolar glycocalyx ultrastructure in vivo. Taken together, SP-A and SP-D bind to model sulfated glycosaminoglycans of the alveolar epithelial glycocalyx in a multivalent and calcium-independent way. These findings provide a potential mechanism for SP-A and SP-D as an integral part of the alveolar epithelial glycocalyx binding and interconnecting free GAGs, proteoglycans, and other glycans in glycoproteins, which may influence glycocalyx composition and structure.NEW & NOTEWORTHY SP-A and SP-D function has been related to innate immunity of the lung based on their binding to sugar residues at pathogen surfaces. However, their function in the healthy alveolus was considered as limited to interaction with surfactant lipids. Here, we demonstrated that these proteins bind to glycosaminoglycans present at typical cell surfaces like the alveolar epithelial glycocalyx. We propose a model where these proteins play an important role in interconnecting alveolar epithelial glycocalyx components.

Forensic application of three interstitial pneumonia markers: search for new pneumonia markers in dead bodies.

In forensic cases, detailed identification of pneumonia is important. Our objective was to statistically determine the applicability of three interstitial lung disease (ILD) markers for forensic diagnosis using serum collected from dead bodies with various postmortem intervals (PMIs). We retrospectively analyzed the levels of postmortem serum Krebs von den Lungen-6 (KL-6) and pulmonary surfactant-associated proteins A and D (SP-A and SP-D) using 221 samples obtained during forensic autopsy at our facility from 2019 to 2023. We evaluated the diagnostic efficacy of ILD markers for various pneumonias against the pathological diagnosis, and examined the assessment of the severity of ILD. When comparing the ILD group with bacterial pneumonia (BP) versus the control group, there was a significant increase in KL-6 in the ILD group. When comparing the severe ILD (SILD) group with the mild ILD (MILD) group, there was a significant increase in KL-6 and SP-D in the SILD group. The optimal cutoff values for differentiating SILD were 607.0 U/mL for KL-6, 55.5 ng/mL for SP-A, and 160.0 ng/mL for SP-D, and the sensitivity/specificity (%) of KL-6, SP-A, and SP-D for SILD were 84.1/95.2, 55.6/85.7, and 66.7/74.6, respectively. This is the first study to examine KL-6 in postmortem serum in forensic medicine. By analyzing dead bodies with various PMIs, our results confirmed statistically that postmortem serum KL-6 specifically detects ILD, postmortem serum SP-A has high sensitivity to lung injury, and postmortem serum SP-D is potentially useful in assessing the severity of ILD.

Disruption of the SP-A/SP-R210L (MYO18Aα) pathway prolongs gestation and reduces fetal survival during lipopolysaccharide-induced parturition in late gestation.

Prolonged labor can lead to infection, fetal distress, asphyxia, and life-threatening harm to both the mother and the baby. Surfactant protein A (SP-A) was shown to contribute to the maintenance of pregnancy and timing of term labor. SP-A modulates the stoichiometric expression of the SP-R210L and SP-R210S isoforms of the SP-R210 receptor on alveolar macrophages (AMs). Lack of SP-R210L dysregulates macrophage inflammatory responses. We asked whether SP-A alters normal and inflammation-induced parturition through SP-R210 using SP-A- and SP-R210L-deficient mice. Labor and delivery of time-pregnant mice were monitored in real time using a time-lapse infrared camera. Intrauterine injection with either vehicle or Escherichia coli lipopolysaccharide (LPS) on embryonic (E) day 18.5 post coitus was used to assess the effect of gene disruption in chorioamnionitis-induced labor. We report that either lack of SP-A or disruption of SP-R210L delays parturition by 0.40 and 0.55 days compared with controls, respectively. LPS induced labor at 0.60, 1.01, 0.40, 1.00, and 1.31 days earlier than PBS controls in wild type (WT), SP-A-deficient, littermate controls, heterozygous, and homozygous SP-R210L-deficient mice, respectively. Lack of SP-A reduced litter size in PBS-treated mice, whereas the total number of pups delivered was similar in all LPS-treated mice. The number of live pups, however, was significantly reduced by 50%-70% in SP-A and SP-R210L-deficient mice compared with controls. Differences in gestational length were not associated with intrauterine growth restriction. The present findings support the novel concept that the SP-A/SP-R210 pathway modulates timely labor and delivery and supports fetal lung barrier integrity during fetal-to-neonatal transition in term pregnancy.NEW & NOTEWORTHY To our knowledge, this study is the first to report that SP-A prevents delay of labor and inflammation-induced stillbirth through the receptor SP-R210L.

Immunoregulatory function of SP-A.

Surfactant protein A (SP-A), a natural immune molecule, plays an important role in lung health. SP-A recognizes and binds microbial surface glycogroups through the C-type carbohydrate recognition domain, and then binds corresponding cell surface receptors (such as C1qRp, CRT-CD91 complex, CD14, SP-R210, Toll-like receptor, SIRP-α, CR3, etc.) through collagen-like region, and subsequently mediates biological effects. SP-A regulates lung innate immunity by promoting surfactant absorption by alveolar type II epithelial cells and phagocytosis of pathogenic microorganisms by alveolar macrophages. SP-A also regulates lung adaptive immunity by inhibiting DC maturation, and T cell proliferation and differentiation. This article reviews latest relationships between SP-A and adaptive and intrinsic immunity.

Association of surfactant protein A2 with acute respiratory failure in children.

BACKGROUND: Interactions among single nucleotide polymorphisms (SNPs) of surfactant protein (SP) are associated with acute respiratory failure (ARF) and its short-term outcome, pulmonary dysfunction at discharge (PDAD) in children. However, genetic association studies using individual SNPs have not been conducted before. We hypothesize that SP genetic variants are associated with pediatric ARF and its short-term complications by themselves. METHODS: We used available genotype and clinical data in the Floros biobank consisting of 248 children aged ≤24 months with ARF; 86 developed PDAD. A logistic regression analysis was performed for each of the 14 selected SNPs, SP-A1 and SP-A2 genotypes. A p-value less than the Bonferroni correction threshold was considered significant. A likelihood ratio test was done to compare two models (one with demographic data and another with genetic variants). RESULTS: Before Bonferroni correction, female sex is associated with a decreased risk of ARF. Black race and the rs721917 of the SFTPD are associated with increased risk of ARF. After Bonferroni correction, the 1A0 1A1 genotype of SFTPA2 was associated with decreased risk of ARF. The likelihood ratio test showed that the model of the genotype information with demographic data was a better fit to predict ARF risk. None of the SP SNPs and SP-A1, SP-A2 genotypes were associated with PDAD. CONCLUSION: Our results indicate that SNPs and genotypes of SPs involved in innate immunity and host defense play an important role in ARF and, in the future, may be used as biomarkers.

Usefulness and performance evaluation of serum KL-6 and SP-A assays in healthy individuals and patients with interstitial lung disease.

BACKGROUND: Interstitial lung abnormalities (ILAs) are associated with the risk of progression to interstitial lung diseases (ILDs). Krebs von den Lungen 6 (KL-6) and surfactant protein (SP)-A have been used as biomarkers of ILDs. In this study, we evaluated the levels of these biomarkers and identified their clinical correlations in healthy individuals to assess their usefulness in the diagnosis of ILAs. METHODS: The patient samples were categorized into three groups: healthy, disease, and ILD groups. We used the automated immunoassay HISCL KL-6 and SP-A assay kits. The analytical performance evaluation involved precision, linearity, comparison, establishment of reference intervals, and determination of the cutoff points. We also analyzed the correlations between presence of abnormalities on chest radiography and computed tomography (CT) or pulmonary function test (PFT) and serum levels in the healthy group. RESULTS: KL-6 and SP-A assays showed good analytical performance. The KL-6 and SP-A cutoff values were 304 U/mL and 43.5 ng/mL between the ILD and healthy groups, respectively, which were lower than the values recommended by the manufacturer. In the clinical correlations with radiological findings, SP-A values in subjects with lung abnormalities on CT scans were significantly higher than those in normal scans. There was no significant difference in KL-6 and SP-A levels among PFT patterns; however, both serum levels in the mixed pattern showed higher values than those in the other patterns. CONCLUSIONS: The results revealed a positive association between increased serum levels of SP-A and KL-6 and clinical characteristics as incidental findings on chest imaging and reduced lung function.

Regulatory roles of SP-A and exosomes in pneumonia-induced acute lung and kidney injuries.

Introduction: Pneumonia-induced sepsis can cause multiple organ dysfunction including acute lung and kidney injury (ALI and AKI). Surfactant protein A (SP-A), a critical innate immune molecule, is expressed in the lung and kidney. Extracellular vesicles like exosomes are involved in the processes of pathophysiology. Here we tested one hypothesis that SP-A regulates pneumonia-induced AKI through the modulation of exosomes and cell death. Methods: Wild-type (WT), SP-A knockout (KO), and humanized SP-A transgenic (hTG, lung-specific SP-A expression) mice were used in this study. Results: After intratracheal infection with Pseudomonas aeruginosa, KO mice showed increased mortality, higher injury scores, more severe inflammation in the lung and kidney, and increased serum TNF-α, IL-1ß, and IL-6 levels compared to WT and hTG mice. Infected hTG mice exhibited similar lung injury but more severe kidney injury than infected WT mice. Increased renal tubular apoptosis and pyroptosis in the kidney of KO mice were found when compared with WT and hTG mice. We found that serum exosomes from septic mice cause ALI and AKI through mediating apoptosis and proptosis when mice were injected intravenously. Furthermore, primary proximal tubular epithelial cells isolated from KO mice showed more sensitivity than those from WT mice after exposure to septic serum exosomes. Discussion: Collectively, SP-A attenuates pneumonia-induced ALI and AKI by regulating inflammation, apoptosis and pyroptosis; serum exosomes are important mediators in the pathogenesis of AKI.

Levels of Clara cell secretory protein and surfactant protein A in municipal solid waste management workers in Ibadan, Southwest Nigeria.

Toxic pneumonitis and related respiratory symptoms are common among waste management workers (WMWs). Products of different cellular responses following exposure to toxic components of wastes can lead to the production of a variety of biomolecules. There is a growing recognition of the importance of biomarkers in risk assessment and a strong advocacy for their determination and use as indicators of health and safety. This study assessed the prevalence of respiratory symptoms and the relevance of pulmonary surfactant protein A (SP-A) and Clara cell 16 protein (CC16) as indicators of occupational inhalation exposure to toxic substances and irritants in WMW. A total of 172 subjects consisting of 112 WMWs and 60 Non-WMWs were recruited by purposive sampling. Data on socio-economic and work-related symptoms were collected using structured questionnaire. CC16 and SP-A were determined by ELISA in serum samples. Clinical history reveals a slightly higher prevalence of respiratory symptoms in WMWs relative to control subjects. Increased permeability of the lung-blood barrier, characterized by significant elevation of serum SP-A and serum CC16, was associated with respiratory symptoms in WMWs. Steady increases in SP-A and CC16, respectively, in relation to occupational duration were observed in WMWs relative to control. Receiver operating characteristic curve and multivariate analyses revealed SP-A and CC16 as important lung biomarkers for assessing sub-clinical effects of occupational exposure. Our data suggest SP-A and CC16 may be relevant indicators for assessing occupational inhalation exposure to toxic substances and irritants among WMWs.

Surfactant protein a attenuates generalized and localized neuroinflammation in neonatal mice.

Surfactant protein A (SP-A) has important roles in innate immunity and modulation of pulmonary and extrapulmonary inflammation. Given SP-A has been detected in rat and human brain, we sought to determine if SP-A has a role in modulating inflammation in the neonatal mouse brain. Neonatal wildtype (WT) and SP-A-deficient (SP-A-/-) mice were subjected to three models of brain inflammation: systemic sepsis, intraventricular hemorrhage (IVH) and hypoxic-ischemic encephalopathy (HIE). Following each intervention, RNA was isolated from brain tissue and expression of cytokine and SP-A mRNA was determined by real-time quantitative RT-PCR analysis. In the sepsis model, expression of most cytokine mRNAs was significantly increased in brains of WT and SP-A-/- mice with significantly greater expression of all cytokine mRNA levels in SP-A-/- mice compared to WT. In the IVH model, expression of all cytokine mRNAs was significantly increased in WT and SP-A-/- mice and levels of most cytokine mRNAs were significantly increased in SP-A-/- mice compared to WT. In the HIE model, only TNF-α mRNA levels were significantly increased in WT brain tissue while all pro-inflammtory cytokine mRNAs were significantly increased in SP-A-/- mice, and all pro-inflammatory cytokine mRNA levels were significantly higher in SP-A-/- mice compared to WT. SP-A mRNA was not detectable in brain tissue of adult WT mice nor in WT neonates subjected to these models. These results suggest that SP-A-/- neonatal mice subjected to models of neuroinflammation are more susceptible to both generalized and localized neuroinflammation compared to WT mice, thus supporting the hypothesis that SP-A attenuates inflammation in neonatal mouse brain.

Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages.

Influenza A virus infection (IAV) often leads to acute lung injury that impairs breathing and can lead to death, with disproportionate mortality in children and the elderly. Surfactant Protein A (SP-A) is a calcium-dependent opsonin that binds a variety of pathogens to help control pulmonary infections by alveolar macrophages. Alveolar macrophages play critical roles in host resistance and susceptibility to IAV infection. The effect of SP-A on IAV infection and antiviral response of macrophages, however, is not understood. Here, we report that SP-A attenuates IAV infection in a dose-dependent manner at the level of endosomal trafficking, resulting in infection delay in a model macrophage cell line. The ability of SP-A to suppress infection was independent of its glycosylation status. Binding of SP-A to hemagglutinin did not rely on the glycosylation status or sugar binding properties of either protein. Incubation of either macrophages or IAV with SP-A slowed endocytic uptake rate of IAV. SP-A interfered with binding to cell membrane and endosomal exit of the viral genome as indicated by experiments using isolated cell membranes, an antibody recognizing a pH-sensitive conformational epitope on hemagglutinin, and microscopy. Lack of SP-A in mice enhanced IFNß expression, viral clearance and reduced mortality from IAV infection. These findings support the idea that IAV is an opportunistic pathogen that co-opts SP-A to evade host defense by alveolar macrophages. Our study highlights novel aspects of host-pathogen interactions that may lead to better understanding of the local mechanisms that shape activation of antiviral and inflammatory responses to viral infection in the lung.

Surfactant Protein A Can Affect Macrophage Phagocytosis: An Important Pathogenic Mechanism of Otitis Media with Effusion.

Otitis media with effusion (OME), also known as secretory otitis media, is a common condition in otorhinolaryngology. The main manifestations include middle ear effusion and conductive hearing loss. Recently, increasing attention has been paid to the etiology of OME, wherein immune dysfunction is one important pathogenic mechanism. However, it is unknown whether changes in surfactant protein A (SPA) secretion affect the phagocytic activity of macrophages in the Eustachian tube, thereby altering pathogen clearance, during the pathogenesis of OME. In our study, an OME animal model was established and evaluated. Differences in SPA levels in Eustachian tube lavage fluid between the experimental and control groups were analyzed. Cell-based experiments revealed that SPA decreased the expression of CD64 and SYK and inhibited phagocytosis by RAW264.7 cells. By using flow cytometry and immunofluorescence, we confirmed that macrophage phagocytosis decreased with increasing SPA levels. Finally, we concluded that SPA affects macrophage function and plays a role in the occurrence and development of OME.

Goose surfactant protein A inhibits the growth of avian pathogenic Escherichia coli via an aggregation-dependent mechanism that decreases motility and increases membrane permeability.

Pulmonary collectins have been reported to bind carbohydrates on pathogens and inhibit infection by agglutination, neutralization, and opsonization. In this study, surfactant protein A (SP-A) was identified from goose lung and characterized at expression- and agglutination-functional levels. The deduced amino acid sequence of goose surfactant protein A (gSP-A) has two characteristic structures: a shorter, collagen-like region and a carbohydrate recognition domain. The latter contains two conserved motifs in its Ca2+-binding site: EPN (Glu-Pro-Asn) and WND (Trp-Asn-Asp). Expression analysis using qRT-PCR and fluorescence IHC revealed that gSP-A was highly expressed in the air sac and present in several other tissues, including the lung and trachea. We went on to produce recombinant gSP-A (RgSP-A) using a baculovirus/insect cell system and purified using a Ni2+ affinity column. A biological activity assay showed that all bacterial strains tested in this study were aggregated by RgSP-A, but only Escherichia coli AE17 (E. coli AE17, O2) and E. coli AE158 (O78) were susceptible to RgSP-A-mediated growth inhibition at 2-6 h. Moreover, the swarming motility of the two bacterial strains were weakened with increasing RgSP-A concentration, and their membrane permeability was compromised at 3 h, as determined by flow cytometry and laser confocal microscopy. Therefore, RgSP-A is capable of reducing bacterial viability of E. coli O2 and O78 via an aggregation-dependent mechanism which involves decreasing motility and increasing the bacterial membrane permeability. These data will facilitate detailed studies into the role of gSP-A in innate immune defense as well as for development of antibacterial agents.

Surfactant Protein A can Affect the Surface Tension of the Eustachian Tube and Macrophage Migration.

OBJECTIVE: To assess the role and possible mechanism of surfactant protein A (SPA) in the pathogenesis of otitis media with effusion (OME). METHODS: This was a multi-part study with both an in vivo mouse model study as well as an in vitro study. The control and study groups (OME group) received phosphate-buffered saline and inactivated Streptococcus pneumoniae, respectively, via external auditory meatus injections. Changes in the surface tension of secretions from the eustachian tube (ET) and SPA expression were measured in both groups. A transwell assay was performed to observe the effect of different concentrations of SPA on the migration ability of macrophages. We examined the differentially expressed genes related to SPA-treated macrophages using RNA-seq analysis. RESULTS: On Day 3, the surface tension of the OME group was higher than that of the control group (p = 0.014). The variation intensity of SPA in the ET of the OME group was significantly lower than that of the control group (p < 0.001). Surface tension was correlated with SPA (r = -0.525, p = 0.037). The expression of SPA and macrophages in the ET was different between the two groups. In vitro experiments revealed that macrophages showed different migration abilities with SPA concentration changes (p < 0.05). RNA-seq and western blotting were performed after macrophages were treated with SPA. The results showed that RhoA and Rac1/2/3 were differentially expressed. CONCLUSIONS: SPA can change the surface tension of secretions from the ET and affect macrophage migration to alter the function of the ET. Although research in this field of OME is nascent, initial work suggests that SPA likely plays an important role in OME progression. LEVEL OF EVIDENCE: NA Laryngoscope, 133:1726-1733, 2023.

Collectins and ficolins in neonatal health and disease.

The immune system starts to develop early in embryogenesis. However, at birth it is still immature and associated with high susceptibility to infection. Adaptation to extrauterine conditions requires a balance between colonization with normal flora and protection from pathogens. Infections, oxidative stress and invasive therapeutic procedures may lead to transient organ dysfunction or permanent damage and perhaps even death. Newborns are primarily protected by innate immune mechanisms. Collectins (mannose-binding lectin, collectin-10, collectin-11, collectin-12, surfactant protein A, surfactant protein D) and ficolins (ficolin-1, ficolin-2, ficolin-3) are oligomeric, collagen-related defence lectins, involved in innate immune response. In this review, we discuss the structure, specificity, genetics and role of collectins and ficolins in neonatal health and disease. Their clinical associations (protective or pathogenic influence) depend on a variety of variables, including genetic polymorphisms, gestational age, method of delivery, and maternal/environmental microflora.