SURFACTANT PROTEIN A: AN INNATE IMMUNE MODULATOR AND THERAPEUTIC IN ASTHMA.

Surfactant Protein A (SP-A) is an innate immune modulator produced by the lung with known protective effects against bacteria and viruses. Its role in asthma, an inflammatory lung disease that affects 10% of the world's population, is not entirely known. In this review, we demonstrate that SP-A confers protection against exposure to interleukin-13, a type 2 cytokine integral to eosinophilic asthma, in a mouse model of SP-A deficiency, a house dust mite model of asthma, and in human bronchial epithelial cells from participants with asthma. We also show that small peptides derived from SP-A, such as the major allele of single nucleotide polymorphism (SNP) rs1965708, which includes the carbohydrate recognition domain of SP-A2 at position 223, reduce airway hyperresponsiveness, airway eosinophils, and mucus in a mouse model of asthma. These data suggest that SP-A has beneficial effects relevant to asthma and that an SP-A peptide may have a new therapeutic use in asthma.

Functional Characterisation of Surfactant Protein A as a Novel Prophylactic Means against Oncogenic HPV Infections.

Human papillomavirus (HPV) infection poses a significant health challenge, particularly in low- and middle-income countries (LMIC), where limited healthcare access and awareness hinder vaccine accessibility. To identify alternative HPV targeting interventions, we previously reported on surfactant protein A (SP-A) as a novel molecule capable of recognising HPV16 pseudovirions (HPV16-PsVs) and reducing infection in a murine cervicovaginal HPV challenge model. Building on these findings, our current study aimed to assess SP-A's suitability as a broad-spectrum HPV-targeting molecule and its impact on innate immune responses. We demonstrate SP-A's ability to agglutinate and opsonise multiple oncogenic HPV-PsVs types, enhancing their uptake and clearance by RAW264.7 murine macrophages and THP-1 human-derived immune cells. The SP-A opsonisation of HPV not only led to increased lysosomal accumulation in macrophages and HaCaT keratinocytes but also resulted in a decreased infection of HaCaT cells, which was further decreased when co-cultured with innate immune cells. An analysis of human innate immune cell cytokine profiles revealed a significant inflammatory response upon SP-A exposure, potentially contributing to the overall inhibition of HPV infection. These results highlight the multi-layered impact of SP-A on HPV, innate immune cells and keratinocytes and lay the basis for the development of alternative prophylactic interventions against diverse HPV types.

Surfactant protein A modulates neuroinflammation in adult mice upon pulmonary infection.

BACKGROUND: One of the most common entry gates for systemic infection is the lung. In humans, pulmonary infections can lead to significant neurological impairment, ranging from acute sickness behavior to long-term disorders. Surfactant proteins (SP), essential parts of the pulmonary innate immune defense, have been detected in the brain of rats and humans. Recent evidence suggests that SP-A, the major protein component of surfactant, also plays a functional role in modulating neuroinflammation. This study aimed to determine whether SP-A deficiency affects the inflammatory response in the brain of adult mice during pulmonary infection. EXPERIMENTAL PROCEDURE: Adult male wild-type (WT, n = 72) and SP-A-deficient (SP-A-/-, n = 72) mice were oropharyngeally challenged with lipopolysaccharide (LPS), Pseudomonas aeruginosa (P. aeruginosa), or PBS (control). Both, behavioral assessment and subsequent brain tissue analysis, were performed 24, 48, and 72 h after challenge. The brain concentrations of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß were determined by ELISA. Quantitative rtPCR was used to detect SP-A mRNA expression in brain homogenates and immunohistochemistry was applied for the detection of SP-A protein expression in brain coronal slices. RESULTS: SP-A mRNA and histological evidence of protein expression were detected in both the lungs and brains of WT mice, with significantly higher amounts in lung samples. SP-A-/- mice exhibited significantly higher baseline concentrations of brain TNF-α, IL-6, and IL-1ß compared to WT mice. Oropharyngeal application of either LPS or P. aeruginosa elicited significantly higher brain levels of TNF-α and IL-1ß in SP-A-/- mice compared to WT mice at all time points. In comparison, behavioral impairment as a measure of sickness behavior, was significantly stronger in WT than in SP-A-/- mice, particularly after LPS application. CONCLUSION: SP-A is known for its anti-inflammatory role in the pulmonary immune response to bacterial infection. Recent evidence suggests that in an abdominal sepsis model SP-A deficiency can lead to increased cytokine levels in the brain. Our results extend this perception and provide evidence for an anti-inflammatory role of SP-A in the brain of adult WT mice after pulmonary infection.

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.

Changes in surfactant protein A and D in ovine ovaries related to follicle development.

Surfactant protein A (SP-A) and Surfactant protein D (SP-D) glycoproteins play a crucial role in maintaining lung homeostasis and lung host defense. Interestingly, these proteins are also expressed in extra-pulmonary tissues, including the female genital tract. The ovarian tissue, where SP-A and SP-D expression increases with follicular development, may serve as the primary site of defense for this tissue. However, their functions in these tissues are not well understood and are currently an active area of research. Therefore, the objective of this study is to investigate the expression of SP-A and SP-D in the ovine ovary throughout the ovarian cycle using immunohistochemistry by semiquantitative intensity classification and Western blotting techniques. These findings revealed the presence of SP-A and SP-D in various compartments of the ovary, such as the follicular epithelium, granulosa cells, cumulus cells, theca cells, oocyte I, follicular fluid, and luteal cells of Graafian follicles, excluding the corpus albicans. SP-A and SP-D likely act as a first line of defense against potential pathogens that infiltrate the ovaries. Further investigation of the differential expression of SP-A and SP-D proteins in ovarian follicles will provide a basis for understanding their interactions with key proteins involved in oogenesis.

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.

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.

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.

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.

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.

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.

The Lung Alveolar Cell (LAC) miRNome and Gene Expression Profile of the SP-A-KO Mice After Infection With and Without Rescue With Human Surfactant Protein-A2 (1A0).

Human surfactant protein (SP)-A1 and SP-A2 exhibit differential qualitative and quantitative effects on the alveolar macrophage (AM), including a differential impact on the AM miRNome. Moreover, SP-A rescue (treatment) of SP-A-knockout (KO) infected mice impoves survival. Here, we studied for the first time the role of exogenous SP-A protein treatment on the regulation of lung alveolar cell (LAC) miRNome, the miRNA-RNA targets, and gene expression of SP-A-KO infected mice of both sexes. Toward this, SP-A-KO mice of both sexes were infected with Klebsiella pneumoniae, and half of them were also treated with SP-A2 (1A0). After 6 h of infection/SP-A treatment, the expression levels and pathways of LAC miRNAs, genes, and target miRNA-mRNAs were studied in both groups. We found 1) significant differences in the LAC miRNome, genes, and miRNA-mRNA targets in terms of sex, infection, and infection plus SP-A2 (1A0) protein rescue; 2) an increase in the majority of miRNA-mRNA targets in both study groups in KO male vs. female mice and involvement of the miRNA-mRNA targets in pathways of inflammation, antiapoptosis, and cell cycle; 3) genes with significant changes to be involved in TP-53, tumor necrosis factor (TNF), and cell cycle signaling nodes; 4) when significant changes in the expression of molecules from all analyses (miRNAs, miRNA-mRNA targets, and genes) were considered, two signaling pathways, the TNF and cell cycle, referred to as "integrated pathways" were shown to be significant; 5) the cell cycle pathway to be present in all comparisons made. Because SP-A could be used therapeutically in pulmonary diseases, it is important to understand the molecules and pathways involved in response to an SP-A acute treatment. The information obtained contributes to this end and may help to gain insight especially in the case of infection.

Silica nanoparticle exposure inhibits surfactant protein A and B in A549 cells through ROS-mediated JNK/c-Jun signaling pathway.

Exposure to silica nanoparticles (SiNPs) is related to the dysregulation of pulmonary surfactant that maintains lung stability and function. Nevertheless, there are limited studies concerning the interaction and influence between SiNPs and pulmonary surfactant, and the damage and mechanism are still unclear. Herein, we used A549 cells to develop an in vitro model, with which we investigated the effect of SiNPs exposure on the expression of pulmonary surfactant and the potential regulatory mechanism. The results showed that SiNPs were of cytotoxicity in regarding of reduced cell viability and promoted the production of excessive reactive oxygen species (ROS). Additionally, the JNK/c-Jun signaling pathway was activated, and the expression of surfactant protein A (SP-A) and surfactant protein B (SP-B) was decreased. After the cells being treated with N-acetyl-L-cysteine (NAC), we found that the ROS content was effectively downregulated, and the expression of proteins related to JNK and c-Jun signaling pathways was suppressed. In contrast, the expression of SP-A and SP-B was enhanced. Furthermore, we treated the cells with JNK inhibitor and c-Jun-siRNA and found that the expression of protein related to JNK and c-Jun signaling pathways, as well as SP-A and SP-B, changed in line with that of NAC treatment. These findings suggest that SiNPs exposure can upregulate ROS and activate the JNK/c-Jun signaling pathway in A549 cells, thereby inhibiting the expression of SP-A and SP-B proteins.

Comparison of the Toponomes of Alveolar Macrophages From Wild Type and Surfactant Protein A Knockout Mice and Their Response to Infection.

Background: Surfactant protein-A (SP-A) plays a critical role in lung innate immunity by regulating alveolar macrophages (AM), expression of inflammatory mediators, and other host defense proteins. The toponome imaging system (TIS), a serial immunostainer, was used to study the AM toponome because it characterizes the localization of multiple markers and identifies marker combinations in each pixel as combinatorial molecular phenotypes (CMPs). We used TIS to study the AM toponome from wild type (WT) and SP-A knockout (KO) mice and changes following Klebsiella pneumoniae exposure. Methods: WT or KO mice received intratracheal K. pneumoniae or vehicle and AM were obtained by bronchoalveolar lavage after one hour. AM were attached to slides and underwent TIS analysis. Images were analyzed to characterize all pixels. AM CMPs from WT vehicle (n=3) and infected (n=3) mice were compared to each other and to AM from KO (n=3 vehicle; n=3 infected). Histograms provided us with a tool to summarize the representation of each marker in a set of CMPs. Results: Using the histograms and other tools we identified markers of interest and observed that: 1) Both comparisons had conserved (present in all group members) CMPs, only in vehicle AM and only in infected AM, or common to both vehicle and infected AM, (i.e., unaffected by the condition). 2) the CMP number decreased with infection in WT and KO versus vehicle controls. 3) More infection-specific CMPs in WT vs KO AM. 4) When AM from WT and KO vehicle or infected were compared, there were more unique CMPs exclusive to the KO AM. 5) All comparisons showed CMPs shared by both groups. Conclusions: The decrease of CMPs exclusive to infected AM in KO mice may underlie the observed susceptibility of KO mice to infection. However, both KO groups had more exclusive CMPs than the corresponding WT groups, perhaps indicating a vigorous effort by KO to overcome deficits in certain proteins and CMPs that are dysregulated by the absence of SP-A. Moreover, the presence of shared CMPs in the compared groups indicates that regulation of these CMPs is not dependent on either infection or the presence or absence of SP-A.

Loss of SP-A in the Lung Exacerbates Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating and common chronic lung disease that is pathologically characterized by the destruction of lung architecture and the accumulation of extracellular matrix in the lung. Previous studies have shown an association between lung surfactant protein (SP) and the pathogenesis of IPF, as demonstrated by mutations and the altered expression of SP in patients with IPF. However, the role of SP in the development of lung fibrosis is poorly understood. In this study, the role of surfactant protein A (SP-A) was explored in experimental lung fibrosis induced with a low or high dose of bleomycin (BLM) and CRISPR/Cas9-mediated genetic deletion of SP-A. Our results showed that lung SP-A deficiency in mice promoted the development of fibrotic damage and exacerbated inflammatory responses to the BLM challenge. In vitro experiments with murine lung epithelial LA-4 cells demonstrated that in response to transforming growth factor-ß1 (TGF-ß1), LA-4 cells had a decreased protein expression of SP-A. Furthermore, exogenous SP administration to LA-4 cells inhibited the TGF-ß1-induced upregulation of fibrotic markers. Overall, these findings suggest a novel antifibrotic mechanism of SP-A in the development of lung fibrosis, which indicates the therapeutic potential of the lung SP-A in preventing the development of IPF.

Signaling Pathways That Mediate Alveolar Macrophage Activation by Surfactant Protein A and IL-4.

Activation of tissue repair program in macrophages requires the integration of IL-4/IL-13 cytokines and tissue-specific signals. In the lung, surfactant protein A (SP-A) is a tissue factor that amplifies IL-4Rα-dependent alternative activation and proliferation of alveolar macrophages (AMs) through the myosin18A receptor. However, the mechanism by which SP-A and IL-4 synergistically increase activation and proliferation of AMs is unknown. Here we show that SP-A amplifies IL-4-mediated phosphorylation of STAT6 and Akt by binding to myosin18A. Blocking PI3K activity or the myosin18A receptor abrogates SP-A´s amplifying effects on IL-4 signaling. SP-A alone activates Akt, mTORC1, and PKCζ and inactivates GSK3α/ß by phosphorylation, but it cannot activate arginase-1 activity or AM proliferation on its own. The combined effects of IL-4 and SP-A on the mTORC1 and GSK3 branches of PI3K-Akt signaling contribute to increased AM proliferation and alternative activation, as revealed by pharmacological inhibition of Akt (inhibitor VIII) and mTORC1 (rapamycin and torin). On the other hand, the IL-4+SP-A-driven PKCζ signaling axis appears to intersect PI3K activation with STAT6 phosphorylation to achieve more efficient alternative activation of AMs. Consistent with IL-4+SP-A-driven activation of mTORC1 and mTORC2, both agonists synergistically increased mitochondrial respiration and glycolysis in AMs, which are necessary for production of energy and metabolic intermediates for proliferation and alternative activation. We conclude that SP-A signaling in AMs activates PI3K-dependent branched pathways that amplify IL-4 actions on cell proliferation and the acquisition of AM effector functions.

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.

Surfactant protein A in particles in exhaled air and plasma.

Respiratory tract lining fluid (RTLF) is an important component of the lung epithelial barrier. Pathological changes in RTLF may cause increased permeability of the epithelial barrier, but changes within RTLF are difficult to assess non-invasively. The aim of this study was to explore if the use of the non-invasive measurement technique, Particles in Exhaled Air (PEx) and blood test were useful in assessing epithelial barrier, and if cigarette smoking affects the relationship. In a general population subcohort from the European Community Respiratory Health Survey III in Iceland (n = 112), we collected RTLF droplets using the PEx technique, in conjunction with blood samples and questionnaire data. We measured surfactant protein A (SP-A) in the collected plasma and PEx samples. Participants were defined as healthy if they did not currently have asthma, were non-smokers and had forced expiratory volume in one second ≥ 80% of predicted value. Of the 112 participants, 97 were healthy and 15 were current smokers. There was no correlation between plasma and PEx SP-A levels. However, the ratio of plasma to PEx SP-A was significantly higher in smokers compared to healthy subjects. The lack of correlation between PEx and plasma SP-A in healthy participants, indicates that SP-A in plasma does not diffuse freely over the lung epithelial barrier. However, the lung epithelial barrier may be injured by smoking, leading to diffusion of SP-A across the barrier into the bloodstream, causing an increased ratio of plasma to PEx SP-A.