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.

SP-R210 isoforms of Myosin18A modulate endosomal sorting and recognition of influenza A virus infection in macrophages.

Influenza A virus (IAV) infection causes acute and often lethal inflammation in the lung. The role of macrophages in this adverse inflammation is partially understood. The surfactant protein A receptor 210 (SP-R210) consists of two isoforms, a long (L) SP-R210L and a short (S) SP-R210S isoform encoded by alternative splicing of the myosin 18A gene. We reported that disruption of SP-R210L enhances cytosolic and endosomal antiviral response pathways. Here, we report that SP-R210L antagonizes type I interferon ß (IFNß), as depletion of SP-R210L potentiates IFNß secretion. SP-R210 antibodies enhance and attenuate IFNß secretion in SP-R210L replete and deficient macrophages, respectively, indicating that SP-R210 isoform stoichiometry alters macrophage function intrinsically. This reciprocal response is coupled to unopposed and restricted expression of viral genes in control and SP-R210L-deficient macrophages, respectively. Human monocytic cells with sub-stoichiometric expression of SP-R210L resist IAV infection, whereas alveolar macrophages with increased abundance of SP-R210L permit viral gene expression similar to murine macrophages. Uptake and membrane binding studies show that lack of SP-R210 isoforms does not impair IAV binding and internalization. Lack of SP-R210L, however, results in macropinocytic retention of the virus that depends on both SP-R210S and interferon-inducible transmembrane protein-3 (IFITM3). Mass spectrometry and Western blot analyses indicate that SP-R210 isoforms modulate differential recruitment of the Rho-family GTPase RAC1 and guanine nucleotide exchange factors. Our study suggests that SP-R210 isoforms modulate RAC-dependent macropinosomal sorting of IAV to discrete endosomal and lysosomal compartments that either permit or prevent endolysosomal escape and inflammatory sensing of viral genomes in macrophages.

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.

All trans-retinoic acid modulates hyperoxia-induced suppression of NF-kB-dependent Wnt signaling in alveolar A549 epithelial cells.

INTRODUCTION: Despite recent advances in perinatal medicine, bronchopulmonary dysplasia (BPD) remains the most common complication of preterm birth. Inflammation, the main cause for BPD, results in arrested alveolarization. All trans-retinoic acid (ATRA), the active metabolite of Vitamin A, facilitates recovery from hyperoxia induced cell damage. The mechanisms involved in this response, and the genes activated, however, are poorly understood. In this study, we investigated the mechanisms of action of ATRA in human lung epithelial cells exposed to hyperoxia. We hypothesized that ATRA reduces hyperoxia-induced inflammatory responses in A549 alveolar epithelial cells. METHODS: A549 cells were exposed to hyperoxia with or without treatment with ATRA, followed by RNA-seq analysis. RESULTS: Transcriptomic analysis of A549 cells revealed ~2,000 differentially expressed genes with a higher than 2-fold change. Treatment of cells with ATRA alleviated some of the hyperoxia-induced changes, including Wnt signaling, cell adhesion and cytochrome P450 genes, partially through NF-κB signaling. DISCUSSION/CONCLUSION: Our findings support the idea that ATRA supplementation may decrease hyperoxia-induced disruption of the neonatal respiratory epithelium and alleviate development of BPD.

Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages.

Macrophages play an important role in maintaining tissue homeostasis, from regulating the inflammatory response to pathogens to resolving inflammation and aiding tissue repair. The surfactant protein A (SP-A) receptor SP-R210 (MYO18A) has been shown to affect basal and inflammatory macrophage states. Specifically, disruption of the longer splice isoform SP-R210L/MYO18Aα renders macrophages hyper-inflammatory, although the mechanism by which this occurs is not well understood. We asked whether disruption of the L isoform led to the hyper-inflammatory state via alteration of global genomic responses. RNA sequencing analysis of L isoform-deficient macrophages (SP-R210L(DN)) revealed basal and influenza-induced upregulation of genes associated with inflammatory pathways, such as TLR, RIG-I, NOD, and cytoplasmic DNA signaling, whereas knockout of both SP-R210 isoforms (L and S) only resulted in increased RIG-I and NOD signaling. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis showed increased genome-wide deposition of the pioneer transcription factor PU.1 in SP-R210L(DN) cells, with increased representation around genes relevant to inflammatory pathways. Additional ChIP-seq analysis of histone H3 methylation marks showed decreases in both repressive H3K9me3 and H3K27me3 marks with a commensurate increase in transcriptionally active (H3K4me3) histone marks in the L isoform deficient macrophages. Influenza A virus (IAV) infection, known to stimulate a wide array of anti-viral responses, caused a differential redistribution of PU.1 binding between proximal promoter and distal sites and decoupling from Toll-like receptor regulated gene promoters in SP-R210L(DN) cells. These finding suggest that the inflammatory differences seen in SP-R210L-deficient macrophages are a result of transcriptional differences that are mediated by epigenetic changes brought about by differential expression of the SP-R210 isoforms. This provides an avenue to explore how the signaling pathways downstream of the receptor and the ligands can modulate the macrophage inflammatory response.

Extracellular matrix-inspired inhalable aerogels for rapid clearance of pulmonary tuberculosis.

Tuberculosis (TB) remains a leading cause of death from a single infectious agent, and limiting the spread of multidrug-resistant TB (MDR-TB) is now an urgent global health priority. Essential to the persistence of this disease is the ability of Mycobacterium tuberculosis (Mtb) to circumvent host defenses by infecting lung macrophages to create a cellular niche for its survival and proliferation. This has urged the development of new therapeutic strategies that act through mechanisms distinct from conventional antibiotics, and thus are effective against MDR bacteria, while being able to efficiently kill persister Mtb cells in infected host macrophages. Here, we report a new class of gel-like microparticle aerosols, or 'aerogels', designed to exploit metabolic vulnerabilities of Mtb pathogens and TB-infected macrophages to enable preferential delivery of synergistic peptide-antibiotic combinations for potent and rapid antitubercular therapy. This is achieved by formulating aerogels through the supramolecular assembly of a de novo designed anti-TB peptide and the extracellular matrix (ECM)-derived polysaccharide, hyaluronic acid (HA). Importantly, HA serves as a nutrient source for Mtb cells during tissue invasion and proliferation, and is recognized by CD44 receptors highly expressed on lung macrophages during TB infection. By exploiting this metabolic substrate for pathogen targeting, HA aerogels are shown to avidly bind and kill both drug-sensitive and drug-resistant mycobacteria, while being efficiently internalized into macrophage host cells in vitro and in vivo to clear Mtb persisters. This multifaceted bioactivity suggests aerogels may serve as a versatile inhalable platform upon which novel biomaterials-enabled therapeutics can be developed to rapidly clear pulmonary MDR-TB.

Cytokine Panels and Pediatric Acute Respiratory Distress Syndrome: A Translational Investigation.

OBJECTIVES: To identify and compare serum and lower respiratory tract fluid biomarkers of lung injury using well-characterized mouse models of lung injury. To explore the relationship between these preclinical biomarkers and clinical outcomes in a discovery cohort of pediatric patients with acute respiratory failure from pneumonia. DESIGN: Prospective, observational cohort study. SETTING: A basic science laboratory and the PICU of a tertiary-care children's hospital. PATIENTS: PICU patients intubated for respiratory failure from a suspected respiratory infection. INTERVENTIONS: Prospective enrollment and collection of lower respiratory tract fluid samples. MEASUREMENTS AND MAIN RESULTS: C57BL6/J mice were intranasally inoculated with escalating doses of influenza A virus or toll-like receptor agonists to simulate varying degrees of lung injury. Serum and bronchoalveolar lavage fluid were measured for the presence of cytokines using commercially available multiplex cytokine assays. Elevated levels of C-C motif chemokine ligand 7 at the peak of inflammation in both bronchoalveolar lavage fluid and serum correlated with lethality, with the bronchoalveolar lavage fluid ratio of C-C motif chemokine ligand 7:C-C motif chemokine ligand 22 providing the best prediction in the mouse models. These preclinical biomarkers were examined in the plasma and lower respiratory tract fluid of a discovery cohort of pediatric patients with acute respiratory failure from pneumonia. The primary clinical outcome measure was ventilator-free days, with secondary outcomes of pediatric acute respiratory distress syndrome severity and mortality. Elevation in peak lower respiratory tract fluid C-C motif chemokine ligand 7:C-C motif chemokine ligand 22 ratios demonstrated a significant negative correlation with ventilator-free days (r = -0.805; p < 0.02). CONCLUSIONS: This study provides evidence that lung immune profiling via lower respiratory tract fluid cytokine analysis is feasible and may provide insight into clinical outcomes. Further validation of markers, including the C-C motif chemokine ligand 7:C-C motif chemokine ligand 22 ratio in this limited study, in a larger cohort of patients is necessary.

Higher serum PD-L1 level predicts increased overall survival with lapatinib versus trastuzumab in the CCTG MA.31 phase 3 trial.

BACKGROUND: The purpose of this retrospective biomarker study of the Canadian Cancer Trials Group (CCTG) MA.31 randomized phase 3 trial (lapatinib vs trastuzumab) of HER2-positive metastatic breast cancer (MBC) was to evaluate the prognostic and predictive biomarker utility of pretreatment serum programmed death ligand 1 (PD-L1) levels. METHODS: CCTG MA.31 accrued 652 HER2-positive patients; 387 had serum available (185 in the trastuzumab arm and 202 in the lapatinib arm). The Ella immunoassay platform (ProteinSimple, San Jose, California) was used to quantitate serum PD-L1 levels. Stepwise forward Cox multivariable analyses were performed for progression-free survival and overall survival (OS). RESULTS: In the whole trial population, continuous pretreatment serum PD-L1 levels were not associated with OS. However, within the trastuzumab arm, a higher continuous pretreatment serum PD-L1 level was significant for shorter OS (hazard ratio [HR], 3.85; P = .04), but within the lapatinib arm, pretreatment serum PD-L1 was not associated with OS (P = .37). In the whole trial, in a multivariable analysis for OS, serum PD-L1 (median cut point) remained a significant independent covariate (HR, 2.38; P = .001). There was a significant interaction between treatment arm and continuous serum PD-L1 (bootstrap method; P = .0025): at or above 214.2 pg/mL (the 89th percentile), serum PD-L1 was associated with significantly shorter OS with trastuzumab treatment versus lapatinib treatment. CONCLUSIONS: In the CCTG MA.31 trial, serum PD-L1 was a significant predictive factor: a higher pretreatment serum PD-L1 level was associated with shorter OS with trastuzumab treatment but with longer OS with lapatinib treatment. Immune evasion may decrease the effectiveness of trastuzumab therapy. Further evaluation of elevated serum PD-L1 in advanced breast cancer is warranted to identify patients with HER2-positive MBC who may benefit from novel immune-targeted therapies in addition to trastuzumab.

Impact of Ozone, Sex, and Gonadal Hormones on Bronchoalveolar Lavage Characteristics and Survival in SP-A KO Mice Infected with Klebsiella pneumoniae.

Surfactant protein A (SP-A) plays an important role in innate immunity. The sex-dependent survival of infected SP-A knockout (KO) mice has been observed. Our goal was to study the impact of ozone (O3) and sex, as well as gonadal hormones, on the bronchoalveolar lavage (BAL) readouts and survival, respectively, of Klebsiella pneumoniae-infected SP-A KO mice. Male and female SP-A KO mice were exposed to O3 or filtered air and infected with K. pneumoniae. We studied markers of inflammation and tissue damage at 4, 24, and 48 h, as well as the survival over 14 days, of gonadectomized (Gx) mice implanted with control pellets (CoP) or hormone (5α-dihydrotestosterone (DHT) in female gonadectomized mice (GxF) or 17ß-estradiol (E2) in male gonadectomized mice (GxM)). We observed: (1) an increase in neutrophil and macrophage inflammatory protein-2 levels as time progressed post-infection, and O3 exposure appeared to increase this response; (2) an increase in lactate dehydrogenase, total protein, oxidized protein, and phospholipids in response to O3 with no consistent sex differences in studied parameters; and (3) a reduction in survival of the GxM and CoP mice, the GxM and E2 mice, and the GxF and DHT mice but not for the GxF and CoP mice after O3. Without SP-A, (a) sex was found to have a minimal impact on BAL cellular composition and tissue damage markers, and (b) the impact of gonadal hormones on survival was found to involve different mechanisms than in the presence of SP-A.

Lower respiratory tract delivery, airway clearance, and preclinical efficacy of inhaled GM-CSF in a postinfluenza pneumococcal pneumonia model.

Inhaled granulocyte/macrophage colony-stimulating factor (GM-CSF) shows promise as a therapeutic to treat viral and bacterial pneumonia, but no mouse model of inhaled GM-CSF has been described. We sought to 1) develop a mouse model of aerosolized recombinant mouse GM-CSF administration and 2) investigate the protection conferred by inhaled GM-CSF during influenza A virus (IAV) infection against secondary bacterial infection with pneumococcus. To assess lower respiratory tract delivery of aerosolized therapeutics, mice were exposed to aerosolized fluorescein (FITC)-labeled dextran noninvasively via an aerosolization tower or invasively using a rodent ventilator. The efficiency of delivery to the lower respiratory tracts of mice was 0.01% noninvasively compared with 0.3% invasively. The airway pharmacokinetics of inhaled GM-CSF fit a two-compartment model with a terminal phase half-life of 1.3 h. To test if lower respiratory tract levels were sufficient for biological effect, mice were infected intranasally with IAV, treated with aerosolized recombinant mouse GM-CSF, and then secondarily infected with Streptococcus pneumoniae. Inhaled GM-CSF conferred a significant survival benefit to mice against secondary challenge with S. pneumoniae (P < 0.05). Inhaled GM-CSF did not reduce airway or lung parenchymal bacterial growth but significantly reduced the incidence of S. pneumoniae bacteremia (P < 0.01). However, GM-CSF overexpression during influenza virus infection did not affect lung epithelial permeability to FITC-dextran ingress into the bloodstream. Therefore, the mechanism of protection conferred by inhaled GM-CSF appears to be locally mediated improved lung antibacterial resistance to systemic bacteremia during IAV infection.

Structural and Functional Determinants of Rodent and Human Surfactant Protein A: A Synthesis of Binding and Computational Data.

Surfactant protein A (SP-A) provides surfactant stability, first line host defense, and lung homeostasis by binding surfactant phospholipids, pathogens, alveolar macrophages (AMs), and epithelial cells. Non-primates express one SP-A protein whereas humans express two: SP-A1 and SP-A2 with core intra- and inter-species differences in the collagen-like domain. Here, we used macrophages and solid phase binding assays to discern structural correlates of rat (r) and human (h) SP-A function. Binding assays using recombinant rSP-A expressed in insect cells showed that lack of proline hydroxylation, truncations of amino-terminal oligomerization domains, and site-directed serine (S) or alanine (A) mutagenesis of cysteine 6 (C6S), glutamate 195 (E195A), and glutamate 171 (E171A) in the carbohydrate recognition domain (CRD) all impaired SP-A binding. Replacement of arginine 197 with alanine found in hSP-A (R197A), however, restored the binding of hydroxyproline-deficient rSP-A to the SP-A receptor SP-R210 similar to native rat and human SP-A. In silico calculation of Ca++ coordination bond length and solvent accessibility surface area revealed that the "humanized" R197A substitution alters topology and solvent accessibility of the Ca++ coordination residues of the CRD domain. Binding assays in mouse AMs that were exposed to either endogenous SP-A or hSP-A1 (6A2) and hSP-A2 (1A0) isoforms in vivo revealed that mouse SP-A is a functional hybrid of hSP-A1 and hSP-A2 in regulating SP-A receptor occupancy and binding affinity. Binding assays using neonatal and adult human AMs indicates that the interaction of SP-A1 and SP-A2 with AMs is developmentally regulated. Furthermore, our data indicate that the auxiliary ion coordination loop encompassing the conserved E171 residue may comprise a conserved site of interaction with macrophages, and SP-R210 specifically, that merits further investigation to discern conserved and divergent SP-A functions between species. In summary, our findings support the notion that complex structural adaptation of SP-A regulate conserved and species specific AM functions in vertebrates.

Major Effect of Oxidative Stress on the Male, but Not Female, SP-A1 Type II Cell miRNome.

Pulmonary surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. In humans there are two proteins, SP-A1 and SP-A2, encoded by SFTPA1 and SFTPA2, respectively, which are produced by the alveolar type II cells (T2C). We sought to investigate the differential influence of SP-A1 and SP-A2 in T2C miRNome under oxidative stress (OxS). SP-A knock out (KO) and hTG male and female mice expressing SP-A1 or SP-A2 as well as gonadectomized (Gx) mice were exposed to O3-induced oxidative stress (OxS) or filtered air (FA). Expression of miRNAs and mRNAs was measured in the T2C of experimental animals. (a) In SP-A1 males after normalizing to KO males, significant changes were observed in the miRNome in terms of sex-OxS effects, with 24 miRNAs being differentially expressed under OxS. (b) The mRNA targets of the dysregulated miRNAs included Ago2, Ddx20, Plcg2, Irs1, Elf2, Jak2, Map2k4, Bcl2, Ccnd1, and Vhl. We validated the expression levels of these transcripts, and observed that the mRNA levels of all of these targets were unaffected in SP-A1 T2C but six of these were significantly upregulated in the KO (except Bcl2 that was downregulated). (c) Gondadectomy had a major effect on the expression of miRNAs and in three of the mRNA targets (Irs1, Bcl2, and Vhl). Ccnd1 was upregulated in KO regardless of Gx. (d) The targets of the significantly changed miRNAs are involved in several pathways including MAPK signaling pathway, cell cycle, anti-apoptosis, and other. In conclusion, in response to OxS, SP-A1 and male hormones appear to have a major effect in the T2C miRNome.

Differential Effects of Human SP-A1 and SP-A2 on the BAL Proteome and Signaling Pathways in Response to Klebsiella pneumoniae and Ozone Exposure.

Surfactant protein A (SP-A) plays critical roles in host defense, regulation of inflammation and surfactant metabolism in the lung. The human SP-A locus consists of two functional genes, SFTPA1 and SFTPA2 encoding surfactant proteins SP-A1 and SP-A2, respectively. Structural and functional differences exist between SP-A1 and SP-A2 in vitro and in vivo. Ozone is a major air pollutant with a negative impact on many biological processes. In this study we used humanized transgenic (hTG) SP-A1 and SP-A2 mice, and SP-A KO mice to study in vivo effects of SP-A1 and SP-A2 on the bronchoalveolar lavage (BAL) proteomic profile and associated signaling pathways in response to ozone or filtered air (FA) exposure and Klebsiella pneumoniae infection. The BAL samples were harvested 24 h after ozone (2 ppm for 3 h) or FA exposure and infection and analyzed by two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-ToF/ToF. We found: that (1) Ozone exposure, but not infection, is a major factor for increases in total BAL protein content. (2) A total of 36 proteins were identified, accounting for 89.62% of the BAL proteins resolved by the 2D-DIGE system. (3) The number of proteins in which levels were altered more than 25% following infection and FA exposure was: SP-A2 > SP-A1 > KO for male mice, and SP-A2 ≈ SP-A1 > KO for female mice. (4) The number of proteins with more than 25% increase/decrease after ozone exposure and infection was: SP-A2 > SP-A1 ≈ KO, with the majority being increases in male mice and decreases in female mice. (5) Eleven out of the 36 proteins, including annexin A5, glutathione S-transferase A4, SP-A1/SP-A2, and 14-3-3 zeta protein, exhibited significant differences among SP-A genotypes. The acute phase response (APR) that includes the NF-kB signaling pathway plays a critical role, followed by Nrf2-mediated oxidative response, and others. These associated with SP-A genotype, sex, and ozone-induced oxidative stress in response to infection. We concluded that human SP-A2 and SP-A1 exhibit differential genotype-and sex-dependent innate immune responses to microbial pathogens and/or ozone-induced oxidative stress by modulating proteomic patterns and signaling pathways in the lung.

Survival of Surfactant Protein-A1 and SP-A2 Transgenic Mice After Klebsiella pneumoniae Infection, Exhibits Sex-, Gene-, and Variant Specific Differences; Treatment With Surfactant Protein Improves Survival.

Surfactant protein A (SP-A) is involved in lung innate host defense and surfactant-related functions. The human SFTPA1 and SFTPA2 genes encode SP-A1 and SP-2 proteins, and each gene has been identified with numerous genetic variants. SP-A1 and SP-A2 differentially enhance bacterial phagocytosis. Sex differences have been observed in pulmonary disease and in survival of wild type and SP-A knockout (KO) mice. The impact of human SP-A variants on survival after infection is unknown. In this study, we determined whether SP-A variants differentially affect survival of male and female mice infected with Klebsiella pneumoniae. Transgenic (TG) mice, where each carries a different human (h) SP-A1 (6A2, 6A4), SP-A2 (1A0, 1A3) variant or both variants SP-A1/SP-A2 (6A2/1A0, co-ex), and SP-A- KO, were utilized. The hTG and KO mice were infected intratracheally with K. pneumoniae bacteria, and groups of KO mice were treated with SP-A1 or SP-A2 either prior to and/or at the time of infection and survival for both experimental groups was monitored over 14 days. The binding of purified SP-A1 and SP-A2 proteins to phagocytic and non-phagocytic cells and expression of cell surface proteins in alveolar macrophages (AM) from SP-A1 and SP-A2 mice was examined. We observed gene-, variant-, and sex-specific (except for co-ex) differences with females showing better survival: (a) Gene-specific differences: co-ex = SP-A2 > SP-A1 > KO (both sexes); (b) Variant-specific survival co-ex (6A2/1A0) = 1A0 > 1A3 = 6A2 > 6A4 (both sexes); (c) KO mice treated with SPs (SP-A1 or SP-A2) proteins exhibit significantly (p < 0.05) better survival; (d) SP-A1 and SP-A2 differentially bind to phagocytic, but not to non-phagocytic cells, and AM from SP-A1 and SP-A2 hTG mice exhibit differential expression of cell surface proteins. Our results indicate that sex and SP-A genetics differentially affect survival after infection and that exogenous SP-A1/SP-A2 treatment significantly improves survival. We postulate that the differential SP-A1/SP-A2 binding to the phagocytic cells and the differential expression of cell surface proteins that bind SP-A by AM from SP-A1 and SP-A2 mice play a role in this process. These findings provide insight into the importance of sex and innate immunity genetics in survival following infection.

Differential effects of innate immune variants of surfactant protein-A1 (SFTPA1) and SP-A2 (SFTPA2) in airway function after Klebsiella pneumoniae infection and sex differences.

BACKGROUND: Surfactant Protein-A (SP-A) is a major protein component of surfactant and plays a role in surfactant-related functions and innate immunity. Human SP-A consists of two functional genes, SFTPA1 and SFTPA2, encoding SP-A1 and SP-A2 proteins, respectively and each is identified with numerous genetic variants. These differentially enhance bacterial phagocytosis, with SP-A2 variants being more effective than SP-A1. METHODS: Lung functions of humanized transgenic (hTG) mice that carry different SP-A1 and SP-A2 variants or both variants SP-A1/SP-A2 (6A2/1A0, co-ex), as well as SP-A knockout (KO), were studied. The animals were connected to a flexiVent system to obtain forced oscillation technique (FOT) measurements and the data were analyzed using various models. Lung function was assessed after infection (baseline) and following inhaled methacholine concentrations (0-50 mg/mL). RESULTS: Here, we investigated the role of SP-A variants on airway function after Klebsiella pneumoniae (Kp) infection (baseline) and following inhaled methacholine. We found that: 1) in the absence of methacholine no significant differences were observed between SP-A1 and SP-A2 variants and/or SP-A knockout (KO) except for sex differences in most of the parameters studied. 2) In response to methacholine, i) sex differences were observed that were reverse of those observed in the absence of methacholine; ii) SP-A2 (1A3) gene variant in males exhibited increased total and central airway resistance (Rrs and Rn) versus all other variants; iii) In females, SP-A2 (1A3) and SP-A1 (6A2) variants had similar increases in total and central airway resistance (Rrs and Rn) versus all other variants; iv) Allele-specific differences were observed, a) with SP-A2 (1A3) exhibiting significantly higher lung functions versus SP-A2 (1A0) in both sexes, except for Crs, and b) SP-A1 (6A2, 6A4) had more diverse changes in lung function in both sexes. CONCLUSION: We conclude that, in response to infection and methacholine, SP-A variants differentially affect lung function and exhibit sex-specific differences consistent with previously reported findings of functional differences of SP-A variants. Thus, the observed changes in respiratory function mechanics provide insight into the role and importance of genetic variation of innate immune molecules, such as SP-A, on mechanical consequences of lung function after infection and inhaled substances.

GM-CSF overexpression after influenza a virus infection prevents mortality and moderates M1-like airway monocyte/macrophage polarization.

BACKGROUND: Influenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Application of high GM-CSF levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established. METHODS: Mice were infected intranasally with influenza A virus (PR8 strain). Supra-physiologic levels of GM-CSF were induced in the airways using the double transgenic GM-CSF (DTGM) or littermate control mice starting on 3 days post-infection (dpi). Assessment of respiratory mechanical parameters was performed using the flexiVent rodent ventilator. RNA sequence analysis was performed on FACS-sorted airway macrophage subsets at 8 dpi. RESULTS: Supra-physiologic levels of GM-CSF conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (BAL) fluid to near baseline levels. Transcriptome analysis, and subsequent validation ELISA assays, revealed that excess GM-CSF re-directs macrophages from an "M1-like" to a more "M2-like" activation state as revealed by alterations in the ratios of CXCL9 and CCL17 in BAL fluid, respectively. Ingenuity pathway analysis predicted that GM-CSF surplus during IAV infection elicits expression of anti-inflammatory mediators and moderates M1 macrophage pro-inflammatory signaling by Type II interferon (IFN-γ). CONCLUSIONS: Our data indicate that application of high levels of GM-CSF in the lung after influenza A virus infection alters pathogenic "M1-like" macrophage inflammation. These results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury.

SP-A2 contributes to miRNA-mediated sex differences in response to oxidative stress: pro-inflammatory, anti-apoptotic, and anti-oxidant pathways are involved.

BACKGROUND: Human innate host defense molecules, surfactant protein A1 (SP-A1), and SP-A2 differentially affect the function and proteome of the alveolar macrophage (AM). We hypothesized that SP-A genes differentially regulate the AM miRNome. METHODS: Humanized transgenic mice expressing SP-A1 and SP-A2 were subjected to O3-induced oxidative stress (OxS) or filtered air (FA), AMs were isolated, and miRNA levels were measured. RESULTS: In SP-A2 males, we found significant changes in miRNome in terms of sex and sex-OxS effects, with 11 miRNAs differentially expressed under OxS. Their mRNA targets included BCL2, CAT, FOXO1, IL6, NF-kB, SOD2, and STAT3. We followed the expression of these transcripts as well as key cytokines, and we found that (a) the STAT3 mRNA significantly increased at 4 h post OxS and returned to baseline at 18 h post OxS. (b) The anti-oxidant protein SOD2 level significantly increased, but the CAT level did not change after 4 h post OxS compared to control. (c) The anti-apoptotic BCL2 mRNA increased significantly (18 h post OxS), but the levels of the other transcripts were decreased. The presence of the SP-A2 gene had a protective role in apoptosis of AMs under OxS compared to mice lacking SP-A (knockout, KO). (d) Pro-inflammatory cytokine IL-6 protein levels were significantly increased in SP-A2 mice compared to KO (4 and 18 h post OxS), which signifies the role of SP-A2 in pro-inflammatory protein expression. (e) SOD2 and CAT mRNAs changed significantly in OxS indicating a plausible role of SP-A2 in the homeostasis of reactive oxygen species. (f) Gonadectomy of transgenic mice showed that sex hormones contribute to significant changes of the miRNome expression. CONCLUSIONS: We conclude that SP-A2 influences the miRNA-mediated sex-specific differences in response to OxS. In males, these differences pertain to inflammatory, anti-apoptotic, and anti-oxidant pathways.

SH3GLB2/endophilin B2 regulates lung homeostasis and recovery from severe influenza A virus infection.

New influenza A viruses that emerge frequently elicit composite inflammatory responses to both infection and structural damage of alveolar-capillary barrier cells that hinders regeneration of respiratory function. The host factors that relinquish restoration of lung health to enduring lung injury are insufficiently understood. Here, we investigated the role of endophilin B2 (B2) in susceptibility to severe influenza infection. WT and B2-deficient mice were infected with H1N1 PR8 by intranasal administration and course of influenza pneumonia, inflammatory, and tissue responses were monitored over time. Disruption of B2 enhanced recovery from severe influenza infection as indicated by swift body weight recovery and significantly better survival of endophilin B2-deficient mice compared to WT mice. Compared to WT mice, the B2-deficient lungs exhibited induction of genes that express surfactant proteins, ABCA3, GM-CSF, podoplanin, and caveolin mRNA after 7 days, temporal induction of CCAAT/enhancer binding protein CEBPα, ß, and δ mRNAs 3-14 days after infection, and differences in alveolar extracellular matrix integrity and respiratory mechanics. Flow cytometry and gene expression studies demonstrated robust recovery of alveolar macrophages and recruitment of CD4+ lymphocytes in B2-deficient lungs. Targeting of endophilin B2 alleviates adverse effects of IAV infection on respiratory and immune cells enabling restoration of alveolar homeostasis.

An Electrochemical Biosensor for Rapid Detection of Pediatric Bloodstream Infections.

Bloodstream infections are major contributing factors of morbidity and mortality among children. Precise and timely identification of causative agents can improve the clinical management and outcome of the infection, potentially saving lives. Electrochemical biosensors previously described by Gao et al. (2017) have the potential to deliver greater speed and discrimination. However, to date there are no data that determine whether the age of the host would cause bacteria to demonstrate different growth characteristics, or whether pediatric samples would behave differently using this electrochemical biosensor. The importance of this knowledge gap is clear: the preclinical testing phase of this line of research is limited by the relative lack of pediatric healthy blood volunteers to complete this work. Therefore, in this study we have applied this novel technology to diagnose bacteria spiked into pediatric blood and compared directly with adult blood samples. Only 180 µL of blood was utilized from both adult and pediatric volunteers and inoculated with Escherichia coli 67, and the signals generated at different time points were compared. We were able to demonstrate that the signals generated by adult and pediatric blood were not significantly different with this detection technology.

SP-R210 (Myo18A) Isoforms as Intrinsic Modulators of Macrophage Priming and Activation.

The surfactant protein (SP-A) receptor SP-R210 has been shown to increase phagocytosis of SP-A-bound pathogens and to modulate cytokine secretion by immune cells. SP-A plays an important role in pulmonary immunity by enhancing opsonization and clearance of pathogens and by modulating macrophage inflammatory responses. Alternative splicing of the Myo18A gene results in two isoforms: SP-R210S and SP-R210L, with the latter predominantly expressed in alveolar macrophages. In this study we show that SP-A is required for optimal expression of SP-R210L on alveolar macrophages. Interestingly, pre-treatment with SP-A prepared by different methods either enhances or suppresses responsiveness to LPS, possibly due to differential co-isolation of SP-B or other proteins. We also report that dominant negative disruption of SP-R210L augments expression of receptors including SR-A, CD14, and CD36, and enhances macrophages' inflammatory response to TLR stimulation. Finally, because SP-A is known to modulate CD14, we used a variety of techniques to investigate how SP-R210 mediates the effect of SP-A on CD14. These studies revealed a novel physical association between SP-R210S, CD14, and SR-A leading to an enhanced response to LPS, and found that SP-R210L and SP-R210S regulate internalization of CD14 via distinct macropinocytosis-like mechanisms. Together, our findings support a model in which SP-R210 isoforms differentially regulate trafficking, expression, and activation of innate immune receptors on macrophages.