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.

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.

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.

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.

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.

Knockdown of Drosha in human alveolar type II cells alters expression of SP-A in culture: a pilot study.

Human surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. SP-A is synthesized and secreted by alveolar type II (ATII) cells, one of the two cell types of the distal lung epithelium (ATII and ATI). We have shown that miRNA interactions with sequence polymorphisms on the SP-A mRNA 3'UTRs mediate differential expression of SP-A1 and SP-A2 gene variants in vitro. In the present study, we describe a physiologically relevant model to study miRNA regulation of SP-A in human ATII. For these studies, we purified and cultured human ATII on an air-liquid interface matrix (A/L) or plastic wells without matrix (P). Gene expression analyses confirmed that cells cultured in A/L maintained the ATII phenotype for over 5 days, whereas P-cultured cells differentiated to ATI. When we transfected ATII with siRNAs to inhibit the expression of Drosha, a critical effector of miRNA maturation, the levels of SP-A mRNA and protein increased in a time dependent manner. We next characterized cultured ATII and ATI by studying expression of 1,066 human miRNAs using miRNA PCR arrays. We detected expression of >300 miRNAs with 24 miRNAs differentially expressed in ATII versus ATI, 12 of which predicted to bind SP-A 3'UTRs, indicating that these may be implicated in SP-A downregulation in ATI. Thus, miRNAs not only affect SP-A expression, but also may contribute to the maintenance of the ATII cell phenotype and/or the trans-differentiation of ATII to ATI cells, and may represent new molecular markers that distinguish ATII and ATI.

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.

Hyperoxia-induced alterations in the pulmonary proteome of juvenile rats.

High inspired concentrations of oxygen (hyperoxia) are often necessary to counteract tissue hypoxia during the treatment of ARDS. Reactive oxygen species generated by hyperoxic therapy may influence the expression of the pulmonary proteome and the application of discovery proteomics to the hyperoxic lung has the potential to divulge mechanisms regulating the expression of specific proteins integral to lung injury and repair. The present study examined the proteome derived from 30-day-old Sprague-Dawley rats exposed to room air (RA) and 95% O2 (Ox) for 24-72 hours using 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with MALDI-ToF/ToF mass spectrometry. A total of 870 protein spots were visualized by 2D-DIGE across all gels. Mass spectral analysis identified 51 proteins representing 187 of the 214 significantly altered spots. Molecular and cellular function analysis grouped the identified proteins into free radical scavenging, cell death, cell-to-cell signaling, and cellular movement categories. The majority of the differences in the protein spots between RA and Ox occurred at 72 hours, with albumin, annexin A6 (AnxA6), and transferrin being increased, and mitochondrial Lon peptidase 1 being decreased by at least 20%. In Ox animals, AnxA6 protein expression increased three-fold without an increase in mRNA expression. Bioinformatic analysis of the AnxA6 transcript revealed the presence of a putative internal ribosome entry site within the 5'-untranslated region. These findings indicate that hyperoxia induces significant alterations in the pulmonary proteome which are temporally related. In addition, hyperoxia selectively enhances the expression of some proteins whose transcripts contain sequence motifs, which impart translational regulation.

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.

Impact of sex and ozone exposure on the course of pneumonia in wild type and SP-A (-/-) mice.

Female mice exhibited higher survival rate than males after pneumonia, with a reversal of this pattern following ozone exposure. Surfactant protein A (SP-A) plays an important role in innate immunity and SP-A (-/-) mice were more susceptible to pneumonia than wild type mice. Here, we investigated underlying mechanisms of the differential susceptibility of mice to pneumonia. Wild type and SP-A (-/-) C57BL/6J male and female mice were exposed to ozone or filtered air (FA) and then infected intratracheally with Klebsiella pneumoniae. Blood, spleen, and lung were analyzed for bacterial counts, lung and spleen weights, and sex hormone and cortisol levels were measured in plasma within two days post-infection. We found: 1) in the absence of ozone-induced oxidative stress, males had higher level of bacterial dissemination compared to females; ozone exposure decreased pulmonary clearance in both sexes and ozone-exposed females were more affected than males; 2) ozone exposure increased lung weight, but decreased spleen weight in both sexes, and in both cases ozone-exposed females were affected the most; 3) plasma cortisol levels in infected mice changed: ozone-exposed>FA-exposed, females>males, and infected>non-infected; 4) no major sex hormone differences were observed in the studied conditions; 5) differences between wild type and SP-A (-/-) mice were observed in some of the studied conditions. We concluded that reduced pulmonary clearance, compromised spleen response to infection, and increased cortisol levels in ozone-exposed females, and the higher level of lung bacterial dissemination in FA-exposed males, contribute to the previously observed survival outcomes.

Sex differences in the response of the alveolar macrophage proteome to treatment with exogenous surfactant protein-A.

BACKGROUND: Male wild type (WT) C57BL/6 mice are less capable of clearing bacteria and surviving from bacterial pneumonia than females. However, if an oxidative stress (acute ozone exposure) occurs before infection, the advantage shifts to males who then survive at higher rates than females. We have previously demonstrated that survival in surfactant protein-A (SP-A) knockout (KO) mice compared to WT was significantly reduced. Because the alveolar macrophage (AM) is pivotal in host defense we hypothesized that SP-A and circulating sex hormones are responsible for these sex differences. We used 2D-DIGE to examine the relationship of sex and SP-A on the AM proteome. The role of SP-A was investigated by treating SP-A KO mice with exogenous SP-A for 6 and 18 hr and studying its effects on the AM proteome. RESULTS: We found: 1) less variance between KO males and females than between the WT counterparts by principal component analysis, indicating that SP-A plays a role in sex differences; 2) fewer changes in females when the total numbers of significantly changing protein spots or identified whole proteins in WT or 18 hr SP-A-treated males or females were compared to their respective KO groups; 3) more proteins with functions related to chaperones or protease balance and Nrf2-regulated proteins changed in response to SP-A in females than in males; and 4) the overall pattern of SP-A induced changes in actin-related proteins were similar in both sexes, although males had more significant changes. CONCLUSIONS: Although there seems to be an interaction between sex and the effect of SP-A, it is unclear what the responsible mechanisms are. However, we found that several of the proteins that were expressed at significantly higher levels in females than in males in WT and/or in KO mice are known to interact with the estrogen receptor and may thus play a role in the SP-A/sex interaction. These include major vault protein, chaperonin subunit 2 (beta) (CCT2), and Rho GDP alpha dissociation inhibitor. We conclude that sex differences exist in the proteome of AM derived from male and female mice and that SP-A contributes to these sex differences.

The kinetics of cardiopulmonary bypass: a dual-platform proteomics study of plasma biomarkers in pediatric patients undergoing cardiopulmonary bypass.

This study was designed to investigate the expression kinetics and patterns of plasma biomarkers throughout the pediatric cardiopulmonary bypass (CPB) procedure to help predict those patients most at risk for complications. This study sampled plasma from pediatric CPB patients at five time points before, during, and after CPB. A dual-platform proteomics approach was then utilized which incorporated two-dimensional difference gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption ionization-time-of-flight/time-of-flight tandem mass spectrometry, and multi-analyte profile (MAP) assays to identify changes in expression of plasma protein biomarkers and characterize the patterns of these changes. A combined total of 134 proteins were identified with significant changes between the two platforms, with 53 coming from 2D-DIGE, 90 from MAP, and nine proteins that were identified using both methods. The proteins were then divided into 12 major groups based on the expression patterns, and two of the most clinically relevant proteins having the greatest changes in expression were selected from each group to use as "predictor biomarkers." A potential model for prediction of patient outcome was then generated using these 24 proteins. The patterns of biomarker expression during pediatric CPB may provide insight into the prediction, prevention, or treatment of complications resulting from CPB, thereby helping to improve the outcomes of pediatric CPB patients and reduce the incidence of complications.

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.

Proteomic profiling of human plasma by iTRAQ reveals down-regulation of ITI-HC3 and VDBP by cigarette smoking.

Biomarkers in noninvasive fluids indicative of cigarette smoke's effects are urgently needed. In this pilot study, we utilized the proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to identify differentially expressed plasma proteins in healthy cigarette smokers compared to healthy nonsmokers; select proteins were further confirmed by immunoblot analysis. Significant, differentially expressed proteins identified in the plasma separated subjects based on their condition as smokers or nonsmokers. Several of the proteins identified in this study are associated with immunity and inflammatory responses and have been shown to be associated with tobacco-related diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. Proteins up-regulated in smokers included complement component 8 polypeptide chains α, ß, and γ, and mannose-binding protein C, and proteins down-regulated included inter-α-trypsin inhibitor heavy chain H3 (ITI-HC3) and vitamin D-binding protein (VDBP). In addition, gelsolin and vitronectin, known tissue leakage proteins, were up- and down-regulated, respectively. Our results demonstrate for the first time that chronic cigarette smoking can influence the expression profile of the human plasma proteome. Proteins identified in this pilot study may serve as candidate biomarkers of diseases resulting from exposure to cigarette smoke in future molecular epidemiological studies.

In vivo rescue of alveolar macrophages from SP-A knockout mice with exogenous SP-A nearly restores a wild type intracellular proteome; actin involvement.

BACKGROUND: Mice lacking surfactant protein-A (SP-A-/-; knockout; KO) exhibit increased vulnerability to infection and injury. Although many bronchoalveolar lavage (BAL) protein differences between KO and wild-type (WT) are rapidly reversed in KO after infection, their clinical course is still compromised. We studied the impact of SP-A on the alveolar macrophage (AM) proteome under basal conditions. Male SP-A KO mice were SP-A-treated (5 micrograms/mouse) and sacrificed in 6 or 18 hr. The AM proteomes of KO, SP-A-treated KO, and WT mice were studied by 2D-DIGE coupled with MALDI-ToF/ToF and AM actin distribution was examined by phalloidon staining. RESULTS: We observed: a) significant differences from KO in WT or exogenous SP-A-treated in 45 of 76 identified proteins (both increases and decreases). These included actin-related/cytoskeletal proteins (involved in motility, phagocytosis, endocytosis), proteins of intracellular signaling, cell differentiation/regulation, regulation of inflammation, protease/chaperone function, and proteins related to Nrf2-mediated oxidative stress response pathway; b) SP-A-induced changes causing the AM proteome of the KO to resemble that of WT; and c) that SP-A treatment altered cell size and F-actin distribution. CONCLUSIONS: These differences are likely to enhance AM function. The observations show for the first time that acute in vivo SP-A treatment of KO mice, under basal or unstimulated conditions, affects the expression of multiple AM proteins, alters F-actin distribution, and can restore much of the WT phenotype. We postulate that the SP-A-mediated expression profile of the AM places it in a state of "readiness" to successfully conduct its innate immune functions and ensure lung health.

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.

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.

Differences in the BAL proteome after Klebsiella pneumoniae infection in wild type and SP-A-/- mice.

BACKGROUND: Surfactant protein-A (SP-A) has been shown to play a variety of roles related to lung host defense function. Mice lacking SP-A are more susceptible to infection than wild type C57BL/6 mice. We studied bronchoalveolar lavage (BAL) protein expression in wild type and SP-A-/- mice infected with Klebsiella pneumoniae by 2D-DIGE. METHODS: Mice were infected intratracheally with K. pneumoniae and after 4 and 24 hours they were subject to BAL. Cell-free BAL was analyzed by 2D-DIGE on two-dimensional gels with pH ranges of 4-7 and 7-11. Under baseline conditions and at 4 and 24 hr post-infection BAL was compared between untreated and infected wild type and SP-A-/- mice. Sixty proteins identified by mass spectrometry were categorized as host defense, redox regulation, and protein metabolism/modification. RESULTS: We found: 1) ~75% of 32 host defense proteins were lower in uninfected SP-A-/- vs wild type, suggesting increased susceptibility to infection or oxidative injury; 2) At 4 hr post-infection > 2/3 of identified proteins were higher in SP-A-/- than wild type mice, almost the exact opposite of untreated mice; 3) At 24 hr post-infection some proteins continued increasing, but many returned to baseline; 4) In infected wild type mice significant changes occurred in 13 of 60 proteins, with 12 of 13 increasing, vs on 4 significant changes in SP-A-/- mice. Infection response patterns between strains demonstrated both commonalities and differences. In several cases changes between 4 and 24 hr followed different patterns between strains. CONCLUSIONS: These indicate that SP-A plays a key role in regulating the BAL proteome, functioning indirectly to regulate lung host defense function, possibly via the macrophage. In the absence of SP-A baseline levels of many host defense molecules are lower. However, many of these indirect deficits in SP-A-/- mice are rapidly compensated for during infection, indicating that SP-A also has a direct role on host defense against K. pneumoniae that may be instrumental in determining clinical course.

Dual-platform proteomics study of plasma biomarkers in pediatric patients undergoing cardiopulmonary bypass.

Plasma samples from pediatric cardiac patients undergoing cardiopulmonary bypass (CPB) procedures were used to identify and characterize patterns of changes in potential biomarkers related to tissue damage and inflammation. These included proteins associated with systemic inflammatory response syndrome. Potential biomarkers were identified using a dual-platform proteomics approach requiring approximately 150 microL of plasma, which included two-dimensional difference gel electrophoresis (2D-DIGE) and a multiplexed immunoassay. Methods used in the dual approach measured levels of 129 proteins in plasma from pediatric CPB patients. Of these, 70 proteins changed significantly (p<0.05) between time points, and 36 of these retained significance after the highly stringent Bonferroni correction [p<0.001 for 2D-DIGE and p<0.00056 for multianalyte profile (MAP) assays]. Many of the changing proteins were associated with tissue damage, inflammation, and oxidative stress. This study uses a novel approach that combines two discovery proteomics techniques to identify a pattern of potential biomarkers changing after CPB. This approach required only 150 microL of plasma per time point and provided quantitative information on 129 proteins. The changes in levels of expression of these proteins may provide insight into the understanding, treatment, and prevention of systemic inflammation, thereby helping to improve the outcomes of pediatric CPB patients.