Association of SNP-SNP interactions of surfactant protein genes with severity of respiratory syncytial virus infection in children.

The severity of respiratory syncytial virus (RSV) may be linked to host genetic susceptibility. Surfactant protein (SP) genetic variants have been associated with RSV severity, but the impact of SNP-SNP (single nucleotide polymorphism) interactions remains unexplored. Therefore, we employed a novel statistical model to investigate the association of SNP-SNP interactions of SFTP genes with RSV severity in two and three-interaction models. We analyzed available genotype and clinical data from prospectively enrolled 405 children diagnosed with RSV, categorizing them into moderate or severe RSV groups. Using Wang's statistical model, we studied significant associations of SNP-SNP interactions with RSV severity in a case-control design. We observed, 1) association of three interactions with increased risk of severe RSV in a two-SNP model. One intragenic interaction was between SNPs of SFTPA2, and the other two were intergenic, involving SNPs of hydrophilic and hydrophobic SPs alone. 2) association of 22 interactions with RSV severity in a three-SNP model. Among these, 20 were unique, with 12 and 10 interactions associated with increased or decreased risk of RSV severity, respectively, and included at least one SNP of either SFTPA1 or SFTPA2. All interactions were intergenic, except one among SNPs of SFTPA1. The remaining interactions were either among SNPs of hydrophilic SPs alone (n=8) or among SNPs of both hydrophilic or hydrophobic SPs (n=11). Our findings indicate that SNPs of all SFTPs may contribute to genetic susceptibility to RSV severity. However, the predominant involvement of SFTPA1 and/or SFTPA2 SNPs in these interactions underscores their significance in RSV severity.

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

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

A Pilot Proteomic Study of Vestibular Fluid From Patients With Vulvodynia.

OBJECTIVE: Many women are affected by vulvodynia, but medical therapies to date have proven ineffective. We performed a pilot study using gel-based proteomics to develop a map of proteins present in vaginal/vestibular secretions and identify proteins that could be considered for future evaluation as potential therapeutic targets. MATERIALS AND METHODS: We collected vestibular fluid from 4 controls and 4 patients with vulvodynia by placing a cotton swab in the vestibule and extracting the absorbed proteins. The proteins underwent 2-dimensional difference gel electrophoresis and mass spectrometry to develop a protein map. Immunohistochemistry was used to validate proteomic findings. RESULTS: A map was constructed of 32 of the more abundant proteins in vestibular fluid and their levels compared in control subjects and vulvodynia patients. Among these were annexin A1, interleukin 1 receptor antagonist, protein S100 A9, and a number of antiproteases and proteases. Many of these proteins differed by at least 50% between groups, but only annexin A1, one of the protease inhibitors, and immunoglobulin G κ chain were significantly different. The results with annexin A1 were validated by similar findings with immunohistochemistry. CONCLUSIONS: The findings of this pilot study demonstrate a set of vestibule mucosa proteins that differ significantly-either increasing or decreasing-in vulvodynia patients compared with controls, and several others that exhibited greater than 1.5-fold change but did not reach statistical significance. This study constitutes a proof-of-principle that an open, unbiased proteomic approach can identify molecular participants in vulvodynia, some of which had not been identified to date by hypothesis-driven studies.

Donor surfactant protein A2 polymorphism and lung transplant survival.

PURPOSE: Gene polymorphisms of surfactant proteins, key players in lung innate immunity, have been associated with various lung diseases. The aim of this study was to investigate the potential association between variations within the surfactant protein (SP)-A gene of the donor lung allograft and recipient post-transplant outcome. METHODS: Lung-transplant patients (n=192) were prospectively followed-up with pulmonary function tests, bronchoscopies with bronchoalveolar lavage and biopsies. Donor lungs were assayed for SP-A1 (6An) and SP-A2 (1An) gene polymorphism using the pyrosequencing method. Unadjusted and adjusted stratified Cox survival models are reported. RESULTS: SP-A1 and SP-A2 genotype frequency and lung transplant recipient and donor characteristics as well as cause of death are noted. Recipients were grouped per donor SP-A2 variants. Individuals that received lungs from donors with the SP-A2 1A0 (n=102) versus 1A1 variant (n=68) or SP-A2 genotype 1A01A0 (n=54) versus 1A0A1 (n=38) had greater survival at 1 year (log-rank p<0.025). No significant association was noted for SP-A1 variants. Stratified adjusted survival models for 1-year survival and diagnosis showed a reduced survival for 1A1 variant and the 1A01A1 genotype. Furthermore, when survival was conditional on 1-year survival no significance was observed, indicating that the survival difference was due to the first year's outcome associated with the 1A1 variant. CONCLUSION: Donor lung SP-A gene polymorphisms are associated with post-transplant clinical outcome. Lungs from donors with the SP-A2 variant 1A1 had a reduced survival at 1 year. The observed donor genetic differences, via innate immunity relate to the post-transplant clinical outcome.

Anti-Inflammatory Effect of Surfactant Lipid in the Vaginal Mucosa: A Pilot Study.

OBJECTIVE: The aim of the study was to test the hypothesis that vaginal administration of surfactant lipids, which may counter-balance the proinflammatory effects of vaginal surfactant protein A, will decrease proinflammatory cytokines and increase anti-inflammatory cytokines in vaginal mucosal fluid in healthy women. MATERIALS AND METHODS: Three groups of healthy cycling women were randomized to receive vaginally a single dose of the following: (1) low-dose calfactant, a type of surfactant lipids, 0.8 mg/ml; (2) high-dose calfactant 8.0 mg/ml; or (3) placebo, at the time of resolution of menses. Vaginal mucosal fluid was collected before administration and also 1 and 8 days after administration of each treatment. After 1 mo, each group was randomized to each alternative treatment; thus, for a 3-month treatment period, each group received each of the 3 treatments. Vaginal fluid was tested using a Multiplex Immunoassay System. Cytokine concentrations on day 1 and day 8 were compared with day zero and tested for significance with the Student's t test. RESULTS: Six healthy subjects completed each treatment. Subjects given high-dose calfactant had, by day 8, a significant reduction in macrophage chemotactic protein-1 and interleukin 15 (IL-15) compared with low-dose calfactant or placebo. High-dose calfactant resulted in an increase in anti-inflammatory cytokines that trended toward significance on day 1 (IL-1RA) or day 8 (IL-10). CONCLUSIONS: This pilot study in healthy women demonstrates that calfactant reduces proinflammatory cytokines and increases anti-inflammatory cytokines in the vagina. We propose that calfactant may be an effective vaginal anti-inflammatory therapy for inflammatory vaginitis and similar disorders for which current therapy is ineffective.

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.

Differential Expression of PGC1α in Intratumor Redox Subpopulations of Breast Cancer.

Our previous studies indicate that the mitochondrial redox state and its intratumor heterogeneity are associated with invasiveness and metastatic potential in human breast cancer cell models and mouse xenografts. To further study the molecular basis of redox heterogeneity, we obtained the fluorescence images of Fp (oxidized flavoproteins containing flavin adenine dinucleotide, i.e., FAD), NADH (reduced nicotinamide adenine dinucleotide), and the Fp redox ratio (FpR = Fp/(Fp + NADH)) of MDA-MB-231 xenografts by the Chance redox scanner, then isolated the intratumoral redox subpopulations by dissection according to the redox ratio image. A total of 12 subpopulations were isolated from 4 tumors (2-4 locations from each tumor). The 12 subpopulations were classified into 3 FpR groups: high FpR (HFpR, n = 4, FpR range 0.78-0.92, average 0.85), medium FpR (MFpR, n = 5, FpR range 0.39-0.68, average 0.52), and low FpR (LFpR, n = 3, FpR range 0.15-0.28, average 0.20). The RT-PCR (reverse transcription polymerase chain reaction) analysis on these redox subpopulations showed that PGC-1α is significantly upregulated in the HFpR redox group compared to the MFpR group (fold change 2.1, p = 0.008), but not significantly different between MFpR and LFpR groups, or between HFpR and LFpR groups. These results indicate that optical redox imaging (ORI)-based redox subpopulations exhibit differential expression of PGC1α gene and suggest that PGC1α might play a role in redox mediation of breast cancer progression.

Human Pulmonary Surfactant Protein SP-A1 Provides Maximal Efficiency of Lung Interfacial Films.

Pulmonary surfactant is a lipoprotein complex that reduces surface tension to prevent alveolar collapse and contributes to the protection of the respiratory surface from the entry of pathogens. Surfactant protein A (SP-A) is a hydrophilic glycoprotein of the collectin family, and its main function is related to host defense. However, previous studies have shown that SP-A also aids in the formation and biophysical properties of pulmonary surfactant films at the air-water interface. Humans, unlike rodents, have two genes, SFTPA1 and SFTPA2. The encoded proteins, SP-A1 and SP-A2, differ quantitatively or qualitatively in function. It has been shown that both gene products are necessary for tubular myelin formation, an extracellular structural form of lung surfactant. The goal of this study was to investigate potential differences in the biophysical properties of surfactants containing human SP-A1, SP-A2, or both. For this purpose, we have studied for the first time, to our knowledge, the biophysical properties of pulmonary surfactant from individual humanized transgenic mice expressing human SP-A1, SP-A2, or both SP-A1 and SP-A2, in the captive bubble surfactometer. We observed that pulmonary surfactant containing SP-A1 reaches lower surface tension after postexpansion interfacial adsorption than surfactants containing no SP-A or only SP-A2. Under interfacial compression-expansion cycling conditions, surfactant films containing SP-A1 also performed better, particularly with respect to the reorganization of the films that takes place during compression. On the other hand, addition of recombinant SP-A1 to a surfactant preparation reconstituted from the hydrophobic fraction of a porcine surfactant made it more resistant to inhibition by serum than the addition of equivalent amounts of SP-A2. We conclude that the presence of SP-A1 allows pulmonary surfactant to adopt a particularly favorable structure with optimal biophysical properties.

14-3-3 isoforms bind directly exon B of the 5'-UTR of human surfactant protein A2 mRNA.

Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5'-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722-L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms ß, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms ß, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5'-UTR mRNA.

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants.

Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5'-untranslated regions (5'-UTR) due to differential splicing. The 5'-UTR variant ACD' is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication.

DNA methylation profile and expression of surfactant protein A2 gene in lung cancer.

Knowledge of the methylation profile of genes allow for the identification of biomarkers that may guide diagnosis and effective treatment of disease. Human surfactant protein A (SP-A) plays an important role in lung homeostasis and immunity, and is encoded by two genes (SFTPA1 and SFTPA2). The goal of this study was to identify differentially methylated CpG sites in the promoter region of the SFTPA2 gene in lung cancer tissue, and to determine the correlation between the promoter's methylation profile and gene expression. For this, we collected 28 pairs of cancerous human lung tissue and adjacent noncancerous (NC) lung tissue: 17 adenocarcinoma (AC), 9 squamous cell carcinoma (SCC), and 2 AC with SCC features, and we evaluated DNA methylation of the SFTPA2 promoter region by bisulfite conversion. Our results identified a higher methylation ratio in one CpG site of the SFTPA2 gene in cancerous tissue versus NC tissue (0.36 versus 0.11, p = 0.001). When assessing AC samples, we also found cancerous tissues associated with a higher methylation ratio (0.43 versus 0.10, p = 0.02). In the SCC group, although cancerous tissue showed a higher methylation ratio (0.22 versus 0.11), this difference was not statistically significant (p = 0.35). Expression of SFTPA2 mRNA and total SP-A protein was significantly lower in cancer tissue when compared to adjacent NC tissue (p < 0.001), and correlated with the hypermethylated status of an SFTPA2 CpG site in AC samples. The findings of this pilot study may hold promise for future use of SFTPA2 as a biomarker for the diagnosis of lung cancer.

Oral administration of surfactant protein-a reduces pathology in an experimental model of necrotizing enterocolitis.

OBJECTIVES: Necrotizing enterocolitis (NEC) frequently results in significant morbidity and mortality in premature infants. Others reported that mice deficient in pulmonary surfactant protein-A (SP-A) born and raised in a nonhygienic environment succumb to significant gastrointestinal tract pathology, and enteral administration of purified SP-A significantly reduced mortality. We hypothesized that oral administration of purified SP-A can ameliorate pathology in an experimental model of neonatal NEC. METHODS: Experimental NEC was induced in newborn Sprague-Dawley rat pups by daily formula gavage and intermittent exposure to hypoxia. Purified human SP-A (5 µg/day) was administered by oral gavage. After 4 days, surviving pups were sacrificed, and intestinal pathology was assessed by histological examination of distal terminal ileal sections. Intestinal levels of inflammatory cytokines (IL-1ß, IFN-γ, and TNF-α) were assessed by enzyme-linked immunosorbent assay and levels of Toll-like receptor 4 (TLR4) by Western analysis. RESULTS: Sixty-one percent of the gavaged rat pups that survived to day 4 met the criteria for experimental NEC after hypoxia, whereas treatment with SP-A significantly reduced mortality and assessment of NEC. Intestinal levels of proinflammatory cytokines were significantly increased in pups exposed to hypoxia. Administration of SP-A to pups exposed to hypoxia significantly reduced IL-1ß and TNF-α levels, but had little effect on elevated levels of IFN-γ. SP-A treatment of hypoxia-exposed pups significantly reduced expression of intestinal TLR4, key in NEC pathogenesis. CONCLUSIONS: In a rat model of experimental neonatal NEC, oral administration of SP-A reduces intestinal levels of proinflammatory cytokines and TLR4 protein and ameliorates adverse outcomes associated with gastrointestinal pathologies.

An 11-nt sequence polymorphism at the 3'UTR of human SFTPA1 and SFTPA2 gene variants differentially affect gene expression levels and miRNA regulation in cell culture.

Surfactant protein A (SP-A) plays a vital role in maintaining normal lung function and in host defense. Two genes encode SP-A in humans (SFTPA1, SFTPA2), and several gene variants have been identified for these. We have previously shown that sequence elements of SFTPA1 and SFTPA2 3' untranslated regions (UTRs) differentially affect translation efficiency in vitro. Polymorphisms at the 3'UTRs of mRNA variants may account for differential binding of miRNAs, a class of small noncoding RNAs that regulate gene expression. In this work, we generated 3'UTR reporter constructs of the SFTPA1 and SFTPA2 variants most frequently found in the population, as well as mutants of a previously described 11-nt indel element (refSNP rs368700152). Reporter constructs were transfected in NCI-H441 cells in the presence or absence of miRNA mimics, and reporter gene expression was analyzed. We found that human miRNA mir-767 negatively affected expression of constructs containing SFTPA1 and SFTPA2 variants, whereas mir-4507 affected only constructs with 3'UTRs of SFTPA1 variants 6A, 6A(3), and 6A(4) (not containing the 11-nt element). Three miRNAs (mir-183, mir-449b, and mir-612) inhibited expression of recombinants of SFTPA2 variants and the SFTPA1 variant 6A(2), all containing the 11-nt element. Similar results were obtained for SP-A expression when these miRNAs were transfected in Chinese hamster ovary cells expressing SFTPA1 or SFTPA2 variants or in NCI-H441 cells (genotype 1A(5)/1A(5)-6A(4)/6A(4)). Moreover, transfection with a specific antagomir (antagomir-183) reversed the effects of mir-183 on SP-A mRNA levels. Our results indicate that sequence variability at the 3'UTR of SP-A variants differentially affects miRNA regulation of gene expression.

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.

Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure.

Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5' untranslated (5'UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5'UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 ß/α isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence- and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA.

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.

IL-18R1 and IL-18RAP SNPs may be associated with bronchopulmonary dysplasia in African-American infants.

INTRODUCTION: The genetic contribution to the development of bronchopulmonary dysplasia (BPD) in prematurely born infants is substantial, but information related to the specific genes involved is lacking. RESULTS: Genotype analysis revealed, after multiple comparisons correction, two significant single-nucleotide polymorphism (SNPs), rs3771150 (IL-18RAP) and rs3771171 (IL-18R1), in African Americans (AAs) with BPD (vs. AAs without BPD; q < 0.05). No associations with Caucasian (CA) BPD, AA or CA respiratory distress syndrome (RDS), or prematurity in either AAs or CAs were identified with these SNPs. Respective frequencies were 0.098 and 0.093 in infants without BPD and 0.38 for each SNP in infants with BPD. In the replication set (82 cases; 102 controls), the P values were 0.012 for rs3771150 and 0.07 for rs3771171. Combining P values using Fisher's method, overall P values were 8.31 × 10(-7) for rs3771150 and 6.33 × 10(-6) for rs3771171. DISCUSSION: We conclude that IL-18RAP and IL-18R1 SNPs identify AA infants at risk for BPD. These genes may contribute to AA BPD pathogenesis via inflammatory-mediated processes and require further study. METHODS: We conducted a case-control SNP association study of candidate genes (n = 601) or 6,324 SNPs in 1,091 prematurely born infants with gestational age <35 weeks, with or without neonatal lung disease including BPD. BPD was defined as a need for oxygen at 28 days.

Gonadal hormones and oxidative stress interaction differentially affects survival of male and female mice after lung Klebsiella pneumoniae infection.

Survival of mice after Klebsiella pneumoniae infection and phagocytosis by alveolar macrophages (AMs), in the presence or absence of ozone (O(3)) exposure prior to infection, is sex dependent. The objective of this work was to study the role of gonadal hormones, 5α-dihydrotestosterone (DHT) and 17ß-estradiol (E(2)), on mouse survival after filtered air (FA) or O(3) exposure. Gonadectomized female (G×F) and male (G×M) mice implanted with control or hormone pellets (DHT in G×F, or E(2) in G×M), exposed to O(3) (2 ppm, 3h) or FA, and infected with K. pneumoniae were monitored for survival. Survival in G×F was identical after FA or O(3) exposure; in G×M O(3) exposure resulted in lower survival compared to FA. In O(3)-exposed females, gonadectomy resulted in increased survival compared to intact females or to G×M+E(2). A similar effect was observed in G×F+DHT. The combined negative effect of oxidative stress and hormone on survival was higher for E(2). Gonadectomy eliminated (females) or minimized (males) the previously observed sex differences in survival in response to oxidative stress, and hormone treatment restored them. These findings indicate that gonadal hormones and/or oxidative stress have a significant effect on mouse survival.

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.