Temporal Changes in Microrna Expression in Blood Leukocytes from Patients with the Acute Respiratory Distress Syndrome.

BACKGROUND: MicroRNA (miRNA) control gene transcription by binding to and repressing the translation of messenger RNA (mRNA). Their role in the acute respiratory distress syndrome (ARDS) is undefined. METHODS: Blood leukocytes from 51 patients enrolled in a prior randomized trial of corticosteroids for ARDS were analyzed. After screening eight patients with microarrays for altered miRNA expression, 25 miRNAs were selected for further analysis using RT-PCR in all 51 patients. RESULTS: On day 0, the 51 patients had APACHE III score of 60.4 ±â€Š17.7 and PaO2/FiO2 of 117 ±â€Š49. 21 miRNA were expressed at increased levels in blood leukocytes at the onset of ARDS compared with healthy controls. These miRNA remained elevated at day 3 and increased further by day 7 (log2 fold change from 0.66 to 5.7 fold, P <0.05 compared to day 0). In a subgroup analysis (37 patients treated with corticosteroids and 14 treated with placebo), the interaction of miRNA expression over time and steroid administration was not significant suggesting that systemic corticosteroids had no effect on the miRNA detected in our study. In contrast, corticosteroids but not placebo decreased IL-6 and C-reactive protein at day 3 (P < 0.001) demonstrating an early systemic anti-inflammatory response whereas both treatment arms had decreased values by day 7 (P <0.001). CONCLUSIONS: Expression of miRNA is increased in blood leukocytes of patients with ARDS at day 0 and day 3 and rises further by day 7, when systemic inflammation is subsiding. These effects appear independent of the administration of steroids, suggesting different inflammatory modifying roles for each in the resolving phases of ARDS.

Activation of heat shock response augments fibroblast growth factor-1 expression in wounded lung epithelium.

We previously showed that coincident exposure to heat shock (HS; 42°C for 2 h) and TNF-α synergistically induces apoptosis in mouse lung epithelium. We extended this work by analyzing HS effects on human lung epithelial responses to clinically relevant injury. Cotreatment with TNF-α and HS induced little caspase-3 and poly(ADP-ribose) polymerase cleavage in human small airway epithelial cells, A549 cells, and BEAS2B cells. Scratch wound closure rates almost doubled when A549 and BEAS2B cells and air-liquid interface cultures of human bronchial epithelial cells were heat shocked immediately after wounding. Microarray, qRT-PCR, and immunoblotting showed fibroblast growth factor 1 (FGF1) to be synergistically induced by HS and wounding. Enhanced FGF1 expression in HS/wounded A549 was blocked by inhibitors of p38 MAPK (SB203580) or HS factor (HSF)-1 (KNK-437) and in HSF1 knockout BEAS2B cells. PCR demonstrated FGF1 to be expressed from the two most distal promoters in wounded/HS cells. Wound closure in HS A549 and BEAS2B cells was reduced by FGF receptor-1/3 inhibition (SU-5402) or FGF1 depletion. Exogenous FGF1 accelerated A549 wound closure in the absence but not presence of HS. In the presence of exogenous FGF1, HS slowed wound closure, suggesting that it increases FGF1 expression but impairs FGF1-stimulated wound closure. Frozen sections from normal and idiopathic pulmonary fibrosis (IPF) lung were analyzed for FGF1 and HSP70 by immunofluorescence confocal microscopy and qRT-PCR. FGF1 and HSP70 mRNA levels were 7.5- and 5.9-fold higher in IPF than normal lung, and the proteins colocalized to fibroblastic foci in IPF lung. We conclude that HS signaling may have an important impact on gene expression contributing to lung injury, healing, and fibrosis.

1918 Influenza receptor binding domain variants bind and replicate in primary human airway cells regardless of receptor specificity.

The 1918 influenza pandemic caused ~50 million deaths. Many questions remain regarding the origin, pathogenicity, and mechanisms of human adaptation of this virus. Avian-adapted influenza A viruses preferentially bind α2,3-linked sialic acids (Sia) while human-adapted viruses preferentially bind α2,6-linked Sia. A change in Sia preference from α2,3 to α2,6 is thought to be a requirement for human adaptation of avian influenza viruses. Autopsy data from 1918 cases, however, suggest that factors other than Sia preference played a role in viral binding and entry to human airway cells. Here, we evaluated binding and entry of five 1918 influenza receptor binding domain variants in a primary human airway cell model along with control avian and human influenza viruses. We observed that all five variants bound and entered cells efficiently and that Sia preference did not predict entry of influenza A virus to primary human airway cells evaluated in this model.

Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages.

PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat-containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1ß secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator.

Validation of normal human bronchial epithelial cells as a model for influenza A infections in human distal trachea.

Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic.

The fish oil ingredient, docosahexaenoic acid, activates cytosolic phospholipase A2 via GPR120 receptor to produce prostaglandin E2 and plays an anti-inflammatory role in macrophages.

Docosahexaenoic acid (DHA) is one of the major ingredients of fish oil and has been reported to have anti-inflammatory properties mediated through the GPR120 receptor. Whether cytosolic phospholipase A2 (cPLA2 ) and lipid mediators produced from cPLA2 activation are involved in the anti-inflammatory role of DHA in macrophages has not been reported. We report here that DHA and the GPR120 agonist, GW9508, activate cPLA2 and cyclooxygenase 2 (COX-2), and cause prostaglandin E2 (PGE2) release in a murine macrophage cell line RAW264.7 and in human primary monocyte-derived macrophages. DHA and GW9508 activate cPLA2 via GPR120 receptor, G protein Gαq and scaffold protein ß-arrestin 2. Extracellular signal-regulated kinase 1/2 activation is involved in DHA- and GW9508-induced cPLA2 activation, but not p38 mitogen-activated protein kinase. The anti-inflammatory role of DHA and GW9508 is in part via activation of cPLA2 , COX-2 and production of PGE2 as a cPLA2 inhibitor or a COX-2 inhibitor partially reverses the DHA- and GW9508-induced inhibition of lipopolysaccharide-induced interleukin-6 secretion. The cPLA2 product arachidonic acid and PGE2 also play an anti-inflammatory role. This effect of PGE2 is partially through inhibition of the nuclear factor-κB signalling pathway and through the EP4 receptor of PGE2 because an EP4 inhibitor or knock-down of EP4 partially reverses DHA inhibition of lipopolysaccharide-induced interleukin-6 secretion. Hence, DHA has an anti-inflammatory effect partially through induction of PGE2.

Low molecular weight hyaluronan activates cytosolic phospholipase A2α and eicosanoid production in monocytes and macrophages.

Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A2 group IVA (cPLA2α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA2α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA2α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E2 production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA2α inhibitor blocked HA-induced AA release and PGE2 production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA2α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE2 production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE2 production and COX2 expression were blocked in Tlr4(-/-) and Myd88(-/-) mice, but not in Cd44(-/-) mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA2α/COX2(high) and COX1/ALOX15/ALOX5/LTA4H(low) gene and PGE2/PGD2/15-HETE(high) and LXA4(low) eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.

Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells.

We studied the changes in expression of microRNAs (miRNAs or miRs) and mRNA in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture. After 28 days in ALI, the epithelial cells differentially expressed basal, ciliated, and goblet cell markers. Using Affymetrix microarrays, 20 human miRNAs were found to be up-regulated, whereas 35 miRNAs were found to be down-regulated in differentiated cells compared with undifferentiated cells. An analysis of changes in global mRNA expression revealed that 1,201 probe sets demonstrated an 8-fold change (FC) or greater at Day 28 of ALI culture. Of these, 816 were up-regulated and 385 were down-regulated. With differentiation, miR-449a increased (FC, 38.15), and was related to changes in mRNA for cell division cycle 25 homolog A (FC, 0.11). MiR-455 decreased (FC, 0.12) and was related to changes in mRNA for the epithelial cell marker, mucin 1 (FC, 136). Transfection with anti-miR-449 or miR-455-3p resulted in changes in target protein expression (cell division cycle 25 homolog A and mucin 1, respectively), whereas transfection with reporter genes with 3'-untranslated regions of these targets confirmed control of expression through that structure. Therefore, changes in specific miRNAs during human airway epithelial cell differentiation control gene and protein expression important for differentiation.

Abnormal nasal nitric oxide production, ciliary beat frequency, and Toll-like receptor response in pulmonary nontuberculous mycobacterial disease epithelium.

RATIONALE: Pulmonary nontuberculous mycobacterial (PNTM) disease has increased over the past several decades, especially in older women. Despite extensive investigation, no consistent immunological abnormalities have been found. Using evidence from diseases such as cystic fibrosis and primary ciliary dyskinesia, in which mucociliary dysfunction predisposes subjects to high rates of nontuberculous mycobacterial disease that increase with age, we investigated correlates of mucociliary function in subjects with PNTM infections and healthy control subjects. OBJECTIVES: To define ex vivo characteristics of PNTM disease. METHODS: From 2009 to 2012, 58 subjects with PNTM infections and 40 control subjects were recruited. Nasal nitric oxide (nNO) was determined at the time of respiratory epithelial collection. Ciliary beat frequency at rest and in response to Toll-like receptor (TLR) and other agonists was determined using high-speed video microscopy. MEASUREMENTS AND MAIN RESULTS: We found decreased nNO production, abnormally low resting ciliary beat frequency, and abnormal responses to agonists of TLR2, -3, -5, -7/8, and -9 in subjects with PNTM compared with healthy control subjects. The low ciliary beat frequency in subjects with PNTM was normalized ex vivo by augmentation of the NO-cyclic guanosine monophosphate pathway without normalization of their TLR agonist responses. CONCLUSIONS: Impaired nNO, ciliary beat frequency, and TLR responses in PNTM disease epithelium identify possible underlying susceptibility mechanisms as well as possible avenues for directed investigation and therapy.

HIV infection and antiretroviral therapy have divergent effects on mitochondria in adipose tissue.

BACKGROUND: Although human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) affect mitochondrial DNA (mtDNA) content and function, comprehensive evaluations of their effects on mitochondria in muscle, adipose tissue, and blood cells are limited. METHODS: Mitochondrial DNA quantification, mitochondrial genome sequencing, and gene expression analysis were performed on muscle, adipose tissue, and peripheral blood mononuclear cell (PBMC) samples from untreated HIV-positive patients, HIV-positive patients receiving nucleoside reverse transcriptase inhibitor (NRTI)-based ART, and HIV-negative controls. RESULTS: The adipose tissue mtDNA/nuclear DNA (nDNA) ratio was increased in untreated HIV-infected patients (ratio, 353) and decreased in those receiving ART (ratio, 162) compared with controls (ratio, 255; P < .05 for both comparisons); the difference between the 2 HIV-infected groups was also significant (P = .002). In HIV-infected participants, mtDNA/nDNA in adipose tissue correlated with the level of activation (CD38+ /HLA-DR+) for CD4+ and CD8+ lymphocytes. No significant differences in mtDNA content were noted in muscle or PMBCs among groups. Exploratory DNA microarray analysis identified differential gene expression between patient groups, including a subset of adipose tissue genes. CONCLUSIONS: HIV infection and ART have opposing effects on mtDNA content in adipose tissue; immune activation may mediate the effects of HIV, whereas NRTIs likely mediate the effects of ART.

Impact of animal strain on gene expression in a rat model of acute cardiac rejection.

BACKGROUND: The expression levels of many genes show wide natural variation among strains or populations. This study investigated the potential for animal strain-related genotypic differences to confound gene expression profiles in acute cellular rejection (ACR). Using a rat heart transplant model and 2 different rat strains (Dark Agouti, and Brown Norway), microarrays were performed on native hearts, transplanted hearts, and peripheral blood mononuclear cells (PBMC). RESULTS: In heart tissue, strain alone affected the expression of only 33 probesets while rejection affected the expression of 1368 probesets (FDR 10% and FC > o= 3). Only 13 genes were affected by both strain and rejection, which was < 1% (13/1368) of all probesets differentially expressed in ACR. However, for PBMC, strain alone affected 265 probesets (FDR 10% and FC > or = 3) and the addition of ACR had little further effect. Pathway analysis of these differentially expressed strain effect genes connected them with immune response, cell motility and cell death, functional themes that overlap with those related to ACR. After accounting for animal strain, additional analysis identified 30 PBMC candidate genes potentially associated with ACR. CONCLUSION: In ACR, genetic background has a large impact on the transcriptome of immune cells, but not heart tissue. Gene expression studies of ACR should avoid study designs that require cross strain comparisons between leukocytes.

Cytosolic phospholipase A2alpha activation induced by S1P is mediated by the S1P3 receptor in lung epithelial cells.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) activation is a regulatory step in the control of arachidonic acid (AA) liberation for eicosanoid formation. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator involved in the regulation of many important proinflammatory processes and has been found in the airways of asthmatic subjects. We investigated the mechanism of S1P-induced AA release and determined the involvement of cPLA(2)alpha in these events in A549 human lung epithelial cells. S1P induced AA release rapidly within 5 min in a dose- and time-dependent manner. S1P-induced AA release was inhibited by the cPLA(2)alpha inhibitors methyl arachidonyl fluorophosphonate (MAFP) and pyrrolidine derivative, by small interfering RNA-mediated downregulation of cPLA(2)alpha, and by inhibition of S1P-induced calcium flux, suggesting a significant role of cPLA(2)alpha in S1P-mediated AA release. Knockdown of the S1P3 receptor, the major S1P receptor expressed on A549 cells, inhibited S1P-induced calcium flux and AA release. The S1P-induced calcium flux and AA release was associated with sphingosine kinase 1 (Sphk1) expression and activity. Furthermore, Rho-associated kinase, downstream of S1P3, was crucial for S1P-induced cPLA(2)alpha activation. Our data suggest that S1P acting through S1P3, calcium flux, and Rho kinase activates cPLA(2)alpha and releases AA in lung epithelial cells. An understanding of S1P-induced cPLA(2)alpha activation mechanisms in epithelial cells may provide potential targets to control inflammatory processes in the lung.

Immune responses to Pneumocystis murina are robust in healthy mice but largely absent in CD40 ligand-deficient mice.

Pneumocystis is a pathogen of immunocompromised hosts but can also infect healthy hosts, in whom infection is rapidly controlled and cleared. Microarray methods were used to examine differential gene expression in the lungs of C57BL/6 and CD40 ligand knockout (CD40L-KO) mice over time following exposure to Pneumocystis murina. Immunocompetent C57BL/6 mice, which control and clear infection efficiently, showed a robust response to infection characterized by the up-regulation of 349 primarily immune response-associated genes. Temporal changes in the expression of these genes identified an early (Week 2), primarily innate response, which waned before the infection was controlled; this was followed by primarily adaptive immune responses that peaked at Week 5, which coincided with clearance of the infection. In conjunction with the latter, there was an increased expression of B cell-associated (Ig) genes at Week 6 that persisted through 11 weeks. In contrast, CD40L-KO mice, which are highly susceptible to developing severe Pneumocystis pneumonia, showed essentially no up-regulation of immune response-associated genes at Days 35-75. Immunohistochemical staining supported these observations by demonstrating an increase in CD4+, CD68+, and CD19+ cells in C57BL/6 but not CD40L-KO mice. Thus, the healthy host demonstrates a robust, biphasic response to infection by Pneumocystis; CD40L is an essential upstream regulator of the adaptive immune responses that efficiently control infection and prevent development of progressive pneumonia.

A focused microarray to study human mitochondrial and nuclear gene expression.

A focused microarray (huMITOchip) was developed to study alterations of human mitochondrial and nuclear gene expression in health and disease. The huMITOchip contains 4,774 probe sets identical to the Affymetrix U 133 plus 2.0 chip covering genes affecting mitochondrial, lipid, cytokine, apoptosis, and muscle function transcripts. Unlike other gene chips, the huMITOchip has 51 probe sets that interrogate 37 genes of the mitochondrial genome. The human mitochondrial gene chip was validated against the Affymetrix U133 plus 2.0 array using an in vitro system of CCL136 muscle cell line stimulated with or without interferon gamma (IFN-gamma). The 37 genes from the mtDNA demonstrated absolute gene expression levels ranging from 0.1 to 3,182. The comparison of the two gene chips yielded an excellent Pearson's correlation coefficient (r = 0.98). At least 17 probe sets were differentially expressed in response to IFN-gamma on both chips, with a high degree of concordance. This is the first report on the development of a focused oligonucleotide microarray containing genes of the mitochondrial genome.

Leukotriene D(4) induces gene expression in human monocytes through cysteinyl leukotriene type I receptor.

BACKGROUND: Cysteinyl leukotrienes (CysLTs) are important mediators of innate immune responsiveness and chronic inflammatory diseases. CysLTs acting through CysLT receptors can influence the migration and activity of cells, such as eosinophils, monocytes, and dendritic cells. OBJECTIVE: We sought to determine the gene expression signature of human monocytes in response to CysLTs and to elucidate the signaling pathways involved in monocyte activation. METHODS: Gene expression was analyzed by using oligonucleotide microarrays. Responsiveness to CysLTs was assessed by using real-time PCR, calcium flux, kinase activation, and chemotaxis assays. RESULTS: CysLT type 1 receptor (CysLTR(1)) transcript 1 is predominantly expressed in human monocytes, and CysLTs signal through CysLTR(1) in these cells. Several immediate-early genes, including early growth response 2 and 3, FBJ murine osteosarcoma viral oncogene homolog B, activating transcription factor 3, and nuclear receptor subfamily 4 were significantly induced by leukotriene (LT) D(4). This effect was mediated by CysLTR(1) coupled to the G protein alpha inhibitory subunit, activation of phospholipase C, and inositol-1,4,5-triphosphate and store-operated calcium channels. LTD(4) induced p38 mitogen-activated protein kinase phosphorylation, a pathway also involved in the regulation of immediate-early gene expression in monocytes. LTD(4) stimulated monocyte chemotactic activity that was fully blocked by a selective CysLTR(1) inhibitor, MK571, and pertussis toxin, suggesting that CysLTR(1) coupled to the G protein alpha inhibitory subunit is a dominant functional pathway in human monocytes. CONCLUSION: Our data show that CysLTs acting through CysLTR(1) can significantly influence the activation and migration of human monocytes and that these effects can be fully inhibited by CysLTR(1) antagonists.

IFN-gamma induces cysteinyl leukotriene receptor 2 expression and enhances the responsiveness of human endothelial cells to cysteinyl leukotrienes.

Cysteinyl leukotrienes (cysLTs) are important mediators of cell trafficking and innate immune responses, involved in the pathogenesis of inflammatory processes, i.e., atherosclerosis, pulmonary fibrosis, and bronchial asthma. The aim of this study was to examine the regulation of cysLT signaling by IFN-gamma in human primary endothelial cells. IFN-gamma increased cysLT receptor 2 (CysLTR2) mRNA expression and CysLTR2-specific calcium signaling in endothelial cells. IFN-gamma signaled through Jak/STAT1, as both AG490, a Jak2 inhibitor, and expression of a STAT1 dominant-negative construct, significantly inhibited CysLTR2 mRNA expression in response to IFN-gamma. To determine mechanisms of IFN-gamma-induced CysLTR2 expression, the human CysLTR2 gene structure was characterized. The CysLTR2 gene has a TATA-less promoter, with multiple transcription start sites. It consists of six variably spliced exons. Eight different CysLTR2 transcripts were identified in endothelial and monocytic cells. Gene reporter assay showed potent basal promoter activity of a putative CysLTR2 promoter region. However, there were no significant changes in gene reporter and mRNA t(1/2) assays in response to IFN-gamma, suggesting transcriptional control of CysLTR2 mRNA up-regulation by IFN-gamma response motifs localized outside of the cloned CysLTR2 promoter region. Stimulation of endothelial cells by cysLTs induced mRNA and protein expression of early growth response genes 1, 2, and 3 and cycloxygenase-2. This response was mediated by CysLTR2 coupled to G(q/11), activation of phospholipase C, and inositol-1,4,5-triphosphate, and was enhanced further 2- to 5-fold by IFN-gamma stimulation. Thus, IFN-gamma induces CysLTR2 expression and enhances cysLT-induced inflammatory responses.

Amplified expression profiling of platelet transcriptome reveals changes in arginine metabolic pathways in patients with sickle cell disease.

BACKGROUND: In sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, which suggests that pathological platelet activation may contribute to sickle cell disease vasculopathy. METHODS AND RESULTS: Studies were therefore undertaken to examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate in the present study the feasibility of comparative platelet transcriptome studies on clinical samples from single donors by the application of RNA amplification followed by microarray-based analysis of 54,000 probe sets. Data mining an existing microarray database, we identified 220 highly abundant genes in platelets and a subset of 72 relatively platelet-specific genes, defined by >10-fold increased expression compared with the median of other cell types in the database with amplified transcripts. The highly abundant platelet transcripts found in the present study included 82% or 70% of platelet-abundant genes identified in 2 previous gene expression studies on nonamplified mRNA from pooled or apheresis samples, respectively. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to those of 12 black control subjects, at a 3-fold cutoff and 5% false-discovery rate, we identified approximately 100 differentially expressed genes, including multiple genes involved in arginine metabolism and redox homeostasis. Further characterization of these pathways with real-time polymerase chain reaction and biochemical assays revealed increased arginase II expression and activity and decreased platelet polyamine levels. CONCLUSIONS: The present studies suggest a potential pathogenic role for platelet arginase and altered arginine and polyamine metabolism in sickle cell disease and provide a novel framework for the study of disease-specific platelet biology.

Gene expression profiles of peripheral blood leukocytes after endotoxin challenge in humans.

To define gene expression profiles that occur during the initial activation of human innate immunity, we administered intravenous endotoxin (n = 8) or saline (n = 4) to healthy subjects and hybridized RNA from blood mononuclear cells (0, 0.5, 6, 24, 168 h) or whole blood (0, 3, 6, 24, 168 h) to oligonucleotide probe arrays. The greatest change in mononuclear cell gene expression occurred at 6 h (439 induced and 428 repressed genes, 1% false discovery rate, and 50% fold change) including increased expression of genes associated with pathogen recognition molecules and signaling cascades linked to receptors associated with cell mobility and activation. Induced defense response genes included cytokines, chemokines, and their respective receptors, acute-phase transcription factors, proteases, arachidonate metabolites, and oxidases. Repressed defense response genes included those associated with co-stimulatory molecules, T and cytotoxic lymphocytes, natural killer (NK) cells, and protein synthesis. Gene expression profiles of whole blood had similar biological themes. Over 100 genes not typically associated with acute inflammation were differentially regulated after endotoxin. By 24 h, gene expression had returned to baseline values. Thus the inflammatory response of circulating leukocytes to endotoxin in humans is characterized by a rapid amplification and subsidence of gene expression. These results indicate that a single intravascular exposure to endotoxin produces a large but temporally short perturbation of the blood transcriptome.

Functional characterization of human cysteinyl leukotriene 1 receptor gene structure.

The 5-lipoxygenase pathway has been strongly implicated in the pathogenesis of chronic inflammatory disorders, such as bronchial asthma and atherosclerosis. Cysteinyl leukotrienes (cysLTs), 5-lipoxygenase pathway products, are recognized now not only as important factors in asthmatic inflammation, but also as mediators of cell trafficking and innate immune responses. To study a role of cysLTs in inflammatory reactions we have characterized the gene structure of human cysteinyl leukotriene receptor type I (cysLT(1)R). The cysLT(1)R gene consists of 5 exons that are variably spliced and a single promoter region with multiple transcription start sites. Four different cysLT(1)R transcripts were identified. RT-PCR showed dominant and wide expression of the transcript I, containing exons 1, 4, and 5, with the strongest presence in blood leukocytes, spleen, thymus, lung, and heart. The expression of cysLT(1)R is functionally regulated at the transcriptional level by IL-4 through a STAT6 response element localized to the proximal cysLT(1)R promoter region. IL-4 stimulation increased cysLT(1)R mRNA (real-time PCR) and surface protein expression (flow cytometry) in a time-dependent fashion. CysLTs (LTD(4) and LTC(4)) induced an increased production of a potent monocyte chemoattractant CCL2 (MCP-1) in IL-4-primed THP-1 cells in a dose-dependent manner. This effect was effectively inhibited by the cysLT(1)R-selective antagonist MK571 in a dose-dependent manner and only partially by a nonselective cysLT(1)R/cysLT(2)R inhibitor BAY-u9773, implying a cysLT(1)R-mediated mechanism. Thus, cysLTs signaling through cysLT(1)R might contribute to inflammatory reactions by cooperating with IL-4 in enhanced CCL2 production in human monocytic cells.