Respiratory syncytial virus infection induces the release of transglutaminase 2 from human airway epithelial cells.

Respiratory syncytial virus (RSV) is an important human pathogen that causes severe lower respiratory tract infections in young children, the elderly, and the immunocompromised, yet no effective treatments or vaccines are available. The precise mechanism underlying RSV-induced acute airway disease and associated sequelae are not fully understood; however, early lung inflammatory and immune events are thought to play a major role in the outcome of the disease. Moreover, oxidative stress responses in the airways play a key role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium (Ca2+) levels, which in turn activate Ca2+-dependent enzymes, including transglutaminase 2 (TG2). Transglutaminase 2 is a multifunctional cross-linking enzyme implicated in various physiological and pathological conditions; however, its involvement in respiratory virus-induced airway inflammation is largely unknown. In this study, we demonstrated that RSV-induced oxidative stress promotes enhanced activation and release of TG2 from human lung epithelial cells as a result of its translocation from the cytoplasm and subsequent release into the extracellular space, which was mediated by Toll-like receptor (TLR)-4 and NF-κB pathways. Antioxidant treatment significantly inhibited RSV-induced TG2 extracellular release and activation via blocking viral replication. Also, treatment of RSV-infected lung epithelial cells with TG2 inhibitor significantly reduced RSV-induced matrix metalloprotease activities. These results suggested that RSV-induced oxidative stress activates innate immune receptors in the airways, such as TLRs, that can activate TG2 via the NF-κB pathway to promote cross-linking of extracellular matrix proteins, resulting in enhanced inflammation.

Proinflammatory Effects of Respiratory Syncytial Virus-Induced Epithelial HMGB1 on Human Innate Immune Cell Activation.

High mobility group box 1 (HMGB1) is a multifunctional nuclear protein that translocates to the cytoplasm and is subsequently released to the extracellular space during infection and injury. Once released, it acts as a damage-associated molecular pattern and regulates immune and inflammatory responses. Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in infants and elderly, for which no effective treatment or vaccine is currently available. This study investigated the effects of HMGB1 on cytokine secretion, as well as the involvement of NF-κB and TLR4 pathways in RSV-induced HMGB1 release in human airway epithelial cells (AECs) and its proinflammatory effects on several human primary immune cells. Purified HMGB1 was incubated with AECs (A549 and small alveolar epithelial cells) and various immune cells and measured the release of proinflammatory mediators and the activation of NF-κB and P38 MAPK. HMGB1 treatment significantly increased the phosphorylation of NF-κB and P38 MAPK but did not induce the release of cytokines/chemokines from AECs. However, addition of HMGB1 to immune cells did significantly induce the release of cytokines/chemokines and activated the NF-κB and P38 MAPK pathways. We found that activation of NF-κB accounted for RSV-induced HMGB1 secretion in AECs in a TLR4-dependent manner. These results indicated that HMGB1 secreted from AECs can facilitate the secretion of proinflammatory mediators from immune cells in a paracrine mechanism, thus promoting the inflammatory response that contributes to RSV pathogenesis. Therefore, blocking the proinflammatory function of HMGB1 may be an effective approach for developing novel therapeutics.

Wounding induces tomato Ve1 R-gene expression.

MAIN CONCLUSION: In tomato, Ve1 gene expression is induced specifically by physical damage or plant wounding, resulting in a defense/stress cascade that mimics responses during Verticillium colonization and wilt. In tomato, Verticillium resistance is determined by the Ve gene locus, which encodes two leucine-rich repeat-receptor-like proteins (Ve1, Ve2); the Ve1 gene is induced differentially while Ve2 is constitutively expressed throughout disease development. These profiles have been observed even during compatible Verticillium interactions, colonization by some bacterial pathogens, and growth of transgenic tomato plants expressing the fungal Ave1 effector, suggesting broader roles in disease and/or stress. Here, we have examined further Ve gene expression in resistant and susceptible plants under abiotic stress, including a water deficit, salinity and physical damage. Using both quantitative RT-PCR and label-free LC-MS methods, changes have been evaluated at both the mRNA and protein levels. The results indicate that Ve1 gene expression responds specifically to physical damage or plant wounding, resulting in a defense/stress cascade that resembles observations during Verticillium colonization. In addition, the elimination or reduction of Ve1 or Ve2 gene function also result in proteomic responses that occur with wilt pathogen and continue to be consistent with an antagonistic relationship between the two genes. Mutational analyses also indicate the plant wounding hormone, systemin, is not required, while jasmonic acid again appears to play a direct role in induction of the Ve1 gene.

Antagonistic function of the Ve R-genes in tomato.

Key message In Verticillium wilt, gene silencing indicates that tomato Ve2-gene expression can have a dramatic effect on many defense/stress protein levels while Ve1-gene induction modulates these effects in a negative fashion. In tomato, Verticillium resistance is dependent on the Ve R-gene locus, which encodes two leucine-rich repeat receptor-like proteins, Ve1 and Ve2. During fungal wilt, Ve1 protein is sharply induced while Ve2 appears expressed constitutively throughout disease development; the disease resistance function usually is attributed to the Ve1 receptor alone. To study Ve2 function, levels of Ve2 mRNA were suppressed using RNAi in both susceptible and resistant Craigella tomato near-isolines and protein changes were evaluated at both the mRNA and protein levels. The results indicate that Ve2-gene expression can have dramatic effects on many defense/stress protein levels while the presence of intact Ve1 protein minimizes these effects in a negative fashion. The data suggest an antagonistic relationship between the Ve proteins in which Ve1 modulates the induction of defense/stress proteins by Ve2.

Tomato Ve resistance locus; defense or growth.

MAIN CONCLUSION: Verticillium colonization does induce a cascade of defense/stress proteins but the Ve1 gene also promotes enhanced root growth, which appears to allow the plant to outgrow the pathogen and avoid symptoms associated with an exaggerated defense response. In tomato, the Ve1 gene provides resistance to the vascular pathogen, Verticillium dahliae, race 1; ve1 plants are susceptible. However, the physiological basis of the resistance is unknown. While developing alternative lines of mutant Ve1 gene transformants to address this question a striking difference was observed in transformation frequency resulting from the inefficient rooting of plantlets from ve1 callus relative to Ve1 callus. Subsequent experiments with resistant and susceptible near-isolines of the cultivar Craigella, as well as Ve1 transformants, showed that in both artificial medium and soil, root growth was significantly enhanced in the resistant cultivar. Parallel studies of Verticillium colonization indicated a significantly lower overall concentration in the resistant plant characteristic of the resistant phenotype, but an almost equal total fungal biomass in both resistant and susceptible roots. Proteomic analyses of the roots of Verticillium-infected plants revealed elevated levels of defense/stress proteins, which correlated with the fungal concentration rather than resistance. Hormone analyses demonstrated a higher cis-ABA level in the resistant isoline consistent with enhanced root growth. Taken together these studies indicate a similar fungal biomass in the roots of both isolines where the Ve1 gene also promotes root production. In the case of the Craigella/Vd1 pathosystem, this appears to allow the host to resist better by outgrowing the pathogen with less wilt rather than reliance only on partial immunity.

Cytokine-Induced Glucocorticoid Resistance from Eosinophil Activation: Protein Phosphatase 5 Modulation of Glucocorticoid Receptor Phosphorylation and Signaling.

The mechanisms contributing to persistent eosinophil activation and poor eosinopenic response to glucocorticoids in severe asthma are poorly defined. We examined the effect of cytokines typically overexpressed in the asthmatic airways on glucocorticoid signaling in in vitro activated eosinophils. An annexin V assay used to measure eosinophil apoptosis showed that cytokine combinations of IL-2 plus IL-4 as well as TNF-α plus IFN-γ, or IL-3, GM-CSF, and IL-5 alone significantly diminished the proapoptotic response to dexamethasone. We found that IL-2 plus IL-4 resulted in impaired phosphorylation and function of the nuclear glucocorticoid receptor (GCR). Proteomic analysis of steroid sensitive and resistant eosinophils identified several differentially expressed proteins, namely protein phosphatase 5 (PP5), formyl peptide receptor 2, and annexin 1. Furthermore, increased phosphatase activity of PP5 correlated with impaired phosphorylation of the GCR. Importantly, suppression of PP5 expression with small interfering RNA restored proper phosphorylation and the proapoptotic function of the GCR. We also examined the effect of lipoxin A4 on PP5 activation by IL-2 plus IL-4. Similar to PP5 small interfering RNA inhibition, pretreatment of eosinophils with lipoxin A4 restored GCR phosphorylation and the proaptoptotic function of GCs. Taken together, our results showed 1) a critical role for PP5 in cytokine-induced resistance to GC-mediated eosinophil death, 2) supported the dependence of GCR phosphorylation on PP5 activity, and 3) revealed that PP5 is a target of the lipoxin A4-induced pathway countering cytokine-induced resistance to GCs in eosinophils.

Activation of Human Peripheral Blood Eosinophils by Cytokines in a Comparative Time-Course Proteomic/Phosphoproteomic Study.

Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.

Respiratory Syncytial Virus Infection Triggers Epithelial HMGB1 Release as a Damage-Associated Molecular Pattern Promoting a Monocytic Inflammatory Response.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infant and elderly populations worldwide. Currently, there is no efficacious vaccine or therapy available for RSV infection. The molecular mechanisms underlying RSV-induced acute airway disease and associated long-term consequences remain largely unknown; however, experimental evidence suggests that the lung inflammatory response plays a fundamental role in the outcome of RSV infection. High-mobility group box 1 (HMGB1) is a nuclear protein that triggers inflammation when released from activated immune or necrotic cells and drives the pathogenesis of various infectious agents. Although HMGB1 has been implicated in many inflammatory diseases, its role in RSV-induced airway inflammation has not been investigated. This study investigates the molecular mechanism of action of extracellularly released HMGB1 in airway epithelial cells (A549 and small airway epithelial cells) to establish its role in RSV infection. Immunofluorescence microscopy and Western blotting results showed that RSV infection of human airway epithelial cells induced a significant release of HMGB1 as a result of translocation of HMGB1 from the cell nuclei to the cytoplasm and subsequent release into the extracellular space. Treating RSV-infected A549 cells with antioxidants significantly inhibited RSV-induced HMGB1 extracellular release. Studies using recombinant HMGB1 triggered immune responses by activating primary human monocytes. Finally, HMGB1 released by airway epithelial cells due to RSV infection appears to function as a paracrine factor priming epithelial cells and monocytes to inflammatory stimuli in the airways. IMPORTANCE: RSV is a major cause of serious lower respiratory tract infections in young children and causes severe respiratory morbidity and mortality in the elderly. In addition, to date there is no effective treatment or vaccine available for RSV infection. The mechanisms responsible for RSV-induced acute airway disease and associated long-term consequences remain largely unknown. The oxidative stress response in the airways plays a major role in the pathogenesis of RSV. HMGB1 is a ubiquitous redox-sensitive multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. This study investigated the mechanism of action of HMGB1 in RSV infection with the aim of identifying new inflammatory pathways at the molecular level that may be amenable to therapeutic interventions.

Myeloid differentiation protein 2 facilitates pollen- and cat dander-induced innate and allergic airway inflammation.

BACKGROUND: The National Health and Nutrition Examination Survey identified several pollens and cat dander as among the most common allergens that induce allergic sensitization and allergic diseases. We recently reported that ragweed pollen extract (RWPE) requires Toll-like receptor 4 (TLR4) to stimulate CXCL-mediated innate neutrophilic inflammation, which in turn facilitates allergic sensitization and airway inflammation. Myeloid differentiation protein 2 (MD2) is a TLR4 coreceptor, but its role in pollen- and cat dander-induced innate and allergic inflammation has not been critically evaluated. OBJECTIVE: We sought to elucidate the role of MD2 in inducing pollen- and cat dander-induced innate and allergic airway inflammation. METHODS: TCM(Null) (TLR4(Null), CD14(Null), MD2(Null)), TLR4(Hi), and TCM(Hi) cells and human bronchial epithelial cells with small interfering RNA-induced downregulation of MD2 were stimulated with RWPE, other pollen allergic extracts, or cat dander extract (CDE), and activation of nuclear factor κB (NF-κB), secretion of the NF-κB-dependent CXCL8, or both were quantified. Wild-type mice or mice with small interfering RNA knockdown of lung MD2 were challenged intranasally with RWPE or CDE, and innate and allergic inflammation was quantified. RESULTS: RWPE stimulated MD2-dependent NF-κB activation and CXCL secretion. Likewise, Bermuda, rye, timothy, pigweed, Russian thistle, cottonwood, walnut, and CDE stimulated MD2-dependent CXCL secretion. RWPE and CDE challenge induced MD2-dependent and CD14-independent innate neutrophil recruitment. RWPE induced MD2-dependent allergic sensitization and airway inflammation. CONCLUSIONS: MD2 plays an important role in induction of allergic sensitization to cat dander and common pollens relevant to human allergic diseases.

Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment.

Neutrophil recruitment is a hallmark of rapid innate immune responses. Exposure of airways of naive mice to pollens rapidly induces neutrophil recruitment. The innate mechanisms that regulate pollen-induced neutrophil recruitment and the contribution of this neutrophilic response to subsequent induction of allergic sensitization and inflammation need to be elucidated. Here we show that ragweed pollen extract (RWPE) challenge in naive mice induces C-X-C motif ligand (CXCL) chemokine synthesis, which stimulates chemokine (C-X-C motif) receptor 2 (CXCR2)-dependent recruitment of neutrophils into the airways. Deletion of Toll-like receptor 4 (TLR4) abolishes CXCL chemokine secretion and neutrophil recruitment induced by a single RWPE challenge and inhibits induction of allergic sensitization and airway inflammation after repeated exposures to RWPE. Forced induction of CXCL chemokine secretion and neutrophil recruitment in mice lacking TLR4 also reconstitutes the ability of multiple challenges of RWPE to induce allergic airway inflammation. Blocking RWPE-induced neutrophil recruitment in wild-type mice by administration of a CXCR2 inhibitor inhibits the ability of repeated exposures to RWPE to stimulate allergic sensitization and airway inflammation. Administration of neutrophils derived from naive donor mice into the airways of Tlr4 knockout recipient mice after each repeated RWPE challenge reconstitutes allergic sensitization and inflammation in these mice. Together these observations indicate that pollen-induced recruitment of neutrophils is TLR4 and CXCR2 dependent and that recruitment of neutrophils is a critical rate-limiting event that stimulates induction of allergic sensitization and airway inflammation. Inhibiting pollen-induced recruitment of neutrophils, such as by administration of CXCR2 antagonists, may be a novel strategy to prevent initiation of pollen-induced allergic airway inflammation.

Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3.

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs' effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils' response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs' TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don't upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.

Fear potentiated startle increases phospholipase D (PLD) expression/activity and PLD-linked metabotropic glutamate receptor mediated post-tetanic potentiation in rat amygdala.

Long-term memory (LTM) of fear stores activity dependent modifications that include changes in amygdala signaling. Previously, we identified an enhanced probability of release of glutamate mediated signaling to be important in rat fear potentiated startle (FPS), a well-established translational behavioral measure of fear. Here, we investigated short- and long-term synaptic plasticity in FPS involving metabotropic glutamate receptors (mGluRs) and associated downstream proteomic changes in the thalamic-lateral amygdala pathway (Th-LA). Aldolase A, an inhibitor of phospholipase D (PLD), expression was reduced, concurrent with significantly elevated PLD protein expression. Blocking the PLD-mGluR signaling significantly reduced PLD activity. While transmitter release probability increased in FPS, PLD-mGluR agonist and antagonist actions were occluded. In the unpaired group (UNP), blocking the PLD-mGluR increased while activating the receptor decreased transmitter release probability, consistent with decreased synaptic potentials during tetanic stimulation. FPS Post-tetanic potentiation (PTP) immediately following long-term potentiation (LTP) induction was significantly increased. Blocking PLD-mGluR signaling prevented PTP and reduced cumulative PTP probability but not LTP maintenance in both groups. These effects are similar to those mediated through mGluR7, which is co-immunoprecipitated with PLD in FPS. Lastly, blocking mGluR-PLD in the rat amygdala was sufficient to prevent behavioral expression of fear memory. Thus, our study in the Th-LA pathway provides the first evidence for PLD as an important target of mGluR signaling in amygdala fear-associated memory. Importantly, the PLD-mGluR provides a novel therapeutic target for treating maladaptive fear memories in posttraumatic stress and anxiety disorders.

Proteomic Analysis in Esophageal Eosinophilia Reveals Differential Galectin-3 Expression and S-Nitrosylation.

BACKGROUND AIMS: Esophageal eosinophilia (EE) can be caused by gastroesophageal reflux disease (GERD), proton-pump inhibitor-responsive EE (PPI-REE) or eosinophilic esophagitis (EoE). This study quantified protein expression and S-nitrosylation (SNO) post-translational modifications in EE to elucidate potential disease biomarkers. METHODS: Proximal and distal esophageal (DE) biopsy proteins in patients with EE and in controls were assayed for protein content and fluorescence-labeled with and without ascorbate treatment. Protein SNO was determined, and selected protein spots were identified by matrix-assisted laser desorption ionization time-of-flight/mass spectrometry. Western blot and ingenuity pathway analysis were performed. RESULTS: Ninety-one of 648 proteins showed differential expression. There were significantly altered levels of abundance for 11 proximal and 14 DE proteins. Hierarchal clustering revealed differential SNO in inflamed tissues, indicating reactive nitrogen/oxygen species involvement. Galectin-3 was upregulated in both proximal (p < 0.04) and distal (p < 0.004) esophageal EE biopsies compared to controls. In distal EE samples, galectin-3 was significantly S-nitrosylated (p < 0.004). Principal component analysis revealed sample group discrimination distally. CONCLUSION: Proteomic analysis in EE esophageal mucosa revealed a distinct abundance and nitrosylation profile, most prominently in distal biopsies. Galectin-3 was upregulated in expression and SNO, which may indicate its potential role in mucosal inflammation. These results call for more studies to be performed to investigate the role of galectin-3 in GERD, PPI-REE and EoE.

Functional proteomics for the characterization of impaired cellular responses to glucocorticoids in asthma.

In chronic airway inflammatory disorders, such as asthma, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression and the potential development of serious side effects. The mechanism of steroid resistance in asthma remains unclear and may be multifactorial. Excluding noncompliance with GC treatment, abnormal steroid pharmacokinetics, and rare genetic defects in the glucocorticoid receptor (GR), the majority of GC insensitivity in asthma can be attributed to secondary defects related to GR function. Airway inflammatory cells obtained from patients with GC-resistant asthma show a number of abnormalities in cell immune responses to GC, which suggests that there is a causative defect in GR signaling in GC-resistant cells that could be further elucidated by a functional and molecular proteomics approach. Since T cells, eosinophils, and monocytes play a major role in the pathogenesis of airway inflammation, most of the work published to date has focused on these cell types as the primary therapeutic targets in GC-insensitive asthma. We herein review several distinct techniques for the assessment of (1) the cellular response to GCs including the effect of GCs on cell viability, adhesion, and mediator release; (2) the functionality of GC receptors, including phosphorylation of the GR, nuclear translocation, and binding activities; and (3) the characterization of proteins differentially expressed in steroid-resistant cells by comparative 2DE-gel electrophoresis-based techniques and mass spectrometry. These comprehensive approaches are expected to reveal novel candidates for biomarkers of steroid insensitivity, which may lead to the development of effective therapeutic interventions for patients with chronic steroid-resistant asthma.

Priming of eosinophils by GM-CSF is mediated by protein kinase CbetaII-phosphorylated L-plastin.

The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CßII (PKCßII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCßII with 4,5-bis(4-fluoroanilino)phtalimide or PKCßII-specific small interfering RNA blocked GM-CSF-induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of α(M)ß(2) integrin, which was sensitive to PKCßII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCßII inhibitors. Similarly, inhibition of PKCßII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2-19) phosphorylated on Ser(5) upregulated α(M)ß(2) integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCßII and L-plastin in linking GM-CSF-induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCßII-mediated L-plastin phosphorylation.

Model studies on iTRAQ modification of peptides: sequence-dependent reaction specificity.

A multiplexed peptide quantification strategy using the iTRAQ reagent has been described for relative measurements of peptides in digested protein mixtures. To validate the chemical specificity of the iTRAQ reaction, we have performed a detailed study of iTRAQ reactivity with two sets of synthetic peptides. The first set of peptides had sequences of Tyr-Xaa-Ser-Glu-Gly-Leu-Ser-Lys and Tyr-Xaa-Ser-Glu-Tyr-Leu-Ser-Lys where Xaa = Ala, Pro, Trp, Tyr, or Glu and was designed to study the extent of O-acylation by iTRAQ, especially hydroxyl-containing residues in different positions. The second set of peptides included Ala-Ser-Glu-His-Ala-Xaa-Tyr-Gly where Xaa = Ser, Thr, or Tyr and was selected to investigate the effect of histidyl residues separated by one amino acid residue from seryl, tyrosyl, or threonyl residues. Our findings indicated that, in addition to variable levels of O-acylation of nonsequence-specific hydroxyl-containing residues, significant sequence-specific O-acylation of seryl, threonyl, and tyrosyl hydroxyls occurred when separated one residue removed from a histidyl residue, that is, (Tyr/Ser)-Xaa-His or His-Xaa-(Tyr/Ser/Thr). This behavior was verified by a separate spiking experiment of one of the first set of peptides into Escherichia coli protein extracts, followed by retention time targeted LC-MS/MS to demonstrate the occurrence of modifications in a complex mixture. These sequence-dependent O-acylation modifications can be confounding factors to accurate MS quantification. Reversal of peptide O-acylation by the iTRAQ reagent can be accomplished by reaction with hydroxylamine with virtually no cleavage of N-acylation and is a recommended modification of the iTRAQ protocol for many applications.

Arsenal of elevated defense proteins fails to protect tomato against Verticillium dahliae.

Although the hypersensitive reaction in foliar plant diseases has been extensively described, little is clear regarding plant defense strategies in vascular wilt diseases affecting numerous economically important crops and trees. We have examined global genetic responses to Verticillium wilt in tomato (Lycopersicon esculentum Mill.) plants differing in Ve1 resistance alleles. Unexpectedly, mRNA analyses in the susceptible plant (Ve1-) based on the microarrays revealed a very heroic but unsuccessful systemic response involving many known plant defense genes. In contrast, the response is surprisingly low in plants expressing the Ve1+ R-gene and successfully resisting the pathogen. Similarly, whole-cell protein analyses, based on 2D gel electrophoresis and mass spectrometry, demonstrate large systemic increases in a variety of known plant defense proteins in the stems of susceptible plants but only modest changes in the resistant plant. Taken together, the results indicate that the large systemic increases in plant defense proteins do not protect the susceptible plant. Indeed, since a number of the highly elevated proteins are known to participate in the plant hypersensitive response as well as natural senescence, the results suggest that some or all of the disease symptoms, including ultimate plant death, actually may be the result of this exaggerated plant response.

Viral-mediated inhibition of antioxidant enzymes contributes to the pathogenesis of severe respiratory syncytial virus bronchiolitis.

RATIONALE: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in children, for which no specific treatment or vaccine is currently available. We have previously shown that RSV induces reactive oxygen species in cultured cells and oxidative injury in the lungs of experimentally infected mice. The mechanism(s) of RSV-induced oxidative stress in vivo is not known. OBJECTIVES: To measure changes of lung antioxidant enzymes expression/activity and activation of NF-E2-related factor 2 (Nrf2), a transcription factor that regulates detoxifying and antioxidant enzyme gene expression, in mice and in infants with naturally acquired RSV infection. METHODS: Superoxide dismutase 1 (SOD 1), SOD 2, SOD 3, catalase, glutathione peroxidase, and glutathione S-transferase, as well as Nrf2 expression, were measured in murine bronchoalveolar lavage, cell extracts of conductive airways, and/or in human nasopharyngeal secretions by Western blot and two-dimensional gel electrophoresis. Antioxidant enzyme activity and markers of oxidative cell injury were measured in either murine bronchoalveolar lavage or nasopharyngeal secretions by colorimetric/immunoassays. MEASUREMENTS AND MAIN RESULTS: RSV infection induced a significant decrease in the expression and/or activity of SOD, catalase, glutathione S-transferase, and glutathione peroxidase in murine lungs and in the airways of children with severe bronchiolitis. Markers of oxidative damage correlated with severity of clinical illness in RSV-infected infants. Nrf2 expression was also significantly reduced in the lungs of viral-infected mice. CONCLUSIONS: RSV infection induces significant down-regulation of the airway antioxidant system in vivo, likely resulting in lung oxidative damage. Modulation of oxidative stress may pave the way toward important advances in the therapeutic approach of RSV-induced acute lung disease.

Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies.

Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique's power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.