Persistent Asthma Is Associated With Carotid Plaque in MESA.

Background Asthma and atherosclerotic cardiovascular disease share an underlying inflammatory pathophysiology. We hypothesized that persistent asthma is associated with carotid plaque burden, a strong predictor of atherosclerotic cardiovascular disease events. Methods and Results The MESA (Multi-Ethnic Study of Atherosclerosis) enrolled adults free of known atherosclerotic cardiovascular disease at baseline. Subtype of asthma was determined at examination 1. Persistent asthma was defined as asthma requiring use of controller medications, and intermittent asthma was defined as asthma without controller medications. B-mode carotid ultrasound was performed to detect carotid plaques (total plaque score [TPS], range 0-12). Multivariable regression modeling with robust variances evaluated the association of asthma subtype and carotid plaque burden. The 5029 participants were a mean (SD) age of 61.6 (10.0) years (53% were women, 26% were Black individuals, 23% were Hispanic individuals, and 12% were Chinese individuals). Carotid plaque was present in 50.5% of participants without asthma (TPS, 1.29 [1.80]), 49.5% of participants with intermittent asthma (TPS, 1.25 [1.76]), and 67% of participants with persistent asthma (TPS, 2.08 [2.35]) (P≤0.003). Participants with persistent asthma had higher interleukin-6 (1.89 [1.61] pg/mL) than participants without asthma (1.52 [1.21] pg/mL; P=0.02). In fully adjusted models, persistent asthma was associated with carotid plaque presence (odds ratio, 1.83 [95% confidence interval, 1.21-2.76]; P<0.001) and TPS (ß=0.66; P<0.01), without attenuation after adjustment for baseline interleukin-6 (P=0.02) or CRP (C-reactive protein) (P=0.01). Conclusions Participants with persistent asthma had higher carotid plaque burden and higher levels of inflammatory biomarkers, compared with participants without asthma. Adjustment for baseline inflammatory biomarkers did not attenuate the association between carotid plaque and asthma subtype, highlighting the increased atherosclerotic cardiovascular disease risk among those with persistent asthma may be multifactorial.

Dual targeting of mTOR/IL-17A and autophagy by fisetin alleviates psoriasis-like skin inflammation.

Psoriasis is a chronic autoimmune inflammatory skin disorder characterized by epidermal hyperplasia and aberrant immune response. In addition to aberrant cytokine production, psoriasis is associated with activation of the Akt/mTOR pathway. mTOR/S6K1 regulates T-lymphocyte activation and migration, keratinocytes proliferation and is upregulated in psoriatic lesions. Several drugs that target Th1/Th17 cytokines or their receptors have been approved for treating psoriasis in humans with variable results necessitating improved therapies. Fisetin, a natural dietary polyphenol with anti-oxidant and anti-proliferative properties, covalently binds mTOR/S6K1. The effects of fisetin on psoriasis and its underlying mechanisms have not been clearly defined. Here, we evaluated the immunomodulatory effects of fisetin on Th1/Th17-cytokine-activated adult human epidermal keratinocytes (HEKa) and anti-CD3/CD28-stimulated inflammatory CD4+ T cells and compared these activities with those of rapamycin (an mTOR inhibitor). Transcriptomic analysis of HEKa revealed 12,713 differentially expressed genes (DEGs) in the fisetin-treated group compared to 7,374 DEGs in the rapamycin-treated group, both individually compared to a cytokine treated group. Gene ontology analysis revealed enriched functional groups related to PI3K/Akt/mTOR signaling pathways, psoriasis, and epidermal development. Using in silico molecular modeling, we observed a high binding affinity of fisetin to IL-17A. In vitro, fisetin significantly inhibited mTOR activity, increased the expression of autophagy markers LC3A/B and Atg5 in HEKa cells and suppressed the secretion of IL-17A by activated CD4+ T lymphocytes or T lymphocytes co-cultured with HEKa. Topical administration of fisetin in an imiquimod (IMQ)-induced mouse psoriasis model exhibited a better effect than rapamycin in reducing psoriasis-like inflammation and Akt/mTOR phosphorylation and promoting keratinocyte differentiation and autophagy in mice skin lesions. Fisetin also significantly inhibited T-lymphocytes and F4/80+ macrophage infiltration into skin. We conclude that fisetin potently inhibits IL-17A and the Akt/mTOR pathway and promotes keratinocyte differentiation and autophagy to alleviate IMQ-induced psoriasis-like disease in mice. Altogether, our findings suggest fisetin as a potential treatment for psoriasis and possibly other inflammatory skin diseases.

Increased IL-6 and Potential IL-6 trans-signalling in the airways after an allergen challenge.

BACKGROUND: In asthma, IL-6 is a potential cause of enhanced inflammation, tissue damage and airway dysfunction. IL-6 signalling is regulated by its receptor, which is composed of two proteins, IL-6R and GP130. In addition to their membrane form, these two proteins may be found as extracellular soluble forms. The interaction of IL-6 with soluble IL-6R (sIL-6R) can trigger IL-6 trans-signalling in cells lacking IL-6R. Conversely, the soluble form of GP130 (sGP130) competes with its membrane form to inhibit IL-6 trans-signalling. OBJECTIVES: We aimed to analyse IL-6 trans-signalling proteins in the airways of subjects after an allergen challenge. METHODS: We used a model of segmental bronchoprovocation with an allergen (SBP-Ag) in human subjects with allergy. Before and 48 h after SBP-Ag, bronchoalveolar lavages (BALs) allowed for the analysis of proteins in BAL fluids (BALFs) by ELISA, and membrane proteins on the surface of BAL cells by flow cytometry. In addition, we performed RNA sequencing (RNA-seq) and used proteomic data to further inform on the expression of the IL-6R subunits by eosinophils, bronchial epithelial cells and lung fibroblasts. Finally, we measured the effect of IL-6 trans-signalling on bronchial fibroblasts, in vitro. RESULTS: IL-6, sIL-6R, sGP130 and the molar ratio of sIL-6R/sGP130 increased in the airways after SBP-Ag, suggesting the potential for enhanced IL-6 trans-signalling activity. BAL lymphocytes, monocytes and eosinophils displayed IL-6R on their surface and were all possible providers of sIL-6R, whereas GP130 was highly expressed in bronchial epithelial cells and lung fibroblasts. Finally, bronchial fibroblasts activated by IL-6 trans-signalling produced enhanced amounts of the chemokine, MCP-1 (CCL2). CONCLUSION AND CLINICAL RELEVANCE: After a bronchial allergen challenge, we found augmentation of the elements of IL-6 trans-signalling. Allergen-induced IL-6 trans-signalling activity can activate fibroblasts to produce chemokines that can further enhance inflammation and lung dysfunction.

Club Cell TRPV4 Serves as a Damage Sensor Driving Lung Allergic Inflammation.

Airway epithelium is the first body surface to contact inhaled irritants and report danger. Here, we report how epithelial cells recognize and respond to aeroallergen alkaline protease 1 (Alp1) of Aspergillus sp., because proteases are critical components of many allergens that provoke asthma. In a murine model, Alp1 elicits helper T (Th) cell-dependent lung eosinophilia that is initiated by the rapid response of bronchiolar club cells to Alp1. Alp1 damages bronchiolar cell junctions, which triggers a calcium flux signaled through calcineurin within club cells of the bronchioles, inciting inflammation. In two human cohorts, we link fungal sensitization and/or asthma with SNP/protein expression of the mechanosensitive calcium channel, TRPV4. TRPV4 is also necessary and sufficient for club cells to sensitize mice to Alp1. Thus, club cells detect junction damage as mechanical stress, which signals danger via TRPV4, calcium, and calcineurin to initiate allergic sensitization.

Fisetin, a 3,7,3',4'-Tetrahydroxyflavone Inhibits the PI3K/Akt/mTOR and MAPK Pathways and Ameliorates Psoriasis Pathology in 2D and 3D Organotypic Human Inflammatory Skin Models.

Psoriasis is a chronic immune-mediated skin disease that involves the interaction of immune and skin cells, and is characterized by cytokine-driven epidermal hyperplasia, deviant differentiation, inflammation, and angiogenesis. Because the available treatments for psoriasis have significant limitations, dietary products are potential natural sources of therapeutic molecules, which can repair the molecular defects associated with psoriasis and could possibly be developed for its management. Fisetin (3,7,3',4'-tetrahydroxyflavone), a phytochemical naturally found in pigmented fruits and vegetables, has demonstrated proapoptotic and antioxidant effects in several malignancies. This study utilized biochemical, cellular, pharmacological, and tissue engineering tools to characterize the effects of fisetin on normal human epidermal keratinocytes (NHEKs), peripheral blood mononuclear cells (PBMC), and CD4+ T lymphocytes in 2D and 3D psoriasis-like disease models. Fisetin treatment of NHEKs dose- and time-dependently induced differentiation and inhibited interleukin-22-induced proliferation, as well as activation of the PI3K/Akt/mTOR pathway. Fisetin treatment of TNF-α stimulated NHEKs also significantly inhibited the activation of p38 and JNK, but had enhanced effect on ERK1/2 (MAPK). In addition, fisetin treatment significantly decreased the secretion of Th1/Th-17 pro-inflammatory cytokines, particularly IFN-γ and IL-17A by 12-O-tetradecanolylphorbol 13-acetate (TPA)-stimulated NHEKs and anti-CD3/CD28-activated human PBMCs. Furthermore, we established the in vivo relevance of fisetin functions, using a 3D full-thickness human skin model of psoriasis (FTRHSP) that closely mimics in vivo human psoriatic skin lesions. Herein, fisetin significantly ameliorated psoriasis-like disease features, and decreased the production of IL-17 by CD4+ T lymphocytes co-cultured with FTRHSP. Collectively, our data identify the prodifferentiative, antiproliferative, and anti-inflammatory effects of fisetin, via modulation of the PI3K-Akt-mTOR and p38/JNK pathways and the production of cytokines in 2D and 3D human skin models of psoriasis. These results suggest that fisetin has a great potential to be developed as an effective and inexpensive agent for the treatment of psoriasis and other related inflammatory skin disorders.

Chitosan-based nanoformulated (-)-epigallocatechin-3-gallate (EGCG) modulates human keratinocyte-induced responses and alleviates imiquimod-induced murine psoriasiform dermatitis.

BACKGROUND: Psoriasis is a chronic and currently incurable inflammatory skin disease characterized by hyperproliferation, aberrant differentiation, and inflammation, leading to disrupted skin barrier function. The use of natural agents that can abrogate these effects could be useful for the treatment of psoriasis. Earlier studies have shown that treatment of keratinocytes and mouse skin with the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) mitigated inflammation and increased the expression of caspase-14 while promoting epidermal differentiation and cornification. However, bioavailability issues have restricted the development of EGCG for the treatment of psoriasis. MATERIALS AND METHODS: To overcome these limitations, we employed a chitosan-based polymeric nanoparticle formulation of EGCG (CHI-EGCG-NPs, hereafter termed nanoEGCG) suitable for topical delivery for treating psoriasis. We investigated and compared the efficacy of nanoEGCG versus native or free EGCG in vitro and in an in vivo imiquimod (IMQ)-induced murine psoriasis-like dermatitis model. The in vivo relevance and efficacy of nanoEGCG formulation (48 µg/mouse) were assessed in an IMQ-induced mouse psoriasis-like skin lesion model compared to free EGCG (1 mg/mouse). RESULTS: Like free EGCG, nanoEGCG treatment induced differentiation, and decreased proliferation and inflammatory responses in cultured keratinocytes, but with a 4-fold dose advantage. Topically applied nanoEGCG elicited a significant (p<0.01) amelioration of psoriasiform pathological markers in IMQ-induced mouse skin lesions, including reductions in ear and skin thickness, erythema and scales, proliferation (Ki-67), infiltratory immune cells (mast cells, neutrophils, macrophages, and CD4+ T cells), and angiogenesis (CD31). We also observed increases in the protein expression of caspase-14, early (keratin-10) and late (filaggrin and loricrin) markers of differentiation, and the activator protein-1 factor (JunB). Importantly, a significant modulation of several psoriasis-related inflammatory cytokines and chemokines was observed compared to the high dose of free EGCG (p<0.05). Taken together, topically applied nanoEGCG displayed a >20-fold dose advantage over free EGCG. CONCLUSION: Based on these observations, our nanoEGCG formulation represents a promising drug-delivery strategy for treating psoriasis and possibly other inflammatory skin diseases.

Control of cytokine-driven eosinophil migratory behavior by TGF-beta-induced protein (TGFBI) and periostin.

Periostin, which is induced by interleukin (IL)-13, is an extracellular matrix (ECM) protein that supports αMß2 integrin-mediated adhesion and migration of IL-5-stimulated eosinophils. Transforming growth factor (TGF)-ß-induced protein (TGFBI) is a widely expressed periostin paralog known to support monocyte adhesion. Our objective was to compare eosinophil adhesion and migration on TGFBI and periostin in the presence of IL-5-family cytokines. Eosinophil adhesion after 1 h and random motility over 20 h in the presence of various concentrations of IL-5, IL-3, or granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified in wells coated with various concentrations of TGFBI or periostin. Results were compared to video microscopy of eosinophils. Cytokine-stimulated eosinophils adhered equivalently well to TGFBI or periostin in a coating concentration-dependent manner. Adhesion was blocked by anti-αMß2 and stimulated at the lowest concentration by GM-CSF. In the motility assay, periostin was more potent than TGFBI, the coating-concentration effect was bimodal, and IL-3 was the most potent cytokine. Video microscopy revealed that under the optimal coating condition of 5 µg/ml periostin, most eosinophils migrated persistently and were polarized and acorn-shaped with a ruffling forward edge and granules gathered together, in front of the nucleus. On 10 µg/ml periostin or TGFBI, more eosinophils adopted a flattened pancake morphology with dispersed granules and nuclear lobes, and slower migration. Conversion between acorn and pancake morphologies were observed. We conclude that TGFBI or periostin supports two modes of migration by IL-5 family cytokine-activated eosinophils. The rapid mode is favored by intermediate protein coatings and the slower by higher coating concentrations. We speculate that eosinophils move by haptotaxis up a gradient of adhesive ECM protein and then slow down to surveil the tissue.

Mepolizumab Attenuates Airway Eosinophil Numbers, but Not Their Functional Phenotype, in Asthma.

RATIONALE: Mepolizumab, an IL-5-blocking antibody, reduces exacerbations in patients with severe eosinophilic asthma. Mepolizumab arrests eosinophil maturation; however, the functional phenotype of eosinophils that persist in the blood and airway after administration of IL-5 neutralizing antibodies has not been reported. OBJECTIVES: To determine the effect of anti-IL-5 antibody on the numbers and phenotypes of allergen-induced circulating and airway eosinophils. METHODS: Airway inflammation was elicited in participants with mild allergic asthma by segmental allergen challenge before and 1 month after a single intravenous 750-mg dose of mepolizumab. Eosinophils were examined in blood, bronchoalveolar lavage, and endobronchial biopsies 48 hours after challenge. MEASUREMENTS AND MAIN RESULTS: Segmental challenge without mepolizumab induced a rise in circulating eosinophils, bronchoalveolar lavage eosinophilia, and eosinophil peroxidase deposition in bronchial mucosa. IL-5 neutralization before allergen challenge abolished the allergen-induced rise in circulating eosinophils and expression of IL-3 receptors, whereas airway eosinophilia and eosinophil peroxidase deposition were blunted but not eliminated. Before mepolizumab treatment, bronchoalveolar lavage eosinophils had more surface IL-3 and granulocyte-monocyte colony-stimulating factor receptors, CD69, CD44, and CD23 and decreased IL-5 and eotaxin receptors than blood eosinophils. This activation phenotype indicated by bronchoalveolar lavage eosinophil surface markers, as well as the release of eosinophil peroxidase by eosinophils in the bronchial mucosa, was maintained after mepolizumab. CONCLUSIONS: Mepolizumab reduced airway eosinophil numbers but had a limited effect on airway eosinophil activation markers, suggesting that these cells retain functionality. This observation may explain why IL-5 neutralization reduces but does not completely eradicate asthma exacerbations. Clinical trial registered with www.clinicaltrials.gov (NCT00802438).

Endogenous Semaphorin-7A Impedes Human Lung Fibroblast Differentiation.

Semaphorin-7A is a glycosylphosphatidylinositol-anchored protein, initially characterized as an axon guidance protein. Semaphorin-7A also contributes to immune cell regulation and may be an essential pro-fibrotic factor when expressed by non-fibroblast cell types (exogenous). In mouse models, semaphorin-7A was shown to be important for TGF-ß1-induced pulmonary fibrosis characterized by myofibroblast accumulation and extracellular matrix deposition, but the cell-specific role of semaphorin-7A was not examined in fibroblasts. The purpose of this study is to determine semaphorin-7A expression by fibroblasts and to investigate the function of endogenously expressed semaphorin-7A in primary human lung fibroblasts (HLF). Herein, we show that non-fibrotic HLF expressed high levels of cell surface semaphorin-7A with little dependence on the percentage of serum or recombinant TGF-ß1. Semaphorin-7A siRNA strongly decreased semaphorin-7A mRNA expression and reduced cell surface semaphorin-7A. Reduction of semaphorin-7A induced increased proliferation and migration of non-fibrotic HLF. Also, independent of the presence of TGF-ß1, the decline of semaphorin-7A by siRNA was associated with increased α-smooth muscle actin production and gene expression of periostin, fibronectin, laminin, and serum response factor (SRF), indicating differentiation into a myofibroblast. Conversely, overexpression of semaphorin-7A in the NIH3T3 fibroblast cell line reduced the production of pro-fibrotic markers. The inverse association between semaphorin-7A and pro-fibrotic fibroblast markers was further analyzed using HLF from idiopathic pulmonary fibrosis (IPF) (n = 6) and non-fibrotic (n = 7) lungs. Using these 13 fibroblast lines, we observed that semaphorin-7A and periostin expression were inversely correlated. In conclusion, our study indicates that endogenous semaphorin-7A in HLF plays a role in maintaining fibroblast homeostasis by preventing up-regulation of pro-fibrotic genes. Therefore, endogenous and exogenous semaphorin-7A may have opposite effects on the fibroblast phenotype.

Dual Inhibition of PI3K/Akt and mTOR by the Dietary Antioxidant, Delphinidin, Ameliorates Psoriatic Features In Vitro and in an Imiquimod-Induced Psoriasis-Like Disease in Mice.

AIM: The treatment of psoriasis remains elusive, underscoring the need for identifying novel disease targets and mechanism-based therapeutic approaches. We recently reported that the PI3K/Akt/mTOR pathway that is frequently deregulated in many malignancies is also clinically relevant for psoriasis. We also provided rationale for developing delphinidin (Del), a dietary antioxidant for the management of psoriasis. This study utilized high-throughput biophysical and biochemical approaches and in vitro and in vivo models to identify molecular targets regulated by Del in psoriasis. RESULTS: A kinome-level screen and Kds analyses against a panel of 102 human kinase targets showed that Del binds to three lipid (PIK3CG, PIK3C2B, and PIK3CA) and six serine/threonine (PIM1, PIM3, mTOR, S6K1, PLK2, and AURKB) kinases, five of which belong to the PI3K/Akt/mTOR pathway. Surface plasmon resonance and in silico molecular modeling corroborated Del's direct interactions with three PI3Ks (α/c2ß/γ), mTOR, and p70S6K. Del treatment of interleukin-22 or TPA-stimulated normal human epidermal keratinocytes (NHEKs) significantly inhibited proliferation, activation of PI3K/Akt/mTOR components, and secretion of proinflammatory cytokines and chemokines. To establish the in vivo relevance of these findings, an imiquimod (IMQ)-induced Balb/c mouse psoriasis-like skin model was employed. Topical treatment of Del significantly decreased (i) hyperproliferation and epidermal thickness, (ii) skin infiltration by immune cells, (iii) psoriasis-related cytokines/chemokines, (iv) PI3K/Akt/mTOR pathway activation, and (v) increased differentiation when compared with controls. Innovation and Conclusion: Our observation that Del inhibits key kinases involved in psoriasis pathogenesis and alleviates IMQ-induced murine psoriasis-like disease suggests a novel PI3K/AKT/mTOR pathway modulator that could be developed to treat psoriasis. Antioxid. Redox Signal. 26, 49-69.

Human eosinophil activin A synthesis and mRNA stabilization are induced by the combination of IL-3 plus TNF.

Eosinophils contribute to immune regulation and wound healing/fibrosis in various diseases, including asthma. Growing appreciation for the role of activin A in such processes led us to hypothesize that eosinophils are a source of this transforming growth factor-ß superfamily member. Tumor necrosis factor-α (TNF) induces activin A by other cell types and is often present at the site of allergic inflammation along with the eosinophil-activating common ß (ßc) chain-signaling cytokines (interleukin (IL)-5, IL-3, granulocyte-macrophages colony-stimulating factor (GM-CSF)). Previously, we established that the combination of TNF plus a ßc chain-signaling cytokine synergistically induces eosinophil synthesis of the remodeling enzyme matrix metalloproteinase-9. Therefore, eosinophils were stimulated ex vivo by these cytokines and in vivo through an allergen-induced airway inflammatory response. In contrast to IL-5+TNF or GM-CSF+TNF, the combination of IL-3+TNF synergistically induced activin A synthesis and release by human blood eosinophils. IL-3+TNF enhanced activin A mRNA stability, which required sustained signaling of pathways downstream of p38 and extracellular signal-regulated kinase mitogen-activated protein kinases. In vivo, following segmental airway allergen challenge of subjects with mild allergic asthma, activin A mRNA was upregulated in airway eosinophils compared with circulating eosinophils, and ex vivo, circulating eosinophils tended to release more activin A in response to IL-3+TNF. These data provide evidence that eosinophils release activin A and that this function is enhanced when eosinophils are present in an allergen-induced inflammatory environment. Moreover, these data provide the first evidence for posttranscriptional control of activin A mRNA. We propose that an environment rich in IL-3+TNF will lead to eosinophil-derived activin A, which has an important role in regulating inflammation and/or fibrosis.

Essential Mechanisms of Differential Activation of Eosinophils by IL-3 Compared to GM-CSF and IL-5.

Compelling evidence has demonstrated that the eosinophils bring negative biological outcomes in several diseases, including eosinophilic asthma and hypereosinophilic syndromes. Eosinophils produce and store a broad range of toxic proteins and other mediators that enhance the inflammatory response and lead to tissue damage. For instance, in asthma, a close relationship has been demonstrated between increased lung eosinophilia, asthma exacerbation, and loss of lung function. The use of an anti-IL-5 therapy in severe eosinophilic asthmatic patients is efficient to reduce exacerbations. However, anti-IL-5-treated patients still display a relatively high amount of functional lung tissue eosinophils, indicating that supplemental therapies are required to damper the eosinophil functions. Our recent published works suggest that compared to IL-5, IL-3 can more strongly and differentially affect eosinophil functions. In this review, we summarize our and other investigations that have compared the effects of the three ß-chain receptor cytokines (IL-5, GM-CSF and IL-3) on eosinophil biology. We focus on how IL-3 differentially activates eosinophils compared to IL-5 or GM-CSF.

IL-3 Maintains Activation of the p90S6K/RPS6 Pathway and Increases Translation in Human Eosinophils.

IL-5 is a major therapeutic target to reduce eosinophilia. However, all of the eosinophil-activating cytokines, such as IL-5, IL-3, and GM-CSF, are typically present in atopic diseases, including allergic asthma. As a result of the functional redundancy of these three cytokines on eosinophils and the loss of IL-5R on airway eosinophils, it is important to take IL-3 and GM-CSF into account to efficiently reduce tissue eosinophil functions. Moreover, these three cytokines signal through a common ß-chain receptor but yet differentially affect protein production in eosinophils. Notably, the increased ability of IL-3 to induce the production of proteins, such as semaphorin-7A, without affecting mRNA levels suggests a unique influence of IL-3 on translation. The purpose of this study was to identify the mechanisms by which IL-3 distinctively affects eosinophil function compared with IL-5 and GM-CSF, with a focus on protein translation. Peripheral blood eosinophils were used to study intracellular signaling and protein translation in cells activated with IL-3, GM-CSF, or IL-5. We establish that, unlike GM-CSF or IL-5, IL-3 triggers prolonged signaling through activation of ribosomal protein S6 (RPS6) and the upstream kinase 90-kDa ribosomal S6 kinase (p90S6K). Blockade of p90S6K activation inhibited phosphorylation of RPS6 and IL-3-enhanced semaphorin-7A translation. Furthermore, in an allergen-challenged environment, in vivo phosphorylation of RPS6 and p90S6K was enhanced in human airway compared with circulating eosinophils. Our findings provide new insights into the mechanisms underlying differential activation of eosinophils by IL-3, GM-CSF, and IL-5. These observations identify IL-3 and its downstream intracellular signals as novel targets that should be considered to modulate eosinophil functions.

Ratios of Four STAT3 Splice Variants in Human Eosinophils and Diffuse Large B Cell Lymphoma Cells.

Signal transducer and activator of transcription 3 (STAT3) is a key mediator of leukocyte differentiation and proliferation. The 3' end of STAT3 transcripts is subject to two alternative splicing events. One results in either full-length STAT3α or in STAT3ß, which lacks part of the C-terminal transactivation domain. The other is at a tandem donor (5') splice site and results in the codon for Ser-701 being included (S) or excluded (ΔS). Despite the proximity of Ser-701 to the site of activating phosphorylation at Tyr-705, ΔS/S splicing has barely been studied. Sequencing of cDNA from purified eosinophils revealed the presence of four transcripts (S-α, ΔS-α, S-ß, and ΔS-ß) rather than the three reported in publically available databases from which ΔS-ß is missing. To gain insight into regulation of the two alternative splicing events, we developed a quantitative(q) PCR protocol to compare transcript ratios in eosinophils in which STAT3 is upregulated by cytokines, activated B cell diffuse large B cell Lymphoma (DLBCL) cells in which STAT3 is dysregulated, and in germinal center B cell-like DLBCL cells in which it is not. With the exception of one line of activated B cell DLCBL cells, the four variants were found in roughly the same ratios despite differences in total levels of STAT3 transcripts. S-α was the most abundant, followed by S-ß. ΔS-α and ΔS-ß together comprised 15.6 ± 4.0 % (mean ± SD, n = 21) of the total. The percentage of STAT3ß variants that were ΔS was 1.5-fold greater than of STAT3α variants that were ΔS. Inspection of Illumina's "BodyMap" RNA-Seq database revealed that the ΔS variant accounts for 10-26 % of STAT3 transcripts across 16 human tissues, with less variation than three other genes with the identical tandem donor splice site sequence. Thus, it seems likely that all cells contain the S-α, ΔS-α, S-ß, and ΔS-ß variants of STAT3.

Activation of SRE and AP1 by olfactory receptors via the MAPK and Rho dependent pathways.

Whereas the activation of MAPKs (mitogen activated kinases) and Rho dependant pathways by GPCR (G protein coupled receptors) has been the subject of many studies, its implication in the signalling of olfactory receptors, which constitute the largest GPCR family, has been far less analysed. Using an in vitro heterologous system, we showed that odorant activated ORs activate SRE containing promoters via the ERK pathway. We also demonstrated that RhoA and Rock kinases but not Rac were involved in ORs-induced SRE/SRF activation and that AP1 was activated, via JNK and p38 MAPKinase. Using real time PCR we found that mOR23, RnI7 and CfOR12A07 induced elevated levels of transcription factors ELK-4, srf, c-fos and c-jun mRNAs whereas mOREG induced an elevated transcription levels of c-fos and c-jun mRNA only. We showed also that odorant activated ORs stimulate the downstream MAPKs and Rho pathways in primary cultures of rat olfactory sensory neurons (OSNs). Similar results were also obtained with OE (olfactory epithelium) extracts prepared from rats exposed to odorants in vivo. Finally, we showed the important role of the AKT and MAPK signalling pathways in OSNs survival. Taken together, these data provide direct evidence that the binding of odorants onto their ORs activates the MAPK and Rho signalling pathways that are involved in OSNs survival events. This suggests that these pathways could be implicated in the regulation of OSNs homeostasis.

Potent synergistic effect of IL-3 and TNF on matrix metalloproteinase 9 generation by human eosinophils.

TNF (designated as TNF-α under previous nomenclature) is the preeminent activator of MMP-9 generation from a variety of cells including eosinophils. We have previously established that TNF strongly synergizes with IFN-γ and IL-4 for eosinophil synthesis of Th1- and Th2-type chemokines respectively. Thus, we sought to determine if TNF-induced synthesis of MMP-9 would be enhanced by the presence of Th1, Th2, or the eosinophil-associated common beta chain (ßc) cytokines. Human blood eosinophils were cultured with TNF alone or in combination with either IFN-γ, IL-4, IL-3, IL-5, or GM-CSF. Concentrations and activities of MMP-9 in eosinophil culture supernates were measured by ELISA and gelatin zymography, mRNA transcription and stabilization by quantitative real-time PCR, and signaling events by immunoblotting and intracellular flow cytometric analysis. Individually, TNF, GM-CSF, or IL-3, but not IL-4 or IFN-γ, induced relatively small (<0.2 ng/ml) but statistically significant quantities of MMP-9. Remarkable synergistic synthesis of MMP-9 (ng/ml levels) occurred in response to TNF plus IL-3, GM-CSF or IL-5, in the order of IL-3>GM-CSF>IL-5. Zymography revealed that eosinophils release MMP-9 in its pro-form. Eosinophil stimulation with the combination of IL-3 plus TNF led to increased steady-state levels of MMP-9 mRNA, prolonged mRNA stabilization, and enhanced activation of ERK1/2 phosphorylation. Inhibition of NF-κB, MEK kinase, or p38 MAP kinase, but not JNK signaling pathways, diminished IL-3/TNF-induced MMP-9 mRNA and protein production. Thus, the synergistic regulation of eosinophil MMP-9 by IL-3 plus TNF likely involves cooperative interaction of multiple transcription factors downstream from ERK, p38, and NF-κB activation as well as post-transcriptional regulation of MMP-9 mRNA stabilization. Our data indicate that within microenvironments rich in ßc-family cytokines and TNF, eosinophils are an important source of proMMP-9 and highlight a previously unrecognized role for synergistic interaction between TNF and ßc-family cytokines, particularly IL-3, for proMMP-9 synthesis.

The peptidyl-prolyl isomerase Pin1 facilitates cytokine-induced survival of eosinophils by suppressing Bax activation.

The mechanisms by which cytokine signals prevent the activation and mitochondrial targeting of the proapoptotic protein Bax are unclear. Here we show, using primary human eosinophils, that in the absence of the prosurvival cytokines granulocyte-macrophage colony-stimulating factor and interleukin 5, Bax spontaneously underwent activation and initiated mitochondrial disruption. Inhibition of Bax resulted in less eosinophil apoptosis, even in the absence of cytokines. Granulocyte-macrophage colony-stimulating factor induced activation of the kinase Erk1/2, which phosphorylated Thr167 of Bax; this facilitated new interaction of Bax with the prolyl isomerase Pin1. Blockade of Pin1 led to cleavage and mitochondrial translocation of Bax and caspase activation, regardless of the presence of cytokines. Our findings indicate that Pin1 is a key mediator of prosurvival signaling and is a regulator of Bax function.