RESUMEN
Eosinophilia is a hallmark of allergic airway inflammation (AAI). Identifying key molecules and specific signaling pathways that regulate eosinophilic inflammation is critical for development of novel therapeutics. Tropomycin receptor kinase A (TrkA) is the high-affinity receptor for nerve growth factor. AAI is associated with increased expression of TrkA by eosinophils; however, the functional role of TrkA in regulating eosinophil recruitment and contributing to AAI is poorly understood. This study identifies, to our knowledge, a novel mechanism of eotaxin-mediated activation of TrkA and its role in regulating eosinophil recruitment by using a chemical-genetic approach to specifically inhibit TrkA kinase activity with 1-NM-PP1 in TrkAF592A-knock-in (TrkA-KI) eosinophils. Blockade of TrkA by 1-NM-PP1 enhanced eosinophil spreading on VCAM-1 but inhibited eotaxin-1 (CCL11)-mediated eosinophil migration, calcium flux, cell polarization, and ERK1/2 activation, suggesting that TrkA is an important player in the signaling pathway activated by eotaxin-1 during eosinophil migration. Further, blockade of matrix metalloprotease with BB-94 inhibited eotaxin-1-induced TrkA activation and eosinophil migration, additively with 1-NM-PP1, indicating a role for matrix metalloproteases in TrkA activation. TrkA inhibition in Alternaria alternata-challenged TrkA-KI mice markedly inhibited eosinophilia and attenuated various features of AAI. These findings are indicative of a distinctive eotaxin-mediated TrkA-dependent signaling pathway, which, in addition to other TrkA-activating mediators, contributes to eosinophil recruitment during AAI and suggests that targeting the TrkA signaling pathway to inhibit eosinophil recruitment may serve as a therapeutic strategy for management of eosinophilic inflammation in allergic airway disease, including asthma.
Asunto(s)
Alternaria/fisiología , Alternariosis/inmunología , Asma/inmunología , Eosinófilos/inmunología , Hipersensibilidad/inmunología , Receptor trkA/metabolismo , Hipersensibilidad Respiratoria/inmunología , Animales , Movimiento Celular , Células Cultivadas , Quimiocina CCL11/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Mutantes , Mutación/genética , Receptor trkA/genética , Transducción de SeñalRESUMEN
Aim/Purpose: Exposure to various allergens has been shown to increase expression of ORMDL3 in the lung in models of allergic asthma. Studies using genetically modified (transgenic or knock out) mice have revealed some of the functions of ORMDL3 in asthma pathogenesis, although amid debate. The goal of this study was to use targeted post-transcriptional downregulation of ORMDL3 in allergen-challenged wild-type (WT) mice by RNA interference to further elucidate the functional role of ORMDL3 in asthma pathogenesis and evaluate a potential therapeutic option.Methods: Allergen (ovalbumin [OVA])-challenged WT mice were administered intranasally (i.n) with a single dose of five short hairpin RNA (shRNA) constructs with different target sequence for murine ORMDL3 cloned in a lentiviral vector or with the empty vector (control). Mice were evaluated for allergen-induced airway hyperresponsiveness (AHR) and various features of airway inflammation after 72 hours.Results: I.n administration of a single dose of ORMDL3 shRNAs to OVA-challenged mice resulted in reduction of ORMDL3 gene expression in the lungs associated with a significant reduction in AHR to inhaled methacholine and in the number of inflammatory cells recruited in the airways, specifically eosinophils, as well as in airway mucus secretion compared to OVA-challenged mice that received the empty vector. Administration of ORMDL3 shRNAs also significantly inhibited levels of IL-13, eotaxin-2 and sphingosine in the lungs. Additionally, ORMDL3 shRNAs significantly inhibited the allergen-mediated increase in monohexyl ceramides C22:0 and C24:0.Conclusions: Post-transcriptional down regulation of ORMDL3 in allergic lungs using i.n-delivered ORMDL3 shRNA (akin to inhaled therapy) attenuates development of key features of airway allergic disease, confirming the involvement of ORMDL3 in allergic asthma pathogenesis and serving as a model for a potential therapeutic strategy.
Asunto(s)
Alérgenos/metabolismo , Inflamación/metabolismo , Pulmón/metabolismo , Proteínas de la Membrana/metabolismo , ARN Interferente Pequeño/metabolismo , Hipersensibilidad Respiratoria/metabolismo , Animales , Asma/tratamiento farmacológico , Asma/metabolismo , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/metabolismo , Líquido del Lavado Bronquioalveolar/química , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Inflamación/tratamiento farmacológico , Pulmón/efectos de los fármacos , Masculino , Cloruro de Metacolina/farmacología , Ratones , Ratones Noqueados , Ratones Transgénicos , Eosinofilia Pulmonar/tratamiento farmacológico , Eosinofilia Pulmonar/metabolismo , Interferencia de ARN/efectos de los fármacos , Hipersensibilidad Respiratoria/tratamiento farmacológicoRESUMEN
Galectin-1 (Gal-1), a glycan-binding protein with broad antiinflammatory activities, functions as a proresolving mediator in autoimmune and chronic inflammatory disorders. However, its role in allergic airway inflammation has not yet been elucidated. We evaluated the effects of Gal-1 on eosinophil function and its role in a mouse model of allergic asthma. Allergen exposure resulted in airway recruitment of Gal-1-expressing inflammatory cells, including eosinophils, as well as increased Gal-1 in extracellular spaces in the lungs. In vitro, extracellular Gal-1 exerted divergent effects on eosinophils that were N-glycan- and dose-dependent. At concentrations ≤0.25 µM, Gal-1 increased eosinophil adhesion to vascular cell adhesion molecule-1, caused redistribution of integrin CD49d to the periphery and cell clustering, but inhibited ERK(1/2) activation and eotaxin-1-induced migration. Exposure to concentrations ≥1 µM resulted in ERK(1/2)-dependent apoptosis and disruption of the F-actin cytoskeleton. At lower concentrations, Gal-1 did not alter expression of adhesion molecules (CD49d, CD18, CD11a, CD11b, L-selectin) or of the chemokine receptor CCR3, but decreased CD49d and CCR3 was observed in eosinophils treated with higher concentrations of this lectin. In vivo, allergen-challenged Gal-1-deficient mice exhibited increased recruitment of eosinophils and CD3(+) T lymphocytes in the airways as well as elevated peripheral blood and bone marrow eosinophils relative to corresponding WT mice. Further, these mice had an increased propensity to develop airway hyperresponsiveness and displayed significantly elevated levels of TNF-α in lung tissue. This study suggests that Gal-1 can limit eosinophil recruitment to allergic airways and suppresses airway inflammation by inhibiting cell migration and promoting eosinophil apoptosis.
Asunto(s)
Asma/etiología , Eosinofilia/etiología , Galectina 1/fisiología , Animales , Apoptosis , Adhesión Celular , Quimiocinas/análisis , Citocinas/análisis , Eosinófilos/fisiología , Galectina 1/análisis , Pulmón/química , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Altered epithelial physical and functional barrier properties along with TH1/TH2 immune dysregulation are features of allergic asthma. Regulation of junction proteins to improve barrier function of airway epithelial cells has the potential for alleviation of allergic airway inflammation. OBJECTIVE: We sought to determine the immunomodulatory effect of knob protein of the adenoviral capsid on allergic asthma and to investigate its mechanism of action on airway epithelial junction proteins and barrier function. METHODS: Airway inflammation, including junction protein expression, was evaluated in allergen-challenged mice with and without treatment with knob. Human bronchial epithelial cells were exposed to knob, and its effects on expression of junction proteins and barrier integrity were determined. RESULTS: Administration of knob to allergen-challenged mice suppressed airway inflammation (eosinophilia, airway hyperresponsiveness, and IL-5 levels) and prevented allergen-induced loss of airway epithelial occludin and E-cadherin expression. Additionally, knob decreased expression of TH2-promoting inflammatory mediators, specifically IL-33, by murine lung epithelial cells. At a cellular level, treatment of human bronchial epithelial cells with knob activated c-Jun N-terminal kinase, increased expression of occludin and E-cadherin, and enhanced epithelial barrier integrity. CONCLUSION: Increased expression of junction proteins mediated by knob leading to enhanced epithelial barrier function might mitigate the allergen-induced airway inflammatory response, including asthma.
Asunto(s)
Proteínas de la Cápside/farmacología , Proteínas de la Cápside/uso terapéutico , Células Epiteliales/efectos de los fármacos , Adenoviridae , Anciano , Animales , Bronquios/citología , Líquido del Lavado Bronquioalveolar/inmunología , Cadherinas/metabolismo , Línea Celular , Citocinas/inmunología , Eosinofilia/inmunología , Células Epiteliales/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos BALB C , Persona de Mediana Edad , Ocludina/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Hipersensibilidad Respiratoria/tratamiento farmacológico , Hipersensibilidad Respiratoria/inmunologíaRESUMEN
Fatty acid binding protein 4 (FABP4), a member of a family of lipid-binding proteins, is known to play a role in inflammation by virtue of its ability to regulate intracellular events such as lipid fluxes and signaling. Studies have indicated a proinflammatory role for FABP4 in allergic asthma although its expression and function in eosinophils, the predominant inflammatory cells recruited to allergic airways, were not investigated. We examined expression of FABP4 in murine eosinophils and its role in regulating cell recruitment in vitro as well as in cockroach antigen (CRA)-induced allergic airway inflammation. CRA exposure led to airway recruitment of FABP4-expressing inflammatory cells, specifically eosinophils, in wild-type (WT) mice. FABP4 expression in eosinophils was induced by TNF-α as well as IL-4 and IL-13. FABP4-deficient eosinophils exhibited markedly decreased cell spreading/formation of leading edges on vascular cell adhesion molecule-1 and significantly decreased adhesion to intercellular adhesion molecule-1 associated with reduced ß2-integrin expression relative to WT cells. Furthermore, FABP4-deficient eosinophils exhibited decreased migration, F-actin polymerization, calcium flux, and ERK(1/2) phosphorylation in response to eotaxin-1. In vivo, CRA-challenged FABP4-deficient mice exhibited attenuated eosinophilia and significantly reduced airway inflammation (improved airway reactivity, lower IL-5, IL-13, TNF-α, and cysteinyl leukotriene C4 levels, decreased airway structural changes) compared with WT mice. In conclusion, expression of FABP4 in eosinophils is induced during conditions of inflammation and plays a proinflammatory role in the development of allergic asthma by promoting eosinophil adhesion and migration and contributing to the development of various aspects of airway inflammation.
Asunto(s)
Movimiento Celular , Eosinófilos/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Hipersensibilidad/metabolismo , Sistema de Señalización de MAP Quinasas , Animales , Adhesión Celular/genética , Citocinas/genética , Citocinas/metabolismo , Eosinófilos/patología , Proteínas de Unión a Ácidos Grasos/genética , Hipersensibilidad/genética , Hipersensibilidad/patología , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismoRESUMEN
BACKGROUND: HSPGs are glycoproteins containing covalently attached heparan sulfate (HS) chains which bind to growth factors, chemokines, etc., and regulate various aspects of inflammation including cell recruitment. We previously showed that deletion of endothelial N-acetylglucosamine N-deacetylase-N-sulfotransferase-1 (Ndst1), an enzyme responsible for N-sulfation during HS biosynthesis, reduces allergic airway inflammation (AAI). Here, we investigated the importance of O-sulfation mediated by uronyl 2-O-sulfotransferase (Hs2st) in development of AAI relative to N-sulfation. METHODS: Mice deficient in endothelial and leukocyte Hs2st (Hs2stf/fTie2Cre+) or Ndst1 (Ndst1f/fTie2Cre+) and WT mice were challenged with Alternaria alternata and evaluated for airway inflammation. Trafficking of murine eosinophils on lung endothelial cells was examined in vitro under conditions of flow. RESULTS: Exposure to Alternaria decreased expression level of Hs2st in WT mice while level of Ndst1 remained unchanged. Compared to WT mice, Alternaria-challenged Hs2stf/fTie2Cre+ mice exhibited significantly increased eosinophils in the bone marrow, bronchoalveolar lavage fluid [BALF] and lung tissue associated with persistent airway hyperresponsiveness, airway mucus hypersecretion and elevated Th2 cytokines. In contrast, Alternaria-challenged Ndst1f/fTie2Cre+ mice exhibited a marked reduction in airway eosinophilia, mucus secretion and smooth muscle mass compared to WT counterparts. While BALF eotaxins were lower in Alternaria-challenged Hs2stf/fTie2Cre+ relative to WT mice, they were not reduced to background levels as in allergen-challenged Ndst1f/fTie2Cre+ mice. Trafficking of murine eosinophils under conditions of flow in vitro was similar on Hs2st-deficient and WT endothelial cells. Expression of ZO-1 in Hs2st-deficient lung blood vessels in control and allergen-challenged mice was significantly lower than in WT counterparts. CONCLUSIONS: Our study demonstrates that allergen exposure reduces expression of Hs2st; loss of uronyl 2-O-sulfation in endothelial and leukocyte HSPG amplifies recruitment of eosinophils likely due to a compromised vascular endothelium resulting in persistent inflammation whereas loss of N-sulfation limits eosinophilia and attenuates inflammation underscoring the importance of site-specific sulfation in HSPG to their role in AAI.
Asunto(s)
Eosinófilos/patología , Proteoglicanos de Heparán Sulfato/metabolismo , Inflamación/metabolismo , Hipersensibilidad Respiratoria/metabolismo , Sulfotransferasas/metabolismo , Alérgenos/farmacología , Alternaria/patogenicidad , Animales , Movimiento Celular , Células Endoteliales/patología , Eosinofilia/etiología , Pulmón/patología , Ratones , Hipersensibilidad Respiratoria/etiologíaRESUMEN
Heparan sulfate (HS) proteoglycans (HSPGs) participate in several aspects of inflammation because of their ability to bind to growth factors, chemokines, interleukins and extracellular matrix proteins as well as promote inflammatory cell trafficking and migration. We investigated whether HSPGs play a role in the development of airway remodeling during chronic allergic asthma using mice deficient in endothelial- and leukocyte-expressed N-deacetylase/N-sulfotransferase-1 (Ndst1), an enzyme involved in modification reactions during HS biosynthesis. Ndst1-deficient and wild-type (WT) mice exposed to repetitive allergen (ovalbumin [OVA]) challenge were evaluated for the development of airway remodeling. Chronic OVA-challenged WT mice exhibited increased HS expression in the lungs along with airway eosinophilia, mucus hypersecretion, peribronchial fibrosis, increased airway epithelial thickness and smooth muscle mass. In OVA-challenged Ndst1-deficient mice, lung eosinophil and macrophage infiltration as well as airway mucus accumulation, peribronchial fibrosis and airway epithelial thickness were significantly lower than in allergen-challenged WT mice along with a trend toward decreased airway smooth muscle mass. Leukocyte and endothelial Ndst 1 deficiency also resulted in significantly decreased expression of IL-13 as well as remodeling-associated mediators such as VEGF, FGF-2 and TGF-ß1 in the lung tissue. At a cellular level, exposure to eotaxin-1 failed to induce TGF-ß1 expression by Ndst1-deficient eosinophils relative to WT eosinophils. These studies suggest that leukocyte and endothelial Ndst1-modified HS contribute to the development of allergen-induced airway remodeling by promoting recruitment of inflammatory cells as well as regulating expression of pro-remodeling factors such as IL-13, VEGF, TGF-ß1 and FGF-2 in the lung.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Alérgenos/inmunología , Células Endoteliales/química , Heparitina Sulfato/metabolismo , Leucocitos/química , Modelos Animales , Animales , Células Endoteliales/metabolismo , Heparitina Sulfato/química , Inflamación/metabolismo , Leucocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteoglicanos/química , Proteoglicanos/metabolismoRESUMEN
BACKGROUND: The cell-surface protein CD38 mediates airway smooth muscle (ASM) contractility by generating cyclic ADP-ribose, a calcium-mobilizing molecule. In human ASM cells, TNF-α augments CD38 expression transcriptionally by NF-κB and AP-1 activation and involving MAPK and PI3K signaling. CD38-/- mice develop attenuated airway hyperresponsiveness following allergen or cytokine challenge. The post-transcriptional regulation of CD38 expression in ASM is relatively less understood. In ASM, microRNAs (miRNAs) regulate inflammation, contractility, and hyperproliferation. The 3' Untranslated Region (3'UTR) of CD38 has multiple miRNA binding sites, including a site for miR-708. MiR-708 is known to regulate PI3K/AKT signaling and hyperproliferation of other cell types. We investigated miR-708 expression, its regulation of CD38 expression and the underlying mechanisms involved in such regulation in human ASM cells. METHODS: Growth-arrested human ASM cells from asthmatic and non-asthmatic donors were used. MiRNA and mRNA expression were measured by quantitative real-time PCR. CD38 enzymatic activity was measured by a reverse cyclase assay. Total and phosphorylated MAPKs and PI3K/AKT as well as enzymes that regulate their activation were determined by Western blot analysis of cell lysates following miRNA transfection and TNF-α stimulation. Dual luciferase reporter assays were performed to determine whether miR-708 binds directly to CD38 3'UTR to alter gene expression. RESULTS: Using target prediction algorithms, we identified several miRNAs with potential CD38 3'UTR target sites and determined miR-708 as a potential candidate for regulation of CD38 expression based on its expression and regulation by TNF-α. TNF-α caused a decrease in miR-708 expression in cells from non-asthmatics while it increased its expression in cells from asthmatics. Dual luciferase reporter assays in NIH-3 T3 cells revealed regulation of expression by direct binding of miR-708 to CD38 3'UTR. In ASM cells, miR-708 decreased CD38 expression by decreasing phosphorylation of JNK MAPK and AKT. These effects were associated with increased expression of MKP-1, a MAP kinase phosphatase and PTEN, a phosphatase that terminates PI3 kinase signaling. CONCLUSIONS: In human ASM cells, TNF-α-induced CD38 expression is regulated by miR-708 directly binding to 3'UTR and indirectly by regulating JNK MAPK and PI3K/AKT signaling and has the potential to control airway inflammation, ASM contractility and proliferation.
Asunto(s)
ADP-Ribosil Ciclasa 1/biosíntesis , Sistema de Señalización de MAP Quinasas/fisiología , Glicoproteínas de Membrana/biosíntesis , MicroARNs/fisiología , Miocitos del Músculo Liso/metabolismo , Fosfohidrolasa PTEN/biosíntesis , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Animales , Células Cultivadas , Humanos , Ratones , Ratones Noqueados , Células 3T3 NIH , Mucosa Respiratoria/metabolismoRESUMEN
Eosinophils are the predominant inflammatory cells recruited to allergic airways. In this article, we show that human and murine eosinophils express SWAP-70, an intracellular RAC-binding signaling protein, and examine its role in mediating eosinophil trafficking and pulmonary recruitment in a murine model of allergic airway inflammation. Compared with wild-type eosinophils, SWAP-70-deficient (Swap-70(-/-)) eosinophils revealed altered adhesive interactions within inflamed postcapillary venules under conditions of blood flow by intravital microscopy, exhibiting enhanced slow rolling but decreased firm adhesion. In static adhesion assays, Swap-70(-/-) eosinophils adhered poorly to VCAM-1 and ICAM-1 and exhibited inefficient leading edge and uropod formation. Adherent Swap-70(-/-) eosinophils failed to translocate RAC1 to leading edges and displayed aberrant cell surface localization/distribution of α4 and Mac-1. Chemokine-induced migration of Swap-70(-/-) eosinophils was significantly decreased, correlating with reduced intracellular calcium levels, defective actin polymerization/depolymerization, and altered cytoskeletal rearrangement. In vivo, recruitment of eosinophils to the lungs of allergen-challenged Swap-70(-/-) mice, compared with wild-type mice, was significantly reduced, along with considerable attenuation of airway inflammation, indicated by diminished IL-5, IL-13, and TNF-α levels; reduced mucus secretion; and improved airway function. These findings suggest that regulation of eosinophil trafficking and migration by SWAP-70 is important for the development of eosinophilic inflammation after allergen exposure.
Asunto(s)
Movimiento Celular , Proteínas de Unión al ADN , Eosinófilos/patología , Factores de Intercambio de Guanina Nucleótido , Proteínas Nucleares , Hipersensibilidad Respiratoria/etiología , Alérgenos , Animales , Adhesión Celular , Humanos , Inflamación , Ratones , Antígenos de Histocompatibilidad Menor , Hipersensibilidad Respiratoria/patología , Vénulas/patologíaRESUMEN
Obesity is an important risk factor for asthma but the mechanistic basis for this association is not well understood. In the current study, the impact of obesity on lung inflammatory responses after allergen exposure was investigated. C57BL/6 mice maintained on a high-fat diet (HFD) or a normal diet (ND) after weaning were sensitized and challenged with cockroach allergen (CRA). Airway inflammation was assessed based on inflammatory cell recruitment, measurement of lung Th1-Th2 cytokines, chemokines, eicosanoids, and other proinflammatory mediators as well as airway hyperresponsiveness (AHR). CRA-challenged mice fed a HFD exhibited significantly decreased allergen-induced airway eosinophilia along with reduced lung IL-5, IL-13, LTC4, CCL11, and CCL2 levels as well as reduced mucus secretion and smooth muscle mass compared to ND fed mice. However, allergen-challenged HFD fed mice demonstrated significantly increased PAI-1 and reduced PGE2 levels in the lung relative to corresponding ND fed mice. Interestingly, saline-exposed HFD fed mice demonstrated elevated baseline levels of TGF-ß1, arginase-1, hypoxia-inducible factor-1α, and lung collagen expression associated with decreased lung function compared to corresponding ND fed mice. These studies indicate that a HFD inhibits airway eosinophilia while altering levels of PAI-1 and PGE2 in response to CRA in mice. Further, a HFD can lead to the development of lung fibrosis even in the absence of allergen exposure which could be due to innate elevated levels of specific profibrotic factors, potentially affecting lung function during asthma.
Asunto(s)
Alérgenos/efectos adversos , Dieta Alta en Grasa/efectos adversos , Eosinofilia/prevención & control , Fibrosis Pulmonar/etiología , Enfermedades Respiratorias/prevención & control , Animales , Arginasa/metabolismo , Asma/etiología , Asma/inmunología , Hiperreactividad Bronquial/inmunología , Quimiocinas/metabolismo , Cucarachas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Eicosanoides/metabolismo , Eosinofilia/inmunología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/inmunología , Pulmón/patología , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/complicaciones , Obesidad/inmunología , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , Enfermedades Respiratorias/inmunología , Factores de Riesgo , Serpina E2/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-D-mannoside ß1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5(-/-)) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5(-/-) eosinophils. Interestingly, allergen-challenged Mgat5(-/-) mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5(-/-) mice. Furthermore, there was significantly increased recruitment of infused Mgat5(-/-) neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5(-/-) neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.
Asunto(s)
Eosinófilos/metabolismo , Inflamación , Infiltración Neutrófila , Neutrófilos/metabolismo , Polisacáridos/química , Animales , Líquido del Lavado Bronquioalveolar , Carbohidratos/química , Movimiento Celular , Quimiotaxis , Hipersensibilidad/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , N-Acetilglucosaminiltransferasas/metabolismo , Células Th2/citologíaRESUMEN
Trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the phosphatidylinositol 3-kinase (PI3K) family of signaling molecules. The role played by the p110δ subunit of PI3K (PI3K p110δ) in regulating eosinophil trafficking and recruitment was investigated using a selective pharmacological inhibitor (IC87114). Treatment with the PI3K p110δ inhibitor significantly reduced murine bone marrow-derived eosinophil (BM-Eos) adhesion to VCAM-1 as well as ICAM-1 and inhibited activation-induced changes in cell morphology associated with reduced Mac-1 expression and aberrant cell surface localization/distribution of Mac-1 and α4. Infused BM-Eos demonstrated significantly decreased rolling and adhesion in inflamed cremaster muscle microvessels of mice treated with IC87114 compared with vehicle-treated mice. Furthermore, inhibition of PI3K p110δ significantly attenuated eotaxin-1-induced BM-Eos migration and prevented eotaxin-1-induced changes in the cytoskeleton and cell morphology. Knockdown of PI3K p110δ with siRNA in BM-Eos resulted in reduced rolling, adhesion, and migration, as well as inhibition of activation-induced changes in cell morphology, validating its role in regulating trafficking and migration. Finally, in a mouse model of cockroach antigen-induced allergic airway inflammation, oral administration of the PI3K p110δ inhibitor significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyperresponsiveness in response to methacholine, reduced mucus secretion, and expression of proinflammatory molecules (found in inflammatory zone-1 and intelectin-1). Overall, these findings indicate the important role played by PI3K p110δ in mediating BM-Eos trafficking and migration by regulating adhesion molecule expression and localization/distribution as well as promoting changes in cell morphology that favor recruitment during inflammation.
Asunto(s)
Asma/inmunología , Eosinofilia/inmunología , Eosinófilos/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Sistema Respiratorio/inmunología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Asma/metabolismo , Células de la Médula Ósea , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocina CCL11/metabolismo , Fosfatidilinositol 3-Quinasa Clase I , Eosinofilia/metabolismo , Eosinófilos/metabolismo , Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno de Macrófago-1/biosíntesis , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinazolinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Sistema Respiratorio/metabolismo , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The role played by the beta-galactoside-binding lectin galectin-3 (Gal-3) in airway remodeling, a characteristic feature of asthma that leads to airway dysfunction and poor clinical outcome in humans, was investigated in a murine model of chronic allergic airway inflammation. Wild-type (WT) and Gal-3 knockout (KO) mice were subjected to repetitive allergen challenge with OVA up to 12 wk, and bronchoalveolar lavage fluid (BALF) and lung tissue collected after the last challenge were evaluated for cellular features associated with airway remodeling. Compared to WT mice, chronic OVA challenge in Gal-3 KO mice resulted in diminished remodeling of the airways with significantly reduced mucus secretion, subepithelial fibrosis, smooth muscle thickness, and peribronchial angiogenesis. The higher degree of airway remodeling in WT mice was associated with higher Gal-3 expression in the BALF as well as lung tissue. Cell counts in BALF and lung immunohistology demonstrated that eosinophil infiltration in OVA-challenged Gal-3 KO mice was significantly reduced compared with that WT mice. Evaluation of cellular mediators associated with eosinophil recruitment and airway remodeling revealed that levels of eotaxin-1, IL-5, IL-13, found in inflammatory zone 1, and TGF-beta were substantially lower in Gal-3 KO mice. Finally, leukocytes from Gal-3 KO mice demonstrated decreased trafficking (rolling) on vascular endothelial adhesion molecules compared with that of WT cells. Overall, these studies demonstrate that Gal-3 is an important lectin that promotes airway remodeling via airway recruitment of inflammatory cells, specifically eosinophils, and the development of a Th2 phenotype as well as increased expression of eosinophil-specific chemokines and profibrogenic and angiogenic mediators.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Alérgenos/inmunología , Galectina 3/inmunología , Inflamación/inmunología , Remodelación de las Vías Aéreas (Respiratorias)/genética , Animales , Western Blotting , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Quimiocina CCL11/genética , Quimiocina CCL11/metabolismo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Eosinófilos/patología , Femenino , Citometría de Flujo , Galectina 3/deficiencia , Galectina 3/genética , Interleucina-13/metabolismo , Interleucina-5/metabolismo , Rodamiento de Leucocito/inmunología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The effect of targeted inactivation of the gene encoding N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in the biosynthesis of heparan sulfate (HS) chains, on the inflammatory response associated with allergic inflammation in a murine model of OVA-induced acute airway inflammation was investigated. OVA-exposed Ndst1(f/f)TekCre(+) (mutant) mice deficient in endothelial and leukocyte Ndst1 demonstrated significantly decreased allergen-induced airway hyperresponsiveness and inflammation characterized by a significant reduction in airway recruitment of inflammatory cells (eosinophils, macrophages, neutrophils, and lymphocytes), diminished IL-5, IL-2, TGF-beta1, and eotaxin levels, as well as decreased expression of TGF-beta1 and the angiogenic protein FIZZ1 (found in inflammatory zone 1) in lung tissue compared with OVA-exposed Ndst1(f/f)TekCre(-) wild-type littermates. Furthermore, murine eosinophils demonstrated significantly decreased rolling on lung endothelial cells (ECs) from mutant mice compared with wild-type ECs under conditions of flow in vitro. Treatment of wild-type ECs, but not eosinophils, with anti-HS Abs significantly inhibited eosinophil rolling, mimicking that observed with Ndst1-deficient ECs. In vivo, trafficking of circulating leukocytes in lung microvessels of allergen-challenged Ndst1-deficient mice was significantly lower than that observed in corresponding WT littermates. Endothelial-expressed HS plays an important role in allergic airway inflammation through the regulation of recruitment of inflammatory cells to the airways by mediating interaction of leukocytes with the vascular endothelium. Furthermore, HS may also participate by sequestering and modulating the activity of allergic asthma-relevant mediators such as IL-5, IL-2, and TGF-beta1.
Asunto(s)
Células Endoteliales/enzimología , Heparitina Sulfato/deficiencia , Leucocitos/enzimología , Hipersensibilidad Respiratoria/patología , Sulfotransferasas/deficiencia , Animales , Quimiotaxis , Inflamación/etiología , Inflamación/patología , Interleucina-2/análisis , Interleucina-5/análisis , Ratones , Hipersensibilidad Respiratoria/etiología , Factor de Crecimiento Transformador beta1/análisisRESUMEN
Allergic inflammation is associated with increased generation and trafficking of inflammatory cells, especially eosinophils, to sites of inflammation. The effect of acute versus chronic airway allergen challenge on hematopoietic activity in the bone marrow (BM) and lungs was investigated using murine models of allergic airway inflammation. Acute allergen challenge induced proliferation of BM cells and significantly increased generation of eosinophil, but not multipotent, granulocyte-macrophage (GM), or B-lymphocyte progenitor cells. However, no hematopoietic activity was observed in the lungs. With chronic challenge, BM cells failed to proliferate, but exhibited increased capacity to generate multipotent as well as eosinophil, GM, and B-lymphocyte progenitors. In addition, increased generation of eosinophil- and GM-specific progenitors was observed in the lungs. Although no differences were observed in their ability to roll on BM endothelium in vitro or in vivo, CD34-enriched hematopoietic/stem progenitor cells (HSPCs) from chronic-, but not acute-, challenged mice demonstrated reduced migration across BM endothelial cells associated with decreased CXCR4 expression. Overall, these studies demonstrate that chronic allergen exposure can alter BM homing due to decreased transendothelial migration enabling noninteracting HSPCs to egress out of the BM and recruit to sites of inflammation such as the airways, resulting in extramedullary hematopoiesis.
Asunto(s)
Alérgenos/inmunología , Hematopoyesis Extramedular/inmunología , Inflamación/inmunología , Pulmón/inmunología , Alérgenos/administración & dosificación , Animales , Antígenos CD34/inmunología , Línea Celular , Movimiento Celular/inmunología , Eosinofilia/inmunología , Eosinofilia/metabolismo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Inflamación/metabolismo , Pulmón/metabolismo , Ratones , Células Madre Multipotentes/inmunología , Células Madre Multipotentes/metabolismo , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/metabolismo , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Receptores CXCR4/metabolismoRESUMEN
Cellular inflammation, with elevated levels of Th1/Th2 cytokines, airway mucus hypersecretion, and thickening of the airway smooth muscle, are characteristic features of the allergic lung. Assessment of pathophysiological changes in allergic lungs serves as an important tool to determine disease progression and understand the underlying mechanisms of pathogenesis. This can be achieved by evaluating the lung tissue for inflammation and airway structural changes along with the measurement of important pro-inflammatory mediators such as Th1/Th2 cytokines and eotaxins. This chapter describes procedures to histologically evaluate inflammatory and pathological changes observed during allergic airway inflammation using lung tissue from mice.
Asunto(s)
Alérgenos/administración & dosificación , Asma/inmunología , Pulmón/inmunología , Hipersensibilidad Respiratoria/inmunología , Coloración y Etiquetado/métodos , Balance Th1 - Th2 , Animales , Asma/inducido químicamente , Asma/metabolismo , Asma/patología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Progresión de la Enfermedad , Pulmón/metabolismo , Pulmón/patología , Ratones , Microtomía/métodos , Moco/inmunología , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/patología , Adhesión en Parafina/métodos , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/metabolismo , Hipersensibilidad Respiratoria/patología , Células TH1/inmunología , Células TH1/patología , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Exposure to environmental tobacco smoke (ETS) is known to contribute to and exacerbate inflammatory diseases of the lung such as chronic obstructive pulmonary disease (COPD) and asthma. The effect of ETS on angiogenesis and leukocyte recruitment, both of which promote lung inflammation, was investigated using lung tissue from mice exposed to aged and diluted sidestream cigarette smoke or fresh air for 12 weeks and transplanted into dorsal skin-fold chambers in nude mice. Lung tissue from mice exposed to cigarette smoke for 12 weeks exhibited significantly increased vascular density (angiogenesis) associated with selectin-mediated increased intravascular leukocyte rolling and adhesion compared to fresh air-exposed lung tissue by intravital microscopy. Further, neutrophils from nicotine-exposed mice displayed significantly increased rolling and adhesion compared to control neutrophils in microvessels of nicotine-exposed lungs versus control lung microvessels, suggesting that nicotine in cigarette smoke can augment leukocyte-endothelial interactions. ETS-induced angiogenesis and leukocyte trafficking may play a key role in airway recruitment of inflammatory cells in ETS-associated disorders such as COPD bronchitis or asthma.
Asunto(s)
Quimiotaxis de Leucocito , Pulmón/irrigación sanguínea , Microvasos/inmunología , Neovascularización Patológica , Contaminación por Humo de Tabaco/efectos adversos , Animales , Adhesión Celular , Inflamación/etiología , Inflamación/patología , Rodamiento de Leucocito , Leucocitos/patología , Ratones , Neutrófilos/patologíaRESUMEN
Arachidonic acid metabolites resulting from the cyclooxygenase (COX), lipoxygenase, and cytochrome P450 oxidase enzymatic pathways play pro- and anti-inflammatory roles in allergic airway inflammation (AAI) and asthma. Expression of COX-2 and soluble epoxide hydrolase (sEH) are elevated in allergic airways and their enzymatic products (e.g., prostaglandins and diols of epoxyeicosatrienoic acids, respectively) have been shown to participate in the pathogenesis of AAI. Here, we evaluated the outcome of inhibiting the COX-2 and sEH enzymatic pathways with a novel dual inhibitor, PTUPB, in A. alternata-induced AAI. Allergen-challenged mice were administered with 10 or 30 mg/kg of PTUPB, celecoxib (selective COX-2 inhibitor), t-TUCB (selective sEH inhibitor) or vehicle daily by gavage and evaluated for various features of AAI. PTUPB and t-TUCB at 30 mg/kg, but not celecoxib, inhibited eosinophilic infiltration and significantly increased levels of anti-inflammatory EETs in the lung tissue of allergen-challenged mice. t-TUCB significantly inhibited allergen-induced IL-4 and IL-13, while a less pronounced reduction was noted with PTUPB and celecoxib. Additionally, t-TUCB markedly inhibited eotaxin-2, an eosinophil-specific chemokine, which was only marginally reduced by PTUPB and remained elevated in celecoxib-treated mice. PTUPB or t-TUCB administration reversed allergen-induced reduction in levels of various lipid mediators in the lungs, with only a minimal effect noted with celecoxib. Despite the anti-inflammatory effects, PTUPB or t-TUCB did not reduce allergen-induced airway hyperresponsiveness (AHR). However, development of structural changes in the allergic airways, such as mucus hypersecretion and smooth muscle hypertrophy, was significantly inhibited by both inhibitors. Celecoxib, on the other hand, inhibited only airway smooth muscle hypertrophy, but not mucus hypersecretion. In conclusion, dual inhibition of COX-2 and sEH offers no additional advantage relative to sEH inhibition alone in attenuating various features associated with A. alternata-induced AAI, while COX-2 inhibition exerts only moderate or no effect on several of these features. Dual sEH/COX-2 inhibition may be useful in treating conditions where eosinophilic inflammation co-exists with pain-associated inflammation.
RESUMEN
BACKGROUND: Accurate identification of eosinophils in the gastrointestinal (GI) tract of dogs with eosinophilic GI disease (EGID) by histological evaluation is challenging. The currently used hematoxylin and eosin (H&E) staining method detects intact eosinophils but does not detect degranulated eosinophils, thus potentially underrepresenting the number of infiltrating eosinophils. OBJECTIVE: To develop a more sensitive method for identifying and quantifying both intact and degranulated eosinophils to diagnose EGID more accurately. METHODS: Endoscopically obtained paraffin-embedded intestinal biopsy specimens from dogs with GI signs were examined. The study groups were dogs with eosinophilic enteritis (EE), lymphoplasmacytic and mixed enteritis, and control dogs with GI signs but no histologic changes on tissue sections. Consecutive sections were immunolabeled with monoclonal antibodies (mAbs) against the eosinophil granule protein eosinophil peroxidase (Epx) and stained by H&E, respectively. The number of eosinophils was manually quantified and classified as intact or degranulated. RESULTS: The number of intact eosinophils detected in Epx mAb-labeled duodenal sections was significantly higher compared with that in H&E-stained sections, with a similar relationship noted in the colon and stomach. The Epx mAb allowed the unique assessment of eosinophil degranulation. The number of intact and degranulated eosinophils was significantly higher in duodenal lamina propria of the EE and mixed group compared to the control group. CONCLUSION: Immunohistochemical detection of Epx provides a more precise method to detect GI tract eosinophils compared to H&E staining and could be used as an alternative and reliable diagnostic tool for assessment of biopsy tissues from dogs with EGID.
Asunto(s)
Enfermedades de los Perros/patología , Enteritis/veterinaria , Eosinofilia/veterinaria , Eosinófilos/patología , Gastritis/veterinaria , Animales , Colorantes/uso terapéutico , Enfermedades de los Perros/diagnóstico , Perros , Duodeno/patología , Enteritis/diagnóstico , Enteritis/patología , Eosinofilia/diagnóstico , Eosinofilia/patología , Femenino , Gastritis/diagnóstico , Gastritis/patología , Inmunohistoquímica/veterinaria , MasculinoRESUMEN
Prevalence of food allergies in the United States is on the rise. Eosinophils are recruited to the intestinal mucosa in substantial numbers in food allergen-driven gastrointestinal (GI) inflammation. Soluble epoxide hydrolase (sEH) is known to play a pro-inflammatory role during inflammation by metabolizing anti-inflammatory epoxyeicosatrienoic acids (EETs) to pro-inflammatory diols. We investigated the role of sEH in a murine model of food allergy and evaluated the potential therapeutic effect of a highly selective sEH inhibitor (trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]-cyclohexyloxy}-benzoic acid [t-TUCB]). Oral exposure of mice on a soy-free diet to soy protein isolate (SPI) induced expression of intestinal sEH, increased circulating total and antigen-specific IgE levels, and caused significant weight loss. Administration of t-TUCB to SPI-challenged mice inhibited IgE levels and prevented SPI-induced weight loss. Additionally, SPI-induced GI inflammation characterized by increased recruitment of eosinophils and mast cells, elevated eotaxin 1 levels, mucus hypersecretion, and decreased epithelial junction protein expression. In t-TUCB-treated mice, eosinophilia, mast cell recruitment, and mucus secretion were significantly lower than in untreated mice and SPI-induced loss of junction protein expression was prevented to variable levels. sEH expression in eosinophils was induced by inflammatory mediators TNF-α and eotaxin-1. Treatment of eosinophils with t-TUCB significantly inhibited eosinophil migration, an effect that was mirrored by treatment with 11,12-EET, by inhibiting intracellular signaling events such as ERK (1/2) activation and eotaxin-1-induced calcium flux. These studies suggest that sEH induced by soy proteins promotes allergic responses and GI inflammation including eosinophilia and that inhibition of sEH can attenuate these responses.