[Show abstract][Hide abstract] ABSTRACT: Zinc oxide (ZnO) nanoparticles are widely used in various products, and the safety evaluation of this manufactured material is important. The present study investigated the inflammatory and fibrotic effects of pulmonary exposure to ZnO nanoparticles in a mouse model of pulmonary fibrosis. Pulmonary fibrosis was induced by constant subcutaneous infusion of bleomycin (BLM). Female C57BL/6Jcl mice were divided into BLM-treated and non-treated groups. In each treatment group, 0, 10, 20 or 30 µg of ZnO nanoparticles were delivered into the lungs through pharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and the lungs were sampled at Day 10 or 14 after administration. Pulmonary exposure by a single bolus of ZnO nanoparticles resulted in severe, but transient inflammatory infiltration and thickening of the alveolar septa in the lungs, along with the increase of total and differential cell counts in BLAF. The BALF level of interleukin (IL)-1β and transforming growth factor (TGF)-β was increased at Day 10 and 14, respectively. At Day 10, the synergistic effect of BLM and ZnO exposure was detected on IL-1β and monocyte chemotactic protein (MCP)-1 in BALF. The present study demonstrated the synergistic effect of pulmonary exposure to ZnO nanoparticles and subcutaneous infusion of BLM on the secretion of pro-fibrotic cytokines in the lungs.
International Journal of Molecular Sciences 12/2015; 16(1):660-76. DOI:10.3390/ijms16010660 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have demonstrated the beneficial activity of activated protein C in allergic diseases including bronchial asthma and rhinitis. However, the exact mechanism of action of activated protein C in allergies is unclear. In this study, we hypothesized that pharmacological doses of activated protein C can modulate allergic inflammation by inhibiting dendritic cells.
Dendritic cells were prepared using murine bone marrow progenitor cells and human peripheral monocytes. Bronchial asthma was induced in mice that received intratracheal instillation of ovalbumin-pulsed dendritic cells.
Activated protein C significantly increased the differentiation of tolerogenic plasmacytoid dendritic cells and the secretion of type I interferons, but it significantly reduced lipopolysaccharide-mediated maturation and the secretion of inflammatory cytokines in myeloid dendritic cells. Activated protein C also inhibited maturation and the secretion of inflammatory cytokines in monocyte-derived dendritic cells. Activated protein C-treated dendritic cells were less effective when differentiating naïve CD4 T-cells from Th1 or Th2 cells, and the cellular effect of activated protein C was mediated by its receptors. Mice that received adoptive transfer of activated protein C-treated ovalbumin-pulsed dendritic cells had significantly less airway hyperresponsiveness, significantly decreased lung concentrations of Th1 and Th2 cytokines, and less plasma concentration of immunoglobulin E when compared to control mice.
These results suggest that dendritic cells mediate the immunosuppressive effect of activated protein C during allergic inflammation.
Journal of Asthma and Allergy 05/2015; 8:29-37. DOI:10.2147/JAA.S75261
[Show abstract][Hide abstract] ABSTRACT: The beneficial effects of edible mushrooms for improving chronic intractable diseases have been documented. However, the antiatherogenic activity of the new medicinal mushroom Grifola gargal is unknown. Therefore, we evaluated whether Grifola gargal can prevent or delay the progression of atherosclerosis. Atherosclerosis was induced in ApoE lipoprotein-deficient mice by subcutaneous infusion of angiotensin II. Grifola gargal extract (GGE) was prepared and intraperitoneally injected. The weight of heart and vessels, dilatation/atheroma formation of thoracic and abdominal aorta, the percentage of peripheral granulocytes, and the blood concentration of MCP-1/CCL2 were significantly reduced in mice treated with GGE compared to untreated mice. By contrast, the percentage of regulatory T cells and the plasma concentration of SDF-1/CXCL12 were significantly increased in mice treated with the mushroom extract compared to untreated mice. In vitro, GGE significantly increased the secretion of SDF-1/CXCL12, VEGF, and TGF-β1 from fibroblasts compared to control. This study demonstrated for the first time that Grifola gargal therapy can enhance regulatory T cells and ameliorate atherosclerosis in mice.
[Show abstract][Hide abstract] ABSTRACT: Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.
Biochemical and Biophysical Research Communications 05/2014; 135(2). DOI:10.1016/j.bbrc.2014.05.033 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Thrombomodulin treatment modulates the properties of dendritic cells (DCs) converting them from immunogenic to tolerogenic and inducing its own expression on DCs. Thrombomodulin binds to the inflammatory mediator, high mobility group protein B1 (HMGB1), antagonizing signalling through its receptor, receptor for advanced glycation end products (RAGE).
To test if soluble thrombomodulin could antagonize HMGB1 signaling via RAGE on DCs. DCs were prepared from mouse bone marrow cells or human monocytes. In some experiments dendritic cells were sorted into thrombomodulin+ and thrombomodulin- populations. Expression of surface maturation markers was determined by flow cytometry following treatment with thrombomodulin in the presence or absence of HMGB1.
Thrombomodulin+ dendritic cells secrete less HMGB1 into the medium. HMGB1 reduces the effects of thrombomodulin on expression of DC maturation markers. Treatment with thrombomodulin reduces the expression of maturation markers such as CD80 and CD86 and increases the expression of thrombomodulin on the DC surface. Treatment of DCs with neutralizing anti-HMGB1 antibody acted synergistically with thrombomodulin in increasing thrombomodulin expression on DCs. Treatment with thrombomodulin can still reduce the expression of surface markers on DCs derived from mice that are deficient in RAGE showing that thrombomodulin can affect DCs by an alternative mechanism.
The results of this study show that thrombomodulin modulates DCs both by antagonizing the interaction of HMGB1 with RAGE and by an independent mechanism.
Allergology International 12/2013; DOI:10.2332/allergolint.13-OA-0595 · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin(+) dendritic cells are tolerogenic while thrombomodulin(-) dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin(+) and thrombomodulin(-) dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin(+) dendritic cells compared to control dendritic cells and thrombomodulin(-) dendritic cells. Thrombomodulin-treated and thrombomodulin(+) dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin(+) dendritic cells produced more lipoxins than thrombomodulin(-) dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin(+) dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin(+) dendritic cells contained more cells actively dividing than those of thrombomodulin(-) dendritic cells. Production of IL-10 is increased in thrombomodulin(+) dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin(+) dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin(+) dendritic cells, which have significantly altered gene expression compared to thrombomodulin(-) dendritic cells in key immune function pathways.
PLoS ONE 08/2013; 8(8):e72392. DOI:10.1371/journal.pone.0072392 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apart from its role in the coagulation system, thrombin plays an important role in the inflammatory response through its protease-activated receptor(PAR)s. However, the role of thrombin in the immune response is not clear.
We evaluated if thrombin has a modulatory role in allergic bronchial asthma.
Bronchial asthma was induced in mice by intraperitoneal sensitization and inhalation challenge with ovalbumin. Thrombin or its inhibitors were administered by inhalation before each allergen challenge.
Mice with low but sustained coagulation activation had reduced allergic inflammation and allergic asthma was inhibited by low doses but worsened by high doses of thrombin. Allergic asthma was worsened by antithrombin, argatroban, hirudin and anti-thrombomodulin antibody. Mice with an increased concentration of an inhibitor of both thrombin and activated protein C had worsened disease. Heterozygous PAR-1 mice had less allergic inflammation but PAR-1 agonist worsened it. Allergic bronchial inflammation was worsened in mice that received adoptive transfer of PAR-1 agonist-treated Th2 cells compared to controls. Low concentrations of thrombin suppressed but high-dose of it enhanced maturation and secretion of cytokines in dendritic cells.
The effects of thrombin on allergic asthma are dose-dependent with detrimental effects at high dose and protective ones at low dose. These data demonstrate that thrombin modulates the outcome in allergic bronchial asthma. This article is protected by copyright. All rights reserved.
Journal of Thrombosis and Haemostasis 08/2013; 11(10). DOI:10.1111/jth.12392 · 5.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Acute lung injury (ALI) is a devastating disease with an overall mortality rate of 30~40%. The coagulation/fibrinolysis system is implicated in the pathogenesis of ALI. Thrombin-activatable fibronolysis inhibitor (TAFI) is an important component of the fibrinolysis system. Recent studies have shown that TAFIa can also regulate inflammatory responses by its ability to inhibit complement C3a and C5a, and osteopontin. Objective: We hypothesized that TAFI might have a protective role in ALI. To demonstrate this hypothesis the development of ALI was compared between wild type and TAFI-deficient mice. Methods: ALI was induced by intratracheal instillation of lipopolysaccharide (LPS). Control mice were treated with saline. Animals were sacrificed 24 hours after LPS. Results: The number of inflammatory cells and the concentration of total protein and inflammatory cytokines were significantly increased in BALF from LPS-treated TAFI-deficient mice compared to their wild type counterparts. Significantly higher concentrations of C5a were found in BALF and plasma in LPS-treated TAFI KO mice compared to wild type mice. Pre-treatment with inhaled C5a receptor antagonist blocked the detrimental effects of TAFI deficiency to levels of wild type mice. Conclusions: TAFI protects against acute lung injury, at least in part, by inhibiting the complement system.
American Journal of Respiratory Cell and Molecular Biology 05/2013; 49(4). DOI:10.1165/rcmb.2012-0454OC · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.
PLoS ONE 05/2013; 8(5):e64281. DOI:10.1371/journal.pone.0064281 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) is being widely used in functional gene research and is an important tool for drug discovery. However, canonical double-stranded short interfering RNAs are unstable and induce undesirable adverse effects, and thus there is no currently RNAi-based therapy in the clinic. We have developed a novel class of RNAi agents, and evaluated their effectiveness in vitro and in mouse models of acute lung injury (ALI) and pulmonary fibrosis. The novel class of RNAi agents (nkRNA®, PnkRNA™) were synthesized on solid phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a central stem and two loops. They are resistant to degradation and suppress their target genes. nkRNA and PnkRNA directed against TGF-β1mRNA ameliorate outcomes and induce no off-target effects in three animal models of lung disease. The results of this study support the pathological relevance of TGF-β1 in lung diseases, and suggest the potential usefulness of these novel RNAi agents for therapeutic application.
PLoS ONE 08/2012; 7(8):e42655. DOI:10.1371/journal.pone.0042655 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulatory T cells (Tregs) are a specific subset of T lymphocytes that regulate the function of other subsets of lymphocytes. Contradictory results have been reported regarding the role of Tregs in lung fibrosis. We wished to clarify the role of Tregs in the early and late stages of bleomycin-induced lung fibrosis in mice by depleting them with anti-CD25+ antibody (PC61). Mice treated with PC61 in early stages had significantly decreased number of CD4+CD25+ T cells compared to mice treated with the isotype control. The number of inflammatory cells, the concentrations of collagen, TGFβ1, the content of collagen and hydroxyproline in lung tissue were significantly reduced in PC61-treated mice compared to mice treated with the isotype control group. Pathological examination of the lung also disclosed reduced fibrotic changes and decreased fibrosis score in the PC61 group compared to control group. By contrast, mice treated with PC61 in late stages of the disease showed more infiltration of inflammatory cells and higher fibrotic score and hydroxyproline content in the lungs than mice treated with the isotype control. Our results suggest that Tregs play a detrimental role in early stages but protective role in late stages of pulmonary fibrosis in mice.
[Show abstract][Hide abstract] ABSTRACT: Idiopathic pulmonary fibrosis is a chronic devastating disease of unknown etiology. No therapy is currently available. A growing body of evidence supports the role of transforming growth factor (TGF)-β1 as the major player in the pathogenesis of the disease. However, attempts to control its expression and to improve the outcome of pulmonary fibrosis have been disappointing. We tested the hypothesis that TGF-β1 is the dominant factor in the acute and chronic phases of pulmonary fibrosis and developed short interfering (si)RNAs directed toward molecules implicated in the disease. This study developed novel sequences of siRNAs targeting the TGF-β1 gene and evaluated their therapeutic efficacy in two models of pulmonary fibrosis: a model induced by bleomycin and a novel model of the disease developed spontaneously in mice overexpressing the full length of human TGF-β1 in the lungs. Intrapulmonary delivery of aerosolized siRNAs of TGF-β1 with sequences common to humans and rodents significantly inhibited bleomycin-induced pulmonary fibrosis in the acute and chronic phases of the disease and in a dose-dependent manner. Aerosolized human-specific siRNA also efficiently inhibited pulmonary fibrosis, improved lung function, and prolonged survival in human TGF-β1 transgenic mice. Mice showed no off-target effects after intratracheal administration of siRNA. These results suggest the applicability of these novel siRNAs as tools for treating pulmonary fibrosis in humans.
American Journal of Respiratory Cell and Molecular Biology 03/2012; 46(3):397-406. DOI:10.1165/rcmb.2011-0158OC · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activated protein C (APC) can regulate immune and inflammatory responses and apoptosis. Protein C transgenic mice develop less diabetic nephropathy but whether exogenous administration of APC suppresses established diabetic nephropathy is unknown.
We investigated the therapeutic potential of APC in mice with streptozotocin-induced diabetic nephropathy.
Diabetes was induced in unilaterally nephrectomized C57/Bl6 mice using intraperitoneal (i.p.) injection of streptozotocin. Four weeks later, the mice were treated with i.p. exogenous APC every other day for 1 month.
APC-treated mice had a significantly improved blood nitrogen urea-to-creatinine ratio, urine total protein to creatinine ratio and proteinuria, and had significantly less renal fibrosis as measured by the levels of collagen and hydroxyproline. The renal tissue concentration of monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and the RNA expression of platelet-derived growth factor (PDGF), transforming growth factor-β1 and connective tissue growth factor (CTGF) were significantly lower in APC-treated mice than in untreated animals. The percentage of apoptotic cells was reduced and the expression of podocin, nephrin and WT-1 in the glomeruli was significantly improved in mice treated with APC compared with untreated mice. The levels of coagulation markers were not affected by APC treatment.
Exogenous APC improves renal function and mitigates pathological changes in mice with diabetic nephropathy by suppressing the expression of fibrogenic cytokines, growth factors and apoptosis, suggesting its potential usefulness for the therapy of this disease.
Journal of Thrombosis and Haemostasis 03/2012; 10(3):337-46. DOI:10.1111/j.1538-7836.2012.04621.x · 5.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bronchial asthma is an inflammatory disease of the airways. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase that besides inhibiting fibrinolysis, also regulates inflammatory processes. The only validated substrate known for TAFI is fibrin. In the present study we evaluated the role of TAFI in bronchial asthma by comparing the development of allergic bronchial asthma between wild-type (WT) and TAFI-deficient mice (KO).
Asthmatic inflammation was induced by sensitization and challenge with ovalbumin in WT (WT/OVA) and TAFI KO (KO/OVA) mice. WT mice (WT/SAL) and TAFI KO (KO/SAL) were used as controls. Cytokines, markers of inflammation, and coagulation were measured in bronchoalveolar lavage fluid (BALF).
Airway hyperresponsiveness was worse in KO/OVA mice than in WT/OVA mice or control mice. Markers of lung injury were significantly increased in BALF from KO/OVA mice compared to WT/OVA mice. Airway hyperresponsiveness and the BALF concentrations of IL-5 and osteopontin were significantly increased in KO/OVA mice compared to WT/OVA mice. Treatment of WT/OVA and KO/OVA mice with a C5a receptor antagonist significantly decreased hyperresponsiveness along with the BALF concentrations of total protein and C5a compared to untreated asthmatic mice.
The results of this study suggest that TAFI plays a protective role in the pathogenesis of allergic inflammation probably by inhibiting the complement system.
Beiträge zur Klinik der Tuberkulose 10/2011; 190(2):189-98. DOI:10.1007/s00408-011-9337-9 · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A moderate intake of alcohol is associated with lower cardiovascular mortality, and the role of circulating progenitor cells in the beneficial effect of alcohol on atherosclerosis is unclear. The hypothesis of this study was that alcohol ameliorates atherosclerosis by modulating the circulating levels of stromal cell-derived growth factor (SDF)-1 and vascular progenitor cells.
Atherosclerosis was induced by infusion of angiotensin II in apolipoprotein-E deficient mice, which were treated with high and low doses of ethanol for 28 days by intraperitoneal injection. Mice treated with low-dose ethanol had significantly less dilatation and fewer atheromatous lesions than mice receiving the high-dose ethanol. The number of circulating fibrocytes was significantly lower in mice treated with high-dose ethanol compared with mice with atherosclerosis untreated with ethanol. The plasma CXCL12/SDF-1 level was significantly increased in mice treated with low-dose ethanol compared with mice treated with a high dose, and the plasma concentration of transforming growth factor-β1 was significantly increased in mice treated with high-dose ethanol compared with control mice. Ethanol regulated the secretion of SDF-1 and vascular endothelial growth factor from fibroblasts in a dose-dependent and bimodal fashion.
The circulating level of CXCL12/SDF-1 may be involved, at least in part, in the differential effects of alcohol consumption on atherosclerosis.