Identification of Eight Different Isoforms of the Glucocorticoid Receptor in Guinea Pig Placenta: Relationship to Preterm Delivery,Sex and Betamethasone Exposure

The placental glucocorticoid receptor (GR) is central to glucocorticoid signalling and for mediating steroid effects on pathways associated with fetal growth and lung maturation but the GR has not been examined in the guinea pig placenta even though this animal is regularly used as a model of preterm birth and excess glucocorticoid exposure. Guinea pig dams received subcutaneous injections of either vehicle or betamethasone at 24 and 12 hours prior to preterm or term caesarean-section delivery. At delivery pup and organ weights were recorded. Placentae were dissected, weighed and analysed using Western blot to examine GR isoform expression in nuclear and cytoplasmic extracts. A comparative examination of the guinea pig GR gene identified it is capable of producing seven of the eight translational GR isoforms which include GRα-A, C1, C2, C3, D1, D2, and D3. GRα-B is not produced in the Guinea Pig. Total GR antibody identified 10 specific bands from term (n = 29) and preterm pregnancies (n = 27). Known isoforms included GRγ, GRα A, GRβ, GRP, GRA and GRα D1-3. There were sex and gestational age differences in placental GR isoform expression. Placental GRα A was detected in the cytoplasm of all groups but was significantly increased in the cytoplasm and nucleus of preterm males and females exposed to betamethasone and untreated term males (KW-ANOVA, P = 0.0001, P = 0.001). Cytoplasmic expression of GRβ was increased in female preterm placentae and preterm and term male placentae exposed to betamethasone (P = 0.01). Nuclear expression of GRβ was increased in all placentae exposed to betamethasone (P = 0.0001). GRα D2 and GRα D3 were increased in male preterm placentae when exposed to betamethasone (P = 0.01, P = 0.02). The current data suggests the sex-specific placental response to maternal betamethasone may be dependent on the expression of a combination of GR isoforms.     

Quantification of Beta Adrenergic Receptor Subtypes in Beta-Arrestin Knockout Mouse Airways

In allergic asthma Beta 2 adrenergic receptors (β₂ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β₂AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β₂AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β₂AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β₂ARs predominate over β₁ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β₂AR expression. These data provide further evidence that β₂ARs are expressed in greater abundance than β₁ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β₂AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference point for future studies exploiting these knockout mice in various disease models including asthma.     

Effect of Lipopolysaccharide on Glucocorticoid Receptor Function in Control Nasal Mucosa Fibroblasts and in Fibroblasts from Patients with Chronic Rhinosinusitis with Nasal Polyps and Asthma

BackgroundChronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the upper airways frequently associated with asthma. Bacterial infection is a feature of CRSwNP that can aggravate the disease and the response to glucocorticoid treatment.ObjectiveWe examined whether the bacterial product lipopolysaccharide (LPS) reduces glucocorticoid receptor (GR) function in control nasal mucosa (NM) fibroblasts and in nasal polyp (NP) fibroblasts from patients with CRSwNP and asthma.MethodsNP (n = 12) and NM fibroblasts (n = 10) were in vitro pre-incubated with LPS (24 hours) prior to the addition of dexamethasone. Cytokine/chemokine secretion was measured by ELISA and Cytometric Bead Array. GRα, GRβ, mitogen-activated protein-kinase phosphatase-1 (MKP-1) and glucocorticoid-induced leucine zipper (GILZ) expression was measured by RT-PCR and immunoblotting, GRα nuclear translocation by immunocytochemistry, and GRβ localization by immunoblotting. The role of MKP-1 and GILZ on dexamethasone-mediated cytokine inhibition was analyzed by small interfering RNA silencing.ResultsPre-incubation of nasal fibroblasts with LPS enhanced the secretion of IL-6, CXCL8, RANTES, and GM-CSF induced by FBS. FBS-induced CXCL8 secretion was higher in NP than in NM fibroblasts. LPS effects on IL-6 and CXCL8 were mediated via activation of p38α/β MAPK and IKK/NF-κB pathways. Additionally, LPS pre-incubation: 1) reduced dexamethasone’s capacity to inhibit FBS-induced IL-6, CXCL8 and RANTES, 2) reduced dexamethasone-induced GRα nuclear translocation (only in NM fibroblasts), 3) did not alter GRα/GRβ expression, 4) decreased GILZ expression, and 5) did not affect dexamethasone’s capacity to induce MKP-1 and GILZ expression. MKP-1 knockdown reduced dexamethasone’s capacity to suppress FBS-induced CXCL8 release.ConclusionThe bacterial product LPS negatively affects GR function in control NM and NP fibroblasts by interfering with the capacity of the activated receptor to inhibit the production of pro-inflammatory mediators. This study contributes to the understanding of how bacterial infection of the upper airways may limit the efficacy of glucocorticoid treatment.     

Human Glucocorticoid Receptor β Regulates Gluconeogenesis and Inflammation in Mouse Liver

While in vitro studies have demonstrated that a glucocorticoid receptor (GR) splice isoform, β-isoform of human GR (hGRβ), acts as a dominant-negative inhibitor of the classic hGRα and confers glucocorticoid resistance, the in vivo function of hGRβ is poorly understood. To this end, we created an adeno-associated virus (AAV) to express hGRβ in the mouse liver under the control of the hepatocyte-specific promoter. Genome-wide expression analysis of mouse livers showed that hGRβ significantly increased the expression of numerous genes, many of which are involved in endocrine system disorders and the inflammatory response. Physiologically, hGRβ antagonized GRα''s function and attenuated hepatic gluconeogenesis through downregulation of phosphoenolpyruvate carboxykinase (PEPCK) in wild-type (WT) mouse liver. Interestingly, however, hGRβ did not repress PEPCK in GR liver knockout (GRLKO) mice. In contrast, hGRβ regulates the expression of STAT1 in the livers of both WT and GRLKO mice. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that hGRβ binds to the intergenic glucocorticoid response element (GRE) of the STAT1 gene. Furthermore, treatment with RU486 inhibited the upregulation of STAT1 mediated by hGRβ. Finally, our array data demonstrate that hGRβ regulates unique components of liver gene expression in vivo by both GRα-dependent and GRα-independent mechanisms.     

Reduced Peripheral Expression of the Glucocorticoid Receptor α Isoform in Individuals with Posttraumatic Stress Disorder: A Cumulative Effect of Trauma Burden

Background

Posttraumatic stress disorder (PTSD) is a serious psychiatric condition that was found to be associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA) axis and changes in glucocorticoid (GC) responsiveness. The physiological actions of GCs are primarily mediated through GC receptors (GR) of which isoforms with different biological activities exist. This study aimed to investigate whether trauma-experience and/or PTSD are associated with altered expression of GR splice variants.

Methods

GRα and GRβ mRNA expression levels were determined by real-time quantitative PCR in whole blood samples of individuals with chronic and severe forms of PTSD (n = 42) as well as in ethnically matched reference subjects (non-PTSD, n = 35).

Results

Individuals suffering from PTSD exhibited significantly lower expression of the predominant and functionally active GRα isoform compared to non-PTSD subjects. This effect remained significant when accounting for gender, smoking, psychotropic medication or comorbid depression. Moreover, the GRα expression level was significantly negatively correlated with the number of traumatic event types experienced, both in the whole sample and within the PTSD patient group. Expression of the less abundant and non-ligand binding GRβ isoform was comparable between patient and reference groups.

Conclusions

Reduced expression of the functionally active GRα isoform in peripheral blood cells of individuals with PTSD seems to be a cumulative effect of trauma burden rather than a specific feature of PTSD since non-PTSD subjects with high trauma load showed an intermediate phenotype between PTSD patients and individuals with no or few traumatic experiences.     

Involvement of β1-Integrin Up-regulation in Basic Fibroblast Growth Factor- and Epidermal Growth Factor-induced Proliferation of Mouse Neuroepithelial Cells

In neural stem cells, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote cell proliferation and self-renewal. In the bFGF- and EGF-responsive neural stem cells, β1-integrin also plays important roles in crucial cellular processes, including proliferation, migration, and apoptosis. The cross-talk of the signaling pathways mediated by these growth factors and β1-integrin, however, has not been fully elucidated. Here we report a novel molecular mechanism through which bFGF or EGF promotes the proliferation of mouse neuroepithelial cells (NECs). In the NECs, total β1-integrin expression levels and proliferation were dose-dependently increased by bFGF but not by EGF. EGF rather than bFGF strongly induced the increase of β1-integrin localization on the NEC surface. bFGF- and EGF-induced β1-integrin up-regulation and proliferation were inhibited after treatment with a mitogen-activated protein kinase kinase inhibitor, U0126, which indicates the dependence on the mitogen-activated protein kinase pathway. Involvement of β1-integrin in bFGF- and EGF-induced proliferation was confirmed by the finding that NEC proliferation and adhesion to fibronectin-coated dishes were inhibited by knockdown of β1-integrin using small interfering RNA. On the other hand, apoptosis was induced in NECs treated with RGD peptide, a small β1-integrin inhibitor peptide with the Arg-Gly-Asp motif, but it was independent of β1-integrin expression levels. Those results suggest that regulation of β1-integrin expression/localization is involved in cellular processes, such as proliferation, induced by bFGF and EGF in NECs. The mechanism underlying the proliferation through β1-integrin would not be expected to be completely identical, however, for bFGF and EGF.     

Regulation of Constitutive Neutrophil Apoptosis Due to House Dust Mite Allergen in Normal and Allergic Rhinitis Subjects

House dust mite (HDM) is a primary allergen in allergic rhinitis (AR) and asthma. Neutrophil apoptosis is associated with allergic diseases and innate immunity to infection. The present study examined how HDM affects constitutive neutrophil apoptosis in normal and AR subjects. Total IgE increased in AR subjects when compared to normal subjects, and patients with AR were HDM-specific IgE positive (+), which is specific IgE to Dermatophagoides pteronissinus and Dermatophagoides farinae. In normal and AR subjects, neutrophil apoptosis was inhibited by extract of Dermatophagoides pteronissinus (DP), but not by extract of Dermatophagoides farina (DF). Aprotinin (serine protease inhibitor) and E64 (cysteine protease inhibitor) have no effect on neutrophil apoptosis due to DP. The anti-apoptotic effect of DP was blocked by TLR4i, an inhibitor of TLR4, rottlerin, an inhibitor of PKCδ, PD98059, an inhibitor of ERK, and BAY-11-7085, an inhibitor of NF-κB. DP induced PKCδ, ERK, and NF-κB activation in a time-dependent manner. DP inhibited the cleavage of procaspase 3 and procaspase 9. The expression of IL-6, IL-8, TNF-α, G-CSF, GM-CSF, and CCL2 increased in the supernatant collected from the normal and AR neutrophils after DP treatment and the supernatant inhibited the apoptosis of normal and AR neutrophils. In summary, DP has anti-apoptotic effects on neutrophils of normal and AR subjects through the TLR4/PKCδ/ERK/NF-κB pathway, and this finding may contribute to solution of the pathogenic mechanism of allergic diseases triggered by DP.     

Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice

Background

The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress–induced inflammation by affecting the NF-κB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be anti-inflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, ragweed pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma.

Methods and Findings

Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on ragweed pollen extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE -induced apoptotic cell death as measured by annexin-v staining, increase in the activation of NF-κB and expression of inflammatory markers such as inducible nitric oxide synthase (iNOS), cycloxygenase (COX)-2, Prostaglandin (PG) E₂, IL-6 and IL-8. Further, BALB/c mice were sensitized with endotoxin-free RWE in the absence and presence of AR inhibitor and followed by evaluation of perivascular and peribronchial inflammation, mucin production, eosinophils infiltration and airway hyperresponsiveness. Our results indicate that inhibition of AR prevents airway inflammation and production of inflammatory cytokines, accumulation of eosinophils in airways and sub-epithelial regions, mucin production in the bronchoalveolar lavage fluid and airway hyperresponsiveness in mice.

Conclusions

These results suggest that airway inflammation due to allergic response to RWE, which subsequently activates oxidative stress-induced expression of inflammatory cytokines via NF-κB-dependent mechanism, could be prevented by AR inhibitors. Therefore, inhibition of AR could have clinical implications, especially for the treatment of airway inflammation, a major cause of asthma pathogenesis.     

Characterization of a panel of six β2-adrenergic receptor antibodies by indirect immunofluorescence microscopy

Background

The β₂-adrenergic receptor (β₂AR) is a primary target for medications used to treat asthma. Due to the low abundance of β₂AR, very few studies have reported its localization in tissues. However, the intracellular location of β₂AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to β-agonist medications. Thus, a method for visualizing β₂AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant β₂AR in primary cell cultures.

Methods

A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat β₂AR expressed in HEK 293 cells. Antibodies capable of recognizing rat β₂AR were identified and used to localize native β₂AR in primary cultures of rat airway smooth muscle and epithelial cells. β₂AR expression was confirmed by performing ligand binding assays using the β-adrenergic antagonist [3H] dihydroalprenolol ^([3H]DHA).

Results

Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human β₂AR (Ab-Bethyl) specifically recognized human but not rat β₂AR. An antibody developed against the C-terminal domain of the mouse β₂AR (Ab-sc570) specifically recognized rat but not human β₂AR. An antibody developed against 78 amino acids of the C-terminus of the human β₂AR (Ab-13989) was capable of recognizing both rat and human β₂ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat β₂AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface.

Conclusion

Antibodies have been identified that recognize human β₂AR, rat β₂AR or both rat and human β₂AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of β₂AR in tissues.     

Aldose Reductase Inhibition Prevents Allergic Airway Remodeling through PI3K/AKT/GSK3β Pathway in Mice

Background

Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR), an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA)-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs) and mouse lung fibroblasts (mLFs).

Methods

Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR) in response to increasing doses of methacholine were assessed. The TGFβ1-induced epithelial-mesenchymal transition (EMT) in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s) of airway remodeling.

Results

In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFβ1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFβ1- induced activation of PI3K/AKT/GSK3β pathway but not the phosphorylation of Smad2/3.

Conclusion

Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFβ1-induced Smad-independent and PI3K/AKT/GSK3β-dependent pathway.     

Role of licochalcone A on thymic stromal lymphopoietin expression: Implications for asthma

Asthma is a common chronic inflammatory disease characterized by the infiltration and accumulation of memory-like Th2 cells and eosinophils. Viral infection has emerged as the most common cause of severe episodes of asthma. For the treatment of bronchial asthma, the root of liquorice (Glycyrrhiza glabra) has been used as a traditional medicine in the East and West. Licochalcone A is the predominant, characteristic chalcone in liquorice root. To determine whether licochalcone A possesses an anti-inflammatory effect, we tested its effect on the expression and production of thymic stromal lymphopoietin (TSLP) in BEAS 2B cells and primary bronchial epithelial cells. We found that polyinosinic-polycytidylic acid (poly-IC)-induced TSLP expression was suppressed by treatment with licochalcone A in a dose- and time-dependent manner. We also found that poly-IC-induced mRNA expression of other proinflammatory mediators such as MCP-1, RANTES, and IL-8 was suppressed by licochalcone A. Furthermore, licochalcone A suppressed poly-IC-induced nuclear factor kappa B (NF-κB) nuclear translocation and DNA-binding activity by suppressing the Iκβ kinase (IKK) activity but not by direct phosphorylation of p65 at serine 276. Collectively, our findings suggest that licochalcone A suppresses poly-IC-induced TSLP expression and production by inhibiting the IKK/NF-κB signaling pathway, which might be involved in the pathogenesis of virus-exacerbated asthma. Further elucidation of the mechanisms underlying these observations can help develop therapeutic strategies for virally induced asthma.     

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists

The β₂-adrenergic receptor (β₂AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β₂AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β₂AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β₂AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-G_s coupling and promote β₂AR internalization and degradation. A biased agonist that can selectively stimulate G_s signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β₂AR, known as pepducins. This screen revealed two classes of G_s-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent G_s-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent G_s-biased pepducins appear to stabilize a G_s-biased conformation of the β₂AR that couples to G_s but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that G_s-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first G_s-biased activator of the β₂AR and provides valuable tools for the study of β₂AR function.     

p110γ/δ Double-Deficiency Induces Eosinophilia and IgE Production but Protects from OVA-Induced Airway Inflammation

The catalytical isoforms p110γ and p110δ of phosphatidylinositide 3-kinase γ (PI3Kγ) and PI3Kδ play an important role in the pathogenesis of asthma. Two key elements in allergic asthma are increased levels of eosinophils and IgE. Dual pharmacological inhibition of p110γ and p110δ reduces asthma-associated eosinophilic lung infiltration and ameliorates disease symptoms, whereas the absence of enzymatic activity in p110γ^KOδ^D910A mice increases IgE and basal eosinophil counts. This suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. Here, we analysed mice genetically deficient for both catalytical subunits (p110γ/δ^-/-) and determined basal IgE and eosinophil levels and the immune response to ovalbumin-induced asthma. Serum concentrations of IgE, IL-5 and eosinophil numbers were significantly increased in p110γ/δ^-/- mice compared to single knock-out and wildtype mice. However, p110γ/δ^-/- mice were protected against OVA-induced infiltration of eosinophils, neutrophils, T and B cells into lung tissue and bronchoalveolar space. Moreover, p110γ/δ^-/- mice, but not single knock-out mice, showed a reduced bronchial hyperresponsiveness. We conclude that increased levels of eosinophils and IgE in p110γ/δ^-/- mice do not abolish the protective effect of p110γ/δ-deficiency against OVA-induced allergic airway inflammation.     

DEK‐targeting aptamer DTA‐64 attenuates bronchial EMT‐mediated airway remodelling by suppressing TGF‐β1/Smad,MAPK and PI3K signalling pathway in asthma

This study is to investigate the inhibitory effects and mechanisms of DEK‐targeting aptamer (DTA‐64) on epithelial mesenchymaltransition (EMT)‐mediated airway remodelling in mice and human bronchial epithelial cell line BEAS‐2B. In the ovalbumin (OVA)‐induced asthmatic mice, DTA‐64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA‐64 reduced collagen deposition, transforming growth factor 1 (TGF‐β1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α‐SMA), as well as weekend matrix metalloproteinases (MMP‐2 and MMP‐9) and NF‐κB p65 activity. In the in vitro experiments, we used TGF‐β1 to induce EMT in the human epithelial cell line BEAS‐2B. DEK overexpression (ovDEK) or silencing (shDEK) up‐regulated or down‐regulated TGF‐β1 expression, respectively, on the contrary, TGF‐β1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF‐β1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF‐β1‐mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA‐64 against EMT of asthmatic mice and BEAS‐2B might partially be achieved through suppressing TGF‐β1/Smad, MAPK and PI3K signalling pathways. DTA‐64 may be a new therapeutic option for the management of airway remodelling in asthma patients.     

Constitutively Active Signaling by the G Protein βγ-Subunit Mediates Intrinsically Increased Phosphodiesterase-4 Activity in Human Asthmatic Airway Smooth Muscle Cells

Signaling by the Gβγ subunit of Gi protein, leading to downstream c-Src-induced activation of the Ras/c-Raf1/MEK-ERK1/2 signaling pathway and its upregulation of phosphodiesterase-4 (PDE4) activity, was recently shown to mediate the heightened contractility in proasthmatic sensitized isolated airway smooth muscle (ASM), as well as allergen-induced airway hyperresponsiveness and inflammation in an in vivo animal model of allergic asthma. This study investigated whether cultured human ASM (HASM) cells derived from asthmatic donor lungs exhibit constitutively increased PDE activity that is attributed to intrinsically upregulated Gβγ signaling coupled to c-Src activation of the Ras/MEK/ERK1/2 cascade. We show that, relative to normal cells, asthmatic HASM cells constitutively exhibit markedly increased intrinsic PDE4 activity coupled to heightened Gβγ-regulated phosphorylation of c-Src and ERK1/2, and direct co-localization of the latter with the PDE4D isoform. These signaling events and their induction of heightened PDE activity are acutely suppressed by treating asthmatic HASM cells with a Gβγ inhibitor. Importantly, along with increased Gβγ activation, asthmatic HASM cells also exhibit constitutively increased direct binding of the small Rap1 GTPase-activating protein, Rap1GAP, to the α-subunit of Gi protein, which serves to cooperatively facilitate Ras activation and, thereby, enable enhanced Gβγ-regulated ERK1/2-stimulated PDE activity. Collectively, these data are the first to identify that intrinsically increased signaling via the Gβγ subunit, facilitated by Rap1GAP recruitment to the α-subunit, mediates the constitutively increased PDE4 activity detected in asthmatic HASM cells. These new findings support the notion that interventions targeted at suppressing Gβγ signaling may lead to novel approaches to treat asthma.     

Effects of intranasal TNFα on granulocyte recruitment and activity in healthy subjects and patients with allergic rhinitis

Background

TNFα may contribute to the pathophysiology of airway inflammation. For example, we have recently shown that nasal administration of TNFα produces late phase co-appearance of granulocyte and plasma exudation markers on the mucosal surface. The objective of the present study was to examine indices of granulocyte presence and activity in response to intranasal TNFα challenge.

Methods

Healthy subjects and patients with allergic rhinitis (examined out of season) were subjected to nasal challenge with TNFα (10 μg) in a sham-controlled and crossover design. Nasal lavages were carried out prior to and 24 hours post challenge. Nasal biopsies were obtained post challenge. Nasal lavage fluid levels of myeloperoxidase (MPO) and eosinophil cationic protein (ECP) were analyzed as indices of neutrophil and eosinophil activity. Moreover, IL-8 and α₂-macroglobulin were analyzed as markers of pro-inflammatory cytokine production and plasma exudation. Nasal biopsy numbers of neutrophils and eosinophils were monitored.

Results

Nasal lavage fluid levels of MPO recorded 24 hours post TNFα challenge were increased in healthy subjects (p = 0.0081) and in patients with allergic rhinitis (p = 0.0081) (c.f. sham challenge). Similarly, α₂-macroglobulin was increased in healthy subjects (p = 0.014) and in patients with allergic rhinitis (p = 0.0034). Lavage fluid levels of ECP and IL-8 were not affected by TNFα challenge. TNFα increased the numbers of subepithelial neutrophils (p = 0.0021), but not the numbers of eosinophils.

Conclusion

TNFα produces a nasal inflammatory response in humans that is characterised by late phase (i.e., 24 hours post challenge) neutrophil activity and plasma exudation.     

Fractalkine Signaling Regulates the Inflammatory Response in an α-Synuclein Model of Parkinson Disease

Background

Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein alpha-synuclein (α-syn). Increasing evidence points to inflammation as a chief mediator; however, the role of α-syn in triggering and sustaining inflammation remains unclear. In models of Alzheimer’s disease (AD), multiple sclerosis (MS) and neurotoxin models of PD, the chemokine CX3CL1 (fractalkine) and its receptor (CX3CR1) have important roles in modulating neuroinflammation.

Methods

To examine the role of fractalkine signaling in α-syn-induced-neuroinflammation and neurodegeneration, we used an in vivo mouse model in which human α-syn is overexpressed by an adeno associated viral vector serotype 2 (AAV2) and in vitro phagocytosis and protein internalization assays with primary microglia treated with aggregated α-syn.

Results

We observed that loss of CX3CR1 expression led to a reduced inflammatory response, with reduced IgG deposition and expression of MHCII 4 weeks post-transduction. Six months post transduction, AAV2 mediated overexpression of α-syn leads to loss of dopaminergic neurons, and this loss was not exacerbated in animals with deletion of CX3CR1. To determine the mechanism by which CX3CR1affects inflammatory responses in α-syn-induced inflammation, phagocytosis was assessed using a fluorescent microsphere assay as well as by microglial uptake of aggregated α-syn. CX3CR1-/- microglia showed reduced uptake of fluorescent beads and aggregated α-syn.

Conclusion

Our results suggest that one mechanism by which CX3CR1-/- attenuates inflammation is at the level of phagocytosis of aggregated α-syn by microglia. These data implicate fractalkine signaling as a potential therapeutic target for regulating inflammatory response in α-syn models PD.     

Cytotoxic effects of GM1 ganglioside and amyloid β-peptide on mouse embryonic neural stem cells

AD (Alzheimer’s disease) is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Aβs (amyloid β-peptides) in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between G_M1 ganglioside and Aβs. Several reports have documented the bifunctional roles of Aβs in NSCs (neural stem cells), but the precise effects of G_M1 and Aβ on NSCs have not yet been clarified. We evaluated the effect of G_M1 and Aβ-(1–40) on mouse NECs (neuroepithelial cells), which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either G_M1 or Aβ-(1–40). On the contrary, a decreased number of NECs were cultured in the presence of a combination of G_M1 and Aβ-(1–40). The exogenously added G_M1 and Aβ-(1–40) were confirmed to incorporate into NECs. The Ras–MAPK (mitogen-activated protein kinase) pathway, important for cell proliferation, was intact in NECs simultaneously treated with G_M1 and Aβ-(1–40), but caspase 3 was activated. NECs treated with G_M1 and Aβ-(1–40) were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, an indicator of cell death. It was found that G_M1 and Aβ-(1–40) interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Aβ-(1–40) and G_M1 co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.     

Central Role of Cellular Senescence in TSLP-Induced Airway Remodeling in Asthma

Background

Airway remodeling is a repair process that occurs after injury resulting in increased airway hyper-responsiveness in asthma. Thymic stromal lymphopoietin (TSLP), a vital cytokine, plays a critical role in orchestrating, perpetuating and amplifying the inflammatory response in asthma. TSLP is also a critical factor in airway remodeling in asthma.

Objectives

To examine the role of TSLP-induced cellular senescence in airway remodeling of asthma in vitro and in vivo.

Methods

Cellular senescence and airway remodeling were examined in lung specimens from patients with asthma using immunohischemical analysis. Both small molecule and shRNA approaches that target the senescent signaling pathways were used to explore the role of cellular senescence in TSLP-induced airway remodeling in vitro. Senescence-Associated β-galactosidase (SA-β-Gal) staining, and BrdU assays were used to detect cellular senescence. In addition, the Stat3-targeted inhibitor, WP1066, was evaluated in an asthma mouse model to determine if inhibiting cellular senescence influences airway remodeling in asthma.

Results

Activation of cellular senescence as evidenced by checkpoint activation and cell cycle arrest was detected in airway epithelia samples from patients with asthma. Furthermore, TSLP-induced cellular senescence was required for airway remodeling in vitro. In addition, a mouse asthma model indicates that inhibiting cellular senescence blocks airway remodeling and relieves airway resistance.

Conclusion

TSLP stimulation can induce cellular senescence during airway remodeling in asthma. Inhibiting the signaling pathways of cellular senescence overcomes TSLP-induced airway remodeling.