Madeleine Rådinger

Effects of the antifungal imidazole ketoconazole on CYP1A and CYP3A in rainbow trout and killifish.

The use of N-substituted imidazoles is widespread, and imidazole and triazole fungicides have been detected in the aquatic environment and shown to bioaccumulate in fish. We have investigated effects of the model imidazole, ketoconazole, on drug-metabolizing cytochrome P450 (CYP) forms. We focused on cytochrome P4501A (CYP1A) and cytochrome P4503A (CYP3A) expression and activities in juvenile rainbow trout and in adult killifish. The CYP1A expression (mRNA, protein) and activity was induced in rainbow trout, whereas in killifish no effect of ketoconazole on CYP1A protein expression was observed. A biphasic dose-response relationship was observed between ketoconazole exposure and hepatic CYP1A-mediated ethoxyresorufin O-deethylase (EROD) activity in rainbow trout in vitro and in vivo, implying that higher doses of ketoconazole inhibit CYP1A activities. Slight induction of CYP3A protein levels was observed in rainbow trout exposed in vivo to ketoconazole. However, the CYP3A-mediated benzyloxy-4-[trifluoromethyl]-coumarin (BFC) O-debenzyloxylase activity was reduced in rainbow trout and killifish treated with ketoconazole. In vitro inhibition studies confirmed that ketoconazole was a potent inhibitor of both CYP3A and CYP1A enzyme activities in these species. This study showed that ketoconazole induced CYP1A and CYP3A expression in rainbow trout. However, the most pronounced effect of ketoconazole was a 60 to 90% decrease in CYP3A catalytic activities in rainbow trout and in killifish.

MicroRNA-155 is essential for TH2-mediated allergen-induced eosinophilic inflammation in the lung

BACKGROUND Allergic asthma is a chronic disease of the conducting airways characterized by T(H)2 inflammation and tissue remodeling after exposure to inhaled allergens. Although the T(H)2 profile is undisputed, the underlying molecular mechanisms leading to this abnormal T(H)2 profile remain largely unclear. MicroRNAs (miRNAs) are short noncoding RNAs that are important regulators of gene expression in the immune system. However, the role of miRNAs, specifically miR-155, in the regulation of allergic airway inflammation is unexplored. OBJECTIVES We sought to assess the contribution of miR-155 in a mouse model of allergic airway inflammation. METHODS To investigate a role for miR-155 in the regulation of allergic inflammation in vivo, we used miR-155 knockout (KO) and wild-type (WT) mice sensitized and exposed to ovalbumin. RESULTS miR-155 deficiency resulted in diminished eosinophilic inflammation and mucus hypersecretion in the lungs of allergen-sensitized and allergen-challenged mice compared with WT control animals. This was supported by a reduction in T(H)2 cell numbers and airway T(H)2 cytokine levels and complete abrogation of allergen-induced airway eotaxin-2/CCL24 and periostin levels in miR-155 KO mice. Intranasal instillation of eotaxin-2/CCL24 before allergen challenge partially restored airway eosinophilia in miR-155 KO mice, and adoptive transfer of CD4(+) T cells resulted in a similar degree of airway eosinophilia in miR-155 KO and WT mice. Furthermore, the transcription factor PU.1, a negative regulator of T(H)2 cytokine production, was upregulated in the airways of allergen-challenged miR-155 KO mice compared with WT mice. CONCLUSIONS Our data provides evidence that miR-155 contributes to the regulation of allergic airway inflammation by modulating T(H)2 responses through the transcription factor PU.1.

Btk-dependent Rac activation and actin rearrangement following FcεRI aggregation promotes enhanced chemotactic responses of mast cells

Mast cells infiltrate the sites of inflammation associated with chronic atopic disease and during helminth and bacterial infection. This process requires receptor-mediated cell chemotaxis across a concentration gradient of their chemotactic ligands. In vivo, mast cells are likely to be exposed to several such agents, which can cooperate in a synergistic manner to regulate mast cell homing. Here, we report that chemotaxis of mouse bone-marrow-derived mast cells (BMMCs) in response to the chemoattractants stem-cell factor (SCF) and prostaglandin (PG)E2, is substantially enhanced following antigen-dependent ligation of the high-affinity receptor for IgE (FcεRI). These responses were associated with enhanced activation of phosphoinositide 3-kinase (PI3K), and downstream activation of the tyrosine protein kinase Btk, with subsequent enhanced phospholipase (PL)Cγ-mediated Ca2+ mobilization, Rac activation and F-actin rearrangement. Antigen-induced chemotaxis, and the ability of antigen to amplify responses mediated by SCF, adenosine and PGE2 were suppressed following inhibition of PI3K, and were impaired in BMMCs derived from Btk−/− mice. There were corresponding decreases in the PLCγ-mediated Ca2+ signal, Rac activation and F-actin rearrangement, which, as they are essential for BMMC chemotaxis, accounts for the impaired migration of Btk-deficient cells. Taken together, these data demonstrate that, by regulating signaling pathways that control F-actin rearrangement, Btk is crucial for the ability of antigen to amplify mast-cell chemotactic responses.

Mast cell exosomes promote lung adenocarcinoma cell proliferation - role of KIT-stem cell factor signaling.

BackgroundHuman cells release nano-sized vesicles called exosomes, containing mRNA, miRNA and specific proteins. Exosomes from one cell can be taken up by another cell, which is a recently discovered cell-to-cell communication mechanism. Also, exosomes can be taken up by different types of cancer cells, but the potential functional effects of mast cell exosomes on tumor cells remain unknown.Methods and resultsExosomes were isolated from the human mast cell line, HMC-1, and uptake of PKH67-labelled exosomes by the lung epithelial cell line, A549, was examined using flow cytometry and fluorescence microscopy. The RNA cargo of the exosomes was analyzed with a Bioanalyzer and absence or presence of the c-KIT mRNA was determined by RT-PCR. The cell proliferation was determined in a BrdU incorporation assay, and proteins in the KIT-SCF signaling pathway were detected by Western blot. Our result demonstrates that exosomes from mast cells can be taken up by lung cancer cells. Furthermore, HMC-1 exosomes contain and transfer KIT protein, but not the c-KIT mRNA to A549 cells and subsequently activate KIT-SCF signal transduction, which increase cyclin D1 expression and accelerate the proliferation in the human lung adenocarcinoma cells.ConclusionsOur results indicate that exosomes can transfer KIT as a protein to tumor cells, which can affect recipient cell signaling events through receptor-ligand interactions.

MicroRNA-155 is a critical regulator of type 2 innate lymphoid cells and IL-33 signaling in experimental models of allergic airway inflammation

Background: Allergic airway inflammation is triggered by allergen exposure through several steps including release of IL‐33, which promotes cytokine (IL‐5, IL‐13) production by type 2 innate lymphoid cells (ILC2s). MicroRNA (miR)‐155 has recently been described to regulate adaptive responses in allergic inflammation. However, the role of miR‐155 in the regulation of ILC2s remains unexplored. Objective: We sought to elucidate the contribution of miR‐155 in ILC2 expansion using experimental murine models of allergic airway inflammation. Methods: To determine the role of miR‐155 in the regulation of ILC2s in allergic airway inflammation, miR‐155 deficient (miR‐155−/−) and wild‐type (WT) mice were subjected to acute or chronic allergen‐induced inflammation or treated with recombinant IL‐33. Results: miR‐155 was 10‐fold upregulated in WT‐derived ILC2s in response to IL‐33. Furthermore, miR‐155−/− mice demonstrated impaired lung IL‐33 levels in response to allergen challenge and the number of ILC2s was significantly reduced in allergen‐challenged miR‐155−/− mice compared with WT mice. Exogenous IL‐33 treatment revealed that miR‐155 is needed for IL‐33–induced ILC2 expansion and eosinophilic airway inflammation. Indeed, ILC2s from IL‐33–challenged miR‐155−/− lungs exhibited impaired proliferation, GATA‐3 expression, and IL‐13 production as compared with IL‐33–challenged WT ILC2s. Conclusions: Our findings for the first time demonstrate that ILC2s and IL‐33 signaling are regulated by miR‐155 in allergic airway inflammation.

Immunophenotyping of Circulating T Helper Cells Argues for Multiple Functions and Plasticity of T Cells In Vivo in Humans - Possible Role in Asthma

Background The immune process driving eosinophilic and non-eosinophilic asthma is likely driven by different subsets of T helper (Th) cells. Recently, in vitro studies and animal studies suggest that Th cell subsets displays plasticity by changing their transcription factor or by expressing multiple transcription factors. Our aim was to determine whether individuals with asthma and elevated circulating eosinophils express signs of different regulatory immune mechanisms compared with asthmatics with low blood eosinophils and non-asthmatic control subjects. In addition, determine the relationship between eosinophilia and circulating Th cell subsets. Methodology/Principal findings Participants were selected from a random epidemiological cohort, the West Sweden Asthma Study. Immunophenotypes of fresh peripheral blood cells obtained from stable asthmatics, with and without elevated eosinophilic inflammation (EOS high and EOS low respectively) and control subjects, were determined by flow cytometry. No differences in the number of Th1 (T-bet), Th2 (GATA-3), Th17 (RORγt) or Treg (FOXP3) cells were observed between the groups when analysing each subset separately. However, in all groups, each of the Th subsets showed expression of additional canonical transcription factors T-bet, GATA-3, RORγt and FOXP3. Furthermore, by in vitro stimulation with anti-CD3/anti-CD28 there was a significant increase of single expressing GATA-3+ and co-expressing T-bet+GATA-3+ cells in the EOS high asthmatics in comparison with control subjects. In addition, T-bet−GATA-3+RORγt+FOXP3+ were decreased in comparison to the EOS low asthmatics. Finally, in a group of control subjects we found that the majority of proliferating Th cells (CD4+CD25+Ki67+) expressed three or four transcription factors. Conclusions The ability of human Th cells to express several regulatory transcription factors suggests that these cells may display plasticity in vivo.

Local proliferation and mobilization of CCR3+ CD34+ eosinophil-lineage-committed cells in the lung

Emerging evidence suggests that haematopoietic CD34+ progenitor cells migrate from bone marrow (BM) to sites of allergen exposure where they can undergo further proliferation and final maturation, potentially augmenting the degree of tissue inflammation. In the current study we used a well‐characterized mouse model of allergen‐induced airway inflammation to determine the role of CCR3 receptor–ligand interactions in the migration and function of CD34+ cells. Allergen exposure significantly increased BM, blood and airway CD34+ CCR3+ cells as well as airway CD34+ CCR3+ stem cell antigen‐1‐positive (Sca‐1+) and CD34+  CD45+ interleukin‐5 receptor‐α‐positive (IL‐5Rα+) cells. A portion of the newly produced CD34+ CCR3+, Sca‐1+ CCR3+ and IL‐5Ralpha+ lung cells showed a significant proliferative capacity in response to allergen when compared with saline‐treated animals. In addition, in vitro colony formation of lung CD34+ cells was increased by IL‐5 or eotaxin‐2 whereas eotaxin‐2 had no effect on BM CD34+ cells. Furthermore, both eotaxin‐1 and eotaxin‐2 induced migration of BM and blood CD34+ CCR3+ cells in vitro. These data suggest that the CCR3/eotaxin pathway is involved in the regulation of allergen‐driven in situ haematopoiesis and the accumulation/mobilization of eosinophil‐lineage‐committed progenitor cells in the lung. Hence, targeting both IL‐5 and CCR3‐mediated signalling pathways may be required to control the inflammation associated with allergen‐induced asthma.

New production of eosinophils and the corresponding Th1/Th2 balance in the lungs after allergen exposure in BALB/c and C57BL/6 mice

Allergic asthma is associated with eosinophilic inflammation in the airways. Animal models commonly used to elucidate allergic inflammation mechanisms include BALB/c and C57BL/6 mice. Our aim was to evaluate lung eosinophilia and the corresponding Th1/Th2 balance in the two strains after allergen exposure. BALB/c and C57BL/6 mice were subjected to ovalbumin‐induced allergic airway inflammation using BrdU to label newly produced cells. The numbers of new eosinophils were evaluated by differential cell count and immunocytochemistry (MBP+BrdU+). Proliferation rate of lung eosinophils was measured by analysis of CD45+CCR3+BrdU+ cells by FACS. Distribution of newly produced eosinophils in the lung and the Th1/Th2 (CD4+T‐bet+/CD4+GATA‐3+) balance was evaluated by immunohistochemistry. Allergen challenge with ovalbumin induced comparable eosinophilia in bone marrow (BM), blood and lung tissue in both strains of mice compared to phosphate‐buffered saline controls, which was confirmed by immunocytochemistry. There was a small increase in the number of lung MBP+BrdU− eosinophils in C57BL/6 mice compared to BALB/c mice, which suggests a basal increase in this strain following sensitization. While there was no difference in eosinophilic proliferation in the lung, the distribution of the newly produced eosinophils differs between the two strains. BALB/c mice showed staining primarily around vessels and airways, whereas C57BL/6 mice showed a more even distribution in the lung tissue. No difference in the Th1/Th2 balance was observed between two strains. This study shows that there is a difference in the distribution of eosinophils in the lung between the C57BL/6 and BALB/c mice, but no difference in eosinophil production or Th1/Th2 balance.

Regulation of allergen‐induced bone marrow eosinophilopoiesis: role of CD4+ and CD8+ T cells

Background:  The mechanisms of the distant stimulation of the bone marrow (BM) after airway allergen exposure remain largely obscure. T cells have been implicated in allergic airway inflammation but their role in allergen‐induced BM eosinophilopoiesis is poorly understood. The aim of this study was to determine the role of CD4+ and CD8+ T cells in allergen‐induced BM eosinophilopoiesis.

Expansion of CD4+CD25+ and CD25- T-Bet, GATA-3, Foxp3 and RORγt Cells in Allergic Inflammation, Local Lung Distribution and Chemokine Gene Expression

Allergic asthma is associated with airway eosinophilia, which is regulated by different T-effector cells. T cells express transcription factors T-bet, GATA-3, RORγt and Foxp3, representing Th1, Th2, Th17 and Treg cells respectively. No study has directly determined the relative presence of each of these T cell subsets concomitantly in a model of allergic airway inflammation. In this study we determined the degree of expansion of these T cell subsets, in the lungs of allergen challenged mice. Cell proliferation was determined by incorporation of 5-bromo-2′-deoxyuridine (BrdU) together with 7-aminoactnomycin (7-AAD). The immunohistochemical localisation of T cells in the lung microenvironments was also quantified. Local expression of cytokines, chemokines and receptor genes was measured using real-time RT-PCR array analysis in tissue sections isolated by laser microdissection and pressure catapulting technology. Allergen exposure increased the numbers of T-bet+, GATA-3+, RORγt+ and Foxp3+ cells in CD4+CD25+ and CD4+CD25- T cells, with the greatest expansion of GATA-3+ cells. The majority of CD4+CD25+ T-bet+, GATA-3+, RORγt+ and Foxp3+ cells had incorporated BrdU and underwent proliferation during allergen exposure. Allergen exposure led to the accumulation of T-bet+, GATA-3+ and Foxp3+ cells in peribronchial and alveolar tissue, GATA-3+ and Foxp3+ cells in perivascular tissue, and RORγt+ cells in alveolar tissue. A total of 28 cytokines, chemokines and receptor genes were altered more than 3 fold upon allergen exposure, with expression of half of the genes claimed in all three microenvironments. Our study shows that allergen exposure affects all T effector cells in lung, with a dominant of Th2 cells, but with different local cell distribution, probably due to a distinguished local inflammatory milieu.