The role of aquaporin-3 (AQP3) in functions of alveolar macrophage (AM) during ovalbumin (OVA)-induced asthma
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ABSTRACT: We compared the gene expression profiles of AMs using microarray analysis between AMs from wild-type (WT) and AQP3-deficient (AQP3-/-) mice before and after OVA challenge RNA was extracted from AMs of WT and AQP3-/- mice before and after OVA challenge. We prepared 4-6 mice for each group and pooled their samples.
Project description:In the present study, we mobilized the latest methodologies in tandem mass spectrometry and cross-link proteomics to decipher the heat-induced structural changes of OVA in the presence or absence of sugar. Our objective was to better understand how these structural changes could lead to a modulation of the allergenicity of OVA, depending on the process it has undergone.
Project description:Abatacept is a recombinant CTLA-4 moleculed fused to a mutated human IgG molecule, which is clinically used in rheumatoid arthritis by inhibiting CD28-costimulation. This study aimed to inverstigate the ability of abatacept -mediated costimulation blockade to induce antigen-specific tolerance during primary immune responses. This is important as some studies have suggested that costimulation blockade can lead to CD4+ T cell anergy which could be beneficial for early therapy of autoimmune diseases such as rheumatoid arthritis. In addition we also investigated the effect that abatacept has on CD11c+ antigen presenting cells. This is important as costimulation blocakde can affect the biderectional interaction between CD4+ T cells and CD11c+ cells influencing the immunological outcome. We used microarrays to identify if abatacept treatment leads to antigen specific anergy using transgenic animals and models of priming and oral tolerance that established a synchronised monoclonal response. In addition this magnified the effect on the CD11c+ antigen presenting cells. This study included 5 experimental groups. DO11.10 RAG2-/- mice have CD4+ T cells specific for the ovalbumin (OVA) peptide OVA323-339. These mice were immunised with CFA/OVA s.c. (primed group) or tolerised by feeding with OVA (50mg/kg) in the drinking water. CD4+ cells were isolated 10 days post immunisation from draining lymph nodes (LNs) of unimmunised (pooled LNs and Spleen), orally tolerised (mesenteric LNs), primed (axillary LNs), primed treated with control IgG (axillary LNs) and primed treated with abatacept (axillary LNs). For CD11c+ cells cells were isolated by pooled secondary lymphoid organs (LNs and spleen).
Project description:A population of non-adherent cells with stem-like characteristics (OVA-BS4 spheres) has been isolated from a primary human epithelial ovarian cancer (EOC) cell line (OVA-BS4) under selective conditions. OVA-BS4 spheres were characterized for their pharmacological properties and their molecular profile by microarray and RT-qPCR.
Project description:Analysis of changes in gene expression after incubation of dendritic cells with immune complexes or medium. Since the dendritic cells are derived from three different mouse strains, either wild type, Fcγ receptor IIb KO (expresses only activating Fcγ receptors) or Fc receptor γ chain KO (expresses only inhibitory Fcγ receptor), the analysis gives important insight into the roles of the activating versus inhibiting Fcγ receptors on dendritic cells. Bone marrow-derived dendritic cells of the three mouse genotypes (see above) were incubated for 4 hours with medium (unstimulated) or OVA-anti OVA immune complexes (IC, stimulated) and changes in gene expression after stimulation with IC were compared between unstimulated vs stimulated with IC and across the three genotypes.
Project description:Asthma bronchiale is an inflammatory disease of the respiratory airways and a major factor of increasing health care costs worldwide. The molecular actors leading to asthma are not fully understood and require further investigation. The aim of this study was to monitor the proteome during asthma development from early inflammatory to late fibrotic stages. A time-course-based ovalbumin (OVA) mouse model was applied to establish an asthma phenotype and the lung proteome was analysed at four time points during asthma development (0 weeks = control, 5 weeks, 8 weeks and 12 weeks of OVA treatment).
Project description:Integrins facilitate intercellular movement and communication. Unlike the promiscuous activities of many integrins, β6 integrin is restricted to epithelia and partners exclusively with integrin αV to modulate acute lung injury (ALI). Given that ALI is a complication of respiratory infection, we used mice lacking β6 integrin (β6 KO) to probe the role of the epithelial layer in controlling the lung microenvironment during infection. We found β6 KO mice were protected from disease caused by influenza and Sendai virus infections. They were also protected from disease caused by Streptococcus pneumoniae infection alone and after prior influenza virus infection, the co-infection representing an often-lethal condition in humans. Resistance in the absence of epithelial β6 integrin was caused by intrinsic priming of the lung microenvironment by type I interferons through a mechanism involving transforming growth factor-β regulation. Expression of β6 on epithelia suppresses the production of interferons, providing an advantage to the pathogen. Acute inhibition of β6 function may therefore provide a means to improve outcomes in lung microbial infections. We used microarrays to explore the gene expression profiles differentially expressed in resident alveolar macrophage cells from wild-type and β6 integrin knockout mice. Cells were purified by sorting from lung digests. The comparison made was CD11c+, autofluorescent+ cells from wild-type controls to the dominant CD11c+, CD11b+, autofluorescent+ cells in the knockout. Note that conventional CD11c+ alveolar macrophages do not exist in the knockout mice.
Project description:This program aims at identifying the lung gene signature associated with OVA-challenged mouse asthma model to facilitate understanding of the disease mechanism and therapeutic compound testing The OVA-challenged profiling data was analyzed by identifying genes that were up- and down-regulated at selected p value and fold change in the lungs of OVA-challenged mice compared to the corresponding PBS-treated controls.
Project description:We examined the effect of PGE receptor EP3 subtype on mouse OVA-induced asthma model. We treat EP3 agonist 3h after OVA challenge. After 24 h of 3rd OVA challenge, whole lung was isolated and the gene expression change was determined.
Project description:Transcriptional profiling of primary human alveolar macrophages (AMs) comparing control untreated AMs with AMs exposed with Serotype 14 Streptococcus pneumoniae (NCTC11902) strain (MOI 10) for 4 hours) Two-condition experiment, control AMs vs. infected AMs. Biological replicates: 3 control replicates, 3 infected replicates MOI 10.
Project description:Mechanisms by which regulatory T (Treg) cells fail to control inflammation in asthma remain poorly understood. We show that a severe asthma-associated polymorphism in the interleukin-4 receptor alpha chain (IL-4Rα-R576) biases induced Treg (iTreg) cells towards a T helper 17 (TH17) cell fate. This skewing reflects the recruitment by IL-4Rα-R576 of the adaptor protein growth factor receptor-bound protein 2 (GRB2), which drives IL-17 expression by an extracellular signal-regulated kinase-, IL-6- and STAT3-dependent mechanism. We showed that the IL-4Rα-R576 mutation elicits TH17 airway responses in vivo, in a house dust mite (HDM)- or ovalbumin (OVA)-driven model of airway inflammation in the mice carry the IL-4Rα-R576 mutation (Il4raR576 mice). Treg cell-specific deletion of genes encoding IL-6Rα or the master TH17 cell regulator Retinoid-related Orphan Receptor γt (RORγt), but not IL-4 and IL-13, protected mice against exacerbated airway inflammation induced by IL-4Rα--576. Analysis of lung tissue Treg cells revealed that the expression of IL-17 and the TH17 cell-associated chemokine receptor CCR6 was largely overlapping and highly enriched in Treg and conventional T (Tconv) cells of allergen-treated Il4raR576 mice. To further characterize the subset of IL-17 producing Foxp3+ Treg in the lung of OVA-treated mice we utilized CCR6 as a marker of Treg cells committed towards the TH17 cell lineage to examine their functional, epigenetic and transcriptional profiles. CCR6+Foxp3EGFP+ Treg cells isolated from OVA-sensitized and challenged Il4raR576 mice, by FACS (Fluorescence Activated Cell Sorting) exhibited decreased methylation of the Foxp3 CNS2 locus comparing to CCR6âFoxp3EGFP+ Treg cells from same animals, indicative of decreased stability. They also exhibited profoundly decreased suppressive function as compared to CCR6â WT and CCR6â Il4raR576 counterparts. Transcriptional profiling of CCR6+Foxp3EGFP+ Treg cells revealed increased relative expression in CCR6+ Il4raR576 Treg cells of genes associated with a TH17 cell signature, including Rorc, Ccr6, Il23r, Il17a, Il17f, Il1r1, Nr1d1, Cstl, and Ahr comparing to CCR6âFoxp3EGFP+ Treg cells from same animals. Three CCR6+Foxp3EGFP+ Il4raR576 replicates and four CCR6âFoxp3EGFP+ Il4raR576 Treg replicates (controls) were sampled