Gene expression changes in C57Bl/6 and CD16-/- murine bone-marrow derived dendritic cells (BMDCs) following overnight stimulation with OVA or OVA-immune complex
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ABSTRACT: Atopic asthma is a chronic inflammatory disease of the lungs that is commonly associated with a Th2 response. The role of allergen-specific IgG in the initiation and development of allergic airway inflammation is still poorly understood; however, a receptor of IgG-immune complexes, CD16, has been demonstrated to promote augmentation of Th2 responses. To identify what genes downstream of CD16 signaling may be contributing to development of a Th2 response, we use ovalbumin (OVA) as our model antigen and compared wildtype and CD16-/- BMDCs that were treated overnight with OVA or OVA-immune complex. C57Bl/6 and CD16-/- BMDCs were treated for 24 hours with OVA or OVA-immune complex and then analyzed for gene expression changes.
Project description:Atopic asthma is a chronic inflammatory disease of the lungs that is commonly associated with a Th2 response. The role of allergen-specific IgG in the initiation and development of allergic airway inflammation is still poorly understood; however, a receptor of IgG-immune complexes, CD16, has been demonstrated to promote augmentation of Th2 responses. To identify what genes downstream of CD16 signaling may be contributing to development of a Th2 response, we use ovalbumin (OVA) as our model antigen and compared wildtype and CD16-/- BMDCs that were treated overnight with OVA or OVA-immune complex.
Project description:There remains a need for analysis of CD4 helper T cells differentiation in vivo. To this end ovalbumin (OVA)-specific CD4 (OTII) T cells transferred into congenic mice were studied. Live attenuated OVA-expressing Salmonella (SalOVA) induce T-bet and IFN-g in OTII cells, while alum-precipitated OVA (alumOVA) induces GATA-3 and IL-4. Although 70% of alumOVA-responding OTII cells express GATA-3, only 7% produce IL-4. Thus Th2-polarization defined solely by IL-4 production does not recognize the diversity of GATA-3-expressing effectors. Low-density arrays were designed to assess the expression of 384 genes by real-time RT-PCR. Extensive early diversification occurred in both responses. SalOVA selectively induced many chemokines and pro-inflammatory cytokines, while alumOVA induced few Th2-associated cytokines. Several cytokines and molecules associated with Th17 cells and follicular helper cells were also induced by both antigens. The transcription factor Helios was exclusively induced in alumOVA-responding OTII cells, and critically not in standard in vitro Th2-polarization systems. Early synchronous up-regulation of Helios and GATA-3 mRNA is paralleled at protein level with largely coincident localization in specific nuclear foci of OTII cells responding to alumOVA. This appears to be consistent with a key role for both transcription regulators in the direction of Th2 responses in vivo. Keywords: In vivo T cell polarization Ovalbumin (OVA)-specific CD4 (OTII) T cells were transferred into C57BL/6 mice that were immunized either with live attenuated OVA-expressing Salmonella (Sal) or with alum-precipitated OVA (alum), or not (Naïve). Gene expression assay was performed on FACS sorted OTII cells (Naïve, Sal, Alum). OTII cells were purified from three independent groups of ten naïve, or SalOVA-immunized or alumOVA-immunized mice.
Project description:The aim of the project was the identification of the transcriptional signature of Tfh cells that emerge following immunization with different adjuvants. For this purpose C57Bl/6 mice, bearing a Foxp3-GFP reporter system, were immunized with OVA-IFA or OVA-CpG. At day 11 post-immunization, Foxp3 negative Tfh cells were FACS-sorted from the draining lymph nodes and processed for single cell RNA sequencing.
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:With PNGase F digestion, PGC enrichment,OVA N-glycans were analyzed using PGC nanoLC-ESI-MS/MS.Datasets were analyzed by the N-glycan database search engine called GlycanGoggle developed in our group.
Project description:specific pathogen-free female BALB/c mice aged 7-8 weeks (Animal Resources Centre, Perth, Western Australia) were systemically sensitised by intraperitoneal injection of 50 M-5g of alum-precipitated chicken egg OVA (Grade V, ?98% pure, Sigma Australia) 21 and 7 days before inhalational challenge, then exposed to aerosolised OVA in a whole body inhalation exposure chamber (Unifab Corporation, Kalamazoo, MI). Chronic low-level challenge involved exposure to ?3 mg/m3 aerosolised OVA for 30 minutes/day on 3 days/week for up to 6 weeks. Particle concentration within the chamber was continuously monitored using a DustTrak 8520 instrument (TSI, St Paul, MN). All experimental procedures complied with the requirements of the Animal Care and Ethics Committee of the University of New South Wales (reference numbers: 06/119B and 08/09B). Mice were sacrificed after 1,2,4 and 6 weeks of OVA exposure. Control groups included naM-ove mice and mice that were not sensitised but were challenged for 6 weeks with aerosolised OVA.
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:The aim of the project was the identification of the transcriptional signature of effector T cells and Tfh cells that emerge following immunization with different adjuvants. For this purpose OVA-specific TCR-transgenic cells (from DO11.10 or OT-II mice) were adoptively transferred into congenic BALB/c or C57Bl/6 mice, subsequently immunized with OVA-IFA or OVA-CpG. At day 11, the OVA-specific T effector and Tfh cells were FACS sorted from the draining lymph nodes for RNA sequencing.
Project description: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.