Project description:IL-23 negatively regulates St2 and Gata3 expression in intestinal CD4+ T cells Colitis was induced by co-transfer of WT and Il23r-/- naïve CD4 T cells into Rag1-/- recipients. Upon development of clinical signs of inflammation WT and Il23r-/- CD4+ T cells were sort-purified from the colon lamina propria on the basis of congenic markers. Total RNA was extracted immediately after the sort without further manipulation.
Project description:The colonic lamina propria contains a distinct population of Foxp3+ T regulatory cells (Tregs) that modulate responses to commensal microbes. Analysis of gene expression revealed that the transcriptome of colonic Tregs is distinct from splenic and other tissue Tregs. Rorγ and Helios in colonic Tregs mark distinct populations: Rorγ+Helios- or Rorγ-Helios+ Tregs. We uncovered an unanticipated role for Rorγ, a transcription factor generally considered to be antagonistic to Foxp3. Rorγ in colonic Tregs accounts for a small but specific part of the colon-specific Treg signature. (1) Total colonic and splenic Foxp3+ Treg comparison: Lymphocytes were isolated from colonic lamina propria and spleens of Foxp3-ires-GFP mice, where GFP reports Foxp3 expression. TCRb+CD4+GFP+ cells were double sorted into Trizol. (2) Colonic Rorγ+ and Rorγ- Treg comparison: Foxp3-ires-Thy1.1 reporter mice were crossed to Rorc-GFP reporter mice to generate mice that report both Foxp3 and Rorγ expression. Rorγ+Foxp3+ Tregs (TCRb+CD4+Thy1.1+GFP+) and Rorγ-Foxp3+ Tregs (TCRb+CD4+Thy1.1+GFP-) from colonic lamina propria were double sorted into Trizol.To reduce variability and increase cell number, cells from multiple mice were pooled for sorting and at least three replicates were generated for all groups. RNA from 1.5-3.0 x104 cells was amplified, labeled and hybridized to Affymetrix Mouse Gene 1.0 ST Arrays.
Project description:To further development of our gene expression approach to CD300a deficiency on dendritic cells (DCs) in colonic lamina propria, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish CD300a deficiency on DCs in colonic lamina propria from those of WT mice. Colonic lamina propria DCs were obtained by cell sorter from WT and CD300a deficient mice raised under SPF and GF condition. Expression of Ifnb1 was significantly higher in CD300a deficient DCs, quantified in the same RNA samples by real-time PCR. Gene expression in WT and CD300a colonic lamina propria DCs raised under SPF and GF conditions were measured. Colonic lamina propria cells were obtained from 5 mice in each conditions. Takara-Bio
Project description:The colonic lamina propria contains a distinct population of Foxp3+ T regulatory cells (Tregs) that modulate responses to commensal microbes. Analysis of gene expression revealed that the transcriptome of colonic Tregs is distinct from splenic and other tissue Tregs. Rorγ and Helios in colonic Tregs mark distinct populations: Rorγ+Helios- or Rorγ-Helios+ Tregs. We uncovered an unanticipated role for Rorγ, a transcription factor generally considered to be antagonistic to Foxp3. Rorγ in colonic Tregs accounts for a small but specific part of the colon-specific Treg signature. Nrp1- Tregs were sorted from Foxp3-cre.Rorcfl/fl mice, which have a Treg-selective deletion of Rorc, or paired WT littermates. For low-input RNAseq, 1,000 TCRb+CD4+YFP(Foxp3)+Nrp1- cells were double-sorted into Trizol, RNA extracted and reverse-transcribed using ArrayScript (Ambion). To reduce variability at least three replicates were generated.
Project description:We report that colonic lamina propria anti-inflammatory macrophages from Il10rb-/- (KO) mice at 12 weeks possessed significantly high levels of proinflammatory transcripts compared to the ones from Il10rb+/- (het) controls. While the transcriptional profile between macrophages from KO and het mice was comparable at 1 weeks and did not possess significant levels of proinflammatory transcripts, we show that the macrophages from KO mice were enriched for proinflammatory transcripts at 3 weeks compared to the ones from 3 week old het controls. These data indicate that the anti-inflammatory signature of colonic lamina propria macrophages from Il10rb-/- mice is compromized at 3 weeks. Overall design: Lamina propria macrophages were FACS sort-purified from 1, 3, and 12 weeks old KO and het mice based on gating strategy; CD45+CD11b+CD11cintCD103-CD64+Ly6C-MHCII+. One mouse each for 3, 12 weeks, and a pool of 3-5 mice at 1 weeks was included in RNA-seq analysis.
Project description:Commensal bacteria shapes gut immune system. Colonization bacteria increase the frequency of regulatory T cells, however, the molecular mechanisms has not yet been unknown. To reveal the mechanism, we isolated Treg cells and Non-Treg cells and performed the global expression analysis. Colonic lamina propria cells were isolated from mice described above. Cells were divided into Treg or Non-Treg cells by FACS AriaIII
Project description:Commensal bacteria shapes gut immune system. Colonization bacteria increase the frequency of regulatory T cells, however, the molecular mechanisms has not yet been unknown. To reveal the mechanism, we isolated Treg cells and Non-Treg cells and performed the global expression analysis. Colonic lamina propria cells were isolated from mice described above. Treg cells and Non-Treg cells (also called as Tconv cells) were sorted by FACS AriaIII
Project description:Leber2015 - Mucosal immunity and gut
microbiome interaction during C. difficile infection
This model is described in the article:
Systems Modeling of
Interactions between Mucosal Immunity and the Gut Microbiome
during Clostridium difficile Infection.
Leber A, Viladomiu M, Hontecillas R,
Abedi V, Philipson C, Hoops S, Howard B, Bassaganya-Riera
PLoS ONE 2015; 10(7): e0134849
Clostridium difficile infections are associated with the use
of broad-spectrum antibiotics and result in an exuberant
inflammatory response, leading to nosocomial diarrhea, colitis
and even death. To better understand the dynamics of mucosal
immunity during C. difficile infection from initiation through
expansion to resolution, we built a computational model of the
mucosal immune response to the bacterium. The model was
calibrated using data from a mouse model of C. difficile
infection. The model demonstrates a crucial role of T helper 17
(Th17) effector responses in the colonic lamina propria and
luminal commensal bacteria populations in the clearance of C.
difficile and colonic pathology, whereas regulatory T (Treg)
cells responses are associated with the recovery phase. In
addition, the production of anti-microbial peptides by inflamed
epithelial cells and activated neutrophils in response to C.
difficile infection inhibit the re-growth of beneficial
commensal bacterial species. Computational simulations suggest
that the removal of neutrophil and epithelial cell derived
anti-microbial inhibitions, separately and together, on
commensal bacterial regrowth promote recovery and minimize
colonic inflammatory pathology. Simulation results predict a
decrease in colonic inflammatory markers, such as neutrophilic
influx and Th17 cells in the colonic lamina propria, and length
of infection with accelerated commensal bacteria re-growth
through altered anti-microbial inhibition. Computational
modeling provides novel insights on the therapeutic value of
repopulating the colonic microbiome and inducing regulatory
mucosal immune responses during C. difficile infection. Thus,
modeling mucosal immunity-gut microbiota interactions has the
potential to guide the development of targeted fecal
transplantation therapies in the context of precision medicine
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Project description:Colorectal cancer (CRC) was induced in Foxp3/eGFP reporter mice by the azoxymethane/dextran sulphate sodium salt (AOM/DSS) protocol. Mice were injected i.p. with the procarcinogen AOM (12.5 mg/kg of body weight). After 1 week, mice received drinking water supplemented with 2.5% DSS for 5 to 7 days, followed by 2 weeks of regular water. The DSS administration was repeated twice with 2% DSS. Mice were sacrificed at week 11 and lamina propia lymphocytes (LPLs) from the colon were isolated. CD4+FOXP3+ (eGFP+) ST2+ or ST2- Tregs were separated from colonic LPLs of CRC induced mice using a FACSAria II cell sorter. Microarray analysis was performed to analyze if ST2+ FOXP3+ Tregs from the colon of CRC mice present a distinct transcription pattern compared to ST2- FOXP3+ Tregs. By this, the role of ST2 for Treg function during intestinal tumorigenesis should be characterized.