Project description:Defense against attaching and effacing (A/E) bacteria requires the sequential generation of IL-23 and IL-22 to induce protective mucosal responses. While the critical source of IL-22 has been identified as CD4+ and Nkp46+ innate lymphoid cells (ILCs), the precise source of IL-23 is unclear. Here, we use genetic techniques to deplete specific classical dendritic cell (cDC) subsets and analyze immunity to the A/E pathogen Citrobacter rodentium. We find that Zbtb46+ cDCs, and specifically Notch2-dependent intestinal CD11b+ cDCs, but not Batf3-dependent CD103+ cDCs, are required for IL-23 production and immunity against C. rodentium. Notch2 controls cDC differentiation at a terminal step mediated by lymphotoxin signaling. Importantly, these results provide the first demonstration of a non-redundant function of CD11b+ cDCs in vivo. Analysis of differentially expressed genes in ESAM+ and ESAM- CD11b+ and DEC205+ splenic classical DC subsets. Splenocytes were harvested from WT C57Bl/6 or WT Cx3cr1-gfp mice and cDC subsets sorted to >95% purity on the FACSAriaII.
Project description:Defense against attaching and effacing (A/E) bacteria requires the sequential generation of IL-23 and IL-22 to induce protective mucosal responses. While the critical source of IL-22 has been identified as CD4+ and Nkp46+ innate lymphoid cells (ILCs), the precise source of IL-23 is unclear. Here, we use genetic techniques to deplete specific classical dendritic cell (cDC) subsets and analyze immunity to the A/E pathogen Citrobacter rodentium. We find that Zbtb46+ cDCs, and specifically Notch2-dependent intestinal CD11b+ cDCs, but not Batf3-dependent CD103+ cDCs, are required for IL-23 production and immunity against C. rodentium. Notch2 controls cDC differentiation at a terminal step mediated by lymphotoxin signaling. Importantly, these results provide the first demonstration of a non-redundant function of CD11b+ cDCs in vivo. Analysis of Notch2-dependent genes in CD11b+ and DEC205+ splenic classical DC subsets. Splenocytes were harvested from littermate WT Notch2 f/f C57Bl/6 or Notch2 CD11c-cre C57Bl/6 mice and DC subsets sorted to >95% purity on the FACSAriaII.
Project description:Analysis of L-Myc-dependent genes in pDCs and classical DC subsets with and without stimulation. Splenocytes were harvested from C57BL/6 wild-type (WT) or 10 generation C57BL/6 backcrossed Mycl1-gfp/gfp (LmycKO) mice and DC subsets sorted to >95% purity on the FACSAriaII.
Project description:Esam/CD4+ dendritic cells are part of the innate immunity essential for priming and activating of CD4+ T cells To identify Runx3 responsive genes Esam dendritic cells were freshly sorted from macs enriched splenic DCs taken from 6 weeks old mice. Four samples from four mice were sorted and analyzed where in each littermates pair consisted of a control and Runx3 conditional KO. Mice lacking Runx3 specifically in the DC compartment were produced by crossing Runx3fl/fl mice onto CD11c-Cre mice. This mating scheme generated Runx3fl/fl/CD11c:Cre (CD11c-DC-Runx3Δ) mice.
Project description:Analysis of stage-specific gene expression in Zbtb46GFP/+ pre-CD8 DCs, pre-CD4 DCs, CD24 cDCs and CD172a cDCs Bone Marrow and Splenocytes were harvested from 8-10 littermate Zbtb46GFP/+ mice and sorted to >95% purity on the FACS AriaFusion.
Project description:CD4+ dendritic cells are part of the innate immunity essential for priming and activating of CD4+ T cells To identify Runx3 responsive genes CD4+ dendritic cells were sorted from freshly isolated macs enriched splenic DCs taken from 6 weeks old mice. Six samples from six mice were sorted and analyzed where in each littermates pair consisted of a control and Runx3 KO.
Project description:Zbtb46 represses G-CSFR and LifR in cDCs Zbtb46 does not significantly affect gene expression in erythroid progenitors WT, Het, and KO cells were sorted from BM or spleen and analyzed. Pre MegE cells were sorted as CD117+ CD150+ CD105-CD41-CD16/32-. Pre CFU-E cells were sorted as CD117+ CD150+ CD105lo CD41- CD16/32-. CFU-E cells were sorted as CD117+ CD150- CD105+ Cd41- CD16/32-. Splenic CD4+ DCs were sorted as B220- CD11c+ MHCII+ CD8- CD172+ CD11b+ CD4+.
Project description:Xbp1 is a major transcription factor in the unfolded protein response. To uncover its function in DCs we generated a conditional KO for Xbp1 in dendritic cells. We here compare the expression of mRNAs in two different splenic DC subpopulations, CD8a and CD11b DCs in both WT and KO mice. Reference: Inositol-requiring enzyme 1-alpha regulates CD8a dendritic cell function via regulated mRNA decay. Osorio et al, Nature Immunology (2014) Primary DC subsets were isolated and sorted from spleens from 3 different WT or CD11c-cre Xbp-1fl/fl mice. RNA was isolated, converted to cDNA and then hybridised on Affymetrix GeneChip Mouse Gene 1.0 ST Arrays (GPL6246).
Project description:Analysis of genes induced in DC precursors and in BM cells and monocytes treated with GM-CSF For progenitor arrays, bone marrow progenitors (CMP, GMP, CDP, and pre-cDC) were harvested from WT C57Bl/6 mice. For culture arrays, BM was cultured in the presence of GM-CSF or M-CSF and adherent and non-adherent cells sorted. For monocyte cultures, sorted BM monocytes were treated with GM-CSF for 0, 24 or 48 hours.
Project description:This SuperSeries is composed of the following subset Series: GSE37028: Microarray analysis of Zbtb46 KO CD4+ Splenic DCs and bone marrow erythroid progenitors GSE37029: Microarray analysis of WT bone marrow myeloid progenitors, BM cultured with GM-CSF and M-CSF, and monocytes treated with GM-CSF Refer to individual Series