ABSTRACT: To evaluate gene expression profiles on different dendritic cell subsets isolated from spleens of mice We used whole genome microarrays to identify genes specific to pDCs and not other DC subsets in the absence of disease pDCs, CD11b+ cDCs, and CD8+ cDCs were FACS-purified from spleens of healthy C57bl/6 mice
Project description:To evaluate gene expression profiles on different dendritic cell subsets isolated from spleens of mice We used whole genome microarrays to identify genes specific to pDCs and not other DC subsets in the absence of disease Overall design: pDCs, CD11b+ cDCs, and CD8+ cDCs were FACS-purified from spleens of healthy C57bl/6 mice
Project description:Committed precursors of conventional dendritic cells (pre-cDCs) derived from the common DC progenitor which differentiate into cDC subpopulations in peripheral tissues have been identified, but committed precursors for plasmacytoid DCs (pDCs) have not been found. Here we show that CDP-derived ‘CCR9- MHCIIlow BST2+ Siglec-H+ pDCs from murine bone marrow which enter the circulation and peripheral tissues have a common DC precursor function in vivo in the steady state. Upon adoptive transfer the fate of CCR9- pDC-like precursors is governed by the tissues they enter. In the bone marrow and liver most transferred CCR9- pDC-like precursors differentiate into CCR9+ pDCs, whereas in peripheral lymphoid organs, lung and intestine they can give rise to CCR9+ pDCs and cDCs. Thus, CCR9- pDC-like cells are novel CDP-derived circulating DC precursors with pDC and cDC potential, whose final differentiation depends on tissue-specific factors allowing adaptation to local requirements. Total RNA obtained from CCR9- pDC-like common DC progenitors and CCR9+ pDCs was compared for differential gene expression. 3 independent isolations were performed for the 2 samples. 6 arrays were run in total.
Project description:Two well-characterized blood dendritic cell populations, conventional (cDC) and plasmacytoid (pDC), exhibit multiple phenotypic, migratory and functional differences that suggest specialized and may be complementary and coordinated functions. To study this possible coordination, cDCs and pDCs from healthy blood donors were sorted and cDCs were stimulated either with LPS or R848 whereas pDCs were CFSE-labelled and maintained in IL3. Then, CFSE labelled-pDCs and stimulated-cDCs were mixed and co-cultured. Following this "conditioning", CFSE-pDCs were sorted again and further analyzed. Conditioned pDCs showed moderate phenotypic maturation and acquired allostimulatory capacity. Microarray and RT-PCR analyses showed the induction of different genes including chemokines and proinflammatory cytokines that were dependent on the stimulation received from the "conditioner" cDC. Additionally, the differential pattern of conditioning was confirmed by protein secretion analyses with the production of specific chemokines and cytokines by conditioned pDCs. Importantly, conditioning of pDCs by activated cDCs required cell-cell contact.
Project description:Hundreds of immune cell types work in coordination to maintain tissue homeostasis. Upon infection, dramatic changes occur with the localization, migration and proliferation of the immune cells to first alert the body of the danger, confine it to limit spreading, and finally extinguish the threat and bring the tissue back to homeostasis. Since current technologies can follow the dynamics of only a limited number of cell types, we have yet to grasp the full complexity of global in vivo cell dynamics in normal developmental processes and disease. Here we devise a computational method, digital cell quantification (DCQ), which combines genomewide gene expression data with an immune cell compendium to infer in vivo dynamical changes in the quantities of 213 immune cell subpopulations. DCQ was applied to study global immune cell dynamics in mice lungs at ten time points during a 7-day time course of flu infection. We find dramatic changes in quantities of 70 immune cell types, including various innate, adaptive and progenitor immune cells. We focus on the previously unreported dynamics of four immune dendritic cell subtypes, and suggest a specific role for CD103+CD11b- cDCs in early stages of disease and CD8+ pDC in late stages of flu infection. To better understand the physiological role of these differential dynamic changes in the DCs, we measured the genome-wide RNA expression of all four DC subpopulations from lung of influenza infected mice at four time points following infections (two mice per time-point). For sorting dendritic cells from lungs, the lungs from infected and control uninfected C57BL/6J mice were immersed in cold PBS, cut into small pieces in 5 ml DMEM media containing 10% Bovine Fetal Serum, the cell suspensions were grinded using 1ml syringe cup on a 70 μm cell strainers (BD Falcon). The cells were washed with ice cold PBS. Remaining red blood cells were lysed using ammonium chloride solution (Sigma). Cells were harvested, immersed 1ml FACS buffer [PBS+2% FBS, 1mM EDTA], Fc receptors were blocked with anti-mouse CD16/CD32, washed with FACS buffer and divided into two tubes for sorting cDC and pDC cells.
Project description:We describe a novel subset of CD8+ DCs in lymphoid organs of naïve mice characterized by expression of the CX3CR1 chemokine receptor. CX3CR1+CD8+ DCs lack hallmarks of classical CD8+ DCs, including IL12 secretion, the capacity to cross-present antigen and their developmental independence of the transcriptional factor BatF3. Gene expression profiling showed that CX3CR1+CD8+ DCs resemble CD8- cDCs. The microarray analysis further revealed a unique plasmacytoid DC (PDC) gene signature of CX3CR1+ CD8+ DCs. A PDC relationship of the cells is further supported by the fact that they harbor characteristic D-J immunoglobulin gene rearrangements and that development of CX3CR1+CD8+ DCs requires E2-2, the critical transcriptional regulator of PDCs. Thus, CX3CR1+ CD8+ DCs represent a unique DC subset, related to but distinct from PDCs. After collagenase D digestion, spleens from Cx3cr1gfp/+ C57BL/6 mice were enriched for CD11c+ cells by magnetic separation according to the manufacturer’s protocol (MiltenyiBiotec GmbH). Splenic CD11chi cells were isolated using the FACS ARIA high-speed sorter (Becton-Dickinson). Total RNA was extracted and subjected to gene expression profiling using the Mouse Genome 430.2 Affymetrix GeneChip
Project description:Comparison of gene expression changes of FACS sorted splenic CD11b+CD8a- and CD11b-CD8a+ cDC subsets reconstituted in vivo following total body irradiation in combination with exogenous retinoic acid or vehicle control. Mice maintained on a control diet were subjected to 6 Gy total body irradiation and then provided exogenous retinoic acid or vehicle control beginning on D(+)3 after irradiation. All treated mice also received adoptive T cell transfer of either Trp-1 CD4+ or Pmel-1 CD8+ T cells, rVV, and IL-2. Splenic cDCs were isolated on D(+)10 after total body irradiation by FACS sorting.
Project description:Plasmacytoid dendritic cells (pDCs) are scarcely present in the inflamed human atherosclerotic plaque, where they are presumed to exert pro-inflammatory functions through release of type I interferons. However, the precise role of pDCs in human atherosclerosis yet remains to be established. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process. We investigated the impact of human plaque pDCs on its local context, applying state of the art transcriptomics analysis on Laser Capture Microdissected fractions of human atherosclerotic plaques, distinctively enriched in pDCs, or pDCs-void.
Project description:Microarray experiments were performed to compare the gene expression profiles exhibited by immature and activated bone-marrow (BM) derived conventional DCs (BM-cDCs) and plasmacytoid DCs (BM-pDCs) from WT and miR155-/- mice. (time points 0, 4 and 24 hours)
Project description:Plasmacytoid dendritic cells (pDCs) rapidly produce type I interferon (IFN-I) in response to viruses and are essential for antiviral immune responses. Although related to classical dendritic cells (cDCs) in their development and expression profile, pDCs possess many distinct features. Unlike cDCs, pDCs develop in the bone marrow (BM) and emerge into peripheral lymphoid organs and tissues as fully differentiated cells. We now report that pDCs specifically express Runx2, a Runt family transcription factor that is essential for bone development. Runx2-deficient murine pDCs developed normally in the BM but were greatly reduced in the periphery. The defect was cell-intrinsic and was associated with the retention of mature Ly49Q+ pDCs in the BM. Runx2 was required for the expression of several pDC-enriched genes including chemokine receptors Ccr2 and Ccr5. Mature pDCs expressed high levels of Ccr5 at the surface, and Ccr5-deficient pDCs in a competitive setting were reduced in the periphery relative to the BM. Thus, Runx2 is required for the emergence of mature BM pDCs into the periphery, in a process that is partially dependent on Ccr5. These results establish Runx2 as a lineage-specific regulator of immune system development. Total BM cells were isolated from Runx2-/- and wildtype fetal liver chimeras 2 months post-reconstitution. BM cells were stained with antibodies against surface markers CD11b, CD11c, BST2, and B220. CD11b- BST2+ B220+ pDCs were purified by flow cytometry on a BD FACS Aria. RNA was purified immediately and prepared for microarray analysis using the Ambion WT labeling kit. Expression was analyzed using Affymetrix Mouse Gene 1.0 ST.
Project description:Plasmacytoid dendritic cells (pDC) efficiently produce large amounts of type I interferon in response to TLR7 and TLR9 ligands, whereas conventional DCs (cDC) predominantly secrete high levels of the cytokines IL-10 and IL-12. The molecular basis underlying this distinct phenotype is not well understood. Here, we identified the MAPK phosphatase Dusp9/MKP-4 by transcriptome analysis as selectively expressed in pDC, but not cDC. We confirmed the constitutive expression of Dusp9 at the protein level in pDC generated in vitro by culture with Flt3L and ex vivo in sorted splenic pDC. Dusp9 expression was low in B220- bone marrow precursors and was up-regulated during pDC differentiation, concomitant with established pDC markers. Higher expression of Dusp9 in pDC correlated with impaired phosphorylation of the MAPK ERK1/2 upon TLR9 stimulation. Notably, Dusp9 was not expressed at detectable levels in human pDC, although these displayed similarly impaired activation of ERK1/2 MAPK compared to cDC. Enforced retroviral expression of Dusp9 in mouse GM-CSF-induced cDC increased the expression of TLR7/9-induced IL-12p40 and IFNwhereas IL-10 levels were diminished. Taken together, our results suggest that the species-specific, selective expression of Dusp9 in murine pDC contributes to the differential cytokine/interferon output of pDC and cDC. pDC and cDC subsets were purified from mouse spleens to high purity and analysed by Affymetrix GeneChips.