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Monocytes Give Rise To Langerhans Cells That Preferentially Migrate To Lymph Nodes At Steady State

ABSTRACT: It is still debated whether the homeostatic function of tissue macrophages is dependent on their origin, and evidence suggests this may depend on the tissue of macrophage residence. Here, we answered this question for Langerhans cells (LCs), an embryonic-derived mononuclear phagocyte that has features of both tissue macrophages and dendritic cells. Using time-course analyses, bone marrow chimeras and fate tracing models, we found that the complete elimination of embryonic-derived LCs at steady state results in their repopulation from circulating monocytes. However, monocyte-derived LCs inefficiently replenished the epidermal niche. Instead, these cells preferentially migrated to skin draining lymph nodes. Mechanistically, we show that the superior migratory capability of monocyte-derived LCs is associated with higher expression of CD207/Langerin, a C-type lectin involved in the capture of skin microbes. Our data shows that ontogeny plays a role in the migratory behavior of epidermal LCs.

ORGANISM(S): Mus musculus

PROVIDER: GSE261079 | GEO | 2024/10/31

REPOSITORIES: GEO

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Project description:miRNA profiling of CD34+ derived, in vitro-generated Langerhans cells (LCs) and interstitial-type dendritic cells (intDCs). Epidermal Langerhans cells (LCs) form a network of dendritic cells (DCs) in basal/suprabasal layers of the skin and are characterized by a unique phenotype (CD207+ CD1abright CD324+ CD11b-). Due to their specific location at body surfaces, LCs are constantly exposed to environmental stimuli. Conversely, interstitial-type DCs (intDCs) are located in adjacent tissues and can be discriminated from LCs phenotypically (CD207- CD1adim CD324- CD11b+). These DCs constitute a second line of defense against pathogens that have crossed the epithelial barrier. During development both DC subsets originally arise from myeloid progenitor cells via monocyte-committed intermediates in response to specific microenvironmental signals. Additionally certain pools of DCs can be replenished by tissue resident cells or monocytes in response to specific microenvironmental signals (2, 4, 5). In vitro generated GM-CSF/IL-4-dependent DCs derived either from CD14+ monocytes or via CD14+CD11b+ monocyte intermediates from CD34+ progenitor cells share many characteristics with intDCs in vivo. On the other hand, LCs generated from CD34+ cells under serum-free TGF-beta1-dependent conditions phenotypically resemble epidermal-resident LCs. Since these two DC subsets are of considerable interest for clinical cell therapy studies, an improved understanding of their development and function is of substantial relevance. To identify differentially expressed miRNAs in DC subsets, we performed a miRNA screen. Therefore, we generated LCs or intDCs from human CD34+ cord blood haematopoietic progenitor cells as described. For intDCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGroÂ® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml) and TNFalpha (2.5 ng/ml) for 3-5 days and subsequently with GM-CSF (100 ng/ml) and IL-4 (2.5 ng/ml) for 4-5 days. For LCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGroÂ® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml), TNFalpha (2.5 ng/ml) and TGF-beta1 (0.5 ng/ml). The well-established immunophenotype of LCs (CD1ahi CD11b- CD207+ CD324+) and intDCs (CD1a+ CD11b+ CD207- CD324-) was confirmed by FACS. These two DC subsets were purified and submitted to differential miRNA profiling. Two conditioned experiment LCs vs. intDCs. Biological replicates: 3 LCs, 3 intDCs, independently differentiated, harvested and purified. 3 replicates each were pooled and hybridized on one array. Supplementary files linked below.

Project description:miRNA profiling of CD34+ derived, in vitro-generated Langerhans cells (LCs) and interstitial-type dendritic cells (intDCs). Epidermal Langerhans cells (LCs) form a network of dendritic cells (DCs) in basal/suprabasal layers of the skin and are characterized by a unique phenotype (CD207+ CD1abright CD324+ CD11b-). Due to their specific location at body surfaces, LCs are constantly exposed to environmental stimuli. Conversely, interstitial-type DCs (intDCs) are located in adjacent tissues and can be discriminated from LCs phenotypically (CD207- CD1adim CD324- CD11b+). These DCs constitute a second line of defense against pathogens that have crossed the epithelial barrier. During development both DC subsets originally arise from myeloid progenitor cells via monocyte-committed intermediates in response to specific microenvironmental signals. Additionally certain pools of DCs can be replenished by tissue resident cells or monocytes in response to specific microenvironmental signals (2, 4, 5). In vitro generated GM-CSF/IL-4-dependent DCs derived either from CD14+ monocytes or via CD14+CD11b+ monocyte intermediates from CD34+ progenitor cells share many characteristics with intDCs in vivo. On the other hand, LCs generated from CD34+ cells under serum-free TGF-beta1-dependent conditions phenotypically resemble epidermal-resident LCs. Since these two DC subsets are of considerable interest for clinical cell therapy studies, an improved understanding of their development and function is of substantial relevance. To identify differentially expressed miRNAs in DC subsets, we performed a miRNA screen. Therefore, we generated LCs or intDCs from human CD34+ cord blood haematopoietic progenitor cells as described. For intDCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGro® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml) and TNFalpha (2.5 ng/ml) for 3-5 days and subsequently with GM-CSF (100 ng/ml) and IL-4 (2.5 ng/ml) for 4-5 days. For LCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGro® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml), TNFalpha (2.5 ng/ml) and TGF-beta1 (0.5 ng/ml). The well-established immunophenotype of LCs (CD1ahi CD11b- CD207+ CD324+) and intDCs (CD1a+ CD11b+ CD207- CD324-) was confirmed by FACS. These two DC subsets were purified and submitted to differential miRNA profiling.

Project description:Langerhans-cell histiocytosis (LCH) is characterized by heterogeneous lesions containing CD207+ Langerhans cells (LCs) and lymphocytes. In this study, we isolated CD207+ cells and CD3+ T cells from LCH lesions to determine cell-specific gene expression. Compared to control epidermal CD207+ cells, the LCH CD207+ cells yielded 2113 differentially-expressed genes (FDR<0.01). Surprisingly, expression of many genes previously associated with LCH, including cell-cycle regulators, pro-inflammatory cytokines and chemokines were not significantly different from control LCs in our study. However, several novel genes whose products activate and recruit T cells to sites of inflammation, including SPP1 (osteopontin), were highly over-expressed in LCH CD207+ cells. Furthermore, several genes associated with immature myeloid dendritic cells were over-expressed in LCH CD207+ cells. Compared to the peripheral CD3+ cells from LCH patients, the LCH lesion CD3+ cells yielded only 162 differentially-regulated genes (FDR<0.01), and the expression profile of the LCH lesion CD3+ cells was consistent with an activated regulatory T cell phenotype with increased expression of FOXP3, CTLA4 as well as SPP1. Based on these results, we propose a new model of LCH pathogenesis in which lesions do not arise from epidermal Langerhans cells, but from accumulation of bone-marrow derived immature myeloid dendritic cells that recruit activated lymphocytes. Cell-specific gene expression from LCH biopsy specimens was evaluated by comparing hybridization signal from amplified cDNA on Affymetrix gene chips (U133A Plus 2.0) (Table1). Three sets of comparisons were performed: 1) Thirteen LCH CD207+ samples were compared to 12 control skin LCH CD207+ samples. 2) Seven LCH lesion CD3+ samples were compared to 7 peripheral blood CD3+ samples from the same patients. 3) Twelve LCH lesion CD3+ samples were compared to 4 pooled control tonsil CD3+ samples (5 individual tonsil CD3+ samples/pool).

Project description:Langerhans cells (LCs) are antigen presenting cells residing in the epidermis. Due to the difficulties with obtaining sufficient quantities of LCs for functional studies, many controversies exist about their origin and function. To gain insights into the molecular mechanisms underpinning LC biology and to elucidate how similar they are to a classical tissue resident DCs of myeloid origin, we undertook a whole transcriptome analysis of human skin migratory CD1a+ LCs and CD11c+ DDCs, stimulated with an epidermal pro-inflammatory cytokine, TNF-α over a time course of 24h. RNA was extracted from 250,000 human skin migratory CD1a+ LCs and CD11c+ DDCs stimulated in culture with 25ng/ml of TNF-α for 0, 2, 8 and 24h. RNA concentration and integrity was determined with an Agilent Bioanalyser (RIN of 7.0 or above) and gene expression analysis was carried out using the Human Genome U-219 Affymetrix platform by ARK-Genomics Centre, The Roslin Institute, Edinburgh. Expression data were normalised using the Robust Multichip Average (RMA) package. 2,334 transcripts regulated by exposure of skin DCs to TNF-α were identified with Bayesian Estimation of Temporal Regulation, a cut-off threshold 0.05 , for genes showing ≥ 1.5 fold difference between the maximum gene expression level and time 0 control in log2(x) RMA normalised gene expression. The interactive 3D diagram presenting the transcriptomic networks in human skin DCs can be viewed at: http://www.macrophages.com/LC_vs_DC RNA was extracted from 250,000 human skin migratory CD1a+ LCs and DDCs stimulated in culture with 25ng/ml of TNF-α for 0, 2, 8 and 24h. RNA concentration and integrity was determined with an Agilent Bioanalyser (RIN of 7.0 or above) and gene expression analysis was carried out using the Human Genome U-219 Affymetrix platform by ARK-Genomics Centre, The Roslin Institute, Edinburgh. Expression data were normalised using the Robust Multichip Average (RMA) package. 2,334 transcripts regulated by exposure of skin DCs to TNF-α were identified with Bayesian Estimation of Temporal Regulation, a cut-off threshold 0.05 , for genes showing ≥ 1.5 fold difference between the maximum gene expression level and time 0 control in log2(x) RMA normalised gene expression. *submitter cannot locate the CEL files

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Monocytes Give Rise To Langerhans Cells That Preferentially Migrate To Lymph Nodes At Steady State

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