fcbb1184ba79d1b225a6b2eac7e6841c pmc_34298983.pdf 03669767a0f0082357e7cc43101b088a528605e6 pmc_34298983.pdf a8412ee0f7c02ef5b06ad1b920f92be99c889a2686a22060ae9181ecee69a193 pmc_34298983.pdf Title: Transfection of Vitamin D3-Induced Tolerogenic Dendritic Cells for the Silencing of Potential Tolerogenic Genes. Identification of CSF1R-CSF1 Signaling as a Glycolytic Regulator Subject: The use of autologous tolerogenic dendritic cells (tolDC) has become a promising strategy to re-establish immune tolerance in autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (VitD3-tolDC) has been widely tested because of their immune regulatory properties. To identify molecules and pathways involved in the generation of VitD3-tolDC, we established an easy and fast gene silencing method based on the use of Viromer blue to introduce siRNA into monocytes on day 1 of culture differentiation. The analysis of the effect of CD209 (DC-SIGN) and CD115 (CSF1R) down-modulation on the phenotype and functionality of transfected VitD3-tolDC revealed a partial role of CD115 in their tolerogenicity. Further investigations showed that CSF1R-CSF1 signaling is involved in the induction of cell metabolic reprogramming, triggering glycolysis to produce high amounts of lactate, a novel suppressive mechanism of T cell proliferation, recently found in autologous tolerogenic dendritic cells (ATDCs). Keywords: tolerance; autoimmunity; vitamin D3; tolerogenic dendritic cells; CSF1R; DC-SIGN Author: María José Mansilla, Iñigo González-Larreategui, Neus Figa-Martín, Jaume Barallat, Federico Fondelli, Ares Sellés-Rius, Bibiana Quirant-Sánchez, Aina Teniente-Serra and Eva Martínez-Cáceres Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jul 9 06:08:17 2021 CEST ModDate: Fri Jul 9 10:08:43 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2642505 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- KORLVE+VnURWPalladioL Type 1 Custom yes yes no 10 0 IHZYNP+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 ZDICAO+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 PAQKXS+URWPalladioL-Roma Type 1 Custom 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PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 475 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: HiPerFect Reagent Allows VitD3-tolDC Transfection after Maturation Stimulus Destination: subsection.2.1 Item: Title: Viromer Blue Allows Monocyte Transfection during Their Differentiation to VitD3-tolDC Destination: subsection.2.2 Item: Title: CD209 Gene Silencing in VitD3-tolDC Alters Neither Their Phenotype nor Their Functionality Destination: subsection.2.3 Item: Title: CD115 Gene Silencing in VitD3-tolDC Partially Inhibits Their Tolerogenic Properties Destination: subsection.2.4 Item: Title: CD115 Signaling Induced by CSF1 Triggers Tolerogenic Properties in Monocytes Destination: subsection.2.5 Item: Title: CD115 Induces Glycolysis Activation on VitD3-tolDC Destination: subsection.2.6 Item: Title: Discussion Destination: section.3 Item: Title: Material and Methods Destination: section.4 Children: Item: Title: Monocyte Isolation Destination: subsection.4.1 Item: Title: Generation of VitD3-tolDC Destination: subsection.4.2 Item: Title: Transfection of Monocytes Using HiPerFect Reagent Destination: subsection.4.3 Item: Title: Transfection of Monocytes Using Viromer Blue Reagent Destination: subsection.4.4 Item: Title: Chemical Inhibition of CSF1R Destination: subsection.4.5 Item: Title: CSF1 and IL34 Treatment Destination: subsection.4.6 Item: Title: Phenotype Analysis Destination: subsection.4.7 Item: Title: Allogeneic Proliferation Assay Destination: subsection.4.8 Item: Title: Gene Expression Analysis Destination: subsection.4.9 Item: Title: Metabolic Analysis of Culture Supernatants Destination: subsection.4.10 Item: Title: Statistical Analysis Destination: subsection.4.11 Item: Title: References Destination: subsection.4.11 Info: Title: Transfection of Vitamin D3-Induced Tolerogenic Dendritic Cells for the Silencing of Potential Tolerogenic Genes. Identification of CSF1R-CSF1 Signaling as a Glycolytic Regulator Author: María José Mansilla, Iñigo González-Larreategui, Neus Figa-Martín, Jaume Barallat, Federico Fondelli, Ares Sellés-Rius, Bibiana Quirant-Sánchez, Aina Teniente-Serra and Eva Martínez-Cáceres Subject: The use of autologous tolerogenic dendritic cells (tolDC) has become a promising strategy to re-establish immune tolerance in autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (VitD3-tolDC) has been widely tested because of their immune regulatory properties. To identify molecules and pathways involved in the generation of VitD3-tolDC, we established an easy and fast gene silencing method based on the use of Viromer blue to introduce siRNA into monocytes on day 1 of culture differentiation. The analysis of the effect of CD209 (DC-SIGN) and CD115 (CSF1R) down-modulation on the phenotype and functionality of transfected VitD3-tolDC revealed a partial role of CD115 in their tolerogenicity. Further investigations showed that CSF1R-CSF1 signaling is involved in the induction of cell metabolic reprogramming, triggering glycolysis to produce high amounts of lactate, a novel suppressive mechanism of T cell proliferation, recently found in autologous tolerogenic dendritic cells (ATDCs). Keywords: tolerance; autoimmunity; vitamin D3; tolerogenic dendritic cells; CSF1R; DC-SIGN Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jul 09 06:08:17 CEST 2021 ModDate: Fri Jul 09 10:08:43 CEST 2021 ID: 0x1a77302461fc5ef681cc64d5d714de52, 0x1a77302461fc5ef681cc64d5d714de52 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: OAMRUE+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: OAMRUE+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: ZDICAO+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: ZDICAO+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: PAQKXS+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: PAQKXS+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: 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