Biomedical subjects

Natalie W Thiex

Publications and source records attributed to Natalie W Thiex.

3 recordsLinked to original sources

PubMed2024

Mannose receptor (MRC1) mediates uptake of dextran by bone marrow-derived macrophages.

Macrophages survey their environment using receptor-mediated endocytosis and pinocytosis. Receptor-mediated endocytosis allows internalization of specific ligands, whereas pinocytosis nonselectively internalizes extracellular fluids and solutes. CRISPR/Cas9 whole-genome screens were used to identify genes regulating constitutive and growth factor-stimulated dextran uptake in murine bone marrow-derived macrophages (BMDM). The mannose receptor c-type 1 (MRC1/CD206) was a top hit in the screen. Targeted gene disruptions of Mrc1 reduced dextran uptake but had little effect on fluid-phase uptake of Lucifer yellow. Other screen hits also differentially affected the uptake of dextran and Lucifer yellow, indicating internalization by separate mechanisms. Visualization of dextran and Lucifer yellow uptake by microscopy showed enrichment of dextran in small puncta, which was inhibitable by mannan, a ligand of MRC1. In contrast, Lucifer yellow predominantly was internalized in larger macropinosomes. In addition, IL4-treated BMDMs internalized more dextran than untreated BMDM correlating with increased MRC1 expression. Therefore, dextran is not an effective marker for pinocytosis in BMDMs since it is internalized by receptor-mediated process. Numerous genes that regulate dextran internalization in primary murine macrophages were identified in the whole-genome screens, which can inform understanding of the regulation of MRC1 expression and MRC1-mediated uptake in macrophages.

Animals↗

PubMed2024

Mannose receptor (MRC1) mediates uptake of dextran in macrophages via receptor-mediated endocytosis.

UNLABELLED: Macrophages maintain surveillance of their environment using receptor-mediated endocytosis and pinocytosis. Receptor-mediated endocytosis allows macrophages to recognize and internalize specific ligands whereas macropinocytosis non-selectively internalizes extracellular fluids and solutes. Here, CRISPR/Cas9 whole-genome screens were used to identify genes regulating constitutive and growth factor-stimulated dextran uptake in murine bone-marrow derived macrophages (BMDM). The endocytic mannose receptor c-type 1 ( Mrc1 , also known as CD206) was a top hit in the screen. Targeted gene disruptions of Mrc1 reduced dextran uptake but had little effect on uptake of Lucifer yellow, a fluid-phase marker. Other screen hits also differentially affected the uptake of dextran and Lucifer yellow, indicating the solutes are internalized by different mechanisms. We further deduced that BMDMs take up dextran via MRC1-mediated endocytosis by showing that competition with mannan, a ligand of MRC1, as well as treatment with Dyngo-4a, a dynamin inhibitor, reduced dextran uptake. Finally, we observed that IL4-treated BMDM internalize more dextran than untreated BMDM by upregulating MRC1 expression. These results demonstrate that dextran is not an effective marker for the bulk uptake of fluids and solutes by macropinocytosis since it is internalized by both macropinocytosis and receptor-mediated endocytosis in cells expressing MRC1. This report identifies numerous genes that regulate dextran internalization in primary murine macrophages and predicts cellular pathways and processes regulating MRC1. This work lays the groundwork for identifying specific genes and regulatory networks that regulate MRC1 expression and MRC1-mediated endocytosis in macrophages. SIGNIFICANCE STATEMENT: Macrophages constantly survey and clear tissues by specifically and non-specifically internalizing debris and solutes. However, the molecular mechanisms and modes of regulation of these endocytic and macropinocytic processes are not well understood. Here, CRISPR/Cas9 whole genome screens were used to identify genes regulating uptake of dextran, a sugar polymer that is frequently used as a marker macropinocytosis, and compared with Lucifer yellow, a fluorescent dye with no known receptors. The authors identified the mannose receptor as well as other proteins regulating expression of the mannose receptor as top hits in the screen. Targeted disruption of Mrc1 , the gene that encodes mannose receptor, greatly diminished dextran uptake but had no effect on cellular uptake of Lucifer yellow. Furthermore, exposure to the cytokine IL4 upregulated mannose receptor expression on the cell surface and increased uptake of dextran with little effect on Lucifer yellow uptake. Studies seeking to understand regulation of macropinocytosis in macrophages will be confounded by the use of dextran as a fluid-phase marker. MRC1 is a marker of alternatively activated/anti-inflammatory macrophages and is a potential target for delivery of therapeutics to macrophages. This work provides the basis for mechanistic underpinning of how MRC1 contributes to the receptor-mediated uptake of carbohydrates and glycoproteins from the tissue milieu and distinguishes genes regulating receptor-mediated endocytosis from those regulating the bona fide fluid-phase uptake of fluids and solutes by macropinocytosis.

Journal Article↗

PubMed2003

Targeting and blocking B7 costimulatory molecules on antigen-presenting cells using CTLA4Ig-conjugated liposomes: in vitro characterization and in vivo factors affecting biodistribution.

PURPOSE: CTLA4Ig, a fusion protein of CTLA-4 and Fc of immunoglobulin (Ig) heavy chain, inhibits the essential costimulatory signal for full T cell activation via blocking the interaction between CD28 and B7 molecules and renders T cell nonresponsiveness. CTLA4Ig has been used to control deleterious T cell activation in many experimental systems. We hypothesized that by conjugating CTLA4Ig to liposomes the efficacy of CTLA4Ig could be enhanced through multivalent ligand effect, superior targetability, and modification of the fate of ligated costimulatory molecules. METHODS AND RESULTS: Consistent with this hypothesis, liposome-conjugated CTLA4Ig bound to B7 and blocked their binding sites more efficiently than free CTLA4Ig, lowering the half maximal dose for B7 blocking by an order of the magnitude. These results were similar both in B7-1 expressing p815 cells and in activated macrophages. Moreover, CTLA4Ig-liposomes underwent rapid internalization upon cell surface binding through B7 molecules. In allogenic mixed lymphocyte reaction assays, the CTLA4Ig-liposomes were tested to show effective inhibition of T cell proliferation. In vivo, however, when CTLA4Ig-liposomes were injected into mice, a significant fraction was localized to the reticuloendothelial system (RES), presumably because of its binding to Fc receptors expressed on tissue macrophages. The Fc receptor-mediated uptake could be alleviated by coinjection of anti-FcR monoclonal antibody. In the mouse engrafted with pancreatic islets of Langerhans underneath the capsule of one kidney, despite the increased localization in RES, enhanced accumulation of CTLA4Ig-conjugated liposome was observed in the engrafted kidney compared to the contralateral kidney. CONCLUSION: We show that the conjugation of CTLA4Ig to liposome could increase the efficiency of the targeting by increasing the binding avidity at cellular level and by increasing the concentration at the target site in in vivo system. The biodistribution and circulation time data suggested that the CTLA4Ig-liposomes could be improved upon minimizing the FcR-mediated uptake by Fc receptor-bearing cells. Thus, the strategy of conjugating CTLA4Ig to liposomes could be exploited for immune intervention in transplantation and autoimmune diseases for the efficient blocking of costimulation.

Abatacept↗