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Lymphatic transport of exosomes as a rapid route of information dissemination to the lymph node.

Srinivasan S, Vannberg FO, Dixon JB - Sci Rep (2016)

Bottom Line: Furthermore, we have demonstrated a differential distribution of exosomes in the draining lymph nodes that is dependent on the lymphatic flow.Lastly, through endpoint analysis of cellular distribution of exosomes in the node, we identified macrophages and B-cells as key players in exosome uptake.Together these results suggest that exosome transfer by lymphatic flow from the periphery to the lymph node could provide a mechanism for rapid exchange of infection-specific information that precedes the arrival of migrating cells, thus priming the node for a more effective immune response.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Georgia Institute of Technology, Atlanta, GA, USA.

ABSTRACT
It is well documented that cells secrete exosomes, which can transfer biomolecules that impact recipient cells' functionality in a variety of physiologic and disease processes. The role of lymphatic drainage and transport of exosomes is as yet unknown, although the lymphatics play critical roles in immunity and exosomes are in the ideal size-range for lymphatic transport. Through in vivo near-infrared (NIR) imaging we have shown that exosomes are rapidly transported within minutes from the periphery to the lymph node by lymphatics. Using an in vitro model of lymphatic uptake, we have shown that lymphatic endothelial cells actively enhanced lymphatic uptake and transport of exosomes to the luminal side of the vessel. Furthermore, we have demonstrated a differential distribution of exosomes in the draining lymph nodes that is dependent on the lymphatic flow. Lastly, through endpoint analysis of cellular distribution of exosomes in the node, we identified macrophages and B-cells as key players in exosome uptake. Together these results suggest that exosome transfer by lymphatic flow from the periphery to the lymph node could provide a mechanism for rapid exchange of infection-specific information that precedes the arrival of migrating cells, thus priming the node for a more effective immune response.

No MeSH data available.


Exosomes transported rapidly and selectively through the lymphatic endothelium in vitro.(a) Schematic of transport experiment, (b) Transport of exosomes across the lymphatic endothelium occurs rapidly (t = 5–30 mins) and is enhanced in the presence of cells, (c) Exosomes are selectively transported into the lymphatic endothelium (versus beads), (d) Orthogonal view of LEC’s (nuclei stained with DAPI, actin stained red) with PKH67 exosomes and at 37 °C. Scale bar, 5 μm and (e) quantitation of exosome and beads in cells by fluorescence intensity at 37 °C and 4 °C.
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f2: Exosomes transported rapidly and selectively through the lymphatic endothelium in vitro.(a) Schematic of transport experiment, (b) Transport of exosomes across the lymphatic endothelium occurs rapidly (t = 5–30 mins) and is enhanced in the presence of cells, (c) Exosomes are selectively transported into the lymphatic endothelium (versus beads), (d) Orthogonal view of LEC’s (nuclei stained with DAPI, actin stained red) with PKH67 exosomes and at 37 °C. Scale bar, 5 μm and (e) quantitation of exosome and beads in cells by fluorescence intensity at 37 °C and 4 °C.

Mentions: To test the hypothesis that transport of exosomes across the lymphatic endothelium is higher than size and density matched beads, the effective permeability of cells (Peff_cell) to the fluorescently labeled exosomes and beads in the basal to apical direction was measured using a transwell system as described previously21 (Fig. 2a). Exosomes, beads and dextran were freely transported across the membrane in the absence of cells (Fig. 2b, dotted lines) and neither exosomes nor beads stuck to the membrane (Supp. Fig. 1d). Additionally, the size ranges of the exosomes collected on the apical side were similar to that on the basal side, further confirming exosome trafficking from the basal to apical sides of the LECs (Supp. Fig. 1e). To understand the kinetics of exosome transport by LECs, transport was assessed every 5 min. The transport of beads was below the detection limit at 30 min and therefore is represented as a solid line at the zero mark indicating no transport. Flux was calculated both in the presence and absence of cells to determine the extent that LECs enhanced or alternatively provided a barrier to selective transport. Dextran, being extremely small (3kDa) freely diffused through the Transwell membrane in the absence of cells, but transport was slightly reduced in the presence of LECs and rapidly reached equilibrium at about 15 min. Exosomes were rapidly detected across the lymphatic endothelium at 5 min and transport in the presence of cells was much higher than in the absence of cells (~2 fold) with transport reaching equilibrium at ~20 min (Fig. 2b, solid lines). In order to quantify this difference, the effective permeability of cells was calculated after incubation with exosomes and beads for 75 mins so transport could attain equilibrium at 37 °C and 4 °C. Exosomes were transported across the lymphatic endothelium ~10 times more as compared to the fluorescent size matched beads (p-value <0.01) at 37 °C (Fig. 2c). When the cells were fixed and examined using confocal microscopy, exosomes were seen within cells at 37 °C (Fig. 2d) whereas beads were not (Supp. Fig. 1b). However, exosome uptake was greatly reduced at 4 °C (Supp. Fig. 1a). The fluorescence in the images that corresponded to exosomes and beads was quantified at 37 °C and 4 °C which showed that exosome transport was reduced by ~80% at 4 °C (Fig. 2e, p-value <0.001). Collectively this data suggests that the lymphatics actively transported exosomes in vitro.


Lymphatic transport of exosomes as a rapid route of information dissemination to the lymph node.

Srinivasan S, Vannberg FO, Dixon JB - Sci Rep (2016)

Exosomes transported rapidly and selectively through the lymphatic endothelium in vitro.(a) Schematic of transport experiment, (b) Transport of exosomes across the lymphatic endothelium occurs rapidly (t = 5–30 mins) and is enhanced in the presence of cells, (c) Exosomes are selectively transported into the lymphatic endothelium (versus beads), (d) Orthogonal view of LEC’s (nuclei stained with DAPI, actin stained red) with PKH67 exosomes and at 37 °C. Scale bar, 5 μm and (e) quantitation of exosome and beads in cells by fluorescence intensity at 37 °C and 4 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834495&req=5

f2: Exosomes transported rapidly and selectively through the lymphatic endothelium in vitro.(a) Schematic of transport experiment, (b) Transport of exosomes across the lymphatic endothelium occurs rapidly (t = 5–30 mins) and is enhanced in the presence of cells, (c) Exosomes are selectively transported into the lymphatic endothelium (versus beads), (d) Orthogonal view of LEC’s (nuclei stained with DAPI, actin stained red) with PKH67 exosomes and at 37 °C. Scale bar, 5 μm and (e) quantitation of exosome and beads in cells by fluorescence intensity at 37 °C and 4 °C.
Mentions: To test the hypothesis that transport of exosomes across the lymphatic endothelium is higher than size and density matched beads, the effective permeability of cells (Peff_cell) to the fluorescently labeled exosomes and beads in the basal to apical direction was measured using a transwell system as described previously21 (Fig. 2a). Exosomes, beads and dextran were freely transported across the membrane in the absence of cells (Fig. 2b, dotted lines) and neither exosomes nor beads stuck to the membrane (Supp. Fig. 1d). Additionally, the size ranges of the exosomes collected on the apical side were similar to that on the basal side, further confirming exosome trafficking from the basal to apical sides of the LECs (Supp. Fig. 1e). To understand the kinetics of exosome transport by LECs, transport was assessed every 5 min. The transport of beads was below the detection limit at 30 min and therefore is represented as a solid line at the zero mark indicating no transport. Flux was calculated both in the presence and absence of cells to determine the extent that LECs enhanced or alternatively provided a barrier to selective transport. Dextran, being extremely small (3kDa) freely diffused through the Transwell membrane in the absence of cells, but transport was slightly reduced in the presence of LECs and rapidly reached equilibrium at about 15 min. Exosomes were rapidly detected across the lymphatic endothelium at 5 min and transport in the presence of cells was much higher than in the absence of cells (~2 fold) with transport reaching equilibrium at ~20 min (Fig. 2b, solid lines). In order to quantify this difference, the effective permeability of cells was calculated after incubation with exosomes and beads for 75 mins so transport could attain equilibrium at 37 °C and 4 °C. Exosomes were transported across the lymphatic endothelium ~10 times more as compared to the fluorescent size matched beads (p-value <0.01) at 37 °C (Fig. 2c). When the cells were fixed and examined using confocal microscopy, exosomes were seen within cells at 37 °C (Fig. 2d) whereas beads were not (Supp. Fig. 1b). However, exosome uptake was greatly reduced at 4 °C (Supp. Fig. 1a). The fluorescence in the images that corresponded to exosomes and beads was quantified at 37 °C and 4 °C which showed that exosome transport was reduced by ~80% at 4 °C (Fig. 2e, p-value <0.001). Collectively this data suggests that the lymphatics actively transported exosomes in vitro.

Bottom Line: Furthermore, we have demonstrated a differential distribution of exosomes in the draining lymph nodes that is dependent on the lymphatic flow.Lastly, through endpoint analysis of cellular distribution of exosomes in the node, we identified macrophages and B-cells as key players in exosome uptake.Together these results suggest that exosome transfer by lymphatic flow from the periphery to the lymph node could provide a mechanism for rapid exchange of infection-specific information that precedes the arrival of migrating cells, thus priming the node for a more effective immune response.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Georgia Institute of Technology, Atlanta, GA, USA.

ABSTRACT
It is well documented that cells secrete exosomes, which can transfer biomolecules that impact recipient cells' functionality in a variety of physiologic and disease processes. The role of lymphatic drainage and transport of exosomes is as yet unknown, although the lymphatics play critical roles in immunity and exosomes are in the ideal size-range for lymphatic transport. Through in vivo near-infrared (NIR) imaging we have shown that exosomes are rapidly transported within minutes from the periphery to the lymph node by lymphatics. Using an in vitro model of lymphatic uptake, we have shown that lymphatic endothelial cells actively enhanced lymphatic uptake and transport of exosomes to the luminal side of the vessel. Furthermore, we have demonstrated a differential distribution of exosomes in the draining lymph nodes that is dependent on the lymphatic flow. Lastly, through endpoint analysis of cellular distribution of exosomes in the node, we identified macrophages and B-cells as key players in exosome uptake. Together these results suggest that exosome transfer by lymphatic flow from the periphery to the lymph node could provide a mechanism for rapid exchange of infection-specific information that precedes the arrival of migrating cells, thus priming the node for a more effective immune response.

No MeSH data available.