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. 2016 Mar 1;196(5):2249-61.
doi: 10.4049/jimmunol.1401545. Epub 2016 Feb 1.

Dectin-1 Controls TLR9 Trafficking to Phagosomes Containing β-1,3 Glucan

Nida S Khan  1 , Pia V Kasperkovitz  2 , Allison K Timmons  3 , Michael K Mansour  4 , Jenny M Tam  3 , Michael W Seward  3 , Jennifer L Reedy  4 , Sravanthi Puranam  3 , Marianela Feliu  5 , Jatin M Vyas  6
Affiliations

Dectin-1 Controls TLR9 Trafficking to Phagosomes Containing β-1,3 Glucan

Nida S Khan et al. J Immunol. .

Abstract

Dectin-1 and TLR9 play distinct roles in the recognition and induction of innate immune responses to Aspergillus fumigatus and Candida albicans. Dectin-1 is a receptor for the major fungal cell wall carbohydrate β-1,3 glucan that induces inflammatory cytokines and controls phagosomal maturation through spleen tyrosine kinase activation. TLR9 is an endosomal TLR that also modulates the inflammatory cytokine response to fungal pathogens. In this study, we demonstrate that β-1,3 glucan beads are sufficient to induce dynamic redistribution and accumulation of cleaved TLR9 to phagosomes. Trafficking of TLR9 to A. fumigatus and C. albicans phagosomes requires Dectin-1 recognition. Inhibition of phagosomal acidification blocks TLR9 accumulation on phagosomes containing β-1,3 glucan beads. Dectin-1-mediated spleen tyrosine kinase activation is required for TLR9 trafficking to β-1,3 glucan-, A. fumigatus-, and C. albicans-containing phagosomes. In addition, Dectin-1 regulates TLR9-dependent gene expression. Collectively, our study demonstrates that recognition of β-1,3 glucan by Dectin-1 triggers TLR9 trafficking to β-1,3 glucan-containing phagosomes, which may be critical in coordinating innate antifungal defense.

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Figures

Figure 1
Figure 1
TLR9 is specifically recruited to β-1,3 glucan phagosomes. (A) Confocal microscopy of RAW macrophages expressing TLR9-GFP (green) incubated with Alexa fluor 647-labeled β-1,3 glucan beads (blue) and Alexa fluor 568-labeled polystyrene beads (red-top panel), or A. fumigatus-RFP (red-middle panel), or Alexa fluor 568-labeled CpG-IgG beads (red-bottom panel) for 30 min. Size bar indicates 5µm. Original magnification X60 or X100. (B) Purified phagosomes containing either β-1,3 glucan beads or CpG-IgG beads were assessed for TLR9-GFP recruitment (black line) by phagoFACS and compared with polystyrene beads control (gray shaded histogram). (C) Kinetics of TLR9-GFP recruitment (black line) to purified β-1,3 glucan-containing phagosomes were assessed by phagoFACS for the indicated times and compared to polystyrene bead-only control (gray shaded histogram). β-1,3 glucan beads were added to cell lysates assessed for TLR9-GFP recruitment (D) β-1,3 glucan beads and CpG-IgG beads were incubated with 2x106 RAW TLR9-GFP cells for 3 h. Lysate control and 1x106 purified phagosomes were analyzed by immunoblot and blotted for GFP. Arrows indicate full-length-TLR9GFP (FL-TLR9-GFP) and cleaved TLR9-GFP as labelled. Data are representative of five independent experiments.
Figure 2
Figure 2
TLR9 trafficking to fungal phagosomes requires Dectin-1. (A) Confocal microscopy of Dectin-1 knockout macrophages expressing TLR9-mcherry (red) in the absence (top panel) or presence (bottom panel) of Dectin-1. (A–D) Cells were incubated with heat-killed C. albicans (A), live A. fumigatus resting conidia (B), heat-killed A. fumigatus resting conidia (C), or live A. fumigatus swollen conidia (D) for 40 min. Scale bar indicates 5µm. Original magnification X100. (Top panels A–D). Data are representative of five independent experiments.
Figure 3
Figure 3
TLR9 recruitment to β-1,3 glucan phagosomes requires acidification. (A) RAW TLR9-GFP cells pre-treated with BafA1 or vehicle control for 15 min and incubated with either β-1,3 glucan beads and CpG-IgG beads for 3 h. TLR9-GFP recruitment to β-1,3 glucan phagosomes was assessed by confocal microscopy. Original magnification X60. Scale bar indicates 5µm. (B) Purified phagosomes and lysate control were analyzed by immunoblot and blotted for TLR9-GFP. Lysate controls indicate FL-TLR9-GFP and cleaved TLR9-GFP. (C) TLR9-GFP recruitment (black line) to purified β-1,3 glucan phagosomes in the presence of BafA1 or vehicle control was assessed by phagoFACS and compared to bead-only control (gray shaded histogram). Data are representative of three independent experiments.
Figure 4
Figure 4
TLR9 recruitment to β-1,3 glucan phagosomes requires Syk activation. (A-C) RAW macrophages expressing TLR9-GFP were pre-treated with R406 or vehicle control for 30 min. Cells were stimulated with either β-1,3 glucan beads or CpG+IgG beads for 3 h. (A) Confocal microscopy of RAW TLR9-GFP recruitment to β-1,3 glucan phagosomes in the presence of R406 or vehicle control. Original magnification X100. Scale bar indicates 5µm. (B) TLR9-GFP recruitment to purified β-1,3 glucan or CpG-IgG phagosomes in the presence of R406 or vehicle were analyzed by immunoblot. Lysate control of indicated cell type were used to indicate FL-TLR9-GFP and cleaved TLR9-GFP. (C) TLR9-GFP recruitment (black line) to purified β-1,3 glucan phagosomes in the presence of R406 or vehicle control was assessed by phagoFACS and compared to bead-only control (gray shaded histogram). Data are representative of three independent experiments.
Figure 5
Figure 5
Dectin-1 dependent Syk activation is required for TLR9 recruitment to β-1,3 glucan and fungal phagosomes. (A-D) Confocal microscopy of Dectin-1-knockout macrophages expressing TLR9-mCherry and GFP-Dectin-1 or GFP-Dectin-1ΔY15 incubated with β-1,3 glucan beads for 3 h (A and B), CpG+IgG beads for 2h (C and D), heat-killed A. fumigatus (E and F), or heat-killed C. albicans (G and H). Original magnification X60 and X100. Scale bar indicates 5µm. Data are representative of five independent experiments.
Figure 6
Figure 6
Dectin-1 regulates TLR9 dependent gene expression. (A–B) Microarray data analysis of TLR9KO and wild-type B6 immortalized macrophages in response to β-1,3 glucan beads. (A) A scatter plot indicating a ratio of fold changes in gene expression between wild-type and TLR9KO macrophages. Off-diagonal dots (red) indicate differentially regulated TLR9 dependent genes that are fold change > 2.0 between macrophages stimulated with β-1,3 glucan beads and unstimulated. (B) A heat map of the 32 genes differentially expressed (fold change > 1.8) between TLR9KO and wild-type macrophages in response to β-1, 3 glucan beads.
Figure 7
Figure 7
Schematic representation of Dectin-1 dependent TLR9 recruitment to fungal- and β-1, 3 glucan-containing phagosomes. (A) Recognition and ligation of Dectin-1 with A. fumigatus, C. albicans, or β-1,3 glucan bead results in phagocytosis and Syk activation triggering the recruitment of TLR9 to the phagosomes and retention of the proteolytically cleaved version of TLR9. (B) Signaling incompetent Dectin-1ΔY15 can mediate phagocytosis of fungi or β-1,3 glucan beads but is incapable of activating Syk, resulting in failed TLR9 recruitment to the phagosome.
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