PMC

Glu-mp is weaker than curdlan at inducing inflammatory cytokine production. (A) HEK293 cells expressing Dectin-1 (black line, left panel) or C57BL/6 BMDC (black line, right panel) bind Alexa555-labelled Glu-mp, as shown by flow cytometric analysis. Binding is not observed in untransfected HEK293 cells (gray, left panel) or Dectin-1-deficient BMDC (gray, right panel). One representative out of three experiments is shown. (B) Production of IL-6, TNF-α, IL-2 and IL-12p40 by C57BL/6 BMDC stimulated with the indicated doses of Glu-mp or with 100 μg/mL curdlan. Cytokine concentration in 16 h culture supernatants was determined by ELISA. Data shown are mean of duplicate wells. One representative out of three experiments is shown.

Block of phagocytosis converts Glu-mp into a potent DC activator. (A) C57BL/6 or Dectin-1-deficient BMDC were treated with or without latrunculin A for 30 min prior to addition of Alexa647-labelled Glu-mp. After 1 h incubation, cells were washed, fixed and visualized by confocal microscopy. Data are the mean particle uptake and SD of three independent experiments. (B) C57BL/6 BMDC were stimulated with different doses of Glu-mp or with 100 μg/mL curdlan in the presence or in the absence of latrunculin A. IL-6, TNF-α, IL-2 and IL-12p40 in culture supernatants were determined by ELISA. Data shown are mean of duplicate wells. One representative out of three independent experiments is shown. (C) TNF-α concentrations were determined by ELISA in the supernatant of C57BL/6, MyD88/TRIF doubly deficient, or Dectin-1-deficient BMDC stimulated with the indicated amounts of Glu-mp in the presence or absence of latrunculin A. Data are represented as fold TNF-α induction, dividing the value obtained with latrunculin A-treated cells by that obtained with untreated cells (where no induction was observed, data are shown as ≤1). Data shown are mean of duplicate wells. One representative out of three independent experiments is shown. (D) MyD88-deficient BMDC were stimulated with different doses of Glu-mp or with 100 μg/mL curdlan in the presence or in the absence of latrunculin A or cytochalasin D. TNF-α concentrations in culture supernatants were determined by ELISA. Data shown are mean of duplicate wells. One representative out of three independent experiments is shown.

Block of Glu-mp phagocytosis promotes sustained MAPK activation. (A) C57BL/6 BMDC were stimulated with or without 100 μg/mL Glu-mp in the presence or absence of latrunculin A, as indicated. Activation of p38, ERK and JNK was analyzed by immunoblotting with antibodies against the phosphorylated form of the kinases. The blots were re-probed with antibodies against total kinase as a loading control. One representative out of four independent experiments is shown. (B) The same experiment conducted with or without (−) 100 μg/mL curdlan. Activation of ERK was analyzed by immunoblotting with antibodies against the phosphorylated form of the kinase. One representative out of four independent experiments is shown. (C) HEK293 cells expressing Dectin-1 were treated with DMSO or with dynasore for 30 min prior to addition of Alexa647-labelled Glu-mp (50 μg/mL). After 1 h incubation, cells were washed, fixed and visualized by confocal microscopy. Data are the mean particle uptake and SD of three independent experiments. (D) HEK293 cells expressing Dectin-1 were stimulated with 50 μg/mL Glu-mp or without stimulus for the indicated periods of time, in the presence of DMSO or dynasore as indicated. Activation of ERK was analyzed by immunoblotting with antibodies against the phosphorylated form of the kinase. The blots were re-probed with antibodies against total kinase as a loading control. One representative out of three independent experiments is shown. (E) LK cells expressing Dectin-1 were stimulated with different doses of curdlan or Glu-mp. IL-2 concentrations in culture supernatants were determined by ELISA. Data shown are mean±SD of triplicate wells. One representative out of three independent experiments is shown.