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. 2023 Mar 8;14:1142492. doi: 10.3389/fimmu.2023.1142492

Figure 1.

Figure 1

Uptake of zymosan by human intestinal epithelial cells. (A) SW480 cells were seeded on glass-bottom chambers as indicated in Methods, and fed overnight with pHrodo-red zymosan (zymosan, red) and counter stained with Hoechst 33342 (blue) prior to confocal live imaging. White arrowheads – intracellular red fluorescent zymosan, Black arrowheads - extracellular intact zymosan, arrow- intracellular fragmented zymosan. Original magnification x20, scale bar 10 µm. (B–E) Zymosan uptake is sensitive to cytochalasin-D. (B, C) SW480 were treated as in A, in the absence (B) or presence (C) of cytochalasin-D (CytoD, 10 µM). Scale bar 20 µm. Arrows and arrowhead indicate intracellular processed and intact zymosan respectively. Wider fields of the images are shown is Supplementary Figure S3 . (D) Phagocytosis was quantified using imageJ as the percentage of red-fluorescence positive cells in at least 4 randomly taken fields as described in Methods. Each dot is the quantification of a single field. Data is representative of three independent experiments performed. ***p ≤ 0.001, Unpaired t-test vs. no inhibitor. (E) SW480 cells were seeded in 96 well plate, treated as in (B, C) as well as with the vehicle (DMSO, 1:1000) in triplicate wells, and phagocytosis was assessed as the relative fluorescence (RFU) by a microplate reader. Data are shown as the individual measure of each biological replica and mean ± SD of biological triplicates from a representative of three independent experiments performed. ns-non significant ****p<0.0001, One-way ANOVA followed by Tukey multiple comparison test. (F–I) Zymosan uptake depends on Dectin-1. (F, G) SW480 were treated as in A, in the absence (F) or presence (G) of laminarin (1 mg/ml) that was added to the medium 1 hour prior to zymosan. Scale bar 50 µm. White arrows and arrowhead indicate intracellular processed and intact zymosan respectively. Black arrowheads indicate extracellular zymosan. (H) Phagocytosis was quantified as in (D). (I) cells were seeded on 96 wells, treated as in (F, G) in triplicate wells, and phagocytosis was analyzed as in (E). (J) Zymosan phagocytosis is resistant to Syk inhibition. SW480 cells were seeded on 96 well plate, in the presence or absence of the Syk inhibitor 574711 (1 and 5 µM), which was added 1 hour prior to the addition of pHrodo-red zymosan. Phagocytosis was assessed as in (E). Data are shown as individual measures and mean ± SD of biological triplicates from a representative of three independent experiments performed. (K) Zymosan-induced IL-8 secretion is sensitive to Syk inhibitor. Cells seeded on the same 96 well plate were pre-treated with Syk inhibitor as in (J) and stimulated overnight with 100 μg/ml of non-labelled zymosan. Supernatants were assessed for IL-8 by ELISA. Data are shown as individual measures and mean ± SD of biological duplicates from a representative of three independent experiments performed. N.D- not detected; ns-non significant *p<0.05; **p<0.01, One-way ANOVA followed by Tukey multiple comparison test.