Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis

Abstract

Background: Intestinal epithelial cells (IECs) are the first to encounter luminal microorganisms and actively participate in intestinal immunity. We reported that IECs express the β-glucan receptor Dectin-1, and respond to commensal fungi and β-glucans. In phagocytes, Dectin-1 mediates LC3-associated phagocytosis (LAP) utilizing autophagy components to process extracellular cargo. Dectin-1 can mediate phagocytosis of β-glucan-containing particles by non-phagocytic cells. We aimed to determine whether human IECs phagocytose β-glucan-containing fungal particles via LAP.

Methods: Colonic (n=18) and ileal (n=4) organoids from individuals undergoing bowel resection were grown as monolayers. Fluorescent-dye conjugated zymosan (β-glucan particle), heat-killed- and UV inactivated C. albicans were applied to differentiated organoids and to human IEC lines. Confocal microscopy was used for live imaging and immuno-fluorescence. Quantification of phagocytosis was carried out with a fluorescence plate-reader.

Results: zymosan and C. albicans particles were phagocytosed by monolayers of human colonic and ileal organoids and IEC lines. LAP was identified by LC3 and Rubicon recruitment to phagosomes and lysosomal processing of internalized particles was demonstrated by co-localization with lysosomal dyes and LAMP2. Phagocytosis was significantly diminished by blockade of Dectin-1, actin polymerization and NAPDH oxidases.

Conclusions: Our results show that human IECs sense luminal fungal particles and internalize them via LAP. This novel mechanism of luminal sampling suggests that IECs may contribute to the maintenance of mucosal tolerance towards commensal fungi.

Keywords: Candida albicans; LC3-associated phagocytosis; dectin-1; intestinal epithelial cells; organoids.

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.

Figure 2

Intestinal organoids uptake zymosan. (A, B) Ileal (A) and colonic (B) organoids were grown as monolayers in expansion medium and let to differentiate for 2 days. pHrodo-red zymosan (red) was added to the medium for 24h. Original magnification x10, scale bar 50 µm, white arrows and arrowhead indicate intracellular processed and intact zymosan respectively. Black arrowheads indicate extracellular zymosan. Shown are representative frames from wider fields, presented in Supplementary Figures S4, S5A , from 3-5 randomly acquired scans of two independent experiments of two organoids. (C) Colonic organoids (from a different individual) were treated as in (B), nuclei were stained with Hoechst 33342 (blue) prior to confocal live imaging. Shown a representative frame from a z-stack analysis. Entire z-stack movie is shown in Supplementary Movie 1 . Arrows and arrowhead indicate intracellular processed and intact zymosan respectively. Original magnification x63, scale bar 10 µm. (D) Laminarin inhibits zymosan uptake by intestinal organoids. Colonic organoids were grown as in B, in the presence or absence of laminarin (1 mg/ml) that was added to the medium 1 hour prior to zymosan in triplicate wells. Shown are intracellular fluorescent zymosan (red) and the organoid cells (DIC), or zoom-in insets where nuclei were stained with Hoechst 33342 (blue) prior to confocal live imaging. Shown are representative frames from 3-6 random fields imaged from each of triplicate wells. The experiment was repeated with organoids from four individuals. Original magnification x20, scale bar 50 and 10 µm, Arrows and arrowhead indicate intracellular processed and intact zymosan respectively. (E) Colonic organoids from three individuals (colon-1 to colon-3) were seeded in 96 well plate, treated as in (D) in triplicate or 6-replicate wells for 48 hours. Phagocytosis was assessed as the relative fluorescence by a microplate reader. Data are shown as the measured value (dots) and mean ± SD of biological replicates from four independent experiments performed. Colon-1 was tested twice (exp.1 and 2), zym=zymosan. ***p<0.001, **p<0.01, *p<0.05 vs. no inhibitor, Student’s t-test was performed individually for each independent experiment. (F, G) Dectin-1 is recruited to internalized zymosan. Ileal organoids were fed with AF488-zymosan (green) overnight and stained with Dectin-1 antibody (magenta) and DAPI. Original magnification x20 scale bar 10 µm (F) and x63 scale bar 5 µm (G). Arrowheads – intact zymosan, arrows- fragmented zymosan. (H) Fluorescence intensity profile along the arrow of an inset from (G) is shown on the graph.

Figure 3

Phagocytosis of C. albicans by human intestinal organoids. Colonic organoids were fed overnight with Rhodamine-green-X labeled HK- C. albicans (A, green), or both pHrodo-red zymosan and HK-C. albicans (B) or UV-inactivated C. albicans (C). Live confocal images were acquired directly or after nuclear stain with Hoechst 33342 (blue). Arrowhead - intact C. albicans, arrow- fragmented C. albicans. Original magnification x40, scale bars 50 µm (A), 10 µm (A-inset and B) and 20 µm (C). (D) Dectin-1 is recruited to phagocytosed C. albicans. Ileal organoids were fed with Rhodamine-green-X labeled HK- C. albicans. Following fixation organoids were stained with Dectin-1 polyclonal antibody. Original magnification x63, scale bar 5 µm. (E) Fluorescence intensity profile along the arrow of an inset from (D) is shown on the graph.

Figure 4

LC3 is recruited to phagosomes in IECs. (A) SW480 LC3-GFP cells were fed with pHrodo-red zymosan overnight. Live imaging shows LAPosomes (arrow) as LC3 (green) around intact zymosan (red) particles, as well as fragmented zymosan and autophagosomes. (B) Fluorescence intensity profile along the arrow of an inset from (A) is shown on the graph. (C) Colonic organoids were fed with pHrodo-red zymosan (red) overnight and stained with LC3 antibody (green) and DAPI (blue). (D–F) Colonic organoids were fed with Rhodamine-green-X HK-C. albicans (green) overnight and stained with LC3 antibody (magenta) and DAPI (blue). Shown is LAP of yeast (D) and hyphal form (E, F) of HK-C. albicans. F is an inset of (E) Original magnification ×40 (A, B), ×63 (C, D) x20 (E, F) scale bar 10 µm (A–D, F) and 50 µm (E).

Figure 5

Rubicon is recruited to the phagosome. Colonic (A) and ileal (B) organoids from the same individual were fed overnight with HK-C. albicans (green) and stained with Rubicon antibody (magenta) and DAPI (blue). (C–E) Colonic (C) and Ileal (D, E) organoids were fed with pHrodo-red zymosan (red) and stained with Rubicon (green) and DAPI (cyan). Original magnification x63, scale bar 5 µm.

Figure 6

DPI inhibits zymosan phagocytosis. (A) Colonic organoid monolayers grown in differentiation medium for 3 days were exposed to pHrodo-red zymosan for 24 hours in the presence or absence of DPI (2 µM) that was added for 60 minutes prior to the addition of zymosan. Shown are intracellular fluorescent zymosan (red) and the organoid cells (DIC) of representative frames from 7-15 random fields imaged. The experiment was repeated 3 times using organoids from two different individuals. Original magnification x20, scale bar 50 µm. (B) Colonic organoids were seeded in 96 well plate, treated as in (A) in the presence or absence of DPI (2 or 10 µM) or in the presence of vehicle (DMSO 1:1000) in 8-replicate wells for 48 hours. Phagocytosis was assessed as the relative fluorescence by a microplate reader. (C) A summary of four experiments performed showing zymosan (zym) phagocytosis inhibition by 2 µM DPI in colonic organoids from two individuals (colon-2 and colon-3). (D) SW480 cells were seeded in 96 well plate and treated as in (B) in triplicates for 24 hours. Data are shown as individual measures (dots) and the mean ± SD of biological 8-replicates (B) and triplicates (D) from a representative of four or three independent experiments performed. **p<0.01, ***p<0.001, ****p<0.0001, One-way ANOVA followed by Tukey multiple comparison test (B, D) or individual t-test vs. no inhibitor for each of the separate experiments shown in (C).

Figure 7

Phagocytosed particles are directed to lysosomal processing. (A) Colonic organoids were incubated overnight with Rhodamine-Green-X labeled HK-C. albicans (green) and stained with lysosomal-NIR reagent (magenta). (B, C) Ileal organoids (B) and SW480 cells (C) were incubated with pHrodo-red zymosan (red) and stained with lysosomal-green reagent. Arrows indicate colocalization of fragmented HK-C. albicans or zymosan and lysosomes. (D) SW480 cells were fed with HK-C. albicans (green) and stained with LAMP2 antibody. (E) Intact and fragmented zymosan particles are surrounded by LAMP2. Ileal organoids were fed with AF488-zymosan (green) overnight, and stained with LAMP2 antibody (red). Arrowhead - intact zymosan, arrow- fragmented zymosan. (F, G) LAPosomes merge with lysosomes. (F) SW480 cells were fed with pHrodo red zymosan (red) and stained with LAMP2 (green) and LC3 (magenta) antibodies and counterstained with DAPI (blue). (G) Ileal organoids were fed with AF488-zymosan (green) and stained with LAMP2 (red) and LC3 (magenta) antibodies and counterstained with DAPI (blue). Original magnification x63 (A, D–G), x40 (B), x20 (C), scale bar 10 µm.

Figure 8

A proposed model for epithelial phagocytosis of fungi. Recognition of commensal fungi by intestinal epithelial cells leads to Dectin-1 mediated phagocytosis. Rubicon and LC3 are recruited to the phagosome and form LAPosomes. Upon fusion with lysosomes fungi are degraded. We propose that degradation products might be presented in the context of MHC class II.