HCD induces myeloid cell accumulation in the intestine.

(a) Total number of L-plastin+ cells in the intestine of 6 dpf WT larvae following HCD (squares) or ZM (triangles) for 6 h. Two-way analysis of variance (ANOVA). (b) Total number of mCherry+ cells in the intestine of Tg(fms:mCherry) larvae following HCD or ZM for 6 h. In a and b, one representative experiment of at least two with ne15 is shown. Error bars represent 95% confidence intervals. Two-way ANOVA. (c) Representative images of distal intestine of Tg( fms:mCherry) larvae after 18 h following ZM or HCD for 6 h. Scale bar, 100 µm. (d) L-plastin+ cells (arrow) localized to the muscularis of intestine layers; intestinal bulb (IB), distal intestine (DI), lumen (L), goblet cells (G), epithelial layer (E), muscularis (M). Scale bars, 20 µm (black); 10 µm (red). (e) Representative flow cytometry plots and quantification of GFP+ and L-Plastin+ cells of adult Tg( mpx:GFP) intestine tissue after 15 h following HCD or Hikari control for 6 h. Each dot represents one individual fish pooled from three experiments (n=11). Two-tailed t-test. One-way ANOVA. Error bars are s.e.m. (f) CD11b+ and CD11c+ cells in the small intestine (distal jejunum and ileum) of Balb/C mice 12 h after gavage with water, oil or oil+ cholesterol. One-way ANOVA. Error bars are s.e.m. (g) Fold change of IL-1β mRNA assessed qRT–PCR in the small intestines (ileum) of Balb/C mice. Relative expression values were normalised to 18S and expressed relative to one control sample. Kruskal–Wallis test. Error bars are s.e.m. In f and g, each dot represents one individual mouse and data are pooled from four experiments (n=12). ***P<0.001, **P<0.01 and *P<0.05.

Treatment with ASC MO using a novel mode of delivery abrogates HCD-induced intestinal myeloid cell accumulation.

(a) Representative fluorescent image of larvae treated with 20 µM FITC-conjugatedASC splice-blocking MO (FITC ASC MO, bottom panel) or left untreated (top panel). Insets highlight green fluorescence of FITC ASC MO in intestinal lumen (l) and in epithelial cells (white arrow). Scale bars, 100 µm (black); 20 µm (white). (b) Representative fluorescent and bright-field images of larvae treated with 20 µM FITC ASC MO (top panel) subsequently treated with 10 mg ml¹ HRP for 2 h. HRP was detected histochemically (bottom panel) and found to be localized in the same region as the MO (indicated by black and white dotted lines). Insets show enlarged images of the region containing specialized enterocytes that have taken up FITC ASC MO and HRP. Scale bars, 100 µm (top); 50 µm (bottom). (c) Histograms depict FITC fluorescence in intestinal cells of larvae treated with 20 µM FITC ASC MO (solid line) or untreated (filled histogram) and double-stained for cytokeratin (left plot), gated on dsRed+ cells in Tg( lyz:dsRed) (middle) or mCherry+ cells in Tg( fms:mCherry) (right). Pool of ne10 larval intestines, representative of e2 experiments. (d) qRT–PCR analysis of FITC+ sorted intestinal cells of zebrafish larvae treated with either 20 µM of ASC-specific control MO or FITC ASC MO (pool of n=50–100 larval intestines per sample). Relative expression values were normalized to 18S and expressed as fold change relative to the control sample. Mean+s.e.m. of triplicates. Representative of two experiments. Mann–Whitney test. (e) Effect of increasing ASC or control MO concentration treatment on the total number of intestinal L-plastin+ cells. (f) Effect of different ASC MOs on the total number of intestinal L-plastin+ cells. (e,f) ne15, one exact experimental replicate and more than three experimental replicates for the highest concentration of MO. Mean+95% confidence intervals. Kruskal–Wallis test. (g) Effect of IL-1β MO treatment the total number of intestinal L-plastin+ cells. ne35, pooled from two experiments. Error bars: s.e.m. One-way analysis of variance. ***P<0.001, **P<0.01 and *P<0.05.

Extended HCD induces steatosis in the liver.

(a) Representative images of paraffin transverse sections stained with haematoxylin and eosin of the liver of 15 dpf larvae fed ZM control or HCD for 10 days. Scale bar, 100 µm. (b) Percentage of fish showing vacuolation in the liver. ne34, pooled from two experimental replicates. (c) Representative image of whole-mount Oil Red O-stained 15 dpf larvae fed ZM control or HCD for 10 days. Scale bar, 500 µm. (d) Percentage of fish showing Oil Red O staining in the liver. n=25, pooled from two experimental replicates. Error bars represent s.e.m. Mann–Whitney test. ***P<0.001 and **P<0.01.

Extended HCD induces sustained inflammation and impaired peristalsis.

(a) Representative images and (b) quantification of the total number of intestinal L-plastin+ cells of 15 dpf Tra^-/-/ Nac^-/- larvae fed HCD or ZM control for 10 days. n=20, one representative experiment of four. Scale bar, 150 µm. Two-tailed t-test. (c) Representative images and (d) average length of anterograde peristalsis with contractions (red arrowhead) over time (20 s) of the distal intestine of larvae fed (n=18). One representative experiment of three. Scale bar, 200 µm. Mann–Whitney test. (e) Total number of intestinal L-plastin+ cells (ne12) and (f) average length of peristaltic wave (ne6) of ezetimibe (25 µM)-treated larvae. One-way analysis of variance (ANOVA). (g) Total number of intestinal L-plastin+ cells (ne8) and (h) average length of peristaltic wave (ne10) of Cathepsin B inhibitor ( Ca-074-Me, 10 µM)-treated larvae. One-way ANOVA. In e–h, one experiment of two is shown. (i) Correlation of peristaltic wavelength with the total number of L-Plastin+ cells in intestine. Pearson’s correlation coefficient: 0.76. Data are pooled from two experiments. ***P<0.001 and **P<0.01. Error bars represent s.e.m.