Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Breast Milk Enhances Intestinal Mucosal Barrier Function and Innate Immunity in a Healthy Pediatric Human Enteroid Model

doi: 10.3389/fcell.2021.685171

Figure Lengend Snippet: Human milk modulates occludin expression. (A) Confocal microscopy images (XY and YZ projections) of 2PD enteroid monolayers NT or apically treated for 24 h with HM (20%; v/v) or IF (20%; w/v). Occludin, green; actin, magenta. Scale bar = 10 μm. (B) Relative fluorescence intensity of occludin quantified by confocal microscopy analysis of monolayers treated with HM (20%; v/v) or IF (20%; w/v) for 24 and 72 h. Mean ± SEM are shown. Data represent three independents with n = 4–6 enteroid monolayers/group per experiment. Each symbol indicates an independent monolayer. p -values were calculated by one-way ANOVA with Šidák’s post hoc analysis. (C) Confocal microscopy images of 5PD enteroid monolayers treated with HM for 48 h. Occludin, green; lysozyme (Lyz), red; trefoil factor 3 (TFF3), red; chromogranin A (ChgA), red; actin, magenta; DNA, blue. Paneth and goblet cells, scale bar = 5 μm; enteroendocrine cells, scale bar = 10 μm. (A,C) Data are representative of three independent experiments with n = 3 enteroid monolayers/group per experiment. p -values were calculated by one-way ANOVA with Šidák’s post hoc analysis. (D) Number of Lyz-positive (E) and TFF3-positive cells quantified by immunofluorescent confocal microscopy analysis of monolayers treated with HM and IF as described in panel (A) . Data represent mean ± SEM of three combined experiments, each including four monolayers/group per experiment. Each symbol indicates an independent monolayer. p -values were calculated by one-way ANOVA with Tukey post hoc analysis. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Basolateral media (100 μl) was sampled at 30 min, 1 and 2 h, and FITC-dextran content was measured by fluorescence intensity using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA, United States).

Techniques: Expressing, Confocal Microscopy, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Human Breast Milk Enhances Intestinal Mucosal Barrier Function and Innate Immunity in a Healthy Pediatric Human Enteroid Model

doi: 10.3389/fcell.2021.685171

Figure Lengend Snippet: Human milk modulates epithelial innate immune function. (A) Relative fluorescence intensity of human DEFA5 quantified by confocal microscopy analysis of 2PD and 5PD monolayers NT or treated with HM (20%; v/v) or IF (20%; w/v) for 48 h. (B) Representative confocal microscopy images (XY projections) of 5PD monolayer showing localization (arrowheads) of DEFA5 in Lyz-negative cells in HM-treated monolayer and (C) number of Lyz-negative cells quantified by immunofluorescent confocal microscopy (≥6 different view fields). DEFA5, green; Lyz, red; actin, magenta; DNA, blue. Scale bar = 10 μm. (D) Representative confocal microscopy images (XY projections) of 5PD monolayer depicting co-localization (arrowheads) of TFF3 (red) and DEFA5 (green); DNA, blue. Scale bar = 50 μm. (E–G) Total amount of MCP-1, GM-CSF, and IL-8 in the apical media of 2PD monolayer treated with HM and IF as described in panel (A) for 24 and 72 h. (H) PCA plot from HM- and IF-treated monolayers, and NT controls for 24 h. PC, principal component. Variables analyzed: TER, occludin, DEFA5, MCP-1, GM-CSF, and IL-8. (A,C,E–G) Mean ± SEM are shown. Data are representative of three independent experiments with n = 6–12 enteroid monolayers/group per experiment. Each symbol indicates an independent monolayer. p -values were calculated by one-way ANOVA with Tukey’s post-test for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Basolateral media (100 μl) was sampled at 30 min, 1 and 2 h, and FITC-dextran content was measured by fluorescence intensity using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA, United States).

Techniques: Fluorescence, Confocal Microscopy