PMC

Wild type and pIgR^−/− cells were seeded in Transwells and treated with 10 μM DAPT and 1 μg/ml LPS or 10⁷ CFU/ml heat-killed E.coli as indicated. (a) IgA transcytosis was analyzed by ELISA, and results were normalized to the WT+DAPT+LPS group (= 100%). (b) Gene expression analysis by qPT-PCR of pIgR was performed and all samples were normalized to Gapdh. Data are presented as fold change relative to untreated (0% CM) cells. All values are indicated as mean ± s.e.m. One-way ANOVA: (a) F = 426.2, P < 0.0001, n ≥ 6 per group, means with different letters are significantly different by Bonferroni's multiple comparison test; (b) F = 11.47, P < 0.0008, n ≥ 5 per group, **P < 0.01 by Bonferroni's multiple comparison test. N.D. = not detected.

Wild type and pIgR^−/− cells were treated with 10 μM DAPT and varying doses of IL-17 (a,d), IL-1β (b,e), or TNFα (c,f). IgA transcytosis was analyzed by ELISA (a–c), and results were normalized to the WT+DAPT+LPS group (= 100%). Gene expression analysis of pIgR was performed (d–f) and all samples were normalized to Gapdh. Data are presented as fold change relative to untreated (0% CM) cells. All values are indicated as mean ± s.e.m. One-way ANOVA: (a) F = 152.3, P < 0.0001, n ≥ 3 per group; (b) F = 187.9, P < 0.0001, n ≥ 3 per group; (c) F = 376.7, P < 0.0001, n ≥ 3 per group; (d) F = 119.5, P < 0.0001, n ≥ 4 per group. (e) F = 49.92, P <0.0001, n ≥ 5 per group; (f) F = 28.29, P < 0.0001, n ≥ 4 per group; Means with different letters are significantly different by Bonferroni's multiple comparison test. N.D. = not detected.

(a,c) Wild type Tlr4 and Tlr4^−/− were treated with 10 μM DAPT and 1 μg/ml LPS. (a) IgA transcytosis was measured by ELISA and results were normalized to the WT group (= 100%). Values are indicated as mean ± s.e.m.; n = 8 per group. Wilcoxon signed rank test: P = 0.0039. (b,d) Wild type Tlr4 and Tlr4^−/− cells were treated with 10 μM DAPT and varying doses of IL-17. (b) IgA transcytosis was analyzed by ELISA and results were normalized to the WT+D+L group (= 100%). Gene expression analysis of pIgR was performed (c–d) and all samples were normalized to Gapdh. Data are presented as fold change relative to untreated (0% CM) cells. All values are indicated as mean ± s.e.m. One-way ANOVA: (b) F = 116.9, P < 0.0001, n ≥ 3 per group; (c) F = 40.92, P < 0.0001, n ≥ 7 per group; (d) F = 6.364, P < 0.0004, n ≥ 3 per group. Means with different letters are significantly different by Bonferroni's multiple comparison test. N.D. = not detected.

(a) Schematic of timeline for Transwell experiments. Wild type cells were treated with +/− 10 μM DAPT +/− 1 μg/ml LPS and were analyzed on day three post-seeding. (b) Cells were fixed and paraffin-embedded on the transwell membranes. Sections were cut and stained with the following: hematoxylin and eosin, anti-ZO-1, anti-villin, and anti-pIgR. Bars = 50 μm. Gene expression analysis was performed by qRT-PCR for pIgR (c), Reg3g (d), and Vil1 (e). All samples were normalized to Gapdh mRNA, and data were presented as fold change relative to untreated (0% CM) cells (mean ± s.e.m.; n ≥ 3 per condition). One-way ANOVA: (c) F = 96.02, P < 0.0001; (d) F = 3.441, P < 0.0376; (e) F = 1.085, P < 0.3762. ***P < 0.001 by Bonferroni's multiple comparison test. (f) Transepithelial electrical resistance was measured on day three. The (resistance × area) is shown for each condition (mean ± s.e.m., n = 6 per group). Statistical analysis by Student's t-test showed no significant difference between the two groups (P < 0.4362).

Two-fold serial dilutions of wild type cells were seeded into Transwells and treated with 10 μM DAPT and 1 μg/ml LPS. (a) IgA transcytosis was performed on day three post-seeding, and measurement of IgA in the supernatants at the six hour time point is shown. (b, c) Cells were fixed and stained on the Transwell membranes (c) with anti-pIgR (green) and bis-benzamide dye (blue). Bars = 200 μm. Quantification of cell density of cells on Transwells was performed using ImageJ software (b). Gene expression analysis was performed by qRT-PCR for pIgR (d), Muc2 (e), Atoh1 (f), and Vil1 (g). All samples were normalized to Gapdh, and data are presented as fold change relative to untreated (0% CM) cells. The dotted lines represent the limit of detection by the ELISA. All values are indicated as mean ± s.e.m. One-way ANOVA: (a) F = 53.11, P < 0.0001, n ≥ 6 per group; (b) F = 183.2, P < 0.0001, n ≥ 15 per group; (d) F = 39.02, P < 0.0001, n ≥ 3 per group; (e) F = 11.07, P < 0.0001, n ≥ 3 per group; (f) F = 3.436, P < 0.0183, n ≥ 3 per group; (g) F = 2.186 , P < 0.0894, n ≥ 3 per group. Means with different letters are significantly different by Bonferroni's multiple comparison test.

(a) Wild type and pIgR^−/− cells were seeded in Transwells and treated with +/− 10 μM DAPT +/−1 μg/ml LPS as indicated. On day three post-seeding, 40 μl of normal mouse IgA (Santa Cruz) was added to the lower compartment of the Transwells, and supernatants from the upper compartment were taken at six hours for the detection of IgA by ELISA. Time course (b) and IgA dose curve (c) experiments were performed on wild type and pIgR^−/− cells treated with 10 μM DAPT and 1μg/ml LPS to determine the optimal conditions for future experiments. An LPS dose curve was performed to determine whether IgA transcytosis (d) and pIgR expression (e) were dose dependent. All LPS-treated cells were also treated with 10 μM DAPT. For IgA transcytosis, results from the ELISA were normalized to the 1 μg/ml LPS treatment group (= 100%). Gene expression analysis by qRT-PCR for pIgR was performed by normalizing to Gapdh, and data are presented as fold change relative to untreated cells. The dotted lines represent the limit of detection by the ELISA. All values are indicated as mean ± s.e.m. One-way ANOVA: (a) F = 573.3, P < 0.0001, n ≥ 3 per group; (c) F = 539.2, P < 0.0001, n ≥ 12 per group; (d) F = 675.7, P < 0.0001, n ≥ 3 per group; (e) F = 46.22, P < 0.0001, n ≥ 6 per group. Means with different letters are significantly different by Bonferroni's multiple comparison test. N.D. = not detected.