Fig 1: Effect of lack of saliva on development of DSS-induced colitis. (A) WT (black and green) and Aqp5-/- mice (blue and red) were fed with 3% DSS (green and red) for 15 days. Mice were 22 weeks old, and the numbers in parenthesis indicate the number of mice in each group. The P values are the difference between WT and Aqp5-/- mice fed with DSS. (B) Time course of development of disease activity of the mice in (A). (C and D) WT and AQP5-/- mice age 8 (C) and 13 (D) weeks were treated with 3% DSS added to drinking water. The number of mice in each group is indicated in parenthesis, and the results are mean ± SEM. The P values are relative to WT mice fed with DSS. (E) Intestinal permeability expressed as fold change relative to the permeability measured before DSS treatment started and was taken as 1 for each group of mice. (F) Example images and averages of intestinal length. (G–I) Example images (G) and the level of ZO1 (H) and Occludin (I) measured by immunofluorescence staining. (J–L) Example H&E and PAS images (J) and analysis of cell proliferation (K) and goblet cell density (L). (M and N) Serum (M) and intestinal MPO (N) levels as reporters of neutrophils activity. (O and P) WT and AQP5-/- mice age 18 weeks (5 mice in each group) were treated with 3% DSS and were gavaged with 50 μL PBS (Black, blue, gray and green) or with 50 μL whole saliva collected from 3 WT mice by stimulation with pilocarpine. The mice were gavaged on days 0, 3, and 6. Mice weight (O) was measured in 2-day intervals, and intestinal permeability (P) was measured on days 1, 3, 6, and 12. The results are mean ± SEM.
Fig 2: Effect of saliva fluid and microbiome fraction on development of DSS-induced colitis. (A) Illustration of the protocol and procedures used to collect the salivary microbiome and fluid fractions. (B and D), WT (B) and Aqp5-/- mice (D) were fed with water (black) or 3% DSS (blue, green and red) for 6 days and allowed to recover for 9 days. The mice were gavaged with PBS (black), the microbiome fraction in PBS (green), or the salivary fluid fraction (red) on days 1, 3, and 6, as indicated. Mice were 18 to 22 weeks old, and the numbers in parenthesis indicate the number of mice in each group. The P values are relative to mice fed with DSS. (C and E) Time course of development of disease activity of the mice in (B and D), respectively. (F) Intestinal permeability expressed as fold change measured on day 8. (G) Example images and averages of intestinal length measured on day 8. (H–J) Example immunofluorescence and bright field images (H) and analysis of ZO1 (I) and Occludin (J) measured on day 8. (K–M) Example H&E and PAS images (K) and analysis of cell proliferation (L) and goblet cell density (M). (N and O) Serum (N) and intestinal MPO (O) levels.
Fig 3: Scavenging saliva fluid MIF and TFF2 ameliorates effects of saliva fluid on development of DSS-induced colitis. (A and B) Standard curves (left) and the level of MIF (A) and TFF2 (B) in saliva from control (black) and DSS-treated mice (red) and saliva treated with the respective antibodies (green). (C and E) WT (C) and Aqp5-/- mice (E) were fed with water (black) or 3% DSS (blue, green and red) for 6 days and allowed to recover for 6 days. The mice were gavaged with the salivary fluid treated with anti-MIF (green) and anti-TFF2 antibodies (red) on days 1, 3, and 6, as indicated. Mice were 18 to 22 weeks old, and the numbers in parenthesis indicate the number of mice in each group. The P values are relative to mice fed with DSS. The results with DSS shown in dotted lines were taken from Figure 7 and are shown to better illustrate the effect of MIF and TFF2 depletion. (D and F) Time course of development of disease activity of the mice in (C and E), respectively. (G) Intestinal length measured on day 8. (H and I) Analysis of cell proliferation (left) and goblet cell density (right). (J and K) Intestinal (J) and serum (K) MPO levels. (L) Intestinal permeability expressed as fold change measured on day 8. (M and N) Analysis of ZO1 (M) and Occludin (N) measured on day 8.
Fig 4: Effect of saliva fluid fractions, MIF, and TFF2 on development of DSS-induced colitis. (A) Coomassie-stained proteins in whole saliva fluid, (1) >100-kDa fraction, (2) >50-kDa fraction, (3) >30-kDa fraction, and (4) <30-kDa fraction. (B and C), WT (B) and Aqp5-/- mice (C) were fed with water (black) or 3% DSS (blue, green, red and dark yellow) for 6 days and allowed to recover for 9 days. The mice were gavaged with the indicated fractions of the salivary fluid on days 1, 3, and 6, as indicated. Mice were 18 to 22 weeks old, and the numbers in parenthesis indicate the number of mice in each group. The P values are relative to mice fed with DSS. The results with DSS shown in dotted lines were taken from Figure 7 and are shown to better illustrate the effect of the fractions. (D) Venn chart of the proteins in each fraction. (E) A list of the proteins in the 50-KDa fraction. MIF and TFF2 are highlighted in red. (F and G) Protocol as in (B and C), except that the mice were gavaged with 50 μL of 0.01 μg/μL MIF (green) or 0.01 μg/μL TFF2 (red). (H and I) Example images (H) and averages of intestinal length (I) measured on day 8. (J–L) Example H&E images (J) and analysis of cell proliferation (K) and goblet cell density (L). (M) Intestinal permeability expressed as fold change measured on day 8. (N–P) Example of immunofluorescence and bright field images (N) and analysis of ZO1 (O) and Occludin (P) measured on day 8. (Q and R) Intestinal (Q) and serum (R) MPO levels.
Supplier Page from Abcam for Myeloperoxidase (MPO) Peroxidation Activity Assay Kit (Fluorometric)