Article Figures & Data
Figures
- FIG 1
Survival of L. casei in DSS-treated mice. Viable, rifampin-resistant L. casei cells in mouse stools were enumerated every second day of the study. The average CFU ± standard errors from fecal samples are shown for each treatment and time point (lower limit of detection, 1,000 CFU/g feces). Fecal samples were recovered from nine mice in each group, except for two BL580- and milk-fed mice on days 14 and 15 due to early termination. Because of diarrhea in some animals, stools were collected from the following numbers of mice on days 14 and 15, respectively: 7 and 6 mice fed BL23 and PBS, 6 and 8 mice fed BL23 and milk, 5 and 5 mice fed BL580 and milk, and 9 and 9 mice fed BL180 and milk.
- FIG 2
Changes in mouse body weight during and after DSS administration. The percent change in body weight was calculated by using the weight on day 4 (the day of DSS initiation) as the reference. The averages ± standard deviations of data from 12 mice (healthy sham control and DSS treated) and 9 mice (L. casei or acidified milk fed) are shown. **, P < 0.01; ***, P < 0.001 according to the Student t test compared to the DSS control group.
- FIG 3
L. casei in milk protects against DSS colitis. Stool consistency (diarrhea) (A), histological scores (B), and disease activity indices (C) were calculated according to the criteria shown in Table 2. A higher score means greater severity. The averages ± standard deviations of data from 12 mice (healthy sham control and DSS treated) and 9 mice (L. casei or acidified milk fed) are shown. *, P < 0.05; ***, P < 0.001 according to the Student t test compared to the DSS control group.
- FIG 4
Intestinal cytokine production differs depending on intestinal location, DSS-induced colitis, and consumption of L. casei or milk. (A to C) PCA of cytokine amounts in colonic and ileal tissues (A) and the colon (B) and ileum (C) of healthy (sham) and DSS-treated mice. (D to F) PCA comparisons of cytokine quantities in colonic tissues of healthy (sham) and DSS-treated control mice and DSS-treated mice fed milk with or without BL23 (D), L. casei BL23 in PBS (E), or the L. casei DltD− (BL580) or L. casei RecA− (BL180) strain in milk (F). The numbers of mice and cytokines used for this analysis are listed in Table S1 in the supplemental material.
- FIG 5
L. casei regulates cytokine production in colon during DSS-induced colitis. The quantities of IL-1α (A), IL-6 (B), IL-17 (C), KC (D), G-CSF (E), and MCP-1 (F) in colonic tissue were measured. Shown are averages ± standard deviations of data from 12 healthy sham-treated mice; 9 mice treated with DSS alone (control), acidified milk, L. casei BL23 in milk, L. casei in PBS, or L. casei BL180 in milk; and 7 mice fed L. casei BL580 in milk. *, P < 0.05; **, P < 0.01; ***, P < 0.001 according to the Mann-Whitney U test compared to the DSS-treated control group.
- FIG 6
Mouse cecal microbiota is altered by DSS, milk, and L. casei. (A) Weighted UniFrac PCoA of the cecal microbiota structure. (B) Taxonomy analysis at the family level. In panel B, the dendrogram on the left shows the Euclidean distance calculated from the bacterial family abundance in each treatment group. The bars on the right indicate the relative abundances of different families with representation of at least 1% in the total DNA sequence reads examined. Asterisks indicate “ unclassified” DNA sequences that are similar to more than one sequence from the reference database and “undefined” sequences that are similar to an unnamed reference sequence.
Tables
- TABLE 1
Primers used for L. casei BL23 mutant construction and PCR validation
Primer Nucleotide sequence Predicted product size (bp) dltD forward 5′-GTCGACAATGGGAAAAAGGC-3′ 498 dltD reverse 5′-TGAGCTCAAATTACGCTTAACC-3′ recA forward 5′-GTCGACAACTAGAAAAAGCCC-3′ 556 recA reverse 5′-AGAGCTCTAACCAGCGTATTGG-3′ pRV300 forward 5′-GATTAAGTTGGGTAACGCC-3′ dltD KO reverse 5′-TGAACATCTGATTGACTTGG-3′ 1,225 recA KO reverse 5′-GGAATGTTATTCAGCATTGG-3′ 1,486 - TABLE 2
Criteria for disease activity index (DAI) scores
Parameter Value for DAI score ofa: 0 1 2 3 4 5 Body wt loss (%)b 0–5 5.1–10 10.1–15 15.1–20 20.1–25 >25 Histology scorec 0–3 4–6 7–9 10–12 13–15 16–18 Blood in stool No Presence of bleeding Gross bleeding Diarrhea scored <0.5 0.5–1.4 1.5–2.4 2.5–3.4 3.5–4.4 4.5–5 ↵a For mice that were sacrificed due to weight loss and sickness, DAI calculations included the last measured body weight and diarrhea and blood scores prior to sacrifice. These mice were assigned the highest histology score (18).
↵b Calculated as (mouse body weight at time of sacrifice/body weight on day 4) × 100.
↵c Sum of Picarella scores for proximal and distal colons. Mice that were sacrificed prior to the planned endpoint were given the highest score.
↵d A score of <0.5 indicates normal stools. The score increased on a gradient to 5 as the amounts of fecal water increased and solids decreased.
Supplemental material
- Supplemental file 1 -
Quantities of cytokines/chemokines measured in colonic tissues (Table S1); correlation analysis between phylotypes, DAI, and percent body weight loss (Table S2); histology of the distal colon (Fig. S1); colonic cytokine levels differed among mice fed L. casei BL23 in milk (Fig. S2); differences in gut microbiota structure between DSS-treated mice and healthy controls (Fig. S3); correlation between L. casei cell viability and mouse body weight (Fig. S4).
PDF, 601K
- Supplemental file 1 -
