Intake of Lactobacillus delbrueckii (pExu: hsp65) Prevents the Inflammation and the Disorganization of the Intestinal Mucosa in a Mouse Model of Mucositis

Abstract

5-Fluorouracil (5-FU) is an antineoplastic drug that causes, as a side effect, intestinal mucositis, acute inflammation in the small bowel. The Heat Shock Protein (Hsp) are highly expressed in inflammatory conditions, developing an important role in immune modulation. Thus, they are potential candidates for the treatment of inflammatory diseases. In the mucositis mouse model, the present study aimed to evaluate the beneficial effect of oral administration of milk fermented by Lactobacillus delbrueckii CIDCA 133 (pExu:hsp65), a recombinant strain. This approach showed increased levels of sIgA in the intestinal fluid, reducing inflammatory infiltrate and intestinal permeability. Additionally, the histological score was improved. Protection was associated with a reduction in the gene expression of pro-inflammatory cytokines such as Tnf, Il6, Il12, and Il1b, and an increase in Il10, Muc2, and claudin 1 (Cldn1) and 2 (Cldn2) gene expression in ileum tissue. These findings are corroborated with the increased number of goblet cells, the electronic microscopy images, and the reduction of intestinal permeability. The administration of milk fermented by this recombinant probiotic strain was also able to reverse the high levels of gene expression of Tlrs caused by the 5-FU. Thus, the rCIDCA 133:Hsp65 strain was revealed to be a promising preventive strategy for small bowel inflammation.

Keywords: DNA delivery; bacterial translocation; gene expression; inflammation; intestinal mucositis; recombinant probiotics; technetium-99m.

Figure 1

Hsp65 expression in transfected eukaryotic cells. Confocal microscopy: (A) negative control: non-transfected Chinese Hamster Ovarian cell line (CHO) cells; (B) negative control: non-transfected eukaryotic cells labeled with primary (Mab anti-Hsp65) and secondary (goat anti-mouse IgG (H + L)) antibodies labeled with Alexa 488. (C,D) CHO cells labeled with primary (Mb_HSP65) and secondary (goat anti-mouse IgG (H + L)) antibodies. In green, the Hsp65 protein is expressed in the cytoplasm of eukaryotic cells. 2D images (A–D) were acquired in both depths (z-stack) using a Zeiss LSM 510 META inverted confocal laser 1358 scanning microscope with 40× or 60× objective.

Figure 2

Small intestine length, bodyweight, food and milk intake analysis: (A) intestine length, (B) bodyweight variation (C) food intake, and (D) milk intake. (A,B) ANOVA followed by the Bonferroni post hoc test and (C,D) Kruskal–Wallis test followed by Dunn’s post hoc test. Different letters (a, b, and c) indicate statistically significant differences between groups (p < 0.05). The symbols (*) (**) show a statistically significant difference (p < 0.05) (p < 0.01), respectively, between rCIDCA 133, MUC (positive control), and rCIDCA 133:Hsp65 groups before and after mucositis induction by an unpaired Student’s t-test (C,D). Geometric symbols show to the number of animals evaluated in each experimental group. • refers to the animals of CTL group, ▲ refers to the animals of MUC group, ■ refers to the animals of rCIDCA 133 group and ▼ refers to the animals of rCIDCA 133:Hsp65 group.

Figure 3

Effect of rCIDCA 133:Hsp65 on inflammatory parameters and epithelial barrier of animals inflamed with 5-fluorouracil (5-FU): (A) Intestinal Myeloperoxidase (MPO) and (B) Eosinophil Peroxidase (EPO) activity, (C) intestinal permeability (%ID/g of 99mTc-DTPA), and (D) levels of Intestinal Secretory IgA (sIgA) (μg/mL). Mice received intraperitoneal 5-FU (300 mg/kg) (MUC, rCIDCA 133, and rCIDCA 133:Hsp65 groups) or 0.9% saline solution (CTL group) and were treated with non-fermented milk or recombinant L. delbrueckii CIDCA 133-fermented milk (n = 6 animals per group). Different letters indicate statistically significant differences (p < 0.05) by ANOVA followed by the Bonferroni post hoc test (A,C,D) and Kruskal–Wallis test followed by Dunn’s post hoc test (B). Geometric symbols show to the number of animals evaluated in each experimental group. • refers to the animals of CTL group, ▲ refers to the animals of MUC group, ■ refers to the animals of rCIDCA 133 group and ▼ refers to the animals of rCIDCA 133:Hsp65 group.

Figure 4

Histopathological and morphometric analysis, intestinal permeability, and evaluation of the relative gene expression of tight junction proteins: (A) intestinal permeability (%ID/g of 99mTc-DTPA), (B) mucosal histopathology, (C) histopathological scores of the ileum of animals (objective: ×20, scale 100 µm), (D) morphometrical analysis of crypt depth, (E) villus height, (F) villus height to crypt depth ratio. Mice received intraperitoneal 5-FU (300 mg/kg) (MUC, rCIDCA 133, and rCIDCA 133:Hsp65 groups) or saline solution (CTL group). They were treated with non-fermented milk supplemented with erythromycin 2.5% (CTL and MUC) or recombinant L. delbrueckii CIDCA 133 (rCIDCA133 and rCIDCA133:Hsp65)-fermented milk supplemented with erythromycin 2.5% (n = 8 animals per group). Different letters (a–d) indicate statistically significant differences (p < 0.05) by ANOVA followed by the Bonferroni post hoc test. Geometric symbols show to the number of animals evaluated in each experimental group. • refers to the animals of CTL group, ▲ refers to the animals of MUC group, ■ refers to the animals of rCIDCA 133 group and ▼ refers to the animals of rCIDCA 133:Hsp65 group.

Figure 5

Assessment of goblet cell integrity and evaluation of the relative gene expression of Muc2 mucin: (A) representative photomicrographs from ileum section stained with Periodic Acid–Schiff (PAS), the arrows show the goblet cells (objective: ×20, scale 100 µm), (B) number of goblet cells/field obtained for experimental groups, and (C) level of relative mRNA expression of Muc2 mucin. Mice received intraperitoneal 5-FU (300 mg/kg) (MUC, rCIDCA 133, and rCIDCA 133:Hsp65 groups) or saline solution (CTL group). They were treated with non-fermented milk supplemented with erythromycin 2,5% (CTL and MUC) or recombinant L. delbrueckii CIDCA 133 (rCIDCA133 and rCIDCA133:Hsp65)-fermented milk supplemented with erythromycin 2,5% (n = 6 animals per group). Different letters (a–d) indicate statistically significant differences (p < 0.05) by ANOVA followed by the Bonferroni post hoc test (B,C). Geometric symbols show to the number of animals evaluated in each experimental group. • refers to the animals of CTL group, ▲ refers to the animals of MUC group, ■ refers to the animals of rCIDCA 133 group and ▼ refers to the animals of rCIDCA 133:Hsp65 group.

Figure 6

Level of relative mRNA expression of (A–F) tight junction proteins (occludin, F11r, claudin 1, claudin 2, claudin 5, and zonulin) determined by qPCR from animal ileum. Different letters (a–d) indicate statistically significant differences (p < 0.05) by ANOVA followed by the Bonferroni post hoc test (A–F). Geometric symbols show to the number of animals evaluated in each experimental group. • refers to the animals of CTL group, ▲ refers to the animals of MUC group, ■ refers to the animals of rCIDCA 133 group and ▼ refers to the animals of rCIDCA 133:Hsp65 group. (G) Evaluation of cell junctions by transmission electron microscopy: dark arrow highlights the reduction in the number and length of microvilli of the MUC group cells compared to the other groups. The white arrows highlight desmosomes’ presence at the lateral junctions between the epithelial cells in the CTL (scale = 1 µm), MUC, rCIDCA133, and rCIDCA 133:Hsp65 (scale = 500 nm) groups.

Figure 7

Relative gene expression of (A–I) Tnf, Il1b, Il6, Il12, Il10, Myd88, Nos2, Toll-like receptors 2 and 4. Mice received intraperitoneal 5-FU (300 mg/kg) (MUC, rCIDCA 133, and rCIDCA 133:Hsp65 groups) or saline solution (CTL group). They were treated with non-fermented milk supplemented with erythromycin 2.5% (CTL and MUC) or recombinant L. delbrueckii CIDCA 133 (rCIDCA133 and rCIDCA133:Hsp65)-fermented milk supplemented with erythromycin 2.5% (n = 5 animals per group). Different letters indicate statistically significant differences (p < 0.05) by ANOVA followed by the Bonferroni post hoc test. Geometric symbols show to the number of animals evaluated in each experimental group. • refers to the animals of CTL group, ▲ refers to the animals of MUC group, ■ refers to the animals of rCIDCA 133 group and ▼ refers to the animals of rCIDCA 133:Hsp65 group.

Figure 8

Biodistribution of 99mTc-Escherichia coli in animals with intestinal mucositis and treated with rCIDCA 133:Hsp65. cpm, counts per minute; MLN, mesenteric lymph node. Different letters indicate statistically significant differences (p < 0.05) by ANOVA followed by the Bonferroni post hoc test.