Host-probiotic interaction: new insight into the role of the endocannabinoid system by in vivo and ex vivo approaches

Abstract

The endocannabinoid system plays an important role in regulating inflammation in several chronic or anomalous gut inflammatory diseases. In vivo and ex vivo studies showed that 30 days treatment with a probiotic mix activated the endocannabinoid system in zebrafish. These results highlight the potential of this probiotic mixture to regulate immune cell function, by inducing gene expression of toll-like receptors and other immune related molecules. Furthermore, TUNEL assay showed a decrease in the number of apoptotic cells, and this finding was supported by a reduction in pro-apoptotic factors and an increase in anti-apoptotic molecules. The results presented here strengthen the molecular mechanisms activated by probiotic mix controlling immune response and inflammation.

Figure 1

(A) Paraphysiological morphology of intestinal wall in untreated control zebrafish (bar = 200 µm). (B) Morphology of intestine of VSL#3-treated zebrafish reveals good integrity of the gut mucosa, without any inflammatory infiltrate or reactive status to probiotics (bar = 200 µm). (C) In section of gut of untreated fish, a high apoptotic rate evidenced by TUNEL is evident among epithelial and mesenchymal cells, with nuclear intense black stain (bar = 200 µm). (D) TUNEL analysis of section of VSL#3-treated intestine revealed that some enterocytes and mesenchymal cells are going into apoptosis, showing their nuclei intensively black stained by the chromogen (arrow heads, bar = 200 µm). Intestinal (E) casp3, (F) baxa and (G) bcl2a mRNA levels by qPCR normalized against act1b and rplp, in control and VSL#3-treated fish. Values indicate mean ± SD. ***p < 0.001 ****p < 0.0001.

Figure 2

(A,C) Section from untreated fish, stained for cnr1 receptor, shows a strong stain restricted to parietal ganglia only (arrow head), with low levels of expression in enterocytes or other cell types (A-bar = 200 µm; C- bar = 50 µm). (B) Strong expression of cnr1 is observed in epithelial cells, in some mesenchymal cells, and in ganglia (arrow heads) of the muscular layer of intestine wall of VSL#3-treated fish (bar = 200 µm). (D) Note the strong presence of the receptor in a ganglion, as demonstrated by high power magnification of a particular area in the same section (bar = 50 µm). (E) Intestinal cnr1 protein levels by IHC in control and VSL#3-treated fish. Values indicate mean ± SD. ***p < 0.001.

Figure 3

Intestinal (A) cnr1, (B) cnr2, (C) trpv1, (D) abhd4, (E) faah and (F) mgll mRNA levels by qPCR normalized against act1b and rplp, in control (CTRL) and VSL#3-treated (VSL#3) fish. Values indicate mean ± SD. *p < 0.05; ***p < 0.001.

Figure 4

(A) Intestine section from untreated fish; note the evident reduction in intensity of tlr3 staining with respect to VSL#3-treated fish, characterized by a weak and irregular enterocyte expression, without any staining of cell free pole membranes (bar = 200 µm). (B) Intestine section from treated fish; note the intense and diffuse presence of tlr3 in the mucosal epithelium. The stain is almost exclusively restricted along the superficial margin of enterocytes (arrow heads), and shows a continuous and constant pattern (bar = 200 µm). (C) In section from untreated fish, note the substantial negativity for the presence of Nos2a, both in the epithelium and in endothelial cells (bar = 200 µm). (D) In intestine section from treated fish, an evident presence of Nos2a is observed, especially in endothelial cells (large arrows) or in scattered mononuclear cells (presumably macrophages) interspersed throughout the mucosal chorion (arrow heads). Note the molecule is also localized in some epithelial cells (small arrow); (bar = 200 µm). (E) Untreated zebrafish show low level of immunoreactivity for Tgfb1a both in the epithelium and in mononuclear cells (bar = 200 µm). (F) In VSL#3-treated fish, intestinal presence of Tgfb1a was strong and diffuse throughout the epithelium (large arrows) and in some mononuclear cells (small arrows); (bar = 200 µm). (G) In untreated fish the localization of the p65 subunit of the Nfkb heterodimer is restricted to scattered epithelial cells (bar = 200 µm). (H) Similarly to Tgfb1a localization, the p65 subunit of the Nfkb heterodimer is present in a large proportion of enterocytes (large arrows). Also note the presence of this marker in many mononuclear cells (small arrows), infiltrating the lamina propria of the intestinal mucosa of VSL#3-treated zebrafish (bar = 200 µm).

Figure 5

Intestinal mRNA levels evaluated by qPCR of (A) cnr1, (B) cnr2, (C) faah, (D) tlr2, (E) casp3 and (F) il1b normalized against act1b and rplp, in different experimental groups from ex vivo experimentation. Values indicate mean ± SD. Different letters denote significant differences among groups.

Figure 6

Intestinal distribution of cnr1 by IHC in different experimental groups from ex vivo experimentation: (A) Control; (B) VSL#3; (C) AM251; (D) VSL#3 + AM251. (E) Cnr1 protein levels by IHC and (F) TUNEL positive cell count in the same ex vivo experimental groups. Values indicate mean ± SD. Different letters denote significant differences among groups.