Visualization of Probiotic-Mediated Ca2+ Signaling in Intestinal Epithelial Cells In Vivo

Abstract

Probiotics, such as lactic acid bacteria (LAB) and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs), because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca²⁺ biosensor Yellow Cameleon (YC3.60) transgenic mouse line and established 5D (x, y, z, time, and Ca²⁺) intravital imaging systems of lymphoid tissues including those in Peyer's patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed B. subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca²⁺ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca²⁺ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.

Keywords: Bacillus subtilis; Ca2+ signaling; Lactococcus; germ-free mouse; intestinal epithelial cell; intravital imaging; probiotic; small intestine.

Figure 1

Structure of intestinal villi in a mouse under ubiquitous YC3.60 expression. (A) Schematic method of intravital imaging of intestinal epithelial cells (IECs). (B) Z-stack analysis of epithelial cells in the intestinal tract. Intravital imaging of small intestinal villi in the jejunum was performed using confocal lazar microscopy. Ratiometric images (yellow fluorescent protein/cyan fluorescent protein at excitation of 458 nm) are shown. Z-stack images of 2-µm intervals up to a depth of 58 µm were obtained. Only representative images are shown. A rainbow parameter indicates relative Ca²⁺ concentration; scale bar, 50 µm. (C) 3D structures of small IECs with intracellular Ca²⁺ concentrations. 3D images based on Z-stack images (B) were obtained using NIS-Elements software. Shown are representative results from three mice.

Figure 2

Intravital Ca²⁺ signaling images mediated by ionomycin in the intestinal tract of a mouse under ubiquitous YC3.60 expression. (A) Representative Ca²⁺ signaling images in the intestinal tract of a mouse under ubiquitous YC3.60 expression without any stimulation. Ratiometric images [yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP) at excitation of 458 nm] are shown. The rainbow parameter indicates relative Ca²⁺ concentration. (B) Distribution of time-integrated intracellular Ca²⁺ concentrations of randomly selected regions. N = 10; frame = 30. (C) Representative Ca²⁺ signaling images in the intestinal tract of a mouse under ubiquitous YC3.60 expression with ionomycin. Ratiometric images (YFP/CFP at excitation of 458 nm) are shown. Five micromolars of ionomycin in PBS were added at the indicated time point. A rainbow parameter indicates relative Ca²⁺ concentration; scale bar, 100 µm; frame = 85. (D) Ratiometric intensities (YFP/CFP at excitation of 458 nm) of indicated regions, represented by yellow arrows in (C) (n = 3), were measured for 2 min at 2-s intervals. Spontaneous Ca²⁺ signals are indicated by white arrows. Shown are representative results from two mice.

Figure 3

Intravital Ca²⁺ signaling images mediated by probiotics in the intestinal tract of a mouse under ubiquitous YC3.60 expression. (A) Representative Ca²⁺ signaling images in the intestinal tract of a mouse under ubiquitous YC3.60 expression and specific pathogen-free (SPF) conditions. Ratiometric images [yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP) at excitation of 458 nm] are shown. The 0.1 ml of Bacillus subtilis natto in PBS (10⁹ cells/ml) was added at the indicated time point. A rainbow parameter indicates relative Ca²⁺ concentration. (B) Time course for fluorescence intensities of YFP/CFP on excitation at 458 nm. Randomly selected regions (n = 10) were measured. Scale bar, 50 µm; frame = 151. Spontaneous Ca²⁺ signals are indicated by arrows. (C) Distribution of time-integrated intracellular Ca²⁺ concentrations of randomly selected regions before (upper panel) and after (lower panel) stimulation; n = 10. (D) Representative Ca²⁺ signaling images in the intestinal tract of a mouse under ubiquitous YC3.60 expression and SPF conditions. Ratiometric images (YFP/CFP at excitation of 458 nm) are shown. The 0.1 ml of Lactococcus lactis in PBS (10⁹ cells/ml) was added at the indicated time point. (E) Time course for fluorescence intensities of YFP/CFP on excitation at 458 nm; frame = 85. (F) Distribution of time-integrated intracellular Ca²⁺ concentrations of randomly selected regions; n = 10. Shown are representative results from three mice.

Figure 4

Intravital Ca²⁺ signaling images mediated by Lactococcus lactis in the intestinal tract of a mouse under ubiquitous YC3.60 expression. (A) Representative Ca²⁺ signaling images in the intestinal tract of a mouse under ubiquitous YC3.60 expression and germ-free conditions. Ratiometric images [yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP) at excitation of 458 nm] are shown. The 0.1 ml of L. lactis in PBS (10⁹ cells/ml) was added at the indicated time point. A rainbow parameter indicates relative Ca²⁺ concentration. Scale bar, 25 µm; frame = 145. Spontaneous Ca²⁺ signals are indicated by arrows. (B) Distribution of time-integrated intracellular Ca²⁺ concentrations of randomly selected regions before (left) and after (right) stimulation. (C) Time course for fluorescence intensities of YFP/CFP on excitation at 458 nm in the indicated region is represented by the yellow arrow in (A); n = 3. Shown are representative results from three mice.

Figure 5

Ca²⁺ signaling images mediated by probiotics in B cells in vitro. (A) Representative Ca²⁺ signaling images in spleen B cells form CD19-Cre/YC3.60 mice. Ratiometric images [YFP/cyan fluorescent protein (CFP) at excitation of 458 nm] are shown. Ten microliters of Bacillus subtilis natto in PBS (10⁹ cells/ml) were added to the cell culture (0.2 ml) at the indicated time point. A rainbow parameter indicates relative Ca²⁺ concentration. (B) The time course for fluorescence intensities of YFP/CFP on excitation at 458 nm in the cells (n = 15). Scale bar, 25 µm; frame = 56. (C) Representative Ca²⁺ signaling images in spleen B cells of CD19-Cre/YC3.60 expression in mice. Ratiometric images (YFP/CFP at excitation of 458 nm) are shown. Ten microliters of Lactococcus lactis in PBS (10⁹ cells/ml) were added to the cell culture at the indicated time point. A rainbow parameter indicates relative Ca²⁺ concentration. (D) The time course for fluorescence intensities of YFP/CFP on excitation at 458 nm in the cells (n = 15). Scale bar, 25 µm; frame = 61. Shown are representative results of three experiments.