Intestinal Mucosal Barrier Improvement with Prebiotics: Histological Evaluation of Longish Glucomannan Hydrolysates-Induced Innate T Lymphocyte Activities in Mice

Abstract

Use of prebiotics is a growing topic in healthcare. A lightweight molecule and water-soluble fiber ingredient, longish glucomannan hydrolysates (LGH), has been developed to improve the intestinal mucosal barrier and confer gut health benefits. This study aims to investigate the implications of continuous LGH intervening in intestinal epithelium integrity and protective immunity against chemical dextran sodium sulfate (DSS)-induced colitis. Twelve male BALB/c mice were randomly arranged into four groups. The LGH/DSS group had results in bodyweight variance, epithelial cell density, and aberrancy score as good as the LGH group, and both were equivalent to the control group. LGH consumption effectively protects the distal intestinal epithelium by activating innate T lymphocytes. Meanwhile, T-cell subsets in subepithelial interspersion take a bystander role in these microenvironmental alterations. Under this stress, the cluster of differentiation 3 (CD3)⁺ T cells infiltrate the epithelium, while CD4⁺ T cells inversely appear in submucosal large lymphoid aggregates/isolated lymphoid follicles (ILFs) in which significant CD3⁺, CD4⁺, and CD8⁺ T-cell populations agglomerate. Moreover, forkhead box P3 (Foxp3) and interleukin 17 (IL-17) are observed in these ILFs. Agglomerated CD4⁺ T-cell lineages may have roles with proinflammatory T helper 17 cells and anti-inflammatory regulatory T cells in balancing responses to intraluminal antigens. Collectively, LGH administration may function in immune modulation to protect against DSS-induced inflammation.

Keywords: T-cell activation; colitis; glucomannan; lymphoid aggregates; prebiotics.

Figure 1

Timing of DSS induction of colitis and LGH administration. Mice were randomly divided into the groups as follows: (1) Control group (n = 3; blue line) with normal chow diet and drinking water; (2) LGH group (n = 3; green line) with LGH gavage feeding; (3) DSS group (n = 3) with 4% DSS (indicated by red line) in drinking water; (4) LGH/DSS group (n = 3) with LGH gavage feeding plus 4% DSS in drinking water. Gray triangle, end of day for sacrifice. DSS, dextran sodium sulfate; LGH, longish glucomannan hydrolysates.

Figure 2

Representative image of TNF-α immunoreactivity for enterocyte nuclei. Immunohistochemical analysis stated the presence and location of TNF-α protein in colon specimen (scale bar, 50 µm). The superficial epithelium was the layer above the red curve. Inset showed the corresponding area at higher magnification (scale bar, 20 µm). Blue arrowhead, the TNF-α-negative enterocyte nucleus; red arrowhead, the TNF-α-positive immunocyte nucleus.

Figure 3

The analysis of body weight and colonic epithelium of mice following LGH administration. (A) Body weights of different groups. Error bars represented the positive SD. The averages of the cell density (B) and the aberrancy score (C) were calculated from 30 sessions for each group. Blue circles, data from the Control group; red circles, data from the DSS group; green circles, data from the LGH group; purple circles, data from the LGH/DSS group. DSS, dextran sodium sulfate; LGH, longish glucomannan hydrolysates. ***, p < 0.001.

Figure 4

Representative histological staining from mouse colons of different groups. Colon tissue sections from control group (a), LGH group (b), DSS group (c), and LGH/DSS group (d) were stained with hematoxylin and eosin (H&E; scale bar, 200 µm). Each inset represented the corresponding area of H&E staining at higher magnification (scale bar, 50 µm). Red arrowheads representatively indicated the cells with folliculate enlargement in the DSS group and with architectural shrunken in the LGH/DSS group. DSS, dextran sodium sulfate; LGH, longish glucomannan hydrolysates.

Figure 5

Representative immunohistochemical staining for the expression of CD3 and CD4 in the colons of different groups. Colon tissue sections from control group, LGH group, DSS group, and LGH/DSS group (scale bar, 200 µm) were stained with CD3 (A) and CD4 (B). Each inset represented the corresponding area (blue square) at higher magnification (scale bar, 50 µm). DSS, dextran sodium sulfate; LGH, longish glucomannan hydrolysates; SM, superficial mucosa; LP, lamina propria; S, submucosa.

Figure 6

Representative immunohistochemical staining for the expression of CD3, CD4, CD8, IL-17, and Foxp3 in the lymphoid aggregates in colons of different groups. (A) DSS and (B) LGH/DSS. Representative images from serial tissue sections showed the target protein in the same region of lymphoid aggregates (inset, indicated by blue square in H&E staining sections of (A) and (B); scale bar, 200 µm). Each inset represented the target protein (CD3, CD4, CD8, IL-17, and Foxp3) at higher magnification (scale bar, 20 µm). DSS, dextran sodium sulfate; LGH, longish glucomannan hydrolysates.