Fecal Microbiota Transplantation Protects the Intestinal Mucosal Barrier by Reconstructing the Gut Microbiota in a Murine Model of Sepsis

Abstract

The gastrointestinal (GI) tract has long been hypothesized to play an integral role in the pathophysiology of sepsis, and gut microbiota (GM) dysbiosis may be the key factor. Previous studies have shown that the gut flora was significantly altered in critically ill patients. This study aimed to observe what kind of GM dysbiosis is in the early stage of sepsis and whether the application of fecal microbiota transplantation (FMT) can reconstruct the GM of septic mice and restore its protective function on the intestinal mucosal barrier. The study investigated the effect of FMT on gut microbiota, mucosal barrier function, inflammatory response, and survival in a murine model of sepsis established by cecal ligation and puncture (CLP). It is found that FMT can not only reduce morbidity and mortality and restore the abundance and diversity of the gut flora in septic mice, but can also improve the intestinal barrier function by reducing epithelial cell apoptosis, improving the composition of the mucus layer, upregulating the expression of tight junction proteins, and reducing intestinal permeability and the inflammatory response. After FMT, Lachnospiraceae contributed the most to intestinal protection through enhancement of the L-lysine fermentation pathway. FMT offers a microbe-mediated survival advantage in a murine model of sepsis. Therefore, an improved understanding of the connection between microbiota, and systemic illness may yield new therapeutic strategies for patients with sepsis.

Keywords: critical care; fecal microbiota transplantation; gut microbiota; intestinal mucosal barrier; sepsis.

Figure 1

Survival analysis, and caspase 3 expressions among the Sham, CLP, and FMT groups. (A) Seven-day mortality observations in the Sham (red), CLP (black), and FMT groups (blue). There were significant differences between the CLP group and the other two groups, P < 0.05, but there was no significant difference between the FMT group and the Sham group; (B) Immunohistochemistry of the colon [under a digital microscope (20 × 10)]. The average integral optical density of caspase 3 between the Sham, CLP, and FMT groups at 12, 24, and 48 h are shown on the right side. (C) Relative expression of caspase 3 compared with GAPDH between the CLP and FMT groups at 24 and 48 h. *P < 0.05, ***P < 0.001, ****P < 0.0001, ns, no statistical difference, respectively.

Figure 2

The serum IL-6, TNF-α, and IL-10 among the Sham, CLP, and FMT groups at 12, 24, and 48 h. The concentration of serum IL-6 (pg/mL) and IL-10 (pg/mL) in the FMT group at 12 h after sepsis modeling were significantly higher than in the Sham group (P < 0.05), and there was no significant difference in TNF-α levels (pg/mL) among the three groups. The serum IL-6 level in the CLP group was significantly higher than in the Sham group and the FMT group (P < 0.001) at 24 h. The TNF-α levels in both the CLP and FMT groups were higher than in the Sham group (P < 0.001 and P < 0.01, respectively). The IL-10 level in the CLP group was lower than in the Sham group and the FMT group (P < 0.001). The serum IL-6 level in the CLP group was higher than in the Sham group at 48 h (P < 0.01). The TNF-α level in the CLP group was higher than in the Sham group and FMT group (P < 0.001). The TNF-α level in the FMT group was higher than in the Sham group (P < 0.05). There was no significant difference among the three groups in the IL-10 level at 48 h.

Figure 3

The mucus layer thickness and MUC2 expression in the Sham, CLP, and FMT groups at 12, 24, and 48 h. (A) The AB-PAS method was used to measure the thickness of the mucus layer in the Sham, CLP, and FMT groups at 12, 24, and 48 h under a digital microscope (20 × 10). The blue color indicates goblet cell secretion and a mucus layer. The histogram on the right shows the statistical results of the comparison of the three groups. There was no significant difference between the Sham group and the FMT group in mucus layer thickness at 24 h. (B) MUC2 expression (green fluorescence) was observed in the three groups at 12, 24, and 48 h. The histogram shows the statistical results of all three groups. There was no significant difference between the Sham group and the FMT group at 12 h. *P < 0.05, **P < 0.01, ****P < 0.0001, ns, no significant difference.

Figure 4

Intestinal epithelial cell junctions. Intestinal epithelial cell junctions in the CLP group and FMT group at 48 h. The images on the left were enlarged 4K, and those on the right were enlarged 10K. ▲ Tight junction and the intermediate junction between intestinal epithelial cells. ■ The space between intestinal epithelial cells.

Figure 5

Comparison of tight junction protein expression. (A) Comparison of occluding expression in the CLP and FMT groups at 12, 24 add 48 h, respectively, and observed under a digital microscope (20 × 10). Significant results are shown in the box plot on the right, ****P < 0.0001; (B) Relative expression of occludin and ZO-1 compared with β-actin expression in the Sham, CLP, and FMT groups at 24 or 48 h. SB, CB, TB, SD, CD, and TD represent the 24 h Sham, CLP, and FMT groups and 48 h Sham, CLP, and FMT groups, respectively.

Figure 6

TLR4, MyD88, and NF-κB expression and mRNA levels. (A) Expression of TLR4, MyD88, and NF-κB compared to GAPDH protein among Sham, CLP, and FMT groups at 24 and 48 h. SB, CB, TB, SD, CD, and TD represent 24 h Sham, CLP, and FMT groups and 48 h Sham, CLP, and FMT groups, respectively. (B) Relative TLR4, MyD88, and NF-κB levels in the colon. *P < 0.05, **P < 0.01.

Figure 7

Microbiota analysis. (A) Krona species at phylum among the Normal, Sham, CLP, and FMT groups. (B) Alpha diversity. *P < 0.05, **P < 0.01 ****P < 0.001. (C) NMDS two-dimensional sorting diagram. The closer the distance between the two points in the figure, the smaller the difference between the microbial communities in the two samples. (D) LEfSe (LAD Effect Size). A longer bar denotes a more significant difference in the taxon. The color of the bar graph indicates the most abundant sample group corresponding to the taxon. (E) Predicted abundance map of KEGG secondary functional pathways. (F) Species composition map of differential MetaCys metabolic pathways. The abscissa shows different groups. The order of the samples in the groups was sorted according to the similarity of the data; the ordinate was the relative abundance of the metabolic pathways, and the species different levels of contribution to the metabolic pathways were displayed in different colors at different levels. (In order to show the results better, we used abbreviations to represent each group: N = normal mice, 7C = CLP group mortality observation, 7T = FMT group mortality observation, SA = Sham group 12 h; CA, CB, and CD = CLP group at 12, 24, and 48 h time points, respectively, and TA, TB, and TD = FMT group at 12, 24, and 48 h time points, respectively).