Early Gut Microbiota Intervention Suppresses DSS-Induced Inflammatory Responses by Deactivating TLR/NLR Signalling in Pigs

Abstract

Recent metagenomic studies suggest that innate and adaptive immune phenotypes can be programmed via gut microbiota-host interactions mediated via activation of pattern recognition receptors (PRRs) on host cells. In this study, we used two extremely different pig lines (the Yorkshire and the Tibetan) to test the hypothesis that the transplantation of gut microbiota could transfer certain immunologic characteristics from donor to recipient. The faecal microbiota of these two pig lines was transplanted in healthy commercial hybrid newborn piglets to establish the "Tibetan-intervened" and "Yorkshire-intervened" porcine models. Then, acute colitis was induced using dextran sulphate sodium (DSS), which activated Toll-/NOD-like receptor (TLR/NLR) signalling in the colonic tissues of the "Yorkshire-intervened" piglets, leading to increases in pro-inflammatory cytokines and immune cells and causing intestinal injuries. Conversely, DSS administration had little influence on the "Tibetan-intervened" piglets, which showed no significant inflammation and no changes in cytokines, immune cells, or signalling molecules, including TLRs, NLRs, MYD88 and NF-κB, after DSS treatment. These results indicate that pigs inoculated with the Tibetan microbiota acquired relatively strong resistance to experimental colitis, suggesting that the genotype of the host contributes to the uniqueness of its intestinal microbial community, whereas the microbiota plays a vital role in programming the immune phenotypes of the host.

Figure 1

Analysis of the faecal microbiota of the Yorkshire and Tibetan pigs. (A) 16S rRNA gene surveys (analysed by unweighted UniFrac PCoA) of the faecal microbiota of the Yorkshire or Tibetan pigs (n = 10). PC1 and PC2 are shown on the x-axis and y-axis, respectively. The percentage of variance explained by each coordinate is shown in parentheses. (B) Relative abundance of the phyla in two pig strains (Yorkshire and Tibetan pigs). Groups within the same bacterial phylum are indicated by different shades of the same colour. Taxa with a mean relative abundance of >1% are shown (n = 5 for each strain). (C–E) Percentage of Bacteroidetes, Spirochaetes and Fibrobacteres in the faeces of Yorkshire and Tibetan pigs (each n = 5) in the classification of phylum. (F) Heatmap of log10-transformed abundance levels of the selected genera (>0.1% in at least one sample) for the individual Yorkshire and Tibetan faecal samples (each n = 5). The pigs with the highest and lowest bacterial levels are in green and red, respectively. (G–J) Percentage of Prevotella, Treponema, Fibrobacter and Lactobacillus in the faeces of Yorkshire and Tibetan pigs (each n = 5) in the classification of genus.

Figure 2

Quantitation of cytokines in the colon tissues by ELISA. The results are presented as the mean ± the SEM. Statistical significance was assessed by performing a Student’s t-test. *P < 0.05; **P < 0.01. Each n = 5.

Figure 3

Relative quantities of mRNAs encoding TLRs, NLRs and associated molecules in the intestinal tissues of the Yorkshire and Tibetan pigs. Total RNA was prepared from the colon tissues of these two pig strains as described in the “Materials and Methods”. Relative mRNA expression was quantified by qRT-PCR. The results are presented as the mean ± the SEM. Statistical significance was assessed by performing a Student’s t-test. *P < 0.05; **P < 0.01. Each n = 5.

Figure 4

Treatment with the faecal microbiota from Tibetan pigs suppresses experimental colitis in the porcine models. The faecal microbiota of the Yorkshire and Tibetan pigs was transplanted in healthy commercial hybrid newborn piglets to establish the “Yorkshire-intervened” (Y-int.) and “Tibetan-intervened” (T-int.) porcine models. (A) Effect of the gut microbiota intervention on the disease activity index (DAI) in Y-int. and T-int. pigs treated with DSS (each n = 6). (B) Gross morphology of the colonic mucosal surface after 5 days of DSS administration. (C) Haematoxylin and eosin staining of the colon after 5 days of DSS administration (original magnification, x40). *P < 0.05.

Figure 5

Immune cells in transplanted pigs was evaluated by Immunohistochemistry. (A) Immunohistochemistry to evaluate CD4⁺ T cells, CD8⁺ T cells, IgA⁺ plasma cells and MAC387⁺ macrophages. (B–F) Quantitative analyses of these positive reactions are presented in the bar graphs (n = 6 for each group). The intensity of staining was quantified using the Image-Pro Plus software. Representative graphs with integrated optical density (IOD) readings are expressed as arbitrary units (AU) ± the SEM. *P < 0.05; **P < 0.01. N.S., no significant difference.

Figure 6

Quantitation of cytokines in colonic tissues from the piglets by ELISA. The faecal microbiota of the Yorkshire and Tibetan pigs was transplanted in healthy commercial hybrid newborn piglets to establish the “Yorkshire-intervened” (Y-int.) and “Tibetan-intervened” (T-int.) porcine models. CTL, control group (without DSS and FMT). The results are presented as the mean ± the SEM. Statistical significance was estimated using two-way ANOVA with DSS treatment and faecal microbiota transplantation as the independent variables. *P < 0.05; **P < 0.01. N.S., no significant difference. Each n = 5.

Figure 7

Relative quantities of TLR, NLR and associated molecule mRNAs in intestinal tissues from microbiota-transplanted piglets (each n = 6). The faecal microbiota of the Yorkshire and Tibetan pigs was transplanted in healthy commercial hybrid newborn piglets to establish the “Yorkshire-intervened” (Y-int.) and “Tibetan-intervened” (T-int.) porcine models. CTL, control group (without DSS and FMT). Total RNA was prepared from the colons as described in the “Materials and Methods”. Relative mRNA expression was quantified by qRT-PCR. The results are presented as the mean ± the SEM. *P < 0.05; **P < 0.01. N.S., no significant difference.