Probiotic Lactobacillus rhamnosus GG Induces Alterations in Ileal Microbiota With Associated CD3-CD19-T-bet+IFNγ+/- Cell Subset Homeostasis in Pigs Challenged With Salmonella enterica Serovar 4,[5],12:i:

Abstract

Salmonella enterica serovar 4,[5],12:i:- (S. 4,[5],12:i:-) is an emerging foodborne pathogen causing salmonellosis in humans and animals. Probiotic Lactobacillus rhamnosus GG (LGG) is an effective strategy for controlling enteric infections through maintaining gut microbiota homeostasis and regulating the intestinal innate immune response. Here, LGG was orally administrated to newly weaned piglets for 1 week before S. 4,[5],12:i:- challenge. S. 4,[5],12:i:- challenge led to disturbed gut microbiota, characterized by increased levels of Psychrobacter, Chryseobacterium indoltheticum, and uncultured Corynebacteriaceae populations, as well as an aberrant correlation network in Prevotellaceae NK3B31 group-centric species. The beneficial effect of LGG correlated with attenuating the expansion of Prevotellaceae NK3B31 group. Fusobacterium only found in the pigs treated with LGG was positively correlated with Lactobacillus animalis and Propionibacterium. Administration of LGG induced the expansion of CD3^-CD19^-T-bet⁺IFNγ⁺ and CD3^-CD19^-T-bet⁺IFNγ^- cell subsets in the peripheral blood at 24 h after a challenge of S. 4,[5],12:i:-. S. 4,[5],12:i:- infection increased the population of intraepithelial CD3^-CD19^-T-bet⁺IFNγ⁺ and CD3^-CD19^-T-bet⁺IFNγ^- cells in the ileum; however, this increase was attenuated via LGG administration. Correlation analysis revealed that LGG enriched Flavobacterium frigidarium and Facklamia populations, which were negatively correlated with intraepithelial CD3^-CD19^-T-bet⁺IFNγ⁺ and CD3^-CD19^-T-bet⁺IFNγ^- cells in the ileum. The present data suggest that probiotic LGG alters gut microbiota with associated CD3^-CD19^-T-bet⁺IFNγ^+/- cell subset homeostasis in pigs challenged with S. enterica 4,[5],12:i:-. LGG may be used in potential gut microbiota-targeted therapy regimens to regulate the specific immune cell function and, consequently, control enteric infections.

Keywords: 12:i:-; IFNγ; Lactobacillus rhamnosus; Salmonella enterica serovar 4; T-bet; [5]; gut microbiota; pig.

FIGURE 1

Ileal mucosal microbiota communities of newly weaned pigs in response to S. 4,[5],12:i:- challenge and LGG pretreatment. (A) Ileal mucosal microbiota communities at the phylum level. The stacked bars show the combined relative abundance of phylum-level taxa per animal. Colors are assigned for all phyla detected. (B) Heatmap showing the spatial distributionsof all OTUs at the genus level, indicating the relative abundance of genera per animal (n = 6 per group) that received oral sterile physiological saline (CN), oral sterile physiological saline followed by S. 4,[5],12:i:- (1 × 10¹⁰ CFU/ml, 10 ml) challenge (SM), or LGG (1 × 10⁹ CFU/ml, 10 ml/day) for 1 week followed by S. 4,[5],12:i:- (LS). Genera are clustered to the left based on relative abundance. The relative abundance of each genus is indicated by a color gradient from blue (low abundance) to red (high abundance). (C) A network diagram showing the OTUs among the three groups. (D) Venn diagrams displaying the overlap degree of OTUs in the ileal mucosal microbiota of the three groups.

FIGURE 2

LGG pretreatment changed the ileal mucosal microbiota structure of pigs challenged with S. 4,[5],12:i:-. (A) Based on the relative abundance of OTUs, two-dimensional PLS-DA score plots distinguishing the ileal mucosal microbiota of pigs in response to S. 4,[5],12:i:- challenge and LGG pretreatment. (B) LEfSe was performed to discover indicator bacteria that distinguished the treatment-specific microbiota features. Taxonomic cladogram indicating the phylogenetic distribution of microbial lineages associated with the three groups (n = 6 per group). Lineages with an LDA value >2.5 are displayed. The diameter of each dot is proportional to its effect size. (C) Differences in relative abundance of OTUs from the three indicated treatment groups are shown using one-way ANOVA. The taxonomy of the OTUs (phylum and family) is depicted on the left. ^∗P < 0.05.

FIGURE 3

Co-occurrence networks of microbiota associated with S. 4,[5],12:i:- challenge and LGG pretreatment. Networks were constructed using OUT pairs present in the microbiota of pigs in the CN (A), SM (B), and LS (C) groups, as well as across the three groups (D), all with an absolute Pearson’s correlation above 0.7 at a 0.05 FDR-corrected significance level. OTUs are colored by phylum affiliation and sized by mean relative abundance. In (D), asterisk (^∗) represents OTUs whose abundances were significantly altered among the three groups. CN, SM, and LS represent OTUs who were only found in the CN, SM, and LS group, respectively.

FIGURE 4

LGG Pretreatment induced the expansion CD3^-CD19^-T-bet⁺IFNγ⁺ and CD3^-CD19^-T-bet⁺IFNγ^- cell subsets in the peripheral blood. Peripheral blood samples were collected from the indicated pigs at 0, 24, and 168 h after S. 4,[5],12:i:- challenge. (A) Representative dot plots show the percentages of T-bet^+/- IFNγ^+/- cell subsets among CD3^-CD19^- cells at 24 h after S. 4,[5],12:i:- challenge. Flow cytometry analysis of the percentages of T-bet^- IFNγ⁺ (B), T-bet⁺ IFNγ⁺ (C), and T-bet⁺ IFNγ^- cells (D) within the peripheral CD3^-CD19^- cell populations in the indicated pigs. The data are presented as mean ± SEM (n = 6 per group). ^∗P < 0.05.

FIGURE 5

LGG Pretreatment Attenuated Salmonella-induced expansion of intraepithelial CD3^-CD19^-T-bet⁺IFNγ⁺ and CD3^-CD19^-T-bet⁺IFNγ^- cell subsets in the ileum. Peyer’s patch lymphocytes (PPLs), intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs) were collected from jejunal and ileal tissues from the indicated pigs 7 days after S. 4,[5],12:i:- challenge. (A) Representative dot plots show the percentages of intraepithelial T-bet^+/- IFNγ^+/- cell subsets among CD3^-CD19^- cells. Flow cytometry analysis of the percentages of T-bet^- IFNγ⁺ (B), T-bet⁺ IFNγ⁺ (C), and T-bet⁺ IFNγ^- cells (D) among CD3^-CD19^- cell populations in the small intestine. The data are presented as mean ± SEM (n = 6 per group). ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 6

Correlation between OTUs and CD3^-CD19^-T-bet^+/-IFNγ^+/- cell subsets in the ileum. The relative abundance of ileal microbiota was assessed for correlations with the levels of T-bet^+/-IFNγ^+/- cells among CD3^-CD19^- cell populations in the small intestine. Rows correspond to OTUs with the IDs and the taxonomy of the OTUs (phylum, family, and genus) is shown on the right. OTUs are clustered to the left based on relative abundance. Columns correspond to CD3^-CD19^-T-bet^+/-IFNγ^+/- cell subsets. Colors red and blue denote positive and negative association, respectively. The intensity of the colors represents the degree of association between the relative abundance of OTU and CD3^-CD19^-T-bet^+/-IFNγ^+/- cell subsets as assessed by Pearson’s correlations. The black asterisks in the blue/red cells indicate that the associations were significant. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 7

Probiotic LGG alters ileal microbiota with associated CD3^-CD19^-T-bet⁺IFNγ^+/- cell subset homeostasis in pigs. S. 4,[5],12:i:- challenge led to disturbed gut microbiota, characterized by increased levels of Psychrobacter, Chryseobacterium indoltheticum, and uncultured Corynebacteriaceae populations, as well as an aberrant correlation network in Prevotellaceae NK3B31 group-centric species. LGG reduces the population of Prevotellaceae NK3B31 group, changes the correlation network in Prevotellaceae NK3B31 group-centric species, and promotes symbiotic synergism of Fusobacterium, Lactobacillus animalis, and Propionibacterium. LGG attenuates the increase in the population of intraepithelial CD3^-CD19^-T-bet⁺IFNγ⁺ and CD3^-CD19^-T-bet⁺IFNγ^- cells resulting from an S. 4,[5],12:i:- infection in the ileum. LGG enriched Flavobacterium frigidarium and Facklamia populations, which are negatively correlated with intraepithelial CD3^-CD19^-T-bet⁺IFNγ⁺ and CD3^-CD19^-T-bet⁺IFNγ^- cells in the ileum.