Release of HMGB1 and Toll-like Receptors 2, 4, and 9 Signaling Are Modulated by Bifidobacterium animalis subsp. lactis BB-12 and Salmonella Typhimurium in a Gnotobiotic Piglet Model of Preterm Infants

Abstract

Gnotobiotic (GN) animals with defined microbiota allow us to study host-microbiota and microbiota-microbiota interferences. Preterm germ-free (GF) piglets were mono-associated with probiotic Bifidobacterium animalis subsp. lactis BB-12 (BB12) to ameliorate/prevent the consequences of infection with the Salmonella Typhimurium strain LT2 (LT2). Goblet cell density; expression of Toll-like receptors (TLRs) 2, 4, and 9; high mobility group box 1 (HMGB1); interleukin (IL)-6; and IL-12/23p40 were analyzed to evaluate the possible modulatory effect of BB12. BB12 prevented an LT2-induced decrease of goblet cell density in the colon. TLRs signaling modified by LT2 was not influenced by the previous association with BB12. The expression of HMGB1, IL-6, and IL12/23p40 in the jejunum, ileum, and colon and their levels in plasma were all decreased by BB12, but these changes were not statistically significant. In the colon, differences in HMGB1 distribution between the GF and LT2 piglet groups were observed. In conclusion, the mono-association of GF piglets with BB12 prior to LT2 infection partially ameliorated the inflammatory response to LT2 infection.

Keywords: Bifidobacterium animalis subsp. lactis BB-12; Salmonella Typhimurium; Toll-like receptors; high mobility group box 1; immunodeficient host; inflammatory cytokines; intestinal barrier; mucin; tight junction proteins.

Figure 1

Goblet cells (blue color) in the ileum of gnotobiotic piglets. Number of goblet cells per mm² in the ileum of the one-week-old piglets: germ-free (GF; (A)), infected with S. Typhimurium LT2 for 24 h (LT2; (B)), associated with B. animalis subsp. lactis BB-12 (BB12; (C)), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2; (D)). Six samples from each group were analyzed, and statistical differences were calculated by a two-way ANOVA with Tukey’s multiple comparison post hoc test. The values are presented as mean + SEM, and p < 0.05 among groups are denoted by different letters above the columns (E). A scale bar (D) depicts 200 μm.

Figure 2

Goblet cells (blue color) in the colon of gnotobiotic piglets. Number of goblet cells per mm² in the colon of the one-week-old gnotobiotic piglets: germ-free (GF; (A)), infected with S. Typhimurium LT2 for 24 h (LT2; (B)), associated with B. animalis subsp. lactis BB-12 (BB12; (C)), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2; (D)). Six samples from each group were analyzed, and statistical differences were calculated by a two-way ANOVA with Tukey’s multiple comparison post hoc test. The values are presented as mean + SEM, and a p < 0.05 among groups is denoted by different letters above the columns (E). A scale bar (D) depicts 100 μm.

Figure 3

Relative expression (fold-change) of TLR2 (A), TLR4 (B), TLR9 (C), MyD88 (D), TRIF (E), and RAGE (F) mRNA in the ileum of the one-week-old gnotobiotic piglets: germ-free (GF), infected with S. Typhimurium LT2 for 24 h (LT2), associated with B. animalis subsp. lactis BB-12 (BB12), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2). Six samples from each group were analyzed, and statistical differences were calculated by a two-way ANOVA with Tukey’s multiple comparison post hoc test. The values are presented as mean + SEM, and a p < 0.05 among groups is denoted by different letters above the columns.

Figure 4

Relative expression (fold-change) of TLR2 (A), TLR4 (B), TLR9 (C), MyD88 (D), TRIF (E), and RAGE (F) mRNA in the colon of the one-week-old gnotobiotic piglets: germ-free (GF), infected with S. Typhimurium LT2 for 24 h (LT2), associated with B. animalis subsp. lactis BB-12 (BB12), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2). Six samples from each group were analyzed, and statistical differences were calculated by a two-way ANOVA with Tukey’s multiple comparison post hoc test. The values are presented as mean + SEM, and a p < 0.05 among groups is denoted by different letters above the columns.

Figure 5

Relative expression (fold-change) of TLR2 (A), TLR4 (B), TLR9 (C), MyD88 (D), TRIF (E), and RAGE (F) mRNA in the mesenteric lymph nodes of the one-week-old piglets: germ-free (GF), infected with S. Typhimurium LT2 for 24 h (LT2), associated with B. animalis subsp. lactis BB-12 (BB12), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2). Six samples from each group were analyzed, and statistical differences were calculated by a two-way ANOVA with Tukey’s multiple comparison post hoc test. The values are presented as mean + SEM, and a p < 0.05 among groups is denoted by different letters above the columns.

Figure 6

Expression of HMGB1 in the colon. Representative micrographs of the germ-free (GF; (A)) and S. Typhimurium LT2-infected piglets for 24 h (LT2; (B)) are depicted. The scale bar (A) corresponds to 50 μm.

Figure 7

Abundance of HMGB1 (A,D,G), IL-6 (B,E,H), and IL-12/23p40 (C,F,I) proteins in the jejunum (A–C), ileum (D–F), and colon (G–I) of the one-week-old piglets: germ-free (GF), infected with S. Typhimurium LT2 for 24 h (LT2), associated with B. animalis subsp. lactis BB-12 (BB12), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2). Six samples from each group were analyzed, and statistical differences were calculated by the Kruskal–Wallis test with Dunn’s multiple comparison post hoc test. The values are presented as boxes and whiskers indicating the lower and upper quartiles, the central line is the median, and the ends of the whiskers depict the minimal and maximal values. A p < 0.05 among groups is denoted with different letters around the columns.

Figure 8

Plasma concentrations of HMGB1 (A), IL-6 (B) and IL-12/23p40 (C) proteins of one-week-old piglets: germ-free (GF), infected with S. Typhimurium LT2 for 24 h (LT2), associated with B. animalis subsp. lactis BB12 (BB12), and associated with BB12 and infected with LT2 for 24 h (BB12 + LT2). Six samples in each group were analyzed and statistical differences were calculated by the Kruskal–Wallis test with Dunn’s multiple comparison post hoc test. The values are presented as boxes and whiskers indicating the lower and upper quartiles, the central line is the median, and the ends of the whiskers depict the minimal and maximal values. A p < 0.05 among groups is denoted with different letters around the columns.

Figure 9

Experiment design. Preterm gnotobiotic piglets (n = 24) were assigned into four groups with six piglets per group: (i) germ-free (GF), (ii) infected with Salmonella Typhimurium strain LT2 (LT2), (iii) associated with probiotic Bifidobacterium animalis subsp. lactis BB-12 (BB12), and (iv) associated with BB12 and infected with LT2 (BB12 + LT2).