Bacterial flagellin stimulates Toll-like receptor 5-dependent defense against vancomycin-resistant Enterococcus infection

Abstract

Treatment of vancomycin-resistant Enterococcus (VRE) infections is limited by the paucity of effective antibiotics. Administration of broad-spectrum antibiotics promotes VRE colonization by down-regulating homeostatic innate immune defenses. Intestinal epithelial cells and Paneth cells express antimicrobial factors on direct or indirect stimulation of the Toll-like receptor (TLR)-myeloid differentiation factor 88-mediated pathway by microbe-derived molecules. Here, we demonstrate that the TLR5 agonist flagellin restores antibiotic-impaired innate immune defenses and restricts colonization with VRE. Flagellin stimulates the expression of RegIIIgamma, a secreted C-type lectin that kills gram-positive bacteria, including VRE. Systemic administration of flagellin induces RegIIIgamma expression in intestinal epithelial cells and Paneth cells along the entire length of the small intestine. Induction of RegIIIgamma requires TLR5 expression in hematopoietic cells and is dependent on interleukin 22 expression. Systemic administration of flagellin to antibiotic-treated mice dramatically reduces VRE colonization. By enhancing mucosal resistance to multidrug-resistant organisms, flagellin administration may provide a clinically useful approach to prevent infections in patients treated with broad-spectrum antibiotics.

Figure 1

Flagellin potently induces RegIIIγ and RegIIIβ in antibiotic-treated mice. A, Mice were treated with metronidazole, neomycin and vancomycin (MNV) for 7 days. Starting on day 5 of antibiotic treatment, mice received 15 μg of flagellin per day via intraperitoneal injection for 3 days. Mice were sacrificed 24 hours after the last flagellin injection. Transcriptional expression of RegIIIγ, RegIIIβ, Ang-4, Defcr-1, Defcr-3, Defcr-5, and MMP-7 was measured by quantitative real-time PCR. Expression levels were normalized to GAPDH and determined relative to MNV-treated mice. Values are representative of at least two experiments and are expressed as mean ± SEM (n=4–9, *p<0.05, **p<0.01, statistical analysis by one-way ANOVA with Bonferroni correction). B. Protein extracts from the distal ileum were analyzed by Western blotting with RegIIIγ-specific antiserum and anti-β-tubulin as a loading control. Shown are representative samples from two experiments (n = 6 for each group). C, On day 7 of MNV treatment, mice received 0, 1, 5, 15, or 50 μg of flagellin intraperitoneally. RegIIIγ mRNA message levels were measured 24 hours later by quantitative real-time PCR and were normalized to GAPDH (n=5 for each group).

Figure 2

Flagellin alters the regional expression of RegIIIγ in the small intestine. Mice were treated with metronidazole, neomycin and vancomycin (MNV) for 7 days, and on day 5, flagellin was given intraperitoneally at 15 μg per day for 3 days. Tissue from the duodenum, jejunum, and ileum were collected for Western blot analysis and immunohistochemistry. A, Protein extracts were analyzed by Western blotting using RegIIIγ-specific anti-serum. Each lane is a representative mouse from the indicated group. B, RegIIIγ was detected in paraffin-embedded tissue using immunohistochemistry with polyclonal RegIIIγ-specific antiserum. Positive cells were colorized brown (400-fold magnification). The data are representative of two independent experiments (n=6).

Figure 3

TLR5-mediated signaling is required in hematopoietic cells for flagellin-induced RegIIIγ expression. Mice were treated with metronidazole, neomycin, and vancomycin (MNV) for 7 days. After 5 days of MNV treatment, mice received 3 doses of 15 μg/day of flagellin via intraperitoneal injection. A, Western blot analysis of protein extracts from the distal ileum was used to assess differences between WT and KO mice using RegIIIγ-specific anti-serum. Each lane is a representative mouse from the indicated group. The data are representative of two independent experiments (n=5). B - E, bone marrow (BM) chimeric mice were treated with MNV and flagellin as described above approximately seven weeks after lethal irradiation and BM transfer. RegIIIγ mRNA levels were measured in the duodenum (B) and ileum (C) by quantitative real-time PCR, normalized to GAPDH, and expressed as mean ± SEM. RegIIIγ protein levels in the duodenum (D) and ileum (E) were assessed by Western blot analysis using RegIIIγ-specific anti-serum. Each lane of the Western blots is a representative mouse from the indicated group. Data are representative of two independent experiments (n=2–3).

Figure 4

Flagellin induces a variety of cytokines. Mice were treated with metronidazole, neomycin, and vancomycin (MNV) for 7 days. Three doses of flagellin (15μg/day) were administered intraperitoneally to mice beginning on day 5 of MNV treatment. Messenger RNA was extracted from the distal ileum to assess cytokine induction after flagellin treatment using quantitative real-time PCR. IL-12 refers to IL-12p35, and IL-23 refers to IL23p19. Data were pooled from two independent experiments with a total of four mice per group. Levels were normalized to GAPDH and are expressed as mean ± SEM (*p < 0.05, statistical analysis by one-way ANOVA with Bonferroni correction)

Figure 5

IL-22 is required for flagellin-mediated RegIIIγ expression. Wild-type (WT) and IL-22-deficient (IL-22-KO) or IL-10Rβ-deficient (IL-10Rβ-KO) mice were administered metronidazole, neomycin, and vancomycin (MNV) for 7 days. Mice received 15μg flagellin intraperitoneally on day 6 and 7 of MNV treatment. Tissue from the duodenum (A, C) and ileum (B, D - F) were collected for mRNA and protein extraction. A, B, and E, Quantitative PCR was used to evaluate RegIIIγ mRNA transcript expression. Levels were normalized to GAPDH and expressed as mean ± SEM (*p<0.05, statistical analysis by one-way ANOVA with Bonferroni correction). C, D, and B, Western blot analysis with RegIIIγ-specific anti-serum was used to assess RegIIIγ protein levels. Each lane is a representative sample from a mouse from the indicated group. A - D, Data were pooled from two independent experiments (n=8–10).

Figure 6

Flagellin reduces susceptibility to VRE colonization in mice treated with broad-spectrum antibiotics. Mice were treated with metronidazole, neomycin and vancomycin (MNV) for 7 days. Starting on day 5 of antibiotic treatment, mice received 15 μg of flagellin per day via intraperitoneal injection for 3 days. On day 7 of antibiotic treatment, mice were inoculated with 10¹⁰ CFUs of VRE by oral gavage. The luminal content (A) and intestinal wall (B) of the distal half of the small intestine were collected for bacterial counts 24 hours post-inoculation. Data was compiled from three independent experiments and expressed as mean ± SEM (n=12–14, *p<0.001, statistical analysis by one-way ANOVA with Bonferroni correction).