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. 2013;5(1):39-49.
doi: 10.1159/000341630. Epub 2012 Sep 14.

Bacterial stimulation of the TLR-MyD88 pathway modulates the homeostatic expression of ileal Paneth cell α-defensins

A Menendez  1 , B P Willing, M Montero, M Wlodarska, C C So, G Bhinder, B A Vallance, B B Finlay
Affiliations

Bacterial stimulation of the TLR-MyD88 pathway modulates the homeostatic expression of ileal Paneth cell α-defensins

A Menendez et al. J Innate Immun. 2013.

Abstract

Paneth cell α-defensins are antimicrobial peptides involved in the control of the intestinal microbiota and immunological homeostasis. In mice, they are encoded by multiple, highly homologous genes (Defa). The transcriptional activity of ileal Defa genes was studied in response to pharmacological and genetic perturbations of the intestinal environment of C57BL/6 mice. Defa gene transcription was sensitive to oral antibiotic administration suggesting that commensal microbes regulate Defa expression. Ileal microbiota analysis showed that decreased transcription of Defa genes correlated with depletion of Lactobacillus. Defa expression was partially restored in vivo by lactobacillus administration to antibiotic-treated mice. Defa transcripts were less abundant in ex vivo, microbiota-free intestinal explants but recovered after explant exposure to UV-killed bacteria, Toll-like receptor (TLR)-2 or TLR4 agonists. Genetic deficiency of several TLRs or MyD88 led to dramatic drops in Defa transcription in vivo. These results show that Paneth cell Defa genes are regulated by commensal bacteria through TLR-MyD88 signaling and provide a further understanding of the dysregulation of intestinal homeostasis that occurs as a result of imbalances in the populations of commensal bacteria.

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Figures

Fig. 1
Fig. 1
Paneth cell Defa genes are not equally expressed. Ileal transcript levels of Defa genes in C57BL/6 mice, relative to the transcript levels of the Rplp0 gene (defined as 1, dashed line). Open bars represent the overall relative expression for each subgroup. Solid bars represent relative expression of each individual gene within a subgroup (this was arbitrarily calculated by dividing the overall relative expression of the subgroup by the number of genes within the subgroup; this correction assumes equal expression between the members of each independent subgroup; 2 genes for Defa3, 20, 21/22, 23, 24 and 26, and 4 genes within the Defa5 group). Data shown are medians and ranges from the average of two qPCRs; n = 5 animals per group.
Fig. 2
Fig. 2
Transcript levels of Defa and Mmp7 genes in the ileum of antibiotic-treated mice, relative to untreated animals. The graph shows medians and ranges, the table shows the Mann-Whitney test p values with respect to the untreated controls; * p < 0.05: significant differences. Results shown are one representative experiment of three; n = 4 females per group.
Fig. 3
Fig. 3
Changes in microbial populations associated with antibiotic treatment. a Average HaeIII-digested T-RFLP profiles of ileal 16S rRNA gene populations of mice untreated, treated with metronidazole or streptomycin, and 2 or 4 days after suspension of streptomycin treatment. Each bacterial phylotype/TRF peak is represented by a different color; peaks of major interest are marked by hatched bars (TRF232 red and TRF329 blue). * p < 0.05: significant differences. b Total (SYBR staining) and culturable (Rogosa agar) bacteria in ileal contents, data are means ± SE.
Fig. 4
Fig. 4
a Oral administration of heat-killed commensal bacteria to streptomycin-treated wild-type C57BL/6 mice partially restores ileal Defa expression in vivo; the data shown are Defa transcript levels relative to untreated animals. The table shows the Mann-Whitney test p values with respect to the streptomycin-treated animals; * p < 0.05: significant differences. b Exposure to commensal bacteria partially restores Defa expression in ileal explants from wild-type C57BL/6 mice. Results shown are relative Defa expressions normalized to relative villin expression for each sample (RelExp Defa/RelExp villin), as a correction for epithelial survival of the individual explants. The graphs show medians and ranges. Results shown are one representative experiment of two; n = 4.
Fig. 5
Fig. 5
TLR2, TLR4 and MyD88 deficiencies cause a drop in the ileal transcript levels of Defa genes. The graph shows medians and ranges; the table shows the Mann-Whitney test p values with respect to the C57BL/6 wild-type (wt) animals; * p < 0.05 and ** p < 0.01: significant differences. Results shown are the average of two qPCRs, n = 6–9 males per group.
Fig. 6
Fig. 6
Treatment with TLR2 and TLR4 agonists (Pam2CSK4, 10 ng/ml; Pam3CSK4; 100 ng/ml and LPS, 10 ng/ml) restores Defa expression in ex vivo ileal explants from wild-type C57BL/6 mice. Results shown are relative Defa expressions normalized to relative villin expression for each sample (Rel Exp Defa/Rel Exp villin), as a correction for epithelial survival of the individual explants. The asterisk marks the lack of detectable Defa23 transcripts in the untreated explants. Results shown are one representative experiment of two; n = 4 or 5 explants per group. Pam2CSK4 and Pam3CSK4 are synthetic diacylated and triacylated lipopeptide agonists of the TLR2/6 and 2/1 heterodimers, respectively.
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