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. 2013 Nov 13;14(5):571-81.
doi: 10.1016/j.chom.2013.10.009.

"V体育官网" Innate and adaptive immunity interact to quench microbiome flagellar motility in the gut

Tyler C Cullender  1 , Benoit Chassaing, Anders Janzon, Krithika Kumar, Catherine E Muller, Jeffrey J Werner, Largus T Angenent, M Elizabeth Bell, Anthony G Hay, Daniel A Peterson, Jens Walter, Matam Vijay-Kumar, Andrew T Gewirtz, Ruth E Ley
Affiliations

Innate and adaptive immunity interact to quench microbiome flagellar motility in the gut

Tyler C Cullender et al. Cell Host Microbe. .

Abstract

Gut mucosal barrier breakdown and inflammation have been associated with high levels of flagellin, the principal bacterial flagellar protein. Although several gut commensals can produce flagella, flagellin levels are low in the healthy gut, suggesting the existence of control mechanisms. We find that mice lacking the flagellin receptor Toll-like receptor 5 (TLR5) exhibit a profound loss of flagellin-specific immunoglobulins (Igs) despite higher total Ig levels in the gut. Ribotyping of IgA-coated cecal microbiota showed Proteobacteria evading antibody coating in the TLR5(-/-) gut. A diversity of microbiome members overexpressed flagellar genes in the TLR5(-/-) host VSports手机版. Proteobacteria and Firmicutes penetrated small intestinal villi, and flagellated bacteria breached the colonic mucosal barrier. In vitro, flagellin-specific Ig inhibited bacterial motility and downregulated flagellar gene expression. Thus, innate-immunity-directed development of flagellin-specific adaptive immune responses can modulate the microbiome's production of flagella in a three-way interaction that helps to maintain mucosal barrier integrity and homeostasis. .

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Figures

Figure 1
Figure 1. Gut flagellin load is inversely proportional to anti-flagellin IgA levels
(A) Total IgA levels, (B) anti-flagellin IgA levels (anti-flagellin IgG equivalents), and (C) flagellin (Salmonella Typhimurium flagellin equivalents) load for WT, TLR5−/−, MyD88−/−, and DSS-treated WT B57Bl/6 mice. (D) Flagellin amounts for conventionally raised RAG1−/− mice (Con-R), and WT and RAG1−/− mice monocolonized with SFB (+SFB). The y-axis label for C applies to D also. Columns represent means ± s.e.m. N= 8 mouse per group; lower-case letters next to the bars indicate significance: bars with different letters indicate significantly different means at P < 0.05 using a two-tailed t-test corrected for multiple comparisons. Relates to Figure S1.
Figure 2
Figure 2. Flagella-motility genes are upregulated in the TLR5−/− microbiome despite similar encoding capacity
Metatranscriptome and metgenome analyses for TLR5−/− and WT cecal microbiomes. Left panel, metatranscriptomes: cDNAs assigned to the 18 most significant COG categories were normalized and hierarchically clustered. Functional categories are indicated to the left of the panels; flagella-associated gene functions are highlighted in red. Dendrogram (bottom) depicts the uncentered correlation similarity metric. Correlation coefficients are represented by color ranging from blue (−5× depletion) to yellow (5× enrichment). Right panel, metagenomes: metagenomic reads from the same 18 COG categories were identically processed, with the exception that correlation coefficients are an order of magnitude less (−0.5× to 0.5×). Relates to Figure S2 and Table S1.
Figure 3
Figure 3. Metatranscriptomic reads annotated as flagellin are phylogenetically diverse
Taxonomic assignments of flagellin gene transcripts are shown to the species level, with abundance of reads corresponding to the key at bottom. Hierarchical classification and the Subsystems database in MG-RAST was used to annotate function. We isolated reads with an annotation of flagellin and assigned taxonomy using BLASTX with default arguments. Note that Bacteroides pectinophilus is a Firmicute based on 16S rRNA gene sequence analysis but misclassified in the phylum Bacteroidetes in the MG-RAST database. Relates to Figure S3, which shows stimulation of TLR5 by flagellins from different species, and to Table S2.
Figure 4
Figure 4. The population sizes for three species of bacteria colonizing gnotobiotic WT and RAG1−/− mice are equivalent despite elevated flagellin production in RAG1−/− mice
(A) Flagellin load (Salmonella Typhimurium flagellin equivalents measured by cell reporter assay) in ceca of gnotobiotic WT and RAG1−/− mice colonized with E. coli (Ec), Bacteroides thetaiotaomicron (Bt), and Bifidobacterium adolescentis (Ba). (B) CFU counts of bacteria cultured from the ceca. Bars are means ± s.e.m., *P < 0.001; two-tailed t-test; ns, non-significant. Relates to Figure S4.
Figure 5
Figure 5. Effect of TLR5 signaling on IgA coating of bacterial populations
(A) Box plots represent the ratio of IgA-coated to non-coated reads for each OTU within the specified phyla, such that a value=1 means the OTUs within a phyla are coated or non-coated with IgA at equal frequency. An open circle represents an outlier. N= 5–8 mice/group. *P < 0.05, two-tailed t-test; n.s., nonsignificant. (B) The frequency of IgA-coated (closed circles) and non-coated (open squares) cells are displayed for the 25 most abundant OTUs in either TLR5−/− or wildtype mice. The two most abundant OTUs for each genotype are indicated by their consensus taxonomy. (C) OTUs with a significant Cook’s distance (and therefore outliers from the trend of equal frequencies of IgA-coating and non-coating) are categorized as having high IgA-coating or low IgA-coating for both TLR5−/− and WT mice. The most specific consensus lineage is shown. Relates to Figure S5.
Figure 6
Figure 6. The addition of anti-flagellin antibody represses bacterial motility
(A) Motility plates containing 0.3% agar stab inoculated with E. coli with or without the addition of antibody (anti-flagellin IgG). Plates incubated at 37°C for 14 hours. Red circles highlight the extent of bacterial movement through the agar. (B) GFP FliC-reporter E. coli PHL628 treated with antibodies or glucose. Means ± s.e.m.’s. for ratios of normalized GFP fluorescence:OD are plotted, n=3/group. Relates to figure S6.
Figure 7
Figure 7. Flagellated bacteria breach the large intestinal mucous barrier and penetrate small intestinal villi of TLR5−/− mice
In all panels, host tissue is on the left, and mucus is on the right. For each pair of images in A, B, and C, the brightness of the images was increased in both panels simultaneously. (A) Flagellin imaged using immunohistochemistry with fluorescently labeled anti-flagellin antibody (GREEN) highlights flagellated rods in the s-layer of the mucus of TLR5−/− (arrows point to examples). Sections are counterstained with Hoechst 33342 (BLUE) to visualize host tissue. The outer (o) mucus layer in the WT also appears to contain less flagellin that in TLR5−/−. “s” indicates the inner, thicker s-layer of the mucus, “o” indicates the outer o-layer where bacteria are normally found. (B) Fluorescent in situ hybridization using the universal probe EUB338 (GREEN) shows clusters of bacteria penetrating the intestinal villi of the TLR5−/− small intestine. For all panels, the scale bar is 50 µm. Relates to Figure S7.
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