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Whole tissue homogenization preferable to mucosal scraping in determining the temporal profile of segmented filamentous bacteria in the ileum of weanling rats.

ABSTRACT: Segmented filamentous bacteria (SFB) are thought to play a role in small intestine immunological maturation. Studies in weanling mice have shown a positive correlation between ileal SFB abundance and plasma and faecal interleukin 17 (IL-17) and immunoglobulin A (IgA) concentrations. Although the first observation of SFB presence was reported in rats, most studies use mice. The size of the mouse ileum is a limitation whereas the rat could be a suitable alternative for sufficient samples. Changes in SFB abundance over time in rats were hypothesized to follow the pattern reported in mice and infants. We characterized the profile of SFB colonization in the ileum tissue and contents and its correlation with two immune markers of gastrointestinal tract (GIT) maturation. We also compared two published ileum collection techniques to determine which yields data on SFB abundance with least variability. Whole ileal tissue and ileal mucosal scrapings were collected from 20- to 32-day-old Sprague-Dawley rats. SFB abundance was quantified from proximal, middle and distal ileal tissues, contents and faeces by quantitative PCR using SFB-specific primers. Antibody-specific ELISAs were used to determine IL-17 and IgA concentrations. Significant differences in SFB abundance were observed from whole and scraped tissues peaking at day 22. Variability in whole ileum data was less, favouring it as a better collection technique. A similar pattern of SFB abundance was observed in ileum contents and faeces peaking at day 24, suggesting faeces can be a proxy for ileal SFB abundance. SFB abundance at day 26 was higher in females than males across all samples. There were significant differences in IgA concentration between days 20, 30 and 32 and none in IL-17 concentration, which was different from reports in mice and infants.

SUBMITTER: Oemcke LA

PROVIDER: S-EPMC8209713 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Segmented filamentous bacteria (SFB) are known modulators of the mammalian immune system. Currently, the technology for investigating SFB culture in vitro is immature, and as a result, the mechanisms of SFB colonization and immune regulation are not yet fully elucidated. In this study, we investigated the gene diversity and host specificity of SFB flagellin genes. The fliC1 and fliC2 genes are relatively conserved, while the fliC3 and fliC4 genes are more variable, especially at the central and C-terminal regions. Host specificity analysis demonstrated that the fliC1 genes do not cluster together based on the host organism, whereas the fliC3 and fliC4 genes were host specific at the nucleotide and deduced amino acid levels. SFB flagellin protein expression in the ileum mucosa and cecal contents was detected by using fluorescence in situ hybridization (FISH) combined with immunohistochemical (IHC) analysis, immunoblotting, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the purified SFB FliC3 protein originating from both mouse and rat was able to activate Toll-like receptor 5 (TLR5)-linked NF-κB signaling, no host specificity was observed. Interestingly, the patterns of interaction with mouse ileum mucosal proteins were different for mouse FliC3 (mFliC3) and rat FliC3 (rFliC3). Gene Ontology (GO) and KEGG analyses indicated that more adherence-related proteins interacted with mFliC3, while more lysosome- and proteolysis-related proteins interacted with rFliC3. In vitro degradation experiments indicated that the stability of rFliC3 was lower than that of mFliC3 when they were incubated with mouse ileum mucosal proteins. In summary, the gene diversity and host specificity of SFB flagellin genes were investigated, and SFB flagellin expression was detected in gut samples.IMPORTANCE Since SFB genomes contain only one copy of each FliC gene, the diversity of FliC is representative of SFB strain diversity. Currently, little is known regarding the diversity and specificity of members of the group of SFB. The work presented herein demonstrates that select SFB strains, exhibiting unique FliC patterns, are present in a variety of mammalian hosts. SFB fliC genes were found to interact with a number of unique targets, providing further evidence for SFB host selection. Together, this work represents a major advancement in identifying SFB and delineating how members of the group of SFB interact with the host. Future examination of FliC genes will likely enhance our knowledge of intestinal colonization by the gut microbiota.

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Whole tissue homogenization preferable to mucosal scraping in determining the temporal profile of segmented filamentous bacteria in the ileum of weanling rats.

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