Project description:Aim: Microbial colonization of the intestine in newborns is a critical event that regulates host physiology with lifelong consequences. However, there is a need to identify microbial molecules and mechanisms that play a role in this process. We aimed to elucidate the effects of flagellin, an important immunomodulatory component involved in bacterial motility, on intestinal epithelial functions. Methods: Germfree mice were colonized with synthetic bacterial communities, including a flagellated mouse gut isolate of Escherichia coli or a ΔfliC mutant thereof that lacks flagella. The offspring of these mice were studied at 7 days of age by single-cell RNA sequencing of epithelial cells isolated from the small intestine. Results: Enterococcus faecalis was the most dominant species in the small intestine of newborn mice. Blautia pseudococcoides was detected at low relative abundances. Colonization by either E. coli strain reduced the relative abundance of E. faecalis to 10% on average, and E. coli became the dominant member, with no substantial differences between mice colonized with the wildtype or mutant strain. The distribution of cell types within the small intestinal epithelium was skewed toward specialized cells (goblet, enteroendocrine, stem cells) at the expense of enterocytes in mice colonized with flagellated bacteria. These mice were characterized by higher expression of genes involved in cell-cell interactions (Npl, Epcam) and responses to infection (Ctsl) in enterocytes and enhanced translation (ribosomal proteins) in stem cells. In contrast, genes involved in lipid homeostasis (transport and metabolism; Apoc 2/4, Dgat2, Fabp 1/2, Mttp) in all cell types and inflammatory and androgen-related pathways in specialized cell types were less expressed in epithelial cells exposed to wildtype E. coli. Conclusion: These findings suggest that early life exposure to flagellated commensal bacteria influences cellular processes underlying tissue metabolism and remodeling in the small intestine.

Project description:M cells are the main site of bacterial translocation in the intestine. We used the in vitro M cell model to study the effect of the commensal bacteria; Lactobacillus salivarius, Eschericha coli and Bacteroides fragilis, on M cell gene expression. Bacterial translocation across the gut mucosa has traditionally been based on the detection of commensals in the mesenteric lymph node. Differential rates of commensal translocation have been reported in vivo, however fewer studies have examined translocation of commensals at the level of the gut epithelial M cell. In this study we employed an in vitro M cell model to quantify translocation of various bacteria. C2BBe1 cells were differentiated into M cells and the gene expression profile and transport kinetics of different bacterial strains, namely Lactobacillus salivarius, Escherichia coli, and Bacteroides fragilis, was assessed. For comparison with M cell uptake, the THP-1 monocytic cell line was used to analyze bacterial internalization and resulting cytokine production. The commensal bacterial strains were translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency while L. salivarius translocated with less efficiency. In contrast, L. salivarius was internalized by THP-1 cells to a higher degree than B. fragilis or E. coli and was associated with a different cytokine profile. Microarray analysis showed both common and differential gene expression amongst the bacteria and control polystyrene beads. In the presence of bacteria, but not beads, upregulated genes were mainly involved in transcription regulation and dephosphorylation, e.g. EGR1, JUN; whereas proinflammatory and stress response genes were primarily upregulated by E. coli and B. fragilis, but not L. salivarius nor beads, e.g. IL8, TNFAIP3. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses. C2bbe1 cells were converted to M cells (C2M) following 21 days of culture on Transwells in the presence of Raji B cells. C2M cells were co-cultured alone, Lactobacillus salivarius, Eschericha coli, Bacteroides fragilis and control beads. Total RNA was extracted and processed for Affymetrix array hybridisation

Project description:M cells are the main site of bacterial translocation in the intestine. We used the in vitro M cell model to study the effect of the commensal bacteria; Lactobacillus salivarius, Eschericha coli and Bacteroides fragilis, on M cell gene expression. Bacterial translocation across the gut mucosa has traditionally been based on the detection of commensals in the mesenteric lymph node. Differential rates of commensal translocation have been reported in vivo, however fewer studies have examined translocation of commensals at the level of the gut epithelial M cell. In this study we employed an in vitro M cell model to quantify translocation of various bacteria. C2BBe1 cells were differentiated into M cells and the gene expression profile and transport kinetics of different bacterial strains, namely Lactobacillus salivarius, Escherichia coli, and Bacteroides fragilis, was assessed. For comparison with M cell uptake, the THP-1 monocytic cell line was used to analyze bacterial internalization and resulting cytokine production. The commensal bacterial strains were translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency while L. salivarius translocated with less efficiency. In contrast, L. salivarius was internalized by THP-1 cells to a higher degree than B. fragilis or E. coli and was associated with a different cytokine profile. Microarray analysis showed both common and differential gene expression amongst the bacteria and control polystyrene beads. In the presence of bacteria, but not beads, upregulated genes were mainly involved in transcription regulation and dephosphorylation, e.g. EGR1, JUN; whereas proinflammatory and stress response genes were primarily upregulated by E. coli and B. fragilis, but not L. salivarius nor beads, e.g. IL8, TNFAIP3. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses.

			Action	DRS
	ERR3308920_1.fastq.gz	Fastqsanger.gz
	ERR3308920_2.fastq.gz	Fastqsanger.gz
	ERR3308940_1.fastq.gz	Fastqsanger.gz
	ERR3308940_2.fastq.gz	Fastqsanger.gz
	ERR3309010_1.fastq.gz	Fastqsanger.gz

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Gut commensal viruses and bacteria

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