(A–E) Elevated small intestinal Enterobacteriaceae levels after ethanol and burn correlate with pro-inflammatory potential. (IMAGE)
Caption
Small intestinal faecal contents were collected from SV (n=7) or EB (n=10) mice 1 day after combined injury. Faecal bacterial DNA was isolated from a portion of each sample while remaining faecal contents were homogenised in PBS to generate faecal slurry. (A) Bacteria were quantified via bacterial qPCR with the level of Enterobacteriaceae in each sample being expressed as a ratio of Enterobacteriaceae/total bacteria and depicted relative to an experiment’s average SV sample. (B) MODE-K cells were treated with small intestinal faecal slurry diluted in culture media for 24 hours and then IL-6 and gene expression were quantified via RT-qPCR with beta actin used as housekeeping. (C) Conditioned media was collected for protein quantification of IL-6 via ELISA and normalised to conditioned media with no treatment. Graphs depict the average result from the faecal slurry of individual mice as a single point in addition to group means±SEM. Statistical analysis of SV versus EB groups via Welch’s t-test depicted as *p<0.05, **p<0.01. (D, E) Correlation of small intestinal faecal samples (n=17) from individual mice comparing Enterobacteriaceae levels to (D) IL-6 gene expression or relative protein concentration following 24 hours of faecal slurry treatment assessed via Spearman correlation analysis. R values displayed with significance of correlation depicted as *p<0.05. (F–I) Ethanol and burn injury reduce faecal SCFA. SCFAs were quantified from caecal contents collected 1 day after ethanol and burn injury via high-performance liquid chromatography mass spectroscopy. Graphs depict concentrations of (A) total SCFAs, (B) acetate, (C) propionate and (D) butyrate in nmol/mg faeces with male and female samples combined to compare SV to EB overall. Individual mice represented by each dot in addition to group mean±SEM, n=10–13. Significance was analysed via Welch’s t-test, n=10–13 animals per group, **p<0.01. EB, ethanol burn; IL-6, interleukin-6; MODE-K, murine duodenal cell clone-K; qPCR, quantitative polymerase chain reaction; RT-qPCR, reverse transcription qPCR; SCFA, short-chain fatty acid; SV, sham vehicle.
Credit
By Caroline J Herrnreiter, Mary Grace Murray, Marisa Luck, et al.
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