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. 2025 Feb 12;16(1):1589.
doi: 10.1038/s41467-025-56698-w.

An optimised faecal microRNA sequencing pipeline reveals fibrosis in Trichuris muris infection

Emma Layton  1   2   3 , Sian Goldsworthy  4 , EnJun Yang  3 , Wei Yee Ong  3   5 , Tara E Sutherland  6 , Allison J Bancroft  1   2   7 , Seona Thompson  1   2   7 , Veonice Bijin Au  3 , Sam Griffiths-Jones  4 , Richard K Grencis  8   9   10 , Anna-Marie Fairhurst  11   12 , Ian S Roberts  13   14
Affiliations

An optimised faecal microRNA sequencing pipeline reveals fibrosis in Trichuris muris infection

Emma Layton et al. Nat Commun. .

Abstract

The intestine is a site of diverse functions including digestion, nutrient absorption, immune surveillance, and microbial symbiosis. Intestinal microRNAs (miRNAs) are detectable in faeces and regulate barrier integrity, host-microbe interactions and the immune response, potentially offering valuable non-invasive tools to study intestinal health. However, current experimental methods are suboptimal and heterogeneity in study design limits the utility of faecal miRNA data. Here, we develop an optimised protocol for faecal miRNA detection and report a reproducible murine faecal miRNA profile in healthy mice. We use this pipeline to study faecal miRNAs during infection with the gastrointestinal helminth, Trichuris muris, revealing roles for miRNAs in fibrosis and wound healing. Intestinal fibrosis was confirmed in vivo using Hyperion® imaging mass cytometry, demonstrating the efficacy of this approach. Further applications of this optimised pipeline to study host-microbe interactions and intestinal disease will enable the generation of hypotheses and therapeutic strategies in diverse contexts.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Faecal miRNA profiles of healthy wild-type mice are reproducible across studies.
The top 20 miRNAs measured by mean normalised counts for each study are represented as 20 chords, and each study represented in a different colour. On the right-hand side, miRNAs are sorted clockwise by their median ranking across the three studies. Liu et al. represents the non-vaccinated group of mice (n = 5, green) and He et al. represents the samples taken at day 0 prior to dextran sulphate sodium treatment (DSS0) (n = 5, pink). Current represents the samples taken from the infected group prior to infection at the baseline time point on day 0 (I0) in the current study (n = 7, blue).
Fig. 2
Fig. 2. Dynamics of most abundant faecal miRNAs in healthy mice over time.
a Representation of sample collection workflow and nomenclature of faecal samples for small RNA sequencing from naïve mice and mice infected with a low dose of T. muris. b Heatmap comparing the ranking of the top 20 miRNAs measured by mean normalised counts in the healthy control group at the baseline (I0) to the other naïve groups control groups at day 21 and 35 (N21 and N35). c–f Expression of miRNAs on day 21 and day 35 (N21 and N35) shown as a percentage change from the value at day 0 (I0). miRNAs are clustered into sub-plots based on expression pattern over time. Mean and SEM are plotted. Statistical significance was calculated on the normalised counts for each group by two-way ANOVA with Geisser-greenhouse correction and Tukey’s multiple comparisons test. Significance values displayed are for each time point versus I0. c miRNAs that increase incrementally, d miRNAs that increase at day 21 before decreasing at day 35, e miRNAs that decrease incrementally and f miRNAs that decrease at day 21 before increasing again at day 35. I0 = 7, N21 = 5 and N35 = 5 individuals. * = adj. p < 0.05, ** = adj. p < 0.01. Exact P values can be found in the Source Data file or Supplementary Data 2 (for b).
Fig. 3
Fig. 3. miRNAs differentially expressed by T. muris infection regulate fibrosis.
a Ingenuity pathway analysis was used to identify the top 16 enriched pathways of the mRNA targets of the faecal miRNAs differentially expressed on day 35 of chronic T. muris infection. Targets represent the number of mRNA targets of the differentially expressed miRNAs in each pathway. The mRNA targets were pre-filtered to only include those with a confidence level of ‘High predicted’ or ‘Experimentally observed’ prior to pathway analysis. P value is a result of Fisher’s exact test (right-tailed). b–d 3T3 fibroblasts were cultured in TGF-β in the presence of 50 nM of negative control miRNA (miR-ctrl), miR-29a mimic or inhibitor, miR-200c mimic or inhibitor, or miR-26b mimic or inhibitor for 36 h. b RNA was harvested and the expression of Col1 quantified by qPCR relative to the expression in the untreated untransfected cells in each biological replicate. Expression levels were normalised to Gapdh using the ∆∆CT method. Statistical significance was calculated by one-way ANOVA with Geisser-greenhouse correction and Dunnet’s multiple comparisons test. The mean and SD are displayed. n = 3 or n = 4 biological replicates for each condition. c Transfected fibroblasts were fixed after 36 h, stained for collagen I (yellow) and a nuclear stain (blue) and imaged by confocal microscopy using a 63x oil immersion objective. Scale bars are 50 µm. d Luminex cytokine profiling of fibroblast cell culture supernatant after 36 h of TGF-β treatment and transfections. Statistical analysis is a result of a one-way ANOVA with Holm–Šídák’s multiple comparisons test. The mean and SD are displayed. n = 3 biological replicates for each condition.
Fig. 4
Fig. 4. Evidence of fibrosis in the caecum of T.muris-infected mice.
Ceca from a naïve and a chronically infected C57BL/6 mouse on day 42 post-gavage of water or low-dose T. muris eggs were randomly selected for assessment with Hyperion® imaging mass cytometry. a Ceca stained for Collagen VI (blue), Collagen I (green), Hyaluronan (yellow) and Heparan sulphate (magenta). b For Collagen I (magenta). c For α-SMA (cyan). d For Collagen I (magenta) and α-SMA (cyan).
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