Multiomic features associated with mucosal healing and inflammation in paediatric Crohn's disease

Abstract

Background: The gastrointestinal microbiota has an important role in mucosal immune homoeostasis and may contribute to maintaining mucosal healing in Crohn's disease (CD).

Aim: To identify changes in the microbiota, metabolome and protease activity associated with mucosal healing in established paediatric CD METHODS: Twenty-five participants aged 3-18 years with CD, disease duration of over 6 months, and maintenance treatment with biological therapy were recruited. They were divided into a low calprotectin group (faecal calprotectin <100 μg/g, "mucosal healing," n = 11), and a high calprotectin group (faecal calprotectin >100 μg/g, "mucosal inflammation," n = 11). 16S gene-based metataxonomics, ¹ H-NMR spectroscopy-based metabolic profiling and protease activity assays were performed on stool samples.

Results: Relative abundance of Dialister species was six-times greater in the low calprotectin group (q = 0.00999). Alpha and beta diversity, total protease activity and inferred metagenomic profiles did not differ between groups. Pentanoate (valerate) and lysine were principal discriminators in a machine-learning model which differentiated high and low calprotectin samples using NMR spectra (R² 0.87, Q² 0.41). Mean relative concentration of pentanoate was 1.35-times greater in the low calprotectin group (95% CI 1.03-1.68, P = 0.036) and was positively correlated with Dialister. Mean relative concentration of lysine was 1.54-times greater in the high calprotectin group (95% CI 1.05-2.03, P = 0.028).

Conclusions: This multiomic study identified an increase in Dialister species and pentanoate, and a decrease in lysine, in patients with "mucosal healing." It supports further investigation of these as potential novel therapeutic targets in CD.

Figure 1. Comparison of microbial diversity metrics and community composition between high and low calprotectin groups.

A) Scatter plots with mean and 95% confidence interval for alpha diversity (Shannon index), species richness, and evenness (Pielou’s index). B) Non-metric multidimensional scaling (NMDS) plot of weighted UniFrac distance with 95% confidence ellipses. Red dots and ellipse = high calprotectin, green dots and ellipse = low calprotectin. P value calculated using PERMANOVA.

Figure 2. Mean relative abundance of Dialister in high and low calprotectin groups.

Bars indicate 95% confidence intervals. q value calculated using two-sided White’s non-parametric t test with Benjamini-Hochberg correction for multiple testing.

Figure 3. Scatter plots of protease activity (expressed as equivalent concentration of trypsin in μg/ml) in patients with high and low calprotectin.

Bars indicate mean and 95% confidence interval. Unpaired t test was performed for all samples, Bonferroni correction was applied for protease subtype activity. A: total protease activity (uninhibited). B-D: protease subtype activity, expressed as reduction in protease activity caused by addition of inhibitors (Δ protease activity); anti-pain dihydrochloride (B); leupeptin (C); anti-pain dihydrochloride, pepstatin, aprotinin (D).

Figure 4

(A) MCCV-PLS-DA score plot derived from 1D ¹H-NMR spectra of faecal water samples, indicating the differentiation between high calprotectin group (red) and low calprotectin group (green). The model is comprised of Kernel Density Estimate (KDE) of the predicted scores (Tpred) for both groups. Dots represent the metabolic profile of each patient from the study cohort when its corresponding NMR spectrum and calprotectin values were available (n=19). The fit and predictability of the model were obtained and expressed as R²Y (explained variance) and Q²Y (capability of prediction) values. (B) MCCV–PLS-DA loading plot. Top depicts the average 1D ¹H-NMR spectrum of the 19 faecal water samples. The bottom depicts the Manhattan plot showing -log₁₀(q) × sign of regression coefficient (β) of the MCCV–PLS-DA model that in conjunction represent the contribution of each variable on it. In green, metabolites are shown that are significantly higher in the low calprotectin group, and in red metabolites significantly higher in the high calprotectin group. Labels: 1.1-1.3 = lysine (1.73(m), 1.46(m), 3.04 (t)), 2 = pentanoate/valerate (1.30(m)). (C) Fragments from the average 1D ¹H-NMR spectra from the high calprotectin (red, n = 8) and low calprotectin (green, n = 11) groups, showing the less overlapped resonances detected as significant in the MCCV-PLS-DA model corresponding to lysine (1.2, 1.73(m), 1.3, 1.46(m)) and pentanoate/valerate (2, 1.30(m)). MCCV = Monte Carlo Cross-Validation. PLS-DA = Partial Least Squares Discriminant Analysis.