Weighted Gene Co-Expression Network Analysis Identifies a Functional Guild and Metabolite Cluster Mediating the Relationship between Mucosal Inflammation and Adherence to the Mediterranean Diet in Ulcerative Colitis

Abstract

Diet influences the pathogenesis and clinical course of inflammatory bowel disease (IBD). The Mediterranean diet (MD) is linked to reductions in inflammatory biomarkers and alterations in microbial taxa and metabolites associated with health. We aimed to identify features of the gut microbiome that mediate the relationship between the MD and fecal calprotectin (FCP) in ulcerative colitis (UC). Weighted gene co-expression network analysis (WGCNA) was used to identify modules of co-abundant microbial taxa and metabolites correlated with the MD and FCP. The features considered were gut microbial taxa, serum metabolites, dietary components, short-chain fatty acid and bile acid profiles in participants that experienced an increase (n = 13) or decrease in FCP (n = 16) over eight weeks. WGCNA revealed ten modules containing sixteen key features that acted as key mediators between the MD and FCP. Three taxa (Faecalibacterium prausnitzii, Dorea longicatena, Roseburia inulinivorans) and a cluster of four metabolites (benzyl alcohol, 3-hydroxyphenylacetate, 3-4-hydroxyphenylacetate and phenylacetate) demonstrated a strong mediating effect (ACME: -1.23, p = 0.004). This study identified a novel association between diet, inflammation and the gut microbiome, providing new insights into the underlying mechanisms of how a MD may influence IBD. See clinicaltrials.gov (NCT04474561).

Keywords: diet; inflammatory bowel disease; metabolomics; microbiome; ulcerative colitis; weighted gene co-expression analysis (WGCNA).

Figure 1

The identification of two distinct trajectories for fecal calprotectin. Participants whose fecal calprotectin increased at week 8 had significantly lower levels at baseline and significantly higher levels at week 8.

Figure 2

Associations between fecal calprotectin (FCP) and the microbial taxa. (A) PCoA plot of baseline microbiome profiles of participants that had an increase in FCP (FCP+; orange) vs. a decrease in FCP (FCP−; blue) from baseline to week 8. PERMANOVA values of the association of baseline microbiome with baseline FCP, week 8 FCP and change in FCP are indicated below the figure. (B) PCoA plot of week 8 microbiome profiles of participants that had an increase in FCP (FCP+; orange) vs. decrease in FCP (FCP−; blue) from baseline to week 8. PERMANOVA values of the association of week 8 microbiome with baseline, week 8 FCP and change in FCP are indicated below the figure. (C) Identification of the specific species of microbes associated with fecal calprotectin at week 8. Asterisks indicate the significance level: ** p < 0.01, * p < 0.05. Abbreviations: FCP, fecal calprotectin; PCoA, principle coordinate analysis; PERMANOVA, permutational multivariate analysis of variance.

Figure 3

Associating module eigengenes with fecal calprotectin and Mediterranean diet score. (A) The Spearman’s rho inter-correlation between cluster eigengenes. Significance level: * p < 0.05, @ p < 0.10, N = not significant. (B) The negative association with fecal calprotectin (green module) encompasses specific nutrients, foods and Bacteroides species. Freq refers to the frequency of food groups (shown in pink); Tot refers to the macronutrient and micronutrients calculated per day (shown in purple); Adj refers to macronutrients and micronutrients calculated per 1000 kilocalories (shown in green); Bacteroides species (shown in orange). Abbreviations: MDS, Mediterranean diet score; FCP, fecal calprotectin; Veg, vegetables; Mg, magnesium; DHA; docosahexaenoic acid, Vit, vitamin; Prot, protein; mcg, micrograms; g, grams; RAE, retinol activity equivalents; BetaCartene, beta-carotene.

Figure 4

Discrete modules for low- and high-risk Co-Abundant Groups (CAGs). FDR, false discovery rates.

Figure 5

Modules enriched for High-Risk CAG taxa. (A) The black module shows taxa (shown in orange) associated with the three clusters predicted to have pathobiont traits. Abbreviations: sp, species. (B) The brown module shows species with pathobiont characteristics that are known to be increased in disease. Abbreviations: CA, cholic acid; AC, acid. (C) The blue module shows microbial taxa and metabolites (SCFA shown in blue, all other metabolites shown in green) often elevated in disease. Abbreviations: Iso_val, isovalerate; iso_But, isobutyrate; pro, propionate; 5-OXO-L proline, 5-oxoproline (pyroglutamic acid).

Figure 6

Low-risk CAG module (red module) with health-associated microbes.

Figure 7

Inter-module association analysis. (A) Stepwise association pattern linking high adherence to the Mediterranean diet and fecal calprotectin mediated by microbiome-dominated modules. (B) Analysis of the sub-network amongst the select features from the grey module indicates specific microbiome and metabolite features that act as mediators in the relationship between fecal calprotectin and the Mediterranean diet score. Abbreviations: MDS, Mediterranean diet score, FCP, fecal calprotectin. Created with BioRender.com (accessed on 5 January 2023).

Figure 8

Weighted gene co-expression network analysis (WGCNA). (A) A specific health-associated microbiome guild of four taxa significantly influences the association between Mediterranean diet score and fecal calprotectin in a 3-step manner. (B) The metabolite cluster mediates the negative association between the Mediterranean diet score and fecal calprotectin. (C) A combination of features (three taxa and four metabolites) demonstrates an even stronger mediating effect between the Mediterranean diet score and fecal calprotectin. Green arrows represent a positive association and red arrows represent a negative association. Abbreviations: MDS, Mediterranean diet score; FCP, fecal calprotectin; ACME, average causal mediation effect; prop. mediated; proportion mediated. Created with BioRender.com (accessed on 5 January 2023).