Evidence for a Modulatory Effect of a 12-Week Pomegranate Juice Intervention on the Transcriptional Response in Inflammatory Bowel Disease Patients Reducing Fecal Calprotectin Levels: Findings From a Proof-of-Principle Study

Abstract

This study aimed at investigating the effects of pomegranate juice (POMJ) consumption on inflammatory biomarkers and gene expression in patients with inflammatory bowel disease (IBD) in clinical remission. In this randomized, placebo-controlled trial, 16 subjects with IBD in remission consumed POMJ or placebo for 12 weeks. POMJ consumption significantly reduced fecal calprotectin (FC) and plasma endotoxin levels. Transcriptomic analysis of peripheral blood mononuclear cells revealed upregulation of genes involved in mucosal immunity, including aryl hydrocarbon receptor (AHR), neutrophil cytosolic factor 4 (NCF4), and nuclear factor, interleukin 3 regulated (NFIL3). Urolithin metabotypes were predominantly of the B type, associated with intestinal dysbiosis. No significant changes were observed in serum inflammatory markers or colonic mucosal cytokine expression. POMJ consumption reduced markers of intestinal inflammation and modulated gene expression related to mucosal immunity and barrier function in patients with IBD. These findings suggest the potential of POMJ as a beneficial dietary intervention for maintaining remission in IBD, highlighting the promise of targeted nutritional strategies in managing chronic inflammatory conditions. Further research is needed to elucidate the long-term clinical implications of these molecular changes. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03000101.

Keywords: ellagitannins; fecal calprotectin; inflammatory bowel disease; pomegranate juice; urolithins.

FIGURE 1

Scheme of the study design. Adapted from Scaioli et al. (2019) [22] licensed under the Creative Commons Attribution 4.0 International License. CD: Crohn's disease; IBD: inflammatory bowel disease; UC: ulcerative colitis.

FIGURE 2

Fecal calprotectin (FC) levels in patients with IBD in clinical remission at baseline (t ₀) and at the end of the intervention (t ₁). (A) FC levels in patients who received (left) pomegranate juice or (right) placebo. The box represents the lower and upper quartiles, and the horizontal line in the middle of the box denotes the median. The reference limit for a raised value is 100 µg/g. p values were reported from comparisons by ratio‐paired t‐test. (B) Probability density distributions of FC levels at (left) baseline (t ₀) and (right) the end of intervention (t ₁) with pomegranate juice or placebo. The violin plots present FC distributions in different patient groups, ulcerative colitis (UC) or Crohn's disease (CD), obtained by a kernel density estimator (KDE), with the median (the dashed bar in the center of the bar) and the interquartile range (the dotted bars representing the 25^th and 75^th percentiles).

FIGURE 3

Circulating levels of CRP, cytokines (IL‐8 and TNF‐α), and endotoxins at baseline (t ₀) and 12 weeks after intervention (t ₁) with pomegranate juice or placebo in patients with IBD in clinical remission. Cytokine concentrations were log₁₀‐transformed. p values were reported from comparisons by ratio‐paired t‐test. CRP: plasma C‐reactive protein; EU: endotoxin units; IL: interleukin; TNF‐α: tumor necrosis factor.

FIGURE 4

Gene expression analysis of peripheral blood mononuclear cells (PBMCs) of patients with IBD in clinical remission receiving either pomegranate juice (POMJ) or placebo for 12 weeks. (A) Principal component analysis of gene expression profiles of PBMCs from patients with UC (ulcerative colitis) or CD (Crohn's disease) treated with POMJ or placebo, at baseline (t ₀) and at the end of the intervention (t ₁). 0.1% of variables were removed based on low variance. (B) Number of up‐ or downregulated genes in PBMCs after 12‐week intervention with POMJ or placebo (log₂ fold‐change ≥ 0.5 or ≤ −0.5, p value ≤ 0.01), compared to baseline. (C) Overlap between up or downregulated genes in POMJ and placebo groups. In the circus plot, each arc on the outside represents a gene list. On the inside, each gene member of that list is assigned a spot on the arc. The dark orange color represents the genes shared by multiple lists and the light orange color represents genes unique to that gene list. Purple lines link genes that are shared by multiple gene lists. DEGs: differentially expressed genes; PC: principal component.

FIGURE 5

Functional analysis of differentially expressed genes (DEGs) in peripheral blood mononuclear cells of patients with IBD in clinical remission after a 12‐week intervention involving consumption of pomegranate juice (POMJ) or placebo, compared to baseline. (A) Significantly altered canonical pathways (−Log p value ≥ 2) in the POMJ group. A positive z‐score denotes pathway activation, and a negative z‐score denotes pathway inhibition. Grey bars represent a pathway with no activity pattern available. (B) Same as in (A), but for the placebo group. (C) Predicted effects (activation‐inhibition) of DEGs on downstream biological functions belonging to functional categories “inflammation,” “inflammatory response,” “inflammatory disorder,” and “chronic inflammatory disorder,” in the POMJ and placebo group. Terms with p value ≤ 0.01 in at least one of the two study groups are reported. Grey circles indicate that no activity pattern is available for the prediction of function activation state. (D) Mechanistic networks reporting molecules up‐ or downregulated in the POMJ group and their interactions with the downstream functions “inflammatory response” and “inflammation of gastrointestinal tract.”.