Inflammatory bowel diseases (IBDs), including Crohn's disease and ulcerative colitis, are chronic inflammatory diseases affecting millions worldwide. Genetic susceptibility, immune dysregulation, and environmental factors contribute to IBD pathogenesis. Diet plays a crucial role, with Western diets, high in food additives, linked to increased IBD incidence. Food additives like emulsifiers, stabilizers, and synthetic colorants are widely used in processed foods. Studies show some additives alter the gut microbiome, increase intestinal permeability, and promote colitis. Synthetic colorants, widely used in the last 50 years, are metabolized to potentially carcinogenic aromatic amines. Allura Red AC (AR), a common azo dye, has pro-inflammatory properties, inducing oxidative stress and COX-2 expression. However, its effects on intestinal inflammation remain unclear. Serotonin (5-HT) regulates GI functions and is elevated in IBD patients. Previous research linked increased gut 5-HT to more severe colitis. This study aimed to investigate the interplay between AR, 5-HT, and gut microbiota in colitis susceptibility using mouse models, intestinal organoids, and human cell cultures. The researchers hypothesized that AR exposure would alter intestinal 5-HT signaling and the gut microbiota, influencing colitis susceptibility.

A growing body of evidence points to the role of diet in IBD development. The Western diet, characterized by high intakes of processed foods, additives, fats, and red meats and low fiber intake, is associated with chronic intestinal inflammation. Several studies have demonstrated that various food additives, including maltodextrin, titanium dioxide, polysorbate-80, and carboxymethylcellulose, as well as artificial sweeteners, can alter the gut microbiome, increase intestinal permeability, reduce mucus barrier thickness, and promote colitis. The use of synthetic colorants has risen sharply, and some azo dyes produce free aromatic amines which are potentially carcinogenic and mutagenic. Allura Red AC (AR), also known as FD&C Red 40 or E129, is a widely used synthetic colorant found in many children's products. Previous studies have shown that AR has pro-inflammatory effects, increasing oxidative stress and COX-2 expression in rats. However, its impact on intestinal inflammation remains poorly understood. Serotonin (5-HT) is a key neurotransmitter and hormone regulating GI function, and its levels are elevated in IBD patients. Previous work established that mice with lower gut 5-HT exhibit reduced colitis severity and that higher mucosal 5-HT enhances colitis susceptibility by modulating gut microbiota. Some azo dyes increase 5-HT levels in the brain and modulate 5-HT synthesis in vitro, but the relationship between AR, 5-HT, and gut microbiota in colitis remains unknown.

The study utilized several mouse models of colitis to investigate the effects of Allura Red AC (AR) exposure. First, C57BL/6 mice were fed either a normal diet or a diet containing 100 ppm AR for 12 weeks, followed by 3.5% dextran sulfate sodium (DSS) for 7 days to induce acute colitis. Colitis severity was assessed by measuring disease activity index (DAI), macroscopic scores, colon length, colon weight, fecal lipocalin-2 (LCN2) levels, histological scores, colonic myeloperoxidase (MPO) levels, and colonic cytokine levels (IL-1β, IL-6, TNF-α). The expression levels of genes related to intestinal epithelial barrier function (ZO-1, occludin, Muc2) were measured. The study also examined the effect of chronic AR exposure in T-cell transfer colitis model using Rag1-/- mice reconstituted with CD4+CD45RBhigh T cells. To investigate the effect of intermittent exposure, a separate group of mice was exposed to AR one day per week for 12 weeks, and colitis was induced by DSS, also to examine whether early life exposure affects colitis susceptibility. A group of 4-week-old mice received AR for 4 weeks before inducing acute colitis. To determine whether AR induces low-grade colitis alone, AR was administered for 14 weeks to separate mice. The researchers also examined the effect of AR on human enteroendocrine (BON) cells and HT-29 cells in vitro. 5-HT secretion and TPH1 mRNA levels were measured after 24h of treatment with AR. To investigate the role of 5-HT in AR-induced colitis, Tph1-/- mice were used, as these mice have reduced gut 5-HT. In addition, the study examined the role of the gut microbiota by using germ-free mice that received cecal contents from AR-exposed or control mice. Furthermore, they examined AR's effects in germ-free mice and performed 16S rRNA sequencing to analyze the microbiota composition of cecal contents. The researchers measured colonic 5-HT levels and pro-inflammatory cytokines. They also investigated the role of Myosin light chain kinase (MLCK) activation in AR-induced intestinal barrier dysfunction using HT-29 cells and murine colon-derived crypt organoids. Western blot and qRT-PCR were used to analyze protein and mRNA expression levels. Immunofluorescence staining was used to count 5-HT cells. Statistical analysis included two-tailed unpaired Student's t-test, one-way ANOVA, two-way ANOVA, and two-tailed Mann-Whitney U test.

Chronic, but not intermittent, exposure to Allura Red AC (AR) at a dietary relevant dose exacerbated DSS-induced colitis in C57BL/6 mice. AR exposure increased disease activity index (DAI), macroscopic scores, colonic weight, fecal LCN2, histological scores, colonic MPO, and pro-inflammatory cytokine levels (IL-1β, IL-6, TNF-α). AR also reduced colonic Muc2 mRNA levels, suggesting mucus layer disruption. Colonic 5-HT levels were elevated in AR-exposed mice, both with and without DSS. AR exposure worsened T cell-induced colitis in Rag1-/- mice. Early life exposure to AR also primed mice for increased susceptibility to DSS-induced colitis. AR alone induced low-grade colonic inflammation in naïve C57BL/6 mice, characterized by increased fecal LCN2, macroscopic and histological scores, colonic MPO levels, 5-HT cell number, colonic 5-HT, and pro-inflammatory cytokine levels. AR also downregulated genes involved in antimicrobial responses (Pparg, Defb3, Il22, Reg3g). AR impaired intestinal epithelial barrier function by activating the MLCK pathway, leading to increased pMLCSer19. In vitro studies showed that AR activated AhR and increased CYP1A1 and CYP1B1 mRNA levels in HT-29 cells. In organoid models, AR reduced the number of organoids with disrupted morphology after TNF-α pretreatment. The effect of AR on colitis was dependent on TPH1, as Tph1-/- mice did not show increased colitis susceptibility after AR exposure. SERT-deficient mice exposed to AR showed increased colitis severity. AR altered the gut microbiota composition, and transplantation of the microbiota from AR-exposed mice to GF mice increased DSS-induced colitis severity. AR alone increased colonic 5-HT levels in GF mice. AR directly promoted 5-HT secretion from EC cells by increasing ROS and activating NF-κB.

This study demonstrates that chronic exposure to Allura Red AC (AR), a common synthetic food colorant, enhances colitis susceptibility in mice, mediated by colonic serotonin (5-HT) through both microbiota-dependent and -independent pathways. The findings highlight the importance of long-term exposure, as intermittent exposure did not have the same effect. The impairment of the intestinal epithelial barrier function through MLCK activation and mucus layer disruption played a significant role in AR-induced colitis. The dependence on TPH1-derived 5-HT underscores the critical role of the host's serotonergic system in AR's effects. The alteration of gut microbiota composition by AR further contributes to increased colitis susceptibility. Early life exposure to AR predisposes mice to more severe colitis later in life. AR's direct stimulation of 5-HT secretion via NF-κB activation and ROS generation suggests a positive feedback loop exacerbating inflammation. These findings raise concerns about the potential long-term health effects of chronic exposure to common food additives, warranting further investigation into the impact of other food colorants and the role of specific gut microbial communities in AR-induced colitis.

This research reveals that chronic exposure to the common food colorant Allura Red AC (AR) promotes experimental colitis in mice through a mechanism involving increased colonic serotonin (5-HT) and disruption of the intestinal epithelial barrier. The effects are observed through both gut microbiota-dependent and independent pathways. The study emphasizes the significance of long-term exposure and the critical role of 5-HT in mediating AR's impact on intestinal inflammation. Future studies should focus on investigating the effects of other common food dyes and exploring the specific roles of gut microbial communities in AR-induced colitis. Further research is crucial to determine if similar effects occur in humans, considering the widespread use of AR in food products consumed by children.

The study primarily used mouse models, which may not fully recapitulate the complexities of human physiology and responses to AR. The dose of AR used may not perfectly reflect the range of human exposure to this colorant. The study focused on a single colorant, Allura Red AC. Further studies are needed to explore the combined effects of other food colorants and potential synergistic interactions. Population-based human studies are necessary to confirm the association between AR consumption and IBD development.