Increase in Epithelial Permeability and Cell Metabolism by High Mobility Group Box 1, Inflammatory Cytokines and TPEN in Caco-2 Cells as a Novel Model of Inflammatory Bowel Disease

High mobility group box 1 protein (HMGB1) is involved in the pathogenesis of inflammatory bowel disease (IBD). Patients with IBD develop zinc deficiency. However, the detailed roles of HMGB1 and zinc deficiency in the intestinal epithelial barrier and cellular metabolism of IBD remain unknown. In the present study, Caco-2 cells in 2D culture and 2.5D Matrigel culture were pretreated with transforming growth factor-β (TGF-β) type 1 receptor kinase inhibitor EW-7197, epidermal growth factor receptor (EGFR) kinase inhibitor AG-1478 and a TNFα antibody before treatment with HMGB1 and inflammatory cytokines (TNFα and IFNγ). EW-7197, AG-1478 and the TNFα antibody prevented hyperpermeability induced by HMGB1 and inflammatory cytokines in 2.5D culture. HMGB1 affected cilia formation in 2.5D culture. EW-7197, AG-1478 and the TNFα antibody prevented the increase in cell metabolism induced by HMGB1 and inflammatory cytokines in 2D culture. Furthermore, ZnSO₄ prevented the hyperpermeability induced by zinc chelator TPEN in 2.5D culture. ZnSO₄ and TPEN induced cellular metabolism in 2D culture. The disruption of the epithelial barrier induced by HMGB1 and inflammatory cytokines contributed to TGF-β/EGF signaling in Caco-2 cells. The TNFα antibody and ZnSO₄ as well as EW-7197 and AG-1478 may have potential for use in therapy for IBD.     

Bifidobacterium longum subsp. infantis CECT 7210 Reduces Inflammatory Cytokine Secretion in Caco-2 Cells Cultured in the Presence of Escherichia coli CECT 515

Previous works have described the activity of Bifidobacterium longum subsp. infantis CECT 7210 (also commercially named B. infantis IM-1^®) against rotavirus in mice and intestinal pathogens in piglets, as well as its diarrhea-reducing effect on healthy term infants. In the present work, we focused on the intestinal immunomodulatory effects of B. infantis IM-1^® and for this purpose we used the epithelial cell line isolated from colorectal adenocarcinoma Caco-2 and a co-culture system of human dendritic cells (DCs) from peripheral blood together with Caco-2 cells. Single Caco-2 cultures and Caco-2: DC co-cultures were incubated with B. infantis IM-1^® or its supernatant either in the presence or absence of Escherichia coli CECT 515. The B. infantis IM-1^® supernatant exerted a protective effect against the cytotoxicity caused by Escherichia coli CECT 515 on single cultures of Caco-2 cells as viability reached the values of untreated cells. B. infantis IM-1^® and its supernatant also decreased the secretion of pro-inflammatory cytokines by Caco-2 cells and the co-cultures incubated in the presence of E. coli CECT 515, with the response being more modest in the latter, which suggests that DCs modulate the activity of Caco-2 cells. Overall, the results obtained point to the immunomodulatory activity of this probiotic strain, which might underlie its previously reported beneficial effects.     

Superoxide Dismutase 3-Transduced Mesenchymal Stem Cells Preserve Epithelial Tight Junction Barrier in Murine Colitis and Attenuate Inflammatory Damage in Epithelial Organoids

Superoxide dismutase 3 (SOD3), also known as extracellular superoxide dismutase, is an enzyme that scavenges reactive oxygen species (ROS). It has been reported that SOD3 exerts anti-inflammatory abilities in several immune disorders. However, the effect of SOD3 and the underlying mechanism in inflammatory bowel disease (IBD) have not been uncovered. Therefore, in the present study, we investigated whether SOD3 can protect intestinal cells or organoids from inflammation-mediated epithelial damage. Cells or mice were treated with SOD3 protein or SOD3-transduced mesenchymal stem cells (MSCs). Caco-2 cells or intestinal organoids stimulated with pro-inflammatory cytokines were used to evaluate the protective effect of SOD3 on epithelial junctional integrity. Dextran sulfate sodium (DSS)-induced colitis mice received SOD3 or SOD3-transduced MSCs (SOD3-MSCs), and were assessed for severity of disease and junctional protein expression. The activation of the mitogen-activated protein kinase (MAPK) pathway and elevated expression of cytokine-encoding genes decreased in TNF-α-treated Caco-2 cells or DSS-induced colitis mice when treated with SOD3 or SOD3-MSCs. Moreover, the SOD3 supply preserved the expression of tight junction (ZO-1, occludin) or adherence junction (E-cadherin) proteins when inflammation was induced. SOD3 also exerted a protective effect against cytokine- or ROS-mediated damage to intestinal organoids. These results indicate that SOD3 can effectively alleviate enteritis symptoms by maintaining the integrity of epithelial junctions and regulating inflammatory- and oxidative stress.     

Bifidobacterium bifidum Enhances the Intestinal Epithelial Tight Junction Barrier and Protects against Intestinal Inflammation by Targeting the Toll-like Receptor-2 Pathway in an NF-κB-Independent Manner

Defective intestinal tight junction (TJ) barrier is a hallmark in the pathogenesis of inflammatory bowel disease (IBD). To date, there are no effective therapies that specifically target the intestinal TJ barrier. Among the various probiotic bacteria, Bifidobacterium, is one of the most widely studied to have beneficial effects on the intestinal TJ barrier. The main purpose of this study was to identify Bifidobacterium species that cause a sustained enhancement in the intestinal epithelial TJ barrier and can be used therapeutically to target the intestinal TJ barrier and to protect against or treat intestinal inflammation. Our results showed that Bifidobacterium bifidum caused a marked, sustained enhancement in the intestinal TJ barrier in Caco-2 monolayers. The Bifidobacterium bifidum effect on TJ barrier was strain-specific, and only the strain designated as BB1 caused a maximal enhancement in TJ barrier function. The mechanism of BB1 enhancement of intestinal TJ barrier required live bacterial cell/enterocyte interaction and was mediated by the BB1 attachment to Toll-like receptor-2 (TLR-2) at the apical membrane surface. The BB1 enhancement of the intestinal epithelial TJ barrier function was mediated by the activation of the p38 kinase pathway, but not the NF-κB signaling pathway. Moreover, the BB1 caused a marked enhancement in mouse intestinal TJ barrier in a TLR-2-dependent manner and protected against dextran sodium sulfate (DSS)-induced increase in mouse colonic permeability, and treated the DSS-induced colitis in a TJ barrier-dependent manner. These studies show that probiotic bacteria BB1 causes a strain-specific enhancement of the intestinal TJ barrier through a novel mechanism involving BB1 attachment to the enterocyte TLR-2 receptor complex and activation of p38 kinase pathway.     

Identification of SLC15A4/PHT1 Gene Products Upregulation Marking the Intestinal Epithelial Monolayer of Ulcerative Colitis Patients

SLC15A4/PHT1 is an endolysosome-resident carrier of oligopeptides and histidine recently come into view as a key path marker of immune/autoimmune/inflammatory pathways in immune cells. Yet, its emerging role in inflammatory processes directly targeting the gastrointestinal epithelial layer, as in the multifactorial pathophysiology of inflammatory bowel disease (IBD), is poorly investigated. Here, the first identification of SLC15A4/PHT1 gene products in human colonic epithelium of ulcerative colitis (UC) patients is reported, showing protein primarily localized in intracellular vesicle-like compartments. Qualitative and quantitative immunohistochemical analyses of colon biopsies revealed overexpression of SLC15A4/PHT1 protein product in the epithelial layer of UC patients. Results were successfully mirrored in vitro, in spontaneously differentiated enterocyte-like monolayers of Caco-2 cells specifically exposed to DSS (dextran sodium sulphate) to mimic IBD inflammatory onsets. SLC15A4/PHT1 expression and cellular localization were characterized confirming its (dys)regulation traits in inflamed vs. healthy epithelia, strongly hinting the hypothesis of SLC15A4/PHT1 increased function associated with epithelial inflammation in IBD patients.     

Gliadin Peptide P31-43 Induces mTOR/NFkβ Activation and Reduces Autophagy: The Role of Lactobacillus paracasei CBA L74 Postbiotc

Celiac disease (CD) is an autoimmune disease characterized by an altered immune response stimulated by gliadin peptides that are not digested and cause damage to the intestinal mucosa. The aim of this study was to investigate whether the postbiotic Lactobacillus paracasei (LP) could prevent the action of gliadin peptides on mTOR, autophagy, and the inflammatory response. Most of the experiments performed were conducted on intestinal epithelial cells Caco-2 treated with a peptic-tryptic digest of gliadin (PTG) and P31-43. Furthermore, we pretreated the Caco-2 with the postbiotic LP before treatment with the previously described stimuli. In both cases, we evaluated the levels of pmTOR, p70S6k, and p4EBP-1 for the mTOR pathway, pNFkβ, and pERK for inflammation and LC 3 and p62 for autophagy. For autophagy, we also used immunofluorescence analysis. Using intestinal organoids derivate from celiac (CD) patients, we analyzed the effect of gliadin after postbiotic pretreatment with LP on inflammation marker NFkβ. Through these experiments, we showed that gliadin peptides are able to induce the increase of the inflammatory response in a more complex model of intestinal epithelial cells. LP postbiotic was able to induce autophagy in Caco-2 cells and prevent gliadin effects. In conclusion, postbiotic pretreatment with LP could be considered for in vivo clinical trials.     

Epithelial Regeneration Ability of Crohn’s Disease Assessed Using Patient-Derived Intestinal Organoids

Little is known about the ability for epithelial regeneration and wound healing in patients with inflammatory bowel diseases. We evaluated the epithelial proliferation and wound healing ability of patients with Crohn’s disease (CD) using patient-derived intestinal organoids. Human intestinal organoids were constructed in a three-dimensional intestinal crypt culture of enteroscopic biopsy samples from controls and CD patients. The organoid-forming efficiency of ileal crypts derived from CD patients was reduced compared with those from control subjects (p < 0.001). Long-term cultured organoids (≥6 passages) derived from controls and CD patients showed an indistinguishable microscopic appearance and culturing behavior. Under TNFα-enriched conditions (30 ng/mL), the organoid reconstitution rate and cell viability of CD patient-derived organoids were significantly lower than those of the control organoids (p < 0.05 for each). The number of EdU+ cells was significantly lower in TNFα-treated organoids derived from CD patients than in TNFα-treated control organoids (p < 0.05). In a wound healing assay, the unhealed area in TNFα-treated CD patient-derived organoids was significantly larger than that of TNFα-treated control organoids (p < 0.001). The wound healing ability of CD patient-derived organoids is reduced in TNFα-enriched conditions, due to reduced cell proliferation. Epithelial regeneration ability may be impaired in patients with CD.     

Beneficial Effects of Natural Mineral Waters on Intestinal Inflammation and the Mucosa-Associated Microbiota

Natural mineral water (NMWs) intake has been traditionally used in the treatment of various gastrointestinal diseases. We investigated the effect of two French NMWs, one a calcium and magnesium sulphate, sodium chloride, carbonic, and ferruginous water (NMW1), the other a mainly bicarbonate water (NMW2) on the prevention of intestinal inflammation. Intestinal epithelial cells stimulated with heat inactivated Escherichia coli or H₂O₂ were treated with NMWs to evaluate the anti-inflammatory effects. Moderate colitis was induced by 1% dextran sulfate sodium (DSS) in Balbc/J mice drinking NMW1, NWW2, or control water. General signs and histological features of colitis, fecal lipocalin-2 and pro-inflammatory KC cytokine levels, global mucosa-associated microbiota, were analyzed. We demonstrated that both NMW1 and NMW2 exhibited anti-inflammatory effects using intestinal cells. In induced-colitis mice, NMW1 was effective in dampening intestinal inflammation, with significant reductions in disease activity scores, fecal lipocalin-2 levels, pro-inflammatory KC cytokine release, and intestinal epithelial lesion sizes. Moreover, NMW1 was sufficient to prevent alterations in the mucosa-associated microbiota. These observations, through mechanisms involving modulation of the mucosa-associated microbiota, emphasize the need of investigation of the potential clinical efficiency of such NMWs to contribute, in human beings, to a state of low inflammation in inflammatory bowel disease.     

T-Cell-Specific CerS4 Depletion Prolonged Inflammation and Enhanced Tumor Burden in the AOM/DSS-Induced CAC Model

To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.     

Novel Role of Ghrelin Receptor in Gut Dysbiosis and Experimental Colitis in Aging

Chronic low-grade inflammation is a hallmark of aging, which is now coined as inflamm-aging. Inflamm-aging contributes to many age-associated diseases such as obesity, type 2 diabetes, cardiovascular disease, and inflammatory bowel disease (IBD). We have shown that gut hormone ghrelin, via its receptor growth hormone secretagogue receptor (GHS-R), regulates energy metabolism and inflammation in aging. Emerging evidence suggests that gut microbiome has a critical role in intestinal immunity of the host. To determine whether microbiome is an integral driving force of GHS-R mediated immune-metabolic homeostasis in aging, we assessed the gut microbiome profiles of young and old GHS-R global knockout (KO) mice. While young GHS-R KO mice showed marginal changes in Bacteroidetes and Firmicutes, aged GHS-R KO mice exhibited reduced Bacteroidetes and increased Firmicutes, featuring a disease-susceptible microbiome profile. To further study the role of GHS-R in intestinal inflammation in aging, we induced acute colitis in young and aged GHS-R KO mice using dextran sulfate sodium (DSS). The GHS-R KO mice showed more severe disease activity scores, higher proinflammatory cytokine expression, and decreased expression of tight junction markers. These results suggest that GHS-R plays an important role in microbiome homeostasis and gut inflammation during aging; GHS-R suppression exacerbates intestinal inflammation in aging and increases vulnerability to colitis. Collectively, our finding reveals for the first time that GHS-R is an important regulator of intestinal health in aging; targeting GHS-R may present a novel therapeutic strategy for prevention/treatment of aging leaky gut and inflammatory bowel disease.     

Identification of an IL-22-Dependent Gene Signature as a Pharmacodynamic Biomarker

Interleukin-22 (IL-22) plays a role in epithelial barrier function and repair, and may provide benefits in conditions like inflammatory bowel disease. However, limited human data are available to assess the clinical effect of IL-22 administration. This study used a human intestinal cell line to identify an IL-22-dependent gene signature that could serve as a pharmacodynamic biomarker for IL-22 therapy. The response to IL-22Fc (UTTR1147A, an Fc-stabilized version of IL-22) was assessed in HT-29 cells by microarray, and the selected responsive genes were confirmed by qPCR. HT-29 cells demonstrated dose-dependent increases in STAT3 phosphorylation and multiple gene expression changes in response to UTTR1147A. Genes were selected that were upregulated by UTTR1147A, but to a lesser extent by IL-6, which also signals via STAT3. IL-1R1 was highly upregulated by UTTR1147A, and differential gene expression patterns were observed in response to IL-22Fc in the presence of IL-1β. An IL-22-dependent gene signature was identified that could serve as a pharmacodynamic biomarker in intestinal biopsies to support the clinical development of an IL-22 therapeutic. The differential gene expression pattern in the presence of IL-1β suggests that an inflammatory cytokine milieu in the disease setting could influence the clinical responses to IL-22.     

High Acetate Concentration Protects Intestinal Barrier and Exerts Anti-Inflammatory Effects in Organoid-Derived Epithelial Monolayer Cultures from Patients with Ulcerative Colitis

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers (n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD.     

Bile Acid Signaling in Inflammatory Bowel Disease

Inflammatory bowel disease is a chronic, idiopathic and complex condition, which most often manifests itself in the form of ulcerative colitis or Crohn’s disease. Both forms are associated with dysregulation of the mucosal immune system, compromised intestinal epithelial barrier, and dysbiosis of the gut microbiome. It has been observed for a long time that bile acids are involved in inflammatory disorders, and recent studies show their significant physiological role, reaching far beyond being emulsifiers helping in digestion of lipids. Bile acids are also signaling molecules, which act, among other things, on lipid metabolism and immune responses, through several nuclear and membrane receptors in hepatocytes, enterocytes and cells of the immune system. Gut microbiota homeostasis also seems to be affected, directly and indirectly, by bile acid metabolism and signaling. This review summarizes recent advances in the field of bile acid signaling, studies of inflamed gut microbiome, and the therapeutic potential of bile acids in the context of inflammatory bowel disease.     

IL-20 Activates ERK1/2 and Suppresses Splicing of X-Box Protein-1 in Intestinal Epithelial Cells but Does Not Improve Pathology in Acute or Chronic Models of Colitis

The cytokine Interleukin (IL)-20 belongs to the IL-10 superfamily. IL-20 levels are reported to increase in the intestines of Ulcerative Colitis (UC) patients, however not much is known about its effects on intestinal epithelial cells. Here, we investigated the influence of IL-20 on intestinal epithelial cell lines and primary intestinal organoid cultures. By using chemical-induced (dextran sodium sulphate; DSS) colitis and a spontaneous model of colitis (Winnie mice), we assess whether recombinant IL-20 treatment is beneficial in reducing/improving pathology. Following stimulation with IL-20, intestinal primary organoids from wild-type and Winnie mice increased the expression of ERK1/2. However, this was lost when cells were differentiated into secretory goblet cells. Importantly, IL-20 treatment significantly reduced endoplasmic reticulum (ER) stress, as measured by spliced-XBP1 in epithelial cells, and this effect was lost in the goblet cells. IL-20 treatment in vivo in the DSS and Winnie models had minimal effects on pathology, but a decrease in macrophage activation was noted. Taken together, these data suggest a possible, but subtle role of IL-20 on epithelial cells in vivo. The therapeutic potential of IL-20 could be harnessed by the development of a targeted therapy or combination therapy to improve the healing of the mucosal barrier.     

In Vitro Metabolism of Six C-Glycosidic Flavonoids from Passiflora incarnata L.

Several medical plants, such as Passiflora incarnata L., contain C-glycosylated flavonoids, which may contribute to their efficacy. Information regarding the bioavailability and metabolism of these compounds is essential, but not sufficiently available. Therefore, the metabolism of the C-glycosylated flavones orientin, isoorientin, schaftoside, isoschaftoside, vitexin, and isovitexin was investigated using the Caco-2 cell line as an in vitro intestinal and epithelial metabolism model. Isovitexin, orientin, and isoorientin showed broad ranges of phase I and II metabolites containing hydroxylated, methoxylated, and sulfated compounds, whereas schaftoside, isoschaftoside, and vitexin underwent poor metabolism. All metabolites were identified via UHPLC-MS or UHPLC-MS/MS using compound libraries containing all conceivable metabolites. Some structures were confirmed via UHPLC-MS experiments with reference compounds after a cleavage reaction using glucuronidase and sulfatase. Of particular interest is the observed cleavage of the C–C bonds between sugar and aglycone residues in isovitexin, orientin, and isoorientin, resulting in unexpected glucuronidated or sulfated luteolin and apigenin derivatives. These findings indicate that C-glycosidic flavones can be highly metabolized in the intestine. In particular, flavonoids with ortho-dihydroxy groups showed sulfated metabolites. The identified glucuronidated or sulfated aglycones demonstrate that enzymes expressed by Caco-2 cells are able to potentially cleave C–C bonds in vitro.     

Baicalin-Induced Autophagy Preserved LPS-Stimulated Intestinal Cells from Inflammation and Alterations of Paracellular Permeability

Several studies have demonstrated a relevant role of intestinal epithelial cells in the immune response and in chronic inflammatory conditions, including ulcers, colitis, and Crohn’s disease. Baicalin (BA), extracted from the root of Scutellaria baicalensis, has various beneficial healthy effects, including anti-inflammatory activity. However, few studies have evaluated BA effects on autophagic signaling in epithelial cell response to inflammatory stimuli. To explore possible beneficial effects of BA, HT-29 cells were exposed to lipopolysaccharide (LPS), in presence or absence of BA, for 4 h. We evaluated mRNA levels of autophagy-related genes and cytokines, triggering inflammatory response. Furthermore, the expression of claudin 1, involved in the regulation of paracellular permeability was analyzed. BA treatment repressed LPS-induced expression of TNF-α and IL-1β. The down-regulation of autophagy-related genes induced by LPS was counteracted by cell pretreatment with BA. Under these conditions, BA reduced the NF-κB activation caused by LPS. Also, BA restored mRNA and protein levels of claudin 1, which were reduced by LPS. In conclusion, in intestinal epithelial cells BA regulates the NF-κB activation and modulates both autophagic and inflammatory processes, leading to an improvement of paracellular permeability. These results suggest that the anti-inflammatory effects of BA can be associated to the regulation of autophagic flux.     

The Progress of Intestinal Epithelial Models from Cell Lines to Gut-On-Chip

Over the past years, several preclinical in vitro and ex vivo models have been developed that helped to understand some of the critical aspects of intestinal functions in health and disease such as inflammatory bowel disease (IBD). However, the translation to the human in vivo situation remains problematic. The main reason for this is that these approaches fail to fully reflect the multifactorial and complex in vivo environment (e.g., including microbiota, nutrition, and immune response) in the gut system. Although conventional models such as cell lines, Ussing chamber, and the everted sac are still used, increasingly more sophisticated intestinal models have been developed over the past years including organoids, InTESTine™ and microfluidic gut-on-chip. In this review, we gathered the most recent insights on the setup, advantages, limitations, and future perspectives of most frequently used in vitro and ex vivo models to study intestinal physiology and functions in health and disease.     

Anthocyanin Absorption and Metabolism by Human Intestinal Caco-2 Cells—A Review

Anthocyanins from different plant sources have been shown to possess health beneficial effects against a number of chronic diseases. To obtain any influence in a specific tissue or organ, these bioactive compounds must be bioavailable, i.e., effectively absorbed from the gut into the circulation and transferred to the appropriate location within the body while still maintaining their bioactivity. One of the key factors affecting the bioavailability of anthocyanins is their transport through the gut epithelium. The Caco-2 cell line, a human intestinal epithelial cell model derived from a colon carcinoma, has been proven to be a good alternative to animal studies for predicting intestinal absorption of anthocyanins. Studies investigating anthocyanin absorption by Caco-2 cells report very low absorption of these compounds. However, the bioavailability of anthocyanins may be underestimated since the metabolites formed in the course of digestion could be responsible for the health benefits associated with anthocyanins. In this review, we critically discuss recent findings reported on the anthocyanin absorption and metabolism by human intestinal Caco-2 cells.