Aryl Hydrocarbon Receptor Activity in Intestinal Epithelial Cells in the Formation of Colonic Tertiary Lymphoid Tissues.

After birth, the development of secondary lymphoid tissues (SLTs) in the colon is dependent on the expression of the Aryl Hydrocarbon Receptor (AhR) in immune cells as a response to the availability of AhR ligands. However, little is known about how AhR activity from intestinal epithelial cells (IECs) may influence the development of tertiary lymphoid tissues (TLTs). As organized structures that develop at sites of inflammation or infection during adulthood, TLTs serve as localized centers of adaptive immune responses, and their presence has been associated with the resolution of inflammation and tumorigenesis in the colon. Here, we investigated the effect of the conditional loss of AhR activity in IECs in the formation and immune cell composition of TLTs in a model of acute inflammation. In females, loss of AhR activity in IECs reduced the formation of TLTs without significantly changing disease outcomes nor immune cell composition within TLTs. In males lacking AhR expression in IECs, increased disease activity index, lower expression of functional-IEC genes, increased number of TLTs, increased T-cell density, and lower B- to T-cell ratio was observed. These findings may represent an unfavorable prognosis when exposed to DSS-induced epithelial damage compared to females. Sex and loss of IEC AhR also resulted in changes in microbial populations in the gut. Collectively, these data suggest that the formation of TLTs in the colon is influenced by sex and AhR expression in IECs.

Oral supplementation of gut microbial metabolite indole-3-acetate alleviates diet-induced steatosis and inflammation in mice.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. There is growing evidence that dysbiosis of the intestinal microbiota and disruption of microbiota-host interactions contribute to the pathology of NAFLD. We previously demonstrated that gut microbiota-derived tryptophan metabolite indole-3-acetate (I3A) was decreased in both cecum and liver of high-fat diet-fed mice and attenuated the expression of inflammatory cytokines in macrophages and Tnfa and fatty acid-induced inflammatory responses in an aryl-hydrocarbon receptor (AhR)-dependent manner in hepatocytes. In this study, we investigated the effect of orally administered I3A in a mouse model of diet-induced NAFLD. Western diet (WD)-fed mice given sugar water (SW) with I3A showed dramatically decreased serum ALT, hepatic triglycerides (TG), liver steatosis, hepatocyte ballooning, lobular inflammation, and hepatic production of inflammatory cytokines, compared to WD-fed mice given only SW. Metagenomic analysis show that I3A administration did not significantly modify the intestinal microbiome, suggesting that I3A's beneficial effects likely reflect the metabolite's direct actions on the liver. Administration of I3A partially reversed WD-induced alterations of liver metabolome and proteome, notably, decreasing expression of several enzymes in hepatic lipogenesis and ß-oxidation. Mechanistically, we also show that AMP-activated protein kinase (AMPK) mediates the anti-inflammatory effects of I3A in macrophages. The potency of I3A in alleviating liver steatosis and inflammation clearly demonstrates its potential as a therapeutic modality for preventing the progression of steatosis to non-alcoholic steatohepatitis (NASH).

Fatty Acid Desaturase 1 Influences Hepatic Lipid Homeostasis by Modulating the PPARα-FGF21 Axis.

The fatty acid desaturase 1 (FADS1), also known as delta-5 desaturase (D5D), is one of the rate-limiting enzymes involved in the desaturation and elongation cascade of polyunsaturated fatty acids (PUFAs) to generate long-chain PUFAs (LC-PUFAs). Reduced function of D5D and decreased hepatic FADS1 expression, as well as low levels of LC-PUFAs, were associated with nonalcoholic fatty liver disease. However, the causal role of D5D in hepatic lipid homeostasis remains unclear. In this study, we hypothesized that down-regulation of FADS1 increases susceptibility to hepatic lipid accumulation. We used in vitro and in vivo models to test this hypothesis and to delineate the molecular mechanisms mediating the effect of reduced FADS1 function. Our study demonstrated that FADS1 knockdown significantly reduced cellular levels of LC-PUFAs and increased lipid accumulation and lipid droplet formation in HepG2 cells. The lipid accumulation was associated with significant alterations in multiple pathways involved in lipid homeostasis, especially fatty acid oxidation. These effects were demonstrated to be mediated by the reduced function of the peroxisome proliferator-activated receptor alpha (PPARα)-fibroblast growth factor 21 (FGF21) axis, which can be reversed by treatment with docosahexaenoic acid, PPARα agonist, or FGF21. In vivo, FADS1-knockout mice fed with high-fat diet developed increased hepatic steatosis as compared with their wild-type littermates. Molecular analyses of the mouse liver tissue largely corroborated the observations in vitro, especially along with reduced protein expression of PPARα and FGF21. Conclusion: Collectively, these results suggest that dysregulation in FADS1 alters liver lipid homeostasis in the liver by down-regulating the PPARα-FGF21 signaling axis.

Effect of diet and intestinal AhR expression on fecal microbiome and metabolomic profiles.

BACKGROUND: Diet, loss of aryl hydrocarbon receptor (AhR) expression and their modification of the gut microbiota community composition and its metabolites affect the development of colorectal cancer (CRC). However, the concordance between fecal microbiota composition and the fecal metabolome is poorly understood. Mice with specific AhR deletion (AhRKO) in intestinal epithelial cell and their wild-type littermates were fed a low-fat diet or a high-fat diet. Shifts in the fecal microbiome and metabolome associated with diet and loss of AhR expression were assessed. Microbiome and metabolome data were integrated to identify specific microbial taxa that contributed to the observed metabolite shifts. RESULTS: Our analysis shows that diet has a more pronounced effect on mouse fecal microbiota composition than the impact of the loss of AhR. In contrast, metabolomic analysis showed that the loss of AhR in intestinal epithelial cells had a more pronounced effect on metabolite profile compared to diet. Integration analysis of microbiome and metabolome identified unclassified Clostridiales, unclassified Desulfovibrionaceae, and Akkermansia as key contributors to the synthesis and/or utilization of tryptophan metabolites. CONCLUSIONS: Akkermansia are likely to contribute to the synthesis and/or degradation of tryptophan metabolites. Our study highlights the use of multi-omic analysis to investigate the relationship between the microbiome and metabolome and identifies possible taxa that can be targeted to manipulate the microbiome for CRC treatment.

Effects of high-fat diet and intestinal aryl hydrocarbon receptor deletion on colon carcinogenesis.

Consumption of a high-fat diet has been associated with an increased risk of developing colorectal cancer (CRC). However, the effects of the interaction between dietary fat content and the aryl hydrocarbon receptor (AhR) on colorectal carcinogenesis remain unclear. Mainly known for its role in xenobiotic metabolism, AhR has been identified as an important regulator for maintaining intestinal epithelial homeostasis. Although previous research using whole body AhR knockout mice has revealed an increased incidence of colon and cecal tumors, the unique role of AhR activity in intestinal epithelial cells (IECs) and modifying effects of fat content in the diet at different stages of sporadic CRC development are yet to be elucidated. In the present study, we have examined the effects of a high-fat diet on IEC-specific AhR knockout mice in a model of sporadic CRC. Although loss of AhR activity in IECs significantly induced the development of premalignant lesions, in a separate experiment, no significant changes in colon mass incidence were observed. Moreover, consumption of a high-fat diet promoted cell proliferation in crypts at the premalignant colon cancer lesion stage and colon mass multiplicity as well as ß-catenin expression and nuclear localization in actively proliferating cells in colon masses. Our data demonstrate the modifying effects of high-fat diet and AhR deletion in IECs on tumor initiation and progression.NEW & NOTEWORTHY Through the use of an intestinal-specific aryl hydrocarbon receptor (AhR) knockout mouse model, this study demonstrates that the expression of AhR in intestinal epithelial cells is required to reduce the formation of premalignant colon cancer lesions. Furthermore, consumption of a high-fat diet and the loss of AhR in intestinal epithelial cells influences the development of colorectal cancer at various stages.

Establishment of a multicomponent dietary bioactive human equivalent dose to delete damaged Lgr5+ stem cells using a mouse colon tumor initiation model.

Multicomponent therapy has gained interest for its potential to synergize and subsequently lower the effective dose of each constituent required to reduce colon cancer risk. We have previously showed that rapidly cycling Lgr5 stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk. In the present study, we quantified the dose-dependent synergistic properties of dietary n-3 polyunsaturated fatty acids (PUFA) and curcumin (Cur) to promote targeted apoptotic deletion of damaged colonic Lgr5 stem cells. For this purpose, both heterogeneous bulk colonocytes and Lgr5 stem cells were isolated from Lgr5-EGFP-IRES-CreER knock-in mice injected with azoxymethane (AOM). Isolated cells were analyzed for DNA damage (γH2AX), apoptosis (cleaved caspase-3), and targeted apoptosis (both γH2AX and cleaved caspase-3) at 12 h post-AOM injection. Comparison of the percentage of targeted apoptosis in Lgr5 stem cells (GFP) across a broad bioactive dose-range revealed an ED50 of 16.0 mg/day n-3 PUFA + 15.9 mg/day Cur. This corresponded to a human equivalent dose of 3.0 g n-3 PUFA + 3.0 g Cur. In summary, our results provide evidence that a low dose (n-3 PUFA + Cur) combination diet reduces AOM-induced DNA damage in Lgr5 stem cells and enhances targeted apoptosis of DNA-damaged cells, implying that a lower human equivalent dose can be utilized in future human clinical trials.

Dietary fat and fiber interact to uniquely modify global histone post-translational epigenetic programming in a rat colon cancer progression model.

Dietary fermentable fiber generates short-chain fatty acids (SCFA), for example, butyrate, in the colonic lumen which serves as a chemoprotective histone deacetylase inhibitor and/or as an acetylation substrate for histone acetylases. In addition, n-3 polyunsaturated fatty acids (n-3 PUFA) in fish oil can affect the chromatin landscape by acting as ligands for tumor suppressive nuclear receptors. In an effort to gain insight into the global dimension of post-translational modification of histones (including H3K4me3 and H3K9ac) and clarify the chemoprotective impact of dietary bioactive compounds on transcriptional control in a preclinical model of colon cancer, we generated high-resolution genome-wide RNA (RNA-Seq) and "chromatin-state" (H3K4me3-seq and H3K9ac-seq) maps for intestinal (epithelial colonocytes) crypts in rats treated with a colon carcinogen and fed diets containing bioactive (i) fish oil, (ii) fermentable fiber (a rich source of SCFA), (iii) a combination of fish oil plus pectin, or (iv) control, devoid of fish oil or pectin. In general, poor correlation was observed between differentially transcribed (DE) and enriched genes (DERs) at multiple epigenetic levels. The combinatorial diet (fish oil + pectin) uniquely affected transcriptional profiles in the intestinal epithelium, for example, upregulating lipid catabolism and beta-oxidation associated genes. These genes were linked to activated ligand-dependent nuclear receptors associated with n-3 PUFA and were also correlated with the mitochondrial L-carnitine shuttle and the inhibition of lipogenesis. These findings demonstrate that the chemoprotective fish oil + pectin combination diet uniquely induces global histone state modifications linked to the expression of chemoprotective genes.

Short Chain Fatty Acids Enhance Aryl Hydrocarbon (Ah) Responsiveness in Mouse Colonocytes and Caco-2 Human Colon Cancer Cells.

Aryl hydrocarbon receptor (AhR) ligands are important for gastrointestinal health and play a role in gut inflammation and the induction of T regulatory cells, and the short chain fatty acids (SCFAs) butyrate, propionate and acetate also induce similar protective responses. Initial studies with butyrate demonstrated that this compound significantly increased expression of Ah-responsive genes such as Cyp1a1/CYP1A1 in YAMC mouse colonocytes and Caco-2 human colon cancer cell lines. Butyrate synergistically enhanced AhR ligand-induced Cyp1a1/CYP1A1 in these cells with comparable enhancement being observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and also microbiota-derived AhR ligands tryptamine, indole and 1,4-dihydroxy-2-naphthoic acid (DHNA). The effects of butyrate on enhancing induction of Cyp1b1/CYP1B1, AhR repressor (Ahrr/AhRR) and TCDD-inducible poly(ADP-ribose)polymerase (Tiparp/TiPARP) by AhR ligands were gene- and cell context-dependent with the Caco-2 cells being the most responsive cell line. Like butyrate and propionate, the prototypical hydroxyamic acid-derived histone deacetylase (HDAC) inhibitors Panobinostat and Vorinostat also enhanced AhR ligand-mediated induction and this was accompanied by enhanced histone acetylation. Acetate also enhanced basal and ligand-inducible Ah responsiveness and histone acetylation, demonstrating that acetate was an HDAC inhibitor. These results demonstrate SCFA-AhR ligand interactions in YAMC and Caco-2 cells where SCFAs synergistically enhance basal and ligand-induced expression of AhR-responsive genes.

Streptococcus gallolyticus subsp. gallolyticus promotes colorectal tumor development.

Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been known to have a strong association with colorectal cancer (CRC). This knowledge has important clinical implications, and yet little is known about the role of Sg in the development of CRC. Here we demonstrate that Sg promotes human colon cancer cell proliferation in a manner that depends on cell context, bacterial growth phase and direct contact between bacteria and colon cancer cells. In addition, we observed increased level of ß-catenin, c-Myc and PCNA in colon cancer cells following incubation with Sg. Knockdown or inhibition of ß-catenin abolished the effect of Sg. Furthermore, mice administered with Sg had significantly more tumors, higher tumor burden and dysplasia grade, and increased cell proliferation and ß-catenin staining in colonic crypts compared to mice receiving control bacteria. Finally, we showed that Sg is present in the majority of CRC patients and is preferentially associated with tumor compared to normal tissues obtained from CRC patients. These results taken together establish for the first time a tumor-promoting role of Sg that involves specific bacterial and host factors and have important clinical implications.

Assessment of histone tail modifications and transcriptional profiling during colon cancer progression reveals a global decrease in H3K4me3 activity.

During colon cancer, epigenetic alterations contribute to the dysregulation of major cellular functions and signaling pathways. Modifications in chromatin signatures such as H3K4me3 and H3K9ac, which are associated with transcriptionally active genes, can lead to genomic instability and perturb the expression of gene sets associated with oncogenic processes. In order to further elucidate early pre-tumorigenic epigenetic molecular events driving CRC, we integrated diverse, genome-wide, epigenetic inputs (by high throughput sequencing of RNA, H3K4me3, and H3K9ac) and compared differentially expressed transcripts (DE) and enriched regions (DER) in an in-vivo rat colon cancer progression model. Carcinogen (AOM) effects were detected genome-wide at the RNA (116 DE genes), K9ac (49 DERs including 24 genes) and K4me3 (7678 DERs including 3792 genes) level. RNA-seq differential expression and pathway analysis indicated that interferon-associated innate immune responses were impacted by AOM exposure. Despite extensive associations between K4me3 DERs and colon tumorigenesis (1210 genes were linked to colorectal carcinoma) including FOXO3, GNAI2, H2AFX, MSH2, NR3C1, PDCD4 and VEGFA, these changes were not reflected at the RNA gene expression level during early cancer progression. Collectively, our results indicate that carcinogen-induced changes in gene K4me3 DERs are harbingers of future transcriptional events, which drive malignant transformation of the colon.

Rapidly cycling Lgr5+ stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk.

The majority of colon tumors are driven by aberrant Wnt signaling in intestinal stem cells, which mediates an efficient route toward initiating intestinal cancer. Natural lipophilic polyphenols and long-chain polyunsaturated fatty acids (PUFAs) generally suppress Wnt- and NF-κB- (nuclear factor-κ light-chain enhancer of activated B-cell) related pathways. However, the effects of these extrinsic agents on colonic leucine-rich repeat-containing G-protein-coupled receptor 5-positive (Lgr5+) stem cells, the cells of origin of colon cancer, have not been documented to date. Therefore, we examined the effect of n-3 PUFA and polyphenol (curcumin) combination on Lgr5+ stem cells during tumor initiation and progression in the colon compared with an n-6 PUFA-enriched control diet. Lgr5-EGFP-IRES-creERT2 knock-in mice were fed diets containing n-6 PUFA (control), n-3 PUFA, n-6 PUFA+curcumin or n-3 PUFA+curcumin for 3 weeks, followed by 6 azoxymethane (AOM) injections, and terminated 17 weeks after the last injection. To further elucidate the effects of the dietary bioactives at the tumor initiation stage, Lgr5+ stem cells were also assessed at 12 and 24 h post AOM injection. Only n-3 PUFA+curcumin feeding reduced nuclear ß-catenin in aberrant crypt foci (by threefold) compared with control at the progression time point. n-3 PUFA+curcumin synergistically increased targeted apoptosis in DNA-damaged Lgr5+ stem cells by 4.5-fold compared with control at 12 h and maximally reduced damaged Lgr5+ stem cells at 24 h, down to the level observed in saline-treated mice. Finally, RNAseq analysis indicated that p53 signaling in Lgr5+ stem cells from mice exposed to AOM was uniquely upregulated only following n-3 PUFA+curcumin cotreatment. These novel findings demonstrate that Lgr5+ stem cells are uniquely responsive to external dietary cues following the induction of DNA damage, providing a therapeutic strategy for eliminating damaged Lgr5+ stem cells to reduce colon cancer initiation.

A New Model to Study the Role of Arachidonic Acid in Colon Cancer Pathophysiology.

A significant increase in cyclooxygenase 2 (COX2) gene expression has been shown to promote cylcooxygenase-dependent colon cancer development. Controversy associated with the role of COX2 inhibitors indicates that additional work is needed to elucidate the effects of arachidonic acid (AA)-derived (cyclooxygenase and lipoxygenase) eicosanoids in cancer initiation, progression, and metastasis. We have recently developed a novel Fads1 knockout mouse model that allows for the investigation of AA-dependent eicosanoid deficiency without the complication of essential fatty acid deficiency. Interestingly, the survival rate of Fads1-null mice is severely compromised after 2 months on a semi-purified AA-free diet, which precludes long-term chemoprevention studies. Therefore, in this study, dietary AA levels were titrated to determine the minimal level required for survival, while maintaining a distinct AA-deficient phenotype. Null mice supplemented with AA (0.1%, 0.4%, 0.6%, 2.0%, w/w) in the diet exhibited a dose-dependent increase (P < 0.05) in AA, PGE2, 6-keto PGF1α, TXB2, and EdU-positive proliferative cells in the colon. In subsequent experiments, null mice supplemented with 0.6% AA diet were injected with a colon-specific carcinogen (azoxymethane) in order to assess cancer susceptibility. Null mice exhibited significantly (P < 0.05) reduced levels/multiplicity of aberrant crypt foci (ACF) as compared with wild-type sibling littermate control mice. These data indicate that (i) basal/minimal dietary AA supplementation (0.6%) expands the utility of the Fads1-null mouse model for long-term cancer prevention studies and (ii) that AA content in the colonic epithelium modulates colon cancer risk. Cancer Prev Res; 9(9); 750-7. ©2016 AACR.

Data describing the effects of dietary bioactive agents on colonic stem cell microRNA and mRNA expression.

With the identification of Lgr5 as a definitive marker for intestinal stem cells, we used the highly novel, recently described, Lgr5-EGFP-IRES-cre ER (T2) knock in mouse model. Mice were injected with azoxymethane (AOM, a colon carcinogen) or saline (control) and fed a chemo-protective diet containing n-3 fatty acids and fermentable fiber (n-3 PUFA+pectin) or a control diet (n-6 PUFA + cellulose). Single cells were isolated from colonic mucosa crypts and three discrete populations of cells were collected via fluorescence activated cell sorting (FACS): Lgr5(high) (stem cells), Lgr5(low) (daughter cells) and Lgr5(negative) (differentiated cells). microRNA profiling and RNA sequencing were performed from the same sample and analyzed. These data refer to 'Comparative effects of diet and carcinogen on microRNA expression in the stem cell niche of the mouse colonic crypt' (Shah et al., 2016) [5].

Homeostatic responses of colonic LGR5+ stem cells following acute in vivo exposure to a genotoxic carcinogen.

Perturbations in DNA damage, DNA repair, apoptosis and cell proliferation in the base of the crypt where stem cells reside are associated with colorectal cancer (CRC) initiation and progression. Although the transformation of leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)(+) cells is an extremely efficient route towards initiating small intestinal adenomas, the role of Lgr5(+) cells in CRC pathogenesis has not been well investigated. Therefore, we further characterized the properties of colonic Lgr5(+) cells compared to differentiated cells in Lgr5-EGFP-IRES-creER(T2) knock-in mice at the initiation stage of carcinogen azoxymethane (AOM)-induced tumorigenesis using a quantitative immunofluorescence microscopy approach. At 12 and 24h post-AOM treatment, colonic Lgr5(+) stem cells (GFP(high)) were preferentially damaged by carcinogen, exhibiting a 4.7-fold induction of apoptosis compared to differentiated (GFP(neg)) cells. Furthermore, with respect to DNA repair, O(6)-methylguanine DNA methyltransferase (MGMT) expression was preferentially induced (by 18.5-fold) in GFP(high) cells at 24h post-AOM treatment compared to GFP(neg) differentiated cells. This corresponded with a 4.3-fold increase in cell proliferation in GFP(high) cells. These data suggest that Lgr5(+) stem cells uniquely respond to alkylation-induced DNA damage by upregulating DNA damage repair, apoptosis and cell proliferation compared to differentiated cells in order to maintain genomic integrity. These findings highlight the mechanisms by which colonic Lgr5(+) stem cells respond to cancer-causing environmental factors.

Comparative effects of diet and carcinogen on microRNA expression in the stem cell niche of the mouse colonic crypt.

There is mounting evidence that noncoding microRNAs (miRNA) are modulated by select chemoprotective dietary agents. For example, recently we demonstrated that the unique combination of dietary fish oil (containing n-3 fatty acids) plus pectin (fermented to butyrate in the colon) (FPA) up-regulates a subset of putative tumor suppressor miRNAs in intestinal mucosa, and down-regulates their predicted target genes following carcinogen exposure as compared to control (corn oil plus cellulose (CCA)) diet. To further elucidate the biological effects of diet and carcinogen modulated miR's in the colon, we verified that miR-26b and miR-203 directly target PDE4B and TCF4, respectively. Since perturbations in adult stem cell dynamics are generally believed to represent an early step in colon tumorigenesis and to better understand how the colonic stem cell population responds to environmental factors such as diet and carcinogen, we additionally determined the effects of the chemoprotective FPA diet on miRNAs and mRNAs in colonic stem cells obtained from Lgr5-EGFP-IRES-creER(T2) knock-in mice. Following global miRNA profiling, 26 miRNAs (P<0.05) were differentially expressed in Lgr5(high) stem cells as compared to Lgr5(negative) differentiated cells. FPA treatment up-regulated miR-19b, miR-26b and miR-203 expression as compared to CCA specifically in Lgr5(high) cells. In contrast, in Lgr5(negative) cells, only miR-19b and its indirect target PTK2B were modulated by the FPA diet. These data indicate for the first time that select dietary cues can impact stem cell regulatory networks, in part, by modulating the steady-state levels of miRNAs. To our knowledge, this is the first study to utilize Lgr5(+) reporter mice to determine the impact of diet and carcinogen on miRNA expression in colonic stem cells and their progeny.

Targeted Deletion of p53 in Lgr5-Expressing Intestinal Stem Cells Promotes Colon Tumorigenesis in a Preclinical Model of Colitis-Associated Cancer.

p53 has been shown to mediate cancer stem-like cell function by suppressing pluripotency and cellular dedifferentiation. However, there have been no studies to date that have addressed the specific effects of p53 loss in colonic adult stem cells. In this study, we investigated the consequences of conditionally ablating p53 in the highly relevant Lgr5(+) stem cell population on tumor initiation and progression in the colon. In a mouse model of carcinogen (AOM)-induced colon cancer, tamoxifen-inducible Lgr5-driven deletion of p53 reduced apoptosis and increased proliferation of crypt stem cells, but had no effect on tumor incidence or size. Conversely, in a mouse model of colitis-associated cancer, in which mice are exposed to AOM and the potent inflammation inducer DSS, stem cell-specific p53 deletion greatly enhanced tumor size and incidence in the colon. These novel findings suggest that the loss of p53 function in stem cells enables colonic tumor formation only when combined with DNA damage and chronic inflammation. Furthermore, we propose that stem cell targeting approaches are valuable for interrogating prevention and therapeutic strategies that aim to specifically eradicate genetically compromised stem cells.

A bioassay to measure energy metabolism in mouse colonic crypts, organoids, and sorted stem cells.

Evidence suggests that targeting cancer cell energy metabolism might be an effective therapeutic approach for selective ablation of malignancies. Using a Seahorse Extracellular Flux Analyzer, we have demonstrated that select environmental agents can alter colonic mitochondrial function by increasing respiration-induced proton leak, thereby inducing apoptosis, a marker of colon cancer risk. To further probe bioenergetics in primary intestinal cells, we developed methodology that can be modified and adapted to measure the bioenergetic profiles of colonic crypts, the basic functional unit of the colon, and colonic organoids, an ex vivo 3D culture of colonic crypts. Furthermore, in combination with the MoFlo Astrios High-Speed Cell Sorter, we were able to measure the bioenergetic profiles of colonic adult stem and daughter cells from Lgr5-EGFP-IRES-creER(T2) transgenic mice. We examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a full arylhydrocarbon receptor agonist, known to affect gastrointestinal function and cancer risk, on the bioenergetic profiles of intestinal epithelial cells. Mouse colonic crypts, organoids, or sorted single cells were seeded onto Matrigel-precoated Seahorse XF24 microplates for extracellular flux analysis. Temporal analyses revealed distinct energy metabolic profiles in crypts and organoids challenged with TCDD. Furthermore, sorted Lgr5(+) stem cells exhibited a Warburg-like metabolic profile. This is noteworthy because perturbations in stem cell dynamics are generally believed to represent the earliest step toward colon tumorigenesis. We propose that our innovative methodology may facilitate future in vivo/ex vivo metabolic studies using environmental agents affecting colonocyte energy metabolism.

Antagonizing arachidonic acid-derived eicosanoids reduces inflammatory Th17 and Th1 cell-mediated inflammation and colitis severity.

During colitis, activation of two inflammatory T cell subsets, Th17 and Th1 cells, promotes ongoing intestinal inflammatory responses. n-6 polyunsaturated fatty acid- (PUFA-) derived eicosanoids, such as prostaglandin E2 (PGE2), promote Th17 cell-mediated inflammation, while n-3 PUFA antagonize both Th17 and Th1 cells and suppress PGE2 levels. We utilized two genetic mouse models, which differentially antagonize PGE2 levels, to examine the effect on Th17 cells and disease outcomes in trinitrobenzene sulfonic acid- (TNBS-) induced colitis. Fat-1 mice contain the ω3 desaturase gene from C. elegans and synthesize n-3 PUFA de novo, thereby reducing the biosynthesis of n-6 PUFA-derived eicosanoids. In contrast, Fads1 Null mice contain a disrupted Δ5 desaturase gene and produce lower levels of n-6 PUFA-derived eicosanoids. Compared to Wt littermates, Fat-1 and Fads1 Null mice exhibited a similar colitic phenotype characterized by reduced colonic mucosal inflammatory eicosanoid levels and mRNA expression of Th17 cell markers (IL-17A, RORγτ, and IL-23), decreased percentages of Th17 cells and, improved colon injury scores (P ≤ 0.05). Thus, during colitis, similar outcomes were obtained in two genetically distinct models, both of which antagonize PGE2 levels via different mechanisms. Our data highlight the critical impact of n-6 PUFA-derived eicosanoids in the promotion of Th17 cell-mediated colonic inflammation.

Differential effects of 2- and 3-series E-prostaglandins on in vitro expansion of Lgr5+ colonic stem cells.

Arachidonic acid (20:4(Δ5,8,11,14), AA)-derived prostaglandin E2 (PGE2) promotes colon cancer development. In contrast, chemoprotective n-3 polyunsaturated fatty acids supplant AA, thereby decreasing PGE2 biosynthesis in colonocytes, with eicosapentaenoic acid (20:5(Δ5,8,11,14,17), EPA) in particular being metabolized to a novel 3-series E-prostaglandin (PGE3), a putative anti-tumorigenic-cyclooxygenase metabolite. Because transformation of adult stem cells is an extremely important route toward initiating intestinal cancer, we utilized the leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescent protein-internal ribosome entry site (IRES)-creER(T2) knock-in mouse model to isolate and culture colonic organoids, in order to document ex vivo responses to exogenous PGE2 and PGE3. Colonic crypts were isolated from transgenic mice and cultured in a Matrigel-based three-dimensional platform. Organoids were treated with exogenous PGE2, PGE3 or dimethyl sulfoxide (vehicle control) for 5 days and the number of viable organoids was recorded daily. Subsequently, samples were processed for immunohistochemistry, flow cytometry and real-time PCR analyses. PGE2 promoted optimal organoid growth and induced significantly higher levels of cell proliferation (P < 0.05) compared with PGE3 and control. In contrast, the Lgr5-green fluorescent protein-positive stem cell number was uniquely elevated by >2-fold in PGE2-treated cultures compared with PGE3 and control. This coincided with the upregulation of stem-cell-related Sox9, Axin2 and Cd44 messenger RNAs. Our results demonstrate that relative to AA-derived PGE2, a known promoter of colon tumorigenesis, EPA-derived PGE3 has diminished ability to support colonic stem cell expansion in mouse colonic organoids.

Inhibitory effects of omega-3 fatty acids on injury-induced epidermal growth factor receptor transactivation contribute to delayed wound healing.

Epidermal growth factor receptor (EGFR)-mediated signaling is required for optimal intestinal wound healing. Since n-3 polyunsaturated fatty acids (PUFA), specifically docosahexaenoic acid (DHA), alter EGFR signaling and suppress downstream activation of key signaling pathways, we hypothesized that DHA would be detrimental to the process of intestinal wound healing. Using a mouse immortalized colonocyte model, DHA uniquely reduced EGFR ligand-induced receptor activation, whereas DHA and its metabolic precursor eicosapentaenoic acid (EPA) reduced wound-induced EGFR transactivation compared with control (no fatty acid or linoleic acid). Under wounding conditions, the suppression of EGFR activation was associated with a reduction in downstream activation of cytoskeletal remodeling proteins (PLCγ1, Rac1, and Cdc42). Subsequently, DHA and EPA reduced cell migration in response to wounding. Mice were fed a corn oil-, DHA-, or EPA-enriched diet prior to intestinal wounding (2.5% dextran sodium sulfate for 5 days followed by termination after 0, 3, or 6 days of recovery). Mortality was increased in EPA-fed mice and colonic histological injury scores were increased in EPA- and DHA-fed mice compared with corn oil-fed (control) mice. Although kinetics of colonic EGFR activation and downstream signaling (PLCγ1, Rac1, and Cdc42) were delayed by both n-3 PUFA, colonic repair was increased in EPA- relative to DHA-fed mice. These results indicate that, during the early response to intestinal wounding, DHA and EPA uniquely delay the activation of key wound-healing processes in the colon. This effect is mediated, at least in part, via suppression of EGFR-mediated signaling and downstream cytoskeletal remodeling.