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-creERT2 knock-in mice. Following global miRNA profiling, 26 miRNAs (P <0.05) were differentially expressed in Lgr5high stem cells as compared to Lgr5negative differentiated cells. FPA treatment up-regulated miR-19b, miR-26b and miR-203 expression as compared to CCA specifically in Lgr5high cells. In contrast, in Lgr5negative 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.
The mechanisms by which n-3 polyunsaturated fatty acids (n-3 PUFA), abundant in fish oil, exert their anti-inflammatory effects have not been rigorously defined. We have previously demonstrated that n-3 PUFA decrease the amount of phosphatidylinositol-(4,5)-bisphosphate, [PI(4,5)P2], in CD4+ T cells, leading to suppressed actin remodeling upon activation. Since discrete pools of PI(4,5)P2 exist in the plasma membrane, we determined whether n-3 PUFA modulate spatial organization of PI(4,5)P2 relative to raft and non-raft domains. We used Förster resonance energy transfer (FRET) to demonstrate that lipid raft mesodomains in the plasma membrane of CD4+ T cells enriched in n-3 PUFA display increased co-clustering of Lck(N10) and LAT(ΔCP), markers of lipid rafts. CD4+ T cells enriched in n-3 PUFA also exhibited a depleted plasma membrane non-raft PI(4,5)P2 pool as detected by decreased co-clustering of Src(N15), a non-raft marker, and PH(PLC-δ), a PI(4,5)P2 reporter. Incubation with exogenous PI(4,5)P2 rescued the effects on the non-raft PI(4,5)P2 pool, and reversed the suppression of T cell proliferation in CD4+ T cells enriched with n-3 PUFA. Furthermore, CD4+ T cells isolated from mice fed a 4% docosahexaenoic acid (DHA)-enriched diet exhibited a decrease in the non-raft pool of PI(4,5)P2, and exogenous PI(4,5)P2 reversed the suppression of T cell proliferation. Finally, these effects were not due to changes to post-translational lipidation, since n-3 PUFA did not alter the palmitoylation status of signaling proteins. These data demonstrate that n-3 PUFA suppress T cell proliferation by altering plasma membrane topography and the spatial organization of PI(4,5)P2.
Fluorescence resonance energy transfer; T cell; membrane; phosphatidylinositol; polyunsaturated fatty acids
Most cancer research now involves one or more assays profiling various biological molecules, e.g., messenger RNA and micro RNA, in samples collected on the same individuals. The main interest with these genomic data sets lies in the identification of a subset of features that are active in explaining the dependence between platforms. To quantify the strength of the dependency between two variables, correlation is often preferred. However, expression data obtained from next-generation sequencing platforms are integer with very low counts for some important features. In this case, the sample Pearson correlation is not a valid estimate of the true correlation matrix, because the sample correlation estimate between two features/variables with low counts will often be close to zero, even when the natural parameters of the Poisson distribution are, in actuality, highly correlated. We propose a model-based approach to correlation estimation between two non-normal data sets, via a method we call Probabilistic Correlations ANalysis, or PCAN. PCAN takes into consideration the distributional assumption about both data sets and suggests that correlations estimated at the model natural parameter level are more appropriate than correlations estimated directly on the observed data. We demonstrate through a simulation study that PCAN outperforms other standard approaches in estimating the true correlation between the natural parameters. We then apply PCAN to the joint analysis of a microRNA (miRNA) and a messenger RNA (mRNA) expression data set from a squamous cell lung cancer study, finding a large number of negative correlation pairs when compared to the standard approaches.
Canonical correlation analysis; Correlation; Generalized linear models; Poisson regression; RNA-sequencing
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 which 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 in the crypt 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.
p53; Lgr5; colon cancer; azoxymethane; dextran sodium sulphate
Combined intakes of specific dietary fiber and fat subtypes protect against colon cancer in animal models. We evaluated associations between self-reported individual and combinations of fiber (insoluble, soluble, and pectins, specifically) and fat (omega-6, omega-3, and docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), specifically) and colorectal cancer (CRC) risk in the Women’s Health Initiative prospective cohort (n = 134,017). During a mean 11.7 years (1993–2010), 1952 incident CRC cases were identified. Cox regression models computed multivariate adjusted hazard ratios to estimate the association between dietary factors and CRC risk. Assessing fiber and fat individually, there was a modest trend for lower CRC risk with increasing intakes of total and insoluble fiber (p-trend 0.09 and 0.08). An interaction (p = 0.01) was observed between soluble fiber and DHA + EPA, with protective effects of DHA + EPA with lower intakes of soluble fiber and an attenuation at higher intakes, however this association was no longer significant after correction for multiple testing. These results suggest a modest protective effect of higher fiber intake on CRC risk, but not in combination with dietary fat subtypes. Given the robust results in preclinical models and mixed results in observational studies, controlled dietary interventions with standardized intakes are needed to better understand the interaction of specific fat and fiber subtypes on colon biology and ultimately CRC susceptibility in humans.
butyrate; colorectal cancer; DHA; EPA; fat; fiber; omega-3; pectin
We have shown that dietary fish oil is protective against experimentally-induced colon cancer and the protective effect is enhanced by co-administration of pectin. However, the underlying mechanism(s) have not been fully elucidated. We hypothesized that fish oil with butyrate, a pectin fermentation product, protects against colon cancer initiation by decreasing cell proliferation and increasing differentiation and apoptosis through a p27Kip1 mediated mechanism. Rats were provided diets of corn or fish oil, with/without butyrate, and terminated 12, 24 or 48 h post azoxymethane (AOM) injection. Proliferation (Ki-67), differentiation (Dolichos Biflorus Agglutinin), apoptosis (TUNEL) and p27Kip1 (cell cycle mediator) were measured in the same cell within crypts in order to examine the coordination of cell cycle as a function of diet. DNA damage (N7-methylguanine) was determined by quantitative immunohistochemical analysis. Dietary fish oil decreased DNA damage by 19% (P=0.001) and proliferation by 50% (P=0.003) and increased differentiation by 56% (P=0.039) compared to corn oil. When combined with butyrate, fish oil enhanced apoptosis 24 h post AOM injection compared to a corn oil/butyrate diet (P=0.039). There was an inverse relationship between crypt height and apoptosis in fish oil/butyrate group (r= −0.53, P=0.040). Corn oil/butyrate group showed a positive correlation between p27Kip1 expression and proliferation (r= 0.61, P=0.035). These results indicate the in vivo effect of butyrate on apoptosis and proliferation is dependent on dietary lipid source. These results demonstrate the presence of an early coordinated colonocyte response by which fish oil and butyrate protects against colon tumorigenesis.
fish oil; butyrate; colon cancer; cell
The International Agency for Research on Cancer recently released an assessment classifying red and processed meat as “carcinogenic to humans” on the basis of the positive association between increased consumption and risk for colorectal cancer. Diet, however, can also decrease the risk for colorectal cancer and be used as a chemopreventive strategy. Bioactive dietary molecules, such as n-3 polyunsaturated fatty acids, curcumin, and fermentable fiber, have been proposed to exert chemoprotective effects, and their molecular mechanisms have been the focus of research in the dietary/chemoprevention field. Using these bioactives as examples, this review surveys the proposed mechanisms by which they exert their effects, from the nucleus to the cellular membrane. In addition, we discuss emerging technologies involving the culturing of colonic organoids to study the physiological effects of dietary bioactives. Finally, we address future challenges to the field regarding the identification of additional molecular mechanisms and other bioactive dietary molecules that can be utilized in our fight to reduce the incidence of colorectal cancer.
n-3 polyunsaturated fatty acids; microRNAs; colonic organoids; membrane organization
This study tested the hypothesis that the fecal bacterial genera of breast-fed (BF) and formula-fed (FF) infants differ and that human milk oligosaccharides (HMO) modulate the microbiota of BF infants.
Fecal samples were obtained from BF (n = 16) or FF (n = 6) infants at 3-month postpartum. Human milk were collected on the same day when feces were collected. The microbiota was assessed by pyrosequencing of bacterial 16S rRNA genes. HMO were measured by HPLC-Chip time-of-flight mass spectrometry.
The overall microbiota of BF differed from that of FF (P = 0.005). Compared to FF, BF had higher relative abundances of Bacteroides, lower proportions of Clostridium XVIII, Lachnospiracea incertae sedis, Streptococcus, Enterococcus and Veillonella (P < 0.05). Bifidobacterium predominated in both BF and FF infants, with no difference in abundance between the two groups. The most abundant HMO were lacto-N-tetraose + lacto-N-neotetraose (LNT + LNnT, 22.6%), followed by 2′-fucosyllactose (2′FL, 14.5%) and lacto-N-fucopentaose I (LNFP I, 9.5%). Partial least squares regression of HMO and microbiota showed several infant fecal bacterial genera could be predicted by their mothers’ HMO profiles and the important HMO for the prediction of bacterial genera were identified by variable importance in the projection scores.
These results strengthen the established relationship between HMO and the infant microbiota, identify statistical means whereby infant bacterial genera can be predicted by milk HMO. Future studies are needed to validate these findings and determine if supplementation of formula with defined HMO could selectively modify the gut microbiota.
gut microbiota; infants; human milk oligosaccharide; breastfed
Inflammatory signaling may play a role in high-fat diet (HFD)-related circadian clock disturbances that contribute to systemic metabolic dysregulation. Therefore, palmitate, the prevalent proinflammatory saturated fatty acid (SFA) in HFD and the anti-inflammatory, poly-unsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), were analyzed for effects on circadian timekeeping and inflammatory responses in peripheral clocks. Prolonged palmitate, but not DHA, exposure increased the period of fibroblast Bmal1-dLuc rhythms. Acute palmitate treatment produced phase shifts of the Bmal1-dLuc rhythm that were larger in amplitude as compared to DHA. These phase-shifting effects were time-dependent and contemporaneous with rhythmic changes in palmitate-induced inflammatory responses. Fibroblast and differentiated adipocyte clocks exhibited cell-specific differences in the time-dependent nature of palmitate-induced shifts and inflammation. DHA and other inhibitors of inflammatory signaling (AICAR, cardamonin) repressed palmitate-induced proinflammatory responses and phase shifts of the fibroblast clock, suggesting that SFA-mediated inflammatory signaling may feed back to modulate circadian timekeeping in peripheral clocks.
•The saturated fatty acid (SFA) palmitate differentially modulates the circadian timekeeping mechanism in peripheral clocks;•Palmitate induces time-dependent phase shifts that coincide with its rhythmic induction of inflammatory signaling;•Time-dependent nature of the palmitate-induced phase shifts and inflammatory signaling is cell specific;•Inhibitors of inflammatory signaling repress the proinflammatory and phase shifting effects of palmitate;•Inflammatory signaling plays a role in the mechanism by which palmitate alters circadian timekeeping in peripheral clocks.
Circadian or 24-hour clocks throughout the body mediate the local temporal coordination of tissue- or cell-specific processes necessary for normal inflammatory responses and metabolic homeostasis. Dysregulation of peripheral clocks and their timekeeping function contribute to obesity-related metabolic disorders (e.g., type 2 diabetes). Our data unveil a novel mechanism by which mutual interactions between peripheral clocks and inflammatory signaling pathways dysregulate circadian timekeeping, and exacerbate proinflammatory responses to saturated fatty acids. These studies will guide the development of chronotherapeutic drug and/or dietary omega-3 fatty acid treatments for managing and preventing metabolic disorders and other inflammation-related pathologies (e.g., cardiovascular disease, stroke, arthritis).
Circadian clocks; Bmal1; Fatty acids; Inflammation; Cytokines; AMPK; NF-κB; AMP-activated protein kinase (AMPK); Cardamonin; Aminoimidazole-4-Carboxamide Riboside (AICAR); Fibroblasts; Adipocytes
Polarized activation of adipose tissue macrophages (ATMs) is crucial for maintaining adipose tissue function and mediating obesity-associated cardiovascular risk and metabolic abnormalities; however, the regulatory network of this key process is not well defined. Here, we identified a PPARγ/microRNA-223 (miR-223) regulatory axis that controls macrophage polarization by targeting distinct downstream genes to shift the cellular response to various stimuli. In BM-derived macrophages, PPARγ directly enhanced miR-223 expression upon exposure to Th2 stimuli. ChIP analysis, followed by enhancer reporter assays, revealed that this effect was mediated by PPARγ binding 3 PPARγ regulatory elements (PPREs) upstream of the pre–miR-223 coding region. Moreover, deletion of miR-223 impaired PPARγ-dependent macrophage alternative activation in cells cultured ex vivo and in mice fed a high-fat diet. We identified Rasa1 and Nfat5 as genuine miR-223 targets that are critical for PPARγ-dependent macrophage alternative activation, whereas the proinflammatory regulator Pknox1, which we reported previously, mediated miR-223–regulated macrophage classical activation. In summary, this study provides evidence to support the crucial role of a PPARγ/miR-223 regulatory axis in controlling macrophage polarization via distinct downstream target genes.
Colorectal cancer is the third major cause of cancer-related mortality in both men and women worldwide. The beneficial role of n-3 polyunsaturated fatty acids (PUFA) in preventing colon cancer is substantiated by experimental, epidemiological, and clinical data. From a mechanistic perspective, n-3 PUFA are pleiotropic and multifaceted with respect to their molecular mechanisms of action. For example, this class of dietary lipid uniquely modulates membrane and nuclear receptors, sensors/ion channels, and membrane structure/cytoskeletal function, thereby regulating signaling processes that influence patterns of gene expression and cell phenotype. In addition, n-3 PUFA can synergize with other potential chemoprotective agents known to reprogram the chromatin landscape, such as the fermentable fiber product, butyrate. Nutri-epigenomics is an emerging field of research that is focused on the interaction between nutrition and epigenetics. Epigenetics refers to a group of heterogeneous processes that regulate transcription without changing the DNA coding sequence, ranging from DNA methylation, to histone tail modifications and transcription factor activity. One implication of the nutri-epigenome is that it may be possible to reprogram epigenetic marks that are associated with increased disease risk by nutritional or lifestyle interventions. This review will focus on the nutri-epigenomic role of n-3 PUFA, particularly DHA, as well as the combinatorial effects of n-3 PUFA and fermentable fiber in relation to colon cancer.
Fish oil; colon cancer; nutri-epigenomics; chemoprevention; fermentable fiber; docosahexaenoic acid
With the identification of Lgr5 as a definitive marker for intestinal stem cells, we used the highly novel, recently described, Lgr5-EGFP-IRES-cre ERT2knock 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): Lgr5high (stem cells), Lgr5low (daughter cells) and Lgr5negative (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) .
Butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis in part via a Fas-mediated (extrinsic) pathway. In previous studies, we demonstrated that docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) enhances the effect of butyrate by increasing mitochondrial lipid oxidation and mitochondrial Ca2+-dependent apoptosis in the colon. In this study, we further examined the mechanism of DHA-butyrate synergism in 1) human colon tumor (HCT-116 isogenic p53+/+ vs. p53−/−) cells and 2) primary cultures of rat colonic crypts. Herein, we show that DHA and butyrate promote apoptosis by enhancing mitochondrial Ca2+ accumulation in both isogenic cell lines. Ca2+ accumulation and apoptosis were inhibited by blockade of mitochondrial uniporter-mediated Ca2+ uptake. In addition, Mito-Q, a mitochondria-targeted antioxidant, also blocked apoptosis induced by DHA and butyrate. In complementary experiments, rats were fed diets supplemented with either corn oil (control, contains no DHA) or fish oil (contains DHA). Colonic crypts were isolated and incubated with or without butyrate, after which the mitochondria-to-cytosol Ca2+ ratio and crypt viability were measured. No significant difference (P > 0.05) in basal mitochondrial Ca2+ levels was observed between fish oil- or corn oil-fed animals. In contrast, when fish oil was the dietary lipid source, crypts incubated with butyrate exhibited a significant increase (3.6-fold, P < 0.001) in mitochondrial Ca2+ compared with corn oil plus butyrate treatment. On the basis of these data, we propose that the combination of DHA and butyrate compared with butyrate alone further enhances colonocyte apoptosis by inducing a p53-independent, oxidation-sensitive, mitochondrial Ca2+-dependent (intrinsic) pathway.
n-3 polyunsaturated fatty acids; chemoprevention; fish oil; fermentable fiber
Colorectal cancer is one of the major causes of cancer-related mortality in both men and women worldwide. Genetic susceptibility and diet are primary determinants of cancer risk and tumor behavior. Experimental, epidemiological, and clinical data substantiate the beneficial role of n–3 polyunsaturated fatty acids (PUFA) in preventing chronic inflammation and colon cancer. From a mechanistic perspective, n–3 PUFA are pleiotropic and multifaceted with respect to their molecular mechanisms of action. For example, this class of dietary lipid uniquely alters membrane structure/ cytoskeletal function, impacting membrane receptor function and downstream signaling cascades, including gene expression profiles and cell phenotype. In addition, n–3 PUFA can synergize with other potential anti-tumor agents, such as fermentable fiber and curcumin. With the rising prevalence of diet-induced obesity, there is also an urgent need to elucidate the link between chronic inflammation in adipose tissue and colon cancer risk in obesity. In this review, we will summarize recent developments linking n–3 PUFA intake, membrane alterations, epigenetic modulation, and effects on obesity-associated colon cancer risk.
(n–3) PUFA; Colon cancer; Membrane rafts; Cytoskeleton; Epigenetics; Obesity
The early postnatal period is a critical window for intestinal and immune maturation. Intestinal development and microbiome diversity and composition differ between breast- (BF) and formula-fed (FF) infants. Mechanistic examination into host-microbe relationships in healthy infants has been hindered by ethical constraints surrounding tissue biopsies. Thus, a statistically rigorous analytical framework to simultaneously examine both host and microbial responses to dietary/environmental factors using exfoliated intestinal epithelial cells was developed. Differential expression of ~1,200 genes, including genes regulating intestinal proliferation, differentiation and barrier function, was observed between BF and FF term infants. Canonical correlation analysis uncovered a relationship between microbiome virulence genes and host immunity and defense genes. Lastly, exfoliated cells from preterm and term infants were compared. Pathways associated with immune cell function and inflammation were up-regulated in preterm, whereas cell growth-related genes were up-regulated in the term infants. Thus, coordinate measurement of the transcriptomes of exfoliated epithelial cells and microbiome allows inquiry into mutualistic host-microbe interactions in the infant, which can be used to prospectively study gut development or, retrospectively, to identify potential triggers of disease in banked samples.
infant; intestine; nutrition; microbiome; gene expression; exfoliated cells
Glycerol-3-phosphate acyltransferase-1 is the first rate limiting step in de novo glycerophospholipid synthesis. We have previously demonstrated that GPAT-1 deletion can significantly alter T cell function resulting in a T cell phenotype similar to that seen in aging. Recent studies have suggested that changes in the metabolic profile of T cells are responsible for defining specific effector functions and T cell subsets. Therefore, we determined whether T cell dysfunction in GPAT-1 −/− CD4+ T cells could be explained by changes in cellular metabolism. We show here for the first time that GPAT-1 −/− CD4+ T cells exhibit several key metabolic defects. Striking decreases in both the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were observed in GPAT-1 −/− CD4+ T cells following CD3/CD28 stimulation indicating an inherent cellular defect in energy production. In addition, the spare respiratory capacity (SRC) of GPAT-1 −/− CD4+ T cells, a key indicator of their ability to cope with mitochondrial stress was significantly decreased. We also observed a significant reduction in mitochondrial membrane potential in GPAT-1 −/− CD4+ T cells compared to their WT counterparts, indicating that GPAT-1 deficiency results in altered or dysfunctional mitochondria. These data demonstrate that deletion of GPAT-1 can dramatically alter total cellular metabolism under conditions of increased energy demand. Furthermore, altered metabolic response following stimulation may be the defining mechanism underlying T cell dysfunction in GPAT-1 −/− CD4+ T cells. Taken together, these results indicate that GPAT-1 is essential for the response to the increased metabolic demands associated with T cell activation.
Docosahexaenoic acid (DHA, 22:6 n-3) from fish oil, and butyrate, a fiber fermentation product, work coordinately to protect against colon tumorigenesis in part by inducing apoptosis. We have recently demonstrated that dietary DHA is incorporated into mitochondrial membrane phospholipids, thereby enhancing oxidative stress induced by butyrate metabolism. In order to elucidate the subcellular origin of oxidation induced by DHA and butyrate, immortalized young adult mouse colonocytes were treated with 0–200 μM DHA or linoleic acid (LA, 18:2 n-6; control) for 72 h with or without 5 mM butyrate for the final 24 h. Cytosolic reactive oxygen species, membrane lipid oxidation, and mitochondrial membrane potential (MP), were measured by live-cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells exhibited a 151% increase in lipid oxidation (P < 0.01), compared with no butyrate treatment, which could be blocked by a mitochondria-specific antioxidant, 10-(6′-ubiquinoyl) decyltriphenylphosphonium bromide (MitoQ) (P < 0.05). Butyrate treatment of LA pretreated cells did not show any significant effect. In the absence of butyrate, DHA treatment, compared with LA, increased resting MP by 120% (P < 0.01). In addition, butyrate-induced mitochondrial membrane potential (MP), dissipation was 21% greater in DHA primed cells as compared with LA at 6 h. This effect was reversed by preincubation with inhibitors of the mitochondrial permeability transition pore, cyclosporin A or bongkrekic acid (1 μM). The functional importance of these events is supported by the demonstration that DHA and butyrate-induced apoptosis is blocked by MitoQ. These data indicate that DHA and butyrate potentiate mitochondrial lipid oxidation and the dissipation of MP which contribute to the induction of apoptosis.
The underlying mechanisms by which n-3 polyunsaturated fatty acids (PUFA) exert a chemopreventive effect in the colon have not been elucidated. Retinoid X receptors (RXR) are a family of nuclear receptors implicated in cancer chemoprevention. Since docosahexaenoic acid (DHA), an n-3 PUFA enriched in fish oil, reduces colonocyte proliferation and enhances apoptosis relative to n-6 PUFA-treated cells, we determined whether DHA can serve as a specific ligand for RXRα activation relative to n-6 PUFA in colonocytes. In a mammalian one-hybrid assay, immortalized young adult mouse colonic (YAMC) cells were co-transfected with a yeast galactose upstream activating sequence (UAS)4-tk-Luciferase (Luc) reporter plasmid, plus either GAL4 DNA-binding domain fused to RXRα, retinoic acid receptor α or GAL4 alone, followed by an n-3, n-6 or n-9 fatty acid incubation. Luc activity levels were dose-dependently elevated only in n-3 PUFA (DHA)-treated RXRα. Since RXR homodimers and RXR/peroxisome proliferator-activated receptor (PPAR) heterodimers bind consensus direct repeat (DR1) motifs, YAMC and NCM460 (a normal human colonic cell line), were respectively, co-transfected with RXRα and DR1-Luc, followed by different PUFA treatment. Luc activity levels were increased (P < 0.05) only in DHA groups. The DHA-dependent induction of DR-1-Luc was reduced to basal levels upon RXRα antagonist-treatment, with no effect on PPARγ antagonist-treatment. A role for select RXR isoforms in colonocyte biology was also determined by examining nuclear receptor mRNA levels in rat colon following dietary lipid and carcinogen exposure over time. RXRα, RXRβ and RXRγ were detected in rat colonic mucosa, and the levels of RXRα and RXRγ were elevated in fish oil (n-3 PUFA) versus corn oil (n-6 PUFA) fed animals after 16 weeks. These data indicate that, RXRα, an obligatory component of various nuclear receptors, preferentially binds n-3 PUFA in colonocytes, and that the nuclear receptor targets for PUFA in the colon are modulated by dietary lipid exposure.
Dietary n-3 PUFAs have been shown to attenuate T-cell-mediated inflammation. To investigate whether dietary n-3 PUFAs promote activation-induced cell death (AICD) in CD4+ T-cells induced in vitro to a polarized T-helper1 (Th1) phenotype, C57BL/6 mice were fed diets containing either 5% corn oil (CO; n-6 PUFA control) or 4% fish oil (FO) plus 1% CO (n-3 PUFA) for 2 weeks. Splenic CD4+ T-cells were cultured with α-interleukin-4 (αIL-4), IL-12, and IL-2 for 2 days and then with recombinant (r) IL-12 and rIL-2 for 3 days in the presence of diet-matched homologous mouse serum (HMS) to prevent loss of cell membrane fatty acids, or with fetal bovine serum. After polarization, Th1 cells were reactivated and analyzed for interferon-γ and IL-4 by intracellular cytokine staining and for apoptosis by Annexin V/propidium iodide. Dietary FO enhanced Th1 polarization by 49% (P = 0.0001) and AICD by 24% (P = 0.0001) only in cells cultured in the presence of HMS. FO enhancement of Th1 polarization and AICD after culture was associated with the maintenance of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) in plasma membrane lipid rafts. In conclusion, n-3 PUFAs enhance the polarization and deletion of proinflammatory Th1 cells, possibly as a result of alterations in membrane micro-domain fatty acid composition.
docosahexaenoic acid; eicosapentaenoic acid; lipid rafts; interferon-γ
The mechanisms by which n-3 polyunsaturated fatty acids (PUFAs) decrease colon tumor formation have not been fully elucidated. Examination of genes up- or down-regulated at various stages of tumor development via the monitoring of gene expression relationships will help to determine the biological processes ultimately responsible for the protective effects of n-3 PUFA. Therefore, using a 3 × × × 2 factorial design, we used Codelink DNA microarrays containing ∼9000 genes to help decipher the global changes in colonocyte gene expression profiles in carcinogen-injected Sprague Dawley rats. Animals were assigned to three dietary treatments differing only in the type of fat (corn oil/n-6 PUFA, fish oil/n-3 PUFA, or olive oil/n-9 monounsaturated fatty acid), two treatments (injection with the carcinogen azoxymethane or with saline), and two time points (12 hours and 10 weeks after first injection). Only the consumption of n-3 PUFA exerted a protective effect at the initiation (DNA adduct formation) and promotional (aberrant crypt foci) stages. Importantly, microarray analysis of colonocyte gene expression profiles discerned fundamental differences among animals treated with n-3 PUFA at both the 12 hours and 10-week time points. Thus, in addition to demonstrating that dietary fat composition alters the molecular portrait of gene expression profiles in the colonic epithelium at both the initiation and promotional stages of tumor development, these findings indicate that the chemopreventive effect of fish oil is due to the direct action of n-3 PUFA and not to a reduction in the content of n-6 PUFA.
Recent studies indicate that the process of Ag presentation induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the immunologic synapse (IS). This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca2+ influx and Th cell activation. Recently, we demonstrated that n−3 polyunsaturated fatty acids (PUFAs) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n−3 PUFAs suppress CD4+ T cell mitochondrial translocation during the early stages of IS formation and downmodulate Ca2+-dependent Th cell activation. CD4+ cells derived from fat-1 mice, a transgenic model that synthesizes n−3 PUFA from n−6 PUFA, were cocultured with anti-CD3–expressing hybridoma cells (145-2C11) for 15 min at 37°C, and mitochondrial translocation to the IS was assessed by confocal microscopy. Fat-1 mice exhibited a significantly (p < 0.05) reduced percentage of T cells with mitochondria which translocated to the IS; fat-1 (30%) versus wild type control (82%). Regarding the effect on the mitochondrial-to-cytosolic Ca2+ ratio, wild type cells showed significant increases at the IS (71%) and total cell (60%) within 30 min of IS formation. In contrast, fat-1 CD4+ T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca2+ ratio was observed in wild type cells that were coincubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca2+ (CRAC) channels, BTP2. These observations provide evidence that n−3 PUFAs modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca2+entry.
Both fish oil (FO) and curcumin have potential as anti-tumour and anti-inflammatory agents. To further explore their combined effects on dextran sodium sulphate (DSS)-induced colitis, C57BL/6 mice were randomised to four diets (2 × 2 design) differing in fatty acid content with or without curcumin supplementation (FO, FO + 2 % curcumin, maize oil (control, MO) or MO + 2 % curcumin). Mice were exposed to one or two cycles of DSS in the drinking-water to induce either acute or chronic intestinal inflammation, respectively. FO-fed mice exposed to the single-cycle DSS treatment exhibited the highest mortality (40 %, seventeen of forty-three) compared with MO with the lowest mortality (3 %, one of twenty-nine) (P = 0·0008). Addition of curcumin to MO increased (P = 0·003) mortality to 37 % compared with the control. Consistent with animal survival data, following the one- or two-cycle DSS treatment, both dietary FO and curcumin promoted mucosal injury/ulceration compared with MO. In contrast, compared with other diets, combined FO and curcumin feeding enhanced the resolution of chronic inflammation and suppressed (P < 0·05) a key inflammatory mediator, NF-κB, in the colon mucosa. Mucosal microarray analysis revealed that dietary FO, curcumin and FO plus curcumin combination differentially modulated the expression of genes induced by DSS treatment. These results suggest that dietary lipids and curcumin interact to regulate mucosal homeostasis and the resolution of chronic inflammation in the colon.
Colitis; Resolution of inflammation; Mucosal repair
Besides their health benefits, dietary omega-3 fatty acids (n-3 PUFAs) can impair host resistance to intracellular pathogens. Previously, we and others have showed that n-3 PUFA–treated macrophages poorly control Mycobacterium tuberculosis infection in vitro.
Wild-type and fat-1 transgenic mice were infected with virulent H37Rv M. tuberculosis via the aerosol route. We evaluated bacteriological and histopathological changes in lungs, as well as differences in activation and antimycobacterial capacity in primary macrophages ex vivo.
fat-1 mice were more susceptible to tuberculosis, as demonstrated by higher bacterial loads and less robust inflammatory responses in lungs. Macrophages obtained from fat-1 mice were more readily infected with M. tuberculosis in vitro, compared with wild-type macrophages. This impaired bacterial control in cells from fat-1 mice correlated with reduced proinflammatory cytokine secretion, impaired oxidative metabolism, and diminished M. tuberculosis–lysotracker colocalization within phagosomes.
We showed that endogenous production of n-3 PUFAs in fat-1 mice increases their susceptibility to tuberculosis, which could be explained in part by diminished activation and antimycobacterial responses in cells from fat-1 mice. These data suggest that n-3 PUFA–supplemented diets might have a detrimental effect on immunity to M. tuberculosis and raise concerns regarding the safety of omega-3 dietary supplementation in humans.
Every 4 years, researchers in the cross-disciplinary field of nutritional immunology convene for a FASEB-sponsored meeting entitled, “Nutritional Immunology: Role in Health and Disease”, which was held this summer in Carefree, AZ, USA. The scope of the conference encompassed a diverse list of research topics, including, but not restricted to, obesity and immune dysfunction, nutrient–gene interactions, mucosal immunity and a discussion of future directions for the field. Here, we summarize some of the findings shared at the conference, specifically focusing on obesity, immunological function of dietary components (n-3 polyunsaturated fatty acids and flavanoids), gut immunity and the microbiota, and relevant emerging technologies and databases.
diet; infectious disease; inflammation; mucosal immunity; n-3 fatty acids; obesity; phytochemicals; Toll-like receptor; vitamin A
This study demonstrates 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
Arachidonic acid (20:4∆5,8,11,14, 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∆5,8,11,14,17, 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)-creERT2 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.