A20, encoded by the TNFAIP3 gene, is a protein linked to Crohn's disease and celiac disease in humans. We now find that mice expressing point mutations in A20's M1 ubiquitin binding motif (ZF7) spontaneously develop proximate enteritis that requires both luminal microbes and T cells. Cellular and transcriptomic profiling reveal expansion of TH17/22 cells and aberrant expression of IL-17A and IL-22 in intestinal lamina propria of A20ZF7 mice. While deletion of IL-17A from A20ZF7/ZF7 mice exacerbates enteritis, deletion of IL-22 abrogates intestinal epithelial cell hyperproliferation, barrier dysfunction, and alarmin expression. A20ZF7/ZF7 TH17/22 cells autonomously express more RORγt and IL-22 after differentiation in vitro. ATAC sequencing identified an enhancer region upstream of the Il22 gene in A20ZF7/ZF7 T cells, and this enhancer demonstrated increased activating histone acetylation coupled with exaggerated Il22 transcription. Finally, CRISPR/Cas9-mediated ablation of A20ZF7 in human T cells increases RORγt expression and IL22 transcription. These studies link A20's M1 ubiquitin binding function with RORγt expression, epigenetic activation of TH17/22 cells, and IL-22 driven enteritis. ### Competing Interest Statement The authors have declared no competing interest.

The incidence of colorectal cancer (CRC) is closely linked to metabolic diseases. Accumulating evidence suggests the regulatory role of AMP-activated protein kinase (AMPK) in cancer metabolic reprogramming. In this study, wild-type and AMPK knockout mice were subjected to azoxymethane-induced and dextran sulfate sodium (AOM/DSS)-promoted colitis-associated CRC induction. A stable AMPK-deficient Caco-2 cell line was also established for the mechanistic studies. The data showed that AMPK deficiency accelerated CRC development, characterized by increased tumor number, tumor size, and hyperplasia in AOM/DSS-treated mice. The aggravated colorectal tumorigenesis resulting from AMPK ablation was associated with reduced α-ketoglutarate production and ten-eleven translocation hydroxylase 2 (TET2) transcription, correlated with the reduced mismatch repair protein mutL homolog 1 (MLH1) protein. Furthermore, in AMPK-deficient Caco-2 cells, the mRNA expression of mismatch repair and tumor suppressor genes, intracellular α-ketoglutarate, and the protein level of TET2 were also downregulated. AMPK deficiency also increased hypermethylation in the CpG islands of Mlh1 in both colonic tissues and Caco-2 cells. In conclusion, AMPK deficiency leads to reduced α-ketoglutarate concentration and elevates the suppressive epigenetic modifications of tumor suppressor genes in gut epithelial cells, thereby increasing the risk of colorectal tumorigenesis. Given the modifiable nature of AMPK activity, it holds promise as a prospective molecular target for the prevention and treatment of CRC.

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of 2 IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20- and Abin-1–deficient IECs were sensitized to TNF-independent, TNFR1-mediated death in response to lymphotoxin α (LTα) homotrimers. Blockade of LTα in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.

ABSTRACT Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of two IBD susceptibility genes, A20 ( Tnfaip3 ) and Abin-1 ( Tnip1 ), in intestinal epithelial cells (IECs) sensitizes mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion is rescued by germ-free derivation or deletion of MyD88 , while deletion of Trif provides only partial protection. Combined deletion of Ripk3 and Casp8 , which inhibits both apoptotic and necroptotic death, completely protects against death after acute deletion of A20 and Abin-1 in IECs. A20 and Abin-1 -deficient IECs are sensitized to TNF-independent, TNFR-1-mediated death in response to lymphotoxin alpha (LTα) homotrimers. Blockade of LTα in vivo reduces weight loss and improves survival when combined with partial deletion of MyD88 . These data show that microbial signals, MyD88 , and LTα all contribute to TNF-independent intestinal injury. SUMMARY Here we show that germ-free derivation, MyD88 deletion, combined Ripk3 and Casp8 deletion, or anti-LTα, all reduce TNF-independent intestinal injury after A20 and Abin-1 deletion.

Oral protein delivery technologies often depend on encapsulating or enclosing the protein cargo to protect it against pH-driven degradation in the stomach or enzymatic digestion in the small intestine. An emergent methodology is to encapsulate therapeutics in microscale, asymmetric, planar microparticles, referred to as microdevices. Previous work has shown that, compared to spherical particles, planar microdevices have longer residence times in the GI tract, but it remains unclear how specific design choices (e.g., material selection, particle diameter) impact microdevice behavior in vivo. Recent advances in microdevice fabrication through picoliter printing have expanded the range of device sizes that can be fabricated in a rapid manner. However, relatively little work has explored how device size governs their behavior in the intestinal environment. In this study, we probe the impact of geometry of planar microdevices on their transit and accumulation in the murine GI tract. Additionally, we present a strategy to label, image, and quantify these distributions in intact tissue in a continuous manner, enabling a more detailed understanding of device distribution and transit kinetics than previously possible. We show that smaller particles (194.6 ± 7 μm.diameter) tend to empty from the stomach faster than midsize (293.2 ± 7 μm.diameter) and larger devices (440.9 ± 9 μm.diameter) and that larger devices distribute more broadly in the GI tract and exit slower than other geometries. In general, we observed an inverse correlation between device diameter and GI transit rate. These results inform the future design of drug delivery systems, using particle geometry as an engineering design parameter to control device accumulation and distribution in the GI tract. Additionally, our image analysis process provides greater insight into the tissue level distribution and transit of particle populations. Using this technique, we demonstrate that microdevices act and translocate independently, as opposed to transiting in one homogeneous mass, meaning that target sites will likely be exposed to devices multiple times over the course of hours post administration. This imaging technique and associated findings enable data-informed design of future particle delivery systems, allowing orthogonal control of transit and distribution kinetics in vivo independent of material and cargo selection.

Selenium (Se) has been recognized as an essential dietary nutrient for decades, and organic Se sources rather than inorganic ones are increasingly advocated as Se supplements. Earthworms have been studied as a feed additive and animal protein source for many yr. The aim of this study was to evaluate the effect of Se-enriched earthworm powder (SEP) on the anti-oxidative ability and immunity of laying hens. A total of 120 27-wk-old laying hens were randomly divided into 4 groups (30 hens per group). Laying hens were fed diets supplemented with SEP having 0, 0.5, or 1 mg/kg of Se or with earthworm powder alone. After 5 wk of supplementation, serum from the hens was tested for nutritional components (protein, globulin, albumin, triglycerides, total cholesterol, and glucose), antioxidative properties (glutathione peroxidase, superoxide dismutase, catalase, and nitric oxide), and immune responses (lysozymes, immunoglobulin G, IL-2, and interferon gamma). We found that SEP with 1.0 mg/kg of Se upregulated the hens' total protein, albumin, glutathione peroxidase, superoxide dismutase, IgG, and IL-2 and downregulated triglycerides, total cholesterol, glucose, and nitric oxide. These results indicate that SEP improves antioxidative levels and immune function of laying hens, indicating potential benefit from use of SEP as a feed additive in the poultry industry.

A20 is an anti-inflammatory protein that is strongly linked to human disease. Here, we find that mice expressing three distinct targeted mutations of A20’s zinc finger 7 (ZF7) ubiquitin-binding motif uniformly developed digit arthritis with features common to psoriatic arthritis, while mice expressing point mutations in A20’s OTU or ZF4 motifs did not exhibit this phenotype. Arthritis in A20 ZF7 mice required T cells and MyD88, was exquisitely sensitive to tumor necrosis factor and interleukin-17A, and persisted in germ-free conditions. A20 ZF7 cells exhibited prolonged IκB kinase activity that drove exaggerated transcription of late-phase nuclear factor-κB response genes in vitro and in prediseased mouse paws in vivo. In addition, mice expressing double-mutant A20 proteins in A20’s ZF4 and ZF7 motifs died perinatally with multi-organ inflammation. Therefore, A20’s ZF4 and ZF7 motifs synergistically prevent inflammatory disease in a non-catalytic manner.

采用荧光光谱法、紫外-可见吸收光谱法与分子对接技术研究了棉酚降解产物四甲氧基棉酚与牛血清白蛋白(BSA)的相互作用.荧光猝灭结果表明四甲氧基棉酚对BSA的猝灭过程属于静态猝灭,结合常数在298 K和310 K时分别为3.68×105 L·mol-1和3.10×105 L·mol-1,结合位点数在298 K和310 K时分别为0.91和0.97.通过分析结合反应的热力学参数(ΔG<0,ΔH<0,ΔS>0),得出四甲氧基棉酚与BSA之间的结合反应为自发进行,主要靠疏水作用力和氢键结合.经紫外吸收光谱和同步荧光光谱研究发现四甲氧基棉酚使BSA的构象发生改变,其作用过程中主要影响色氨酸残基.分子对接实验进一步得到了四甲氧基棉酚在BSA上的结合位点位于IIA活性口袋.

Grape pomace (GP), a by-product of the wine and juice industry, is rich in bioflavonoids and dietary fibers. We hypothesized that GP has protective effects against colitis-associated colorectal cancer (CRC). Nine-week-old female mice were fed a control diet (CON) or CON with 5% grape pomace (GP) for 2 weeks, when mice were subjected to azoxymethane (AOM)/dextran sulfate sodium (DSS) induced-CRC induction. GP supplementation ameliorated the disease activity index (DAI) score, reduced tumor number, tumor size and pathological scores in AOM/DSS treated mice. Furthermore, dietary GP suppressed colonic expression of inflammatory cytokines, IL-1β and TNF-α, and inhibited NF-κB inflammatory signaling, while increased anti-inflammatory cytokine TGF-β mRNA expression. Colorectal inflammation is known to enhance Wnt signaling and cell proliferation. In agreement, the content of β-catenin, a key downstream mediator of Wnt signaling, was reduced as was the expression of Cyclin D1, phosphorylation and content of p53 and PCNA level in GP-fed mice. In addition, GP reduced the expression of ALDH1, a marker of cell stemness, and increased the expression of Cdx2, a key transcription factor initiating epithelial cell differentiation, DNA methylation of the promoter region of Cdx2 gene and hypermethylation of CpG island methylator phenotype (CIMP), which commonly occurs during CRC carcinogenesis, was alleviated in the GP group. In conclusion, GP supplementation suppressed colitis-associated CRC carcinogenesis, which was associated with the suppression of inflammation and cell proliferation and the enhancement of DNA demethylation in Cdx2 and CIMP genes in the colon. These data suggest that dietary GP supplementation has preventive effects against colorectal carcinogenesis.

Diet plays an important role in shaping the gut microbiota, which has been called “a separate organ” due to its profound effects on host health. Potato, being a rich source of phytonutrients and bioactive food components, becomes a functional food for improving gut microbiota and gut health. Potato phytonutrients and bioactive food components contribute to gastrointestinal health through direct interactions with the epithelium or indirectly through modulation of the gut microbiota, and microbially derived metabolites, such as short chain fatty acids (SCFAs). In addition, gut microbiota degrade polyphenols and phytonutrients, and increase their bioavailability and thus the functionality of complex macronutrients. This review discusses the potential role of potato in shaping gut microbiota, strengthening intestinal epithelial barrier function, and thereby improving gastrointestinal health.

BackgroundThe incidence of colorectal cancer (CRC) is closely related to metabolic diseases. AMP‐activated protein kinase (AMPK) is known as a key metabolic regulator. The objective of study is to assess the regulatory role of AMPK in metabolic alteration, epigenetic modification and colorectal tumorigenesis.MethodsAMPKα1‐floxed mice was cross‐bred with Lgr5Cre mice to specifically knockout AMPK α1 gene in intestinal stem cells and their derived epithelial cells. The wild‐type and AMPK knockout mice were subjected to azoxymethane (AOM)/DSS induced‐CRC induction.ResultsAMPK stem cell specific knockout caused colonic hyperproliferation and pathological features in mice without CRC induction; augmented adenocarcinoma number and size as well as dysperplasia in AOM/DSS‐indiced mice model. The aggravated colorectal tumorigenesis induced by AMPK ablation likely resulted from alteration of metabolites, as indicated by reduced production of acetyl‐CoA, a substrate required for histone acetylation, and α‐ketoglutarate, an obligate substrate for ten‐eleven translocation hydroxylases (TETs) mediated DNA demethylation, in colonic tissues. Consistently, α‐ketoglutarate producing isocitrate dehydrogenase 1 (IDH1) content and the activity TET1 were markably decreased in AMPK deficient mice, correlated with reduced metastasis inhibitors, Adamts1 (A disintegrin and metalloproteinase with thrombospondin motif 1) and Mal (myelin and lymphocyte), as well as hypermethylation of Mal and Mgmt (O‐6‐methylguanine‐DNA methyltransferase) promotor regions.ConclusionData suggested critical roles of AMPK in integrating metabolism with epigenetic modifications and colorectal tumorigenesis.Support or Funding InformationUSDA‐NIFA#2018‐67017‐27517 and WSU ERI competitive grantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Colorectal cancer (CRC) is one of the most common cause of cancer death. Phytochemicals, especially anthocyanins/anthocyanidins (A/A), have gathered attention of the scientific community owing to their anti-inflammatory, antioxidant, and cancer-inhibitory properties. In this review, we discussed the possible mechanisms whereby A/A exhibit intestinal anticarcinogenic characteristics. Anthocyanins/anthocyanidins inhibit the pro-inflammatory NF-κB pathway, attenuate Wnt signaling and suppress abnormal epithelial cell proliferation. In addition, A/A induce mitochondrial-mediated apoptosis and downregulate Akt/mTOR (mammalian target of rapamycin) pathway. Furthermore, activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) also contributes to the anti-carcinogenic effects of A/A. Finally, downregulation of metalloproteinases (MMPs) by A/A inhibits tumor invasion and metastasis. In conclusion, A/A exert their anti-tumor effects against colorectal carcinogenesis via multiple mechanisms, providing insights into the use of A/A as a natural chemopreventive intervention on major colorectal carcinogenesis.

This study evaluated the fate of Listeria innocua, a non-pathogenic species closely related to Listeria monocytogenes, on Fuji apple fruit surfaces during commercial cold storage with and without continuous low doses of gaseous ozone. Unwaxed Fuji apples of commercially acceptable maturity were inoculated with 6.0-7.0 Log(10) CFU L. innocua/apple, and subjected to refrigerated air (RA, 33 degrees F), controlled atmosphere (CA, 33 degrees F, 2% O-2, 1% CO2), or CA with low doses of ozone gas (50.0-87.0 ppb) storage in a commercial facility for 30 weeks. A set of uninoculated apples was simultaneously subjected to the above storage conditions for total plate count and yeasts and molds enumeration. L. innocua survival under RA and CA storage was similar, which led to 2.5-3.0 Log(10) CFU/apple reduction during storage. Continuous gaseous ozone application decreased L. innocua population on Fuji apples to similar to 1.0 Log(10) CFU/apple after 30-week storage, and suppressed apple native flora. CA storage delayed apple fruit ripening through reduction of apple firmness and titratable acidity loss, and low dose gaseous ozone application had no negative influence on apple visual quality, including both external and internal disorders. In summary, L. innocua decreased on Fuji apple surfaces during commercial long-term RA and CA storage. Ozone gas has the potential to be used as a supplemental intervention method to control Listeria spp. and to ensure fresh apple safety. (c) 2018 Elsevier Ltd. All rights reserved.

Gut epithelium covers the inner layer of the gastrointestinal tract and provides a physical barrier to separate the host from its external environment, and its barrier function is critical for maintaining host health. AMP-activated protein kinase (AMPK) as a master regulator of energy metabolism plays a critical role in epithelial barrier function. AMPK activation promotes epithelial differentiation and facilitates cell polarity establishment, both of which strengthen epithelial barrier. In addition, AMPK promotes the assembly of tight junctions and adherens junctions by direct phosphorylation of proteins composing apical junctions, junctional anchors, and cytoskeletons. Pharmacological and nutraceutical compounds, as well as physiological states triggering AMPK activation strengthen epithelial barrier function. This review summarized recent progress in delineating the regulatory roles of AMPK in apical junction formation and barrier function of intestinal epithelium.

Background. Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Present data has reported the role of anthocyanin-rich food/extract in increasing fecal short-chain fatty acids (SCFA) concentrations and NK cells infiltration in the large intestine mucosa, thus contributing to prevent preneoplastic lesions formation. Here we hypothesized whether carcinogeninduced aberrant crypt foci (ACF) progression in BALB/c mice fed a diet containing dietary Hibiscus sabdariffa L. (HS) is suppressed by modulation of fecal SCFA and NK cells infiltration. Furthermore, we also investigate whether such supplementation might induce hepatoprotective effects. Methods. Nutritional composition, total phenolic and total monomeric anthocyanin content were assessed in HS calyces. Preneoplastic colorectal lesions were induced in male BALB/c mice by injecting 1,2-dimethylhydrazine (20 mg/kg body weight) intraperitoneally. Mice were fed control or supplemented diet containing either 5 or 10% dietary HS for 14 weeks. ACF counts, fecal SCFA concentrations and leukocytes infiltration were assessed. Results. Polyphenol and anthocyanin contents in HS calyces were found to be 57.84 mg GAE/g dw HS and 7.81 mg cyanidin-3 glucoside/ dw HS, respectively. Supplementation with 5 or 10% dietary HS attenuated colonic ACF development in the distal colon (P < 0.01). Total ACF counts per mouse was reduced by almost 29.0% in HS supplemented groups when compared to control (P < 0.01). Fecal butyric and propionic acids concentrations, in addition to NK cell infiltration, were increased with 10% dietary HS supplementation. Hepatic catalase activity was enhanced in 10% dietary HStreated mice when compared to control group (P < 0.01). Conclusions. Colonic preneoplastic lesions in carcinogenic-induced male BALB/c mice are mitigated by HS dietary treatment probably due to modulation of SCFA and NK cell infiltration. We might also infer that 10% dietary HS is quite more effective when compared to 5%.

SCOPE:Butyrate, the fermentation end product of gut microbiota in the colon, is known for its antitumor effects, but the mechanisms remained to be defined. α-ketoglutarate (α-KG) mediates DNA demethylation and aberrant epigenetic modifications are associated with carcinogenesis. The objectives of this study are to evaluate the effects of butyrate on α-KG mediated epigenetic modification in colorectal adenocarcinoma HT-29 and Caco-2 cells.METHODS AND RESULTS:Butyrate suppressed proliferation, potentiated differentiation, and induced apoptosis in both HT-29 and Caco-2 cells, associated with enhanced expression of isocitrate dehydrogenase 1 (IDH1) and pyruvate dehydrogenase. Furthermore, butyrate upregulated acetyl-CoA and α-KG, concomitant with enhanced histone acetylation and DNA demethylation in the promoter of DNA mismatch repair (MMR) gene. Knocking down IDH1 abolished the positive effects of butyrate on CRC apoptosis and MMR protein expression, in conjunction with reduced α-KG content. Importantly, α-KG supplementation recovered the beneficial effects of butyrate in IDH1-deficient cells.CONCLUSION:In summary, butyrate inhibits indices of colorectal carcinogenesis in an α-KG-dependent manner.

Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent in vivo gut development, which requires intense cross-regulation of the epithelial layer with the underlying mesenchymal tissue. In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPKα1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α1 in intestinal health.