Microbiota-Dependent Hepatic Lipogenesis Mediated by Stearoyl CoA Desaturase 1 (SCD1) Promotes Metabolic Syndrome in TLR5-Deficient Mice.

The gut microbiota plays a key role in host metabolism. Toll-like receptor 5 (TLR5), a flagellin receptor, is required for gut microbiota homeostasis. Accordingly, TLR5-deficient (T5KO) mice are prone to develop microbiota-dependent metabolic syndrome. Here we observed that T5KO mice display elevated neutral lipids with a compositional increase of oleate [C18:1 (n9)] relative to wild-type littermates. Increased oleate contribution to hepatic lipids and liver SCD1 expression were both microbiota dependent. Analysis of short-chain fatty acids (SCFAs) and (13)C-acetate label incorporation revealed elevated SCFA in ceca and hepatic portal blood and increased liver de novo lipogenesis in T5KO mice. Dietary SCFAs further aggravated metabolic syndrome in T5KO mice. Deletion of hepatic SCD1 not only prevented hepatic neutral lipid oleate enrichment but also ameliorated metabolic syndrome in T5KO mice. Collectively, these results underscore the key role of the gut microbiota-liver axis in the pathogenesis of metabolic diseases.

Inhibition of MDR1 gene expression and enhancing cellular uptake for effective colon cancer treatment using dual-surface-functionalized nanoparticles.

Nanomedicine options for colon cancer therapy have been limited by the lack of suitable carriers capable of delivering sufficient drug into tumors to cause lethal toxicity. To circumvent this limitation, we fabricated a camptothecin (CPT)-loaded poly(lactic-co-glycolic acid) nanoparticle (NP) with dual-surface functionalization-Pluronic F127 and chitosan-for inhibiting multi-drug resistant gene 1 (MDR1) expression and enhancing tumor uptake. The resultant spherical NPs-P/C had a desirable particle size (∼268 nm), slightly positive zeta-potential, and the ability to efficiently down-regulate the expression of MDR1. In vitro cytotoxicity tests revealed that the 24 and 48 h IC50 values of NPs-P/C1 were 2.03 and 0.67 µm, respectively, which were much lower than those for free CPT and other NPs. Interestingly, NPs-P/C1 showed the highest cellular uptake efficiency (approximately 85.5%) among the different drug formulations. Most importantly, treatment of colon tumor-bearing mice with various drug formulations confirmed that the introduction of Pluronic F127 and chitosan to the NP surface significantly enhanced the therapeutic efficacy of CPT, induced tumor cell apoptosis, and reduced systemic toxicity. Collectively, these findings suggest that our one-step-fabricated, dual-surface-functionalized NPs may hold promise as a readily scalable and effective drug carrier with clinical potential in colon cancer therapy.

Longitudinal study of circulating protein biomarkers in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) are chronic and progressive inflammatory disorders of the gastrointestinal tract. In IBD, protein serological biomarkers could be relevant tools for assessing disease activity, performing early-stage diagnosis and managing the treatment. Using the interleukin-10 knockout (IL-10(-/-)) mouse, a model that develops a time-dependent IBD-like disorder that predominates in the colon; we performed longitudinal studies of circulating protein biomarkers in IBD. Circulating protein profiles in serum samples collected from 30-, 93-, to 135-day-old IL-10(-/-) mice were investigated using two-dimensional differential gel electrophoresis and MALDI-TOF/TOF tandem mass spectrometry. A total of 15 different proteins were identified and confirmed by ELISA and Western blot to be differentially accumulated in serum samples from mid- to late-stage IL-10(-/-) mice compared to early non-inflamed IL-10(-/-) mice. The use of another model of colitis and an extra-intestinal inflammation model validated this biomarker panel and demonstrated that comprised some global inflammatory markers, some intestinal inflammation-specific markers and some chronic intestinal inflammation markers. Statistical analyses using misclassification error rate charts validated the use of these identified proteins as powerful biomarkers of colitis. Unlike standard biomarker screening studies, our analyses identified a panel of proteins that allowed the definition of protein signatures that reflect colitis status. BIOLOGICAL SIGNIFICANCE: Crohn's disease (CD) and ulcerative colitis (UC) are the most common inflammatory bowel diseases (IBDs) occurring in humans. The major current diagnosis tool is colonoscopy, which is invasive and could lead to false diagnosis. The emergence of serological biomarkers enables the use of new diagnosis tools such as protein signatures for IBD diagnosis/management. Using 2D-DIGE coupled to mass spectrometry, our longitudinal study in a mouse model of colitis identified a signature of protein biomarkers for specific stages of disease.

Mammalian gut immunity.

The mammalian intestinal tract is the largest immune organ in the body and comprises cells from non-hemopoietic (epithelia, Paneth cells, goblet cells) and hemopoietic (macrophages, dendritic cells, T-cells) origin, and is also a dwelling for trillions of microbes collectively known as the microbiota. The homeostasis of this large microbial biomass is prerequisite to maintain host health by maximizing beneficial symbiotic relationships and minimizing the risks of living in such close proximity. Both microbiota and host immune system communicate with each other to mutually maintain homeostasis in what could be called a "love-hate relationship." Further, the host innate and adaptive immune arms of the immune system cooperate and compensate each other to maintain the equilibrium of a highly complex gut ecosystem in a stable and stringent fashion. Any imbalance due to innate or adaptive immune deficiency or aberrant immune response may lead to dysbiosis and low-grade to robust gut inflammation, finally resulting in metabolic diseases.

Nanoparticles with surface antibody against CD98 and carrying CD98 small interfering RNA reduce colitis in mice.

BACKGROUND & AIMS: Nanoparticles have been explored as carriers of small interfering RNAs (siRNAs) and might be developed to treat patients with inflammatory bowel disease (IBD). Overexpression of CD98 on the surface of colonic epithelial cells and macrophages promotes the development and progression of IBD. We developed an orally delivered hydrogel that releases nanoparticles with single-chain CD98 antibodies on their surface (scCD98 functionalized) and loaded with CD98 siRNA (siCD98). We tested the ability of the nanoparticles to reduce levels of CD98 in the colons of mice with colitis. METHODS: scCD98-functionalized siCD98-loaded nanoparticles were fabricated using a complex coacervation technique. We investigated the cellular uptake and lysosome escape profiles of the nanoparticles in Colon-26 cells and RAW 264.7 macrophages using fluorescence microscopy. Colitis was induced by transfer of CD4(+)CD45RB(high) T cells to Rag(-/-) mice or administration of dextran sodium sulfate to C57BL/6 mice. Mice were then given hydrogel (chitosan and alginate) containing scCD98-functionalized nanoparticles loaded with siCD98 or scrambled siRNA (control) via gavage. RESULTS: The scCD98-functionalized nanoparticles were approximately 200 nm in size and had high affinity for CD98-overexpressing cells. The scCD98-functionalized siCD98-loaded nanoparticles significantly reduced levels of CD98 in Colon-26 cells and RAW 264.7 macrophages, along with production of inflammatory cytokines (tumor necrosis factor α, interleukin-6, and interleukin-12). In mice with colitis, administration of the scCD98-functionalized siCD98-loaded nanoparticles reduced colon expression of CD98. Importantly, the severity of colitis was also reduced compared with controls (based on loss of body weight, myeloperoxidase activity, inflammatory cytokine production, and histological analysis). Approximately 24.1% of colonic macrophages (CD11b(+)CD11c(-)F4/80(+)) in the mice had taken up fluorescently labeled siRNA-loaded nanoparticles within 12 hours of administration. CONCLUSIONS: Nanoparticles containing surface CD98 antibody and loaded with siCD98 reduce expression of this protein by colonic epithelial cells and macrophages, and oral administration decreases the severity of colitis in mice. This nanoparticle in hydrogel (chitosan/alginate) formulation might be developed to treat patients with IBD.

Dextran sodium sulfate inhibits the activities of both polymerase and reverse transcriptase: lithium chloride purification, a rapid and efficient technique to purify RNA.

BACKGROUND: Dextran sodium sulfate (DSS) is commonly used in mouse studies to induce a very reproducible colitis that effectively mimics the clinical and histological features of human inflammatory bowel disease (IBD) patients, especially ulcerative colitis. However, the mechanisms of action of DSS remain poorly understood, and observations by our laboratory and other groups indicate that DSS contamination of colonic tissues from DSS-treated mice potently inhibits the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) amplification of mRNA. RESULTS: A prior study used poly-A-mediated mRNA purification to remove DSS from RNA extracts, but we herein report a second efficient and cost-effective approach to counteract this inhibition, using lithium chloride precipitation to entirely remove DSS from RNAs. We also explored how DSS interferes with qRT-PCR process, and we report for the first time that DSS can alter the binding of reverse transcriptase to previously primed RNA and specifically inhibits the enzymatic activities of reverse transcriptase and Taq polymerase in vitro. This likely explains why DSS-treated colonic RNA is not suitable to qRT-PCR amplification without a previous purification step. CONCLUSION: In summary, we provide a simple method to remove DSS from colonic RNAs, and we demonstrate for the first time that DSS can inhibit the activities of both polymerase and reverse transcriptase. In order to reliably analyze gene expression in the colonic mucosa of DSS-treated mice, the efficiency rate of qRT-PCR must be the same between all the different experimental groups, including the water-treated control group, suggesting that whatever the duration and the percentage of the DSS treatment, RNAs must be purified.

Knockout of Ste20-like proline/alanine-rich kinase (SPAK) attenuates intestinal inflammation in mice.

Inflammatory bowel diseases are characterized by epithelial barrier disruption and alterations in immune regulation. Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but the underlying mechanisms need to be defined. Herein, SPAK knockout (KO) C57BL/6 mice exhibited significant increases in intestinal transepithelial resistance, a marked decrease in paracellular permeability to fluorescence isothiocyanate-dextran, and altered apical side tight junction sodium ion selectivity, compared with wild-type mice. Furthermore, the expression of junction protein, claudin-2, decreased. In contrast, expressions of occludin, E-cadherin, ß-catenin, and claudin-5 increased significantly, whereas no obvious change of claudin-1, claudin-4, zonula occludens protein 1, and zonula occludens protein 2 expressions was observed. In murine models of colitis induced by dextran sulfate sodium and trinitrobenzene sulfuric acid, KO mice were more tolerant than wild-type mice, as demonstrated by colonoscopy features, histological characteristics, and myeloperoxidase activities. Consistent with these findings, KO mice showed increased IL-10 levels and decreased proinflammatory cytokine secretion, ameliorated bacterial translocation on treatment with dextran sulfate sodium, and regulation of with no lysine (WNK) kinase activity. Together, these features may reduce epithelial permeability. In conclusion, SPAK deficiency increases intestinal innate immune homeostasis, which is important for control or attenuation of pathological responses in inflammatory bowel diseases.

Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-chain-length fatty acids in the colon.

Inflammatory bowel diseases (IBDs), primarily ulcerative colitis and Crohn's disease, are inflammatory disorders caused by multiple factors. Research on IBD has often used the dextran sodium sulfate (DSS)-induced colitis mouse model. DSS induces in vivo but not in vitro intestinal inflammation. In addition, no DSS-associated molecule (free glucose, sodium sulfate solution, free dextran) induces in vitro or in vivo intestinal inflammation. We find that DSS but not dextran associated molecules established linkages with medium-chain-length fatty acids (MCFAs), such as dodecanoate, that are present in the colonic lumen. DSS complexed to MCFAs forms nanometer-sized vesicles ~200 nm in diameter that can fuse with colonocyte membranes. The arrival of nanometer-sized DSS/MCFA vesicles in the cytoplasm may activate intestinal inflammatory signaling pathways. We also show that the inflammatory activity of DSS is mediated by the dextran moieties. The deleterious effect of DSS is localized principally in the distal colon, therefore it will be important to chemically modify DSS to develop materials beneficial to the colon without affecting colon-targeting specificity.