Scott T. Magness

High-Fat Diet: Bacteria Interactions Promote Intestinal Inflammation Which Precedes and Correlates with Obesity and Insulin Resistance in Mouse

Background Obesity induced by high fat (HF) diet is associated with inflammation which contributes to development of insulin resistance. Most prior studies have focused on adipose tissue as the source of obesity-associated inflammation. Increasing evidence links intestinal bacteria to development of diet-induced obesity (DIO). This study tested the hypothesis that HF western diet and gut bacteria interact to promote intestinal inflammation, which contributes to the progression of obesity and insulin resistance. Methodology/Principal Findings Conventionally raised specific-pathogen free (CONV) and germ-free (GF) mice were given HF or low fat (LF) diet for 2–16 weeks. Body weight and adiposity were measured. Intestinal inflammation was assessed by evaluation of TNF-α mRNA and activation of a NF-κBEGFP reporter gene. In CONV but not GF mice, HF diet induced increases in body weight and adiposity. HF diet induced ileal TNF-α mRNA in CONV but not GF mice and this increase preceded obesity and strongly and significantly correlated with diet induced weight gain, adiposity, plasma insulin and glucose. In CONV mice HF diet also resulted in activation of NF-κBEGFP in epithelial cells, immune cells and endothelial cells of small intestine. Further experiments demonstrated that fecal slurries from CONV mice fed HF diet are sufficient to activate NF-κBEGFP in GF NF-κBEGFP mice. Conclusions/Significance Bacteria and HF diet interact to promote proinflammatory changes in the small intestine, which precede weight gain and obesity and show strong and significant associations with progression of obesity and development of insulin resistance. To our knowledge, this is the first evidence that intestinal inflammation is an early consequence of HF diet which may contribute to obesity and associated insulin resistance. Interventions which limit intestinal inflammation induced by HF diet and bacteria may protect against obesity and insulin resistance.

Restriction of intestinal stem cell expansion and the regenerative response by YAP

A remarkable feature of regenerative processes is their ability to halt proliferation once an organ’s structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)—a protein known for its powerful growth-inducing and oncogenic properties—has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN–APC–GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.

A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations

Activation of hepatic stellate cells (HSCs) and other resident mesenchymal cells into myofibroblasts expressing alpha smooth muscle actin (αSMA) and collagen I is a key event in liver fibrogenesis. However, the temporal expression profiles of αSMA and collagen I genes in these cells is unknown. To address this question, we studied αSMA and collagen α1(I) transcriptional patterns in primary cultures of HSCs, and additionally, in an in vivo model of secondary biliary fibrosis using transgenic mice that express the Discomsoma sp. red fluorescent protein (RFP) and the enhanced green fluorescent protein (EGFP) reporter genes under direction of the mouse αSMA and collagen α1(I) promoter/enhancers, respectively. The αSMA‐RFP mice were crossed with collagen‐EGFP mice to generate double transgenic mice. Reporter gene expression in cultured HSCs demonstrated that both transgenes were induced at day 3 with continued expression through day 14. Interestingly, αSMA and collagen α1(I) transgenes were not coexpressed in all cells. Flow cytometry analysis showed three different patterns of gene expression: αSMA‐RFP positive cells, collagen‐EGFP positive cells, and cells expressing both transgenes. αSMA‐only and αSMA/collagen expressing cells showed higher expression levels of synaptophysin, reelin, MMP13, TIMP1, and ICAM‐1 compared to collagen‐only expressing cells, as assessed by real‐time PCR. Following bile duct ligation, αSMA and collagen α1(I) transgenes were differentially expressed by peribiliary, parenchymal and vascular fibrogenic cells. Peribiliary cells preferentially expressed collagen α1(I), while parenchymal myofibroblasts expressed both αSMA and collagen α1(I). In conclusion, these data demonstrate heterogeneity of gene expression in myofibroblastic cells during active fibrogenesis. These reporter mice provide a useful tool to further characterize fibrogenic cell types and to evaluate antifibrotic drugs. (HEPATOLOGY 2004.)

Distinct SOX9 levels differentially mark stem/progenitor populations and enteroendocrine cells of the small intestine epithelium.

SOX transcription factors have the capacity to modulate stem/progenitor cell proliferation and differentiation in a dose-dependent manner. SOX9 is expressed in the small intestine epithelial stem cell zone. Therefore, we hypothesized that differential levels of SOX9 may exist, influencing proliferation and/or differentiation of the small intestine epithelium. Sox9 expression levels in the small intestine were investigated using a Sox9 enhanced green fluorescent protein (Sox9(EGFP)) transgenic mouse. Sox9(EGFP) levels correlate with endogenous SOX9 levels, which are expressed at two steady-state levels, termed Sox9(EGFPLO) and Sox9(EGFPHI). Crypt-based columnar cells are Sox9(EGFPLO) and demonstrate enriched expression of the stem cell marker, Lgr5. Sox9(EGFPHI) cells express chromogranin A and substance P but do not express Ki67 and neurogenin3, indicating that Sox9(EGFPHI) cells are postmitotic enteroendocrine cells. Overexpression of SOX9 in a crypt cell line stopped proliferation and induced morphological changes. These data support a bimodal role for SOX9 in the intestinal epithelium, where low SOX9 expression supports proliferative capacity, and high SOX9 expression suppresses proliferation.

Isolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay

BACKGROUND & AIMS Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. METHODS We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5-green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. RESULTS CD44(+)CD24(lo)CD166(+) cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell-associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44(+)CD24(lo)CD166(+) GRP78(lo/-) putative stem cells from mouse small intestine included Lgr5-GFP(hi) and Lgr5-GFP(med/lo) cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. CONCLUSIONS We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44(+)CD24(lo)CD166(+), GRP78(lo/-), and c-Kit(-) facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44(+)CD24(-/lo)CD166(+) also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.

Gnotobiotic IL-10−/−;NF-κBEGFP Mice Reveal the Critical Role of TLR/NF-κB Signaling in Commensal Bacteria-Induced Colitis

Commensal bacteria and TLR signaling have been associated with the maintenance of intestinal homeostasis in dextran sodium sulfate-induced intestinal injury. The aim of this study was to determine the in vivo role of TLR/NF-κB activation in a model of commensal bacteria-induced T cell-mediated colitis. A NF-κB reporter gene mouse (NF-κBEGFP) (EGFP, enhanced GFP) was crossed to the colitogenic susceptible strain IL-10−/− and derived into germfree conditions using embryo-transfer technology. Germfree IL-10wt/wt;NF-κBEGFP and IL-10−/−;NF-κBEGFP mice (wt, wild type) were dual associated with the nonpathogenic commensal bacteria strains Enterococcus faecalis and Escherichia coli. EGFP was detected using macroimaging, confocal microscopy, and flow cytometry. IL-10−/−;MyD88−/− mice were used to assess E. faecalis/E. coli-induced TLR-dependent signaling and IL-23 gene expression. Dual-associated IL-10−/−;NF-κBEGFP mice developed severe inflammation by 7 wk. Macroscopic analysis showed elevated EGFP expression throughout the colon of bacteria-associated IL-10−/−;NF-κBEGFP mice. Confocal microscopy analysis revealed EGFP-positive enterocytes during the early phase of bacterial colonization (1 wk) in both IL-10wt/wt and IL-10−/− mice, while the signal shifted toward lamina propria T cells, dendritic cells, neutrophils, and macrophages in IL-10−/− mice during colitis (7 wk). The NF-κB inhibitor BAY 11-7085 attenuated E. faecalis/E. coli-induced EGFP expression and development of colitis. Additionally, E. faecalis/E. coli-induced NF-κB signaling and IL-23 gene expression were blocked in bone marrow-derived dendritic cells derived from IL-10−/−;MyD88−/− mice. We conclude that bacteria-induced experimental colitis involves the activation of TLR-induced NF-κB signaling derived mostly from mucosal immune cells. Blocking TLR-induced NF-κB activity may represent an attractive strategy to treat immune-mediated intestinal inflammation.

Activation of two distinct Sox9-EGFP-expressing intestinal stem cell populations during crypt regeneration after irradiation.

Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.

A nomenclature for intestinal in vitro cultures

Many advances have been reported in the long-term culture of intestinal mucosal cells in recent years. A significant number of publications have described new culture media, cell formations, and growth patterns. Furthermore, it is now possible to study, e.g., the capabilities of isolated stem cells or the interactions between stem cells and mesenchyme. However, at the moment there is significant variation in the way these structures are described and named. A standardized nomenclature would benefit the ability to communicate and compare findings from different laboratories using the different culture systems. To address this issue, members of the NIH Intestinal Stem Cell Consortium herein propose a systematic nomenclature for in vitro cultures of the small and large intestine. We begin by describing the structures that are generated by preparative steps. We then define and describe structures produced in vitro, specifically: enterosphere, enteroid, reconstituted intestinal organoid, induced intestinal organoid, colonosphere, colonoid, and colonic organoid.

In Vivo Pattern of Lipopolysaccharide and Anti-CD3-Induced NF-κB Activation Using a Novel Gene-Targeted Enhanced GFP Reporter Gene Mouse

NF-κB is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-κB has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the kinetics and cellular localization of NF-κB-induced transcription, we created a transgenic mouse expressing the enhanced GFP (EGFP) under the transcriptional control of NF-κB cis elements (cis-NF-κBEGFP). A gene-targeting approach was used to insert a single copy of a NF-κB-dependent EGFP reporter gene 5′ of the X-linked hypoxanthine phosphoribosyltransferase locus in mouse embryonic stem cells. Embryonic fibroblasts, hepatic stellate cells, splenocytes, and dendritic cells isolated from cis-NF-κBEGFP mice demonstrated a strong induction of EGFP in response to LPS, anti-CD3, or TNF-α that was blocked by the NF-κB inhibitors BAY 11-0782 and NEMO-binding peptide. Chromatin immunoprecipitation analysis demonstrated RelA binding to the cis-NF-κBEGFP promoter. Adenoviral delivery of NF-κB-inducing kinase strongly induced EGFP expression in the liver of cis-NF-κBEGFP mice. Similarly, mice injected with anti-CD3 or LPS showed increased EGFP expression in mononuclear cells, lymph node, spleen, and liver as measured by flow cytometry and/or fluorescence microscopy. Using whole organ imaging, LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. Confocal analysis indicated EGFP expression was primarily found in lamina propria mononuclear cells. In summary, the cis-NF-κBEGFP mouse will serve as a valuable tool to address multiple questions regarding the cell-specific and real-time activation of NF-κB during normal and diseased states.