Altered microbial biogeography in an innate model of colitis.

Changes in the spatial organization, or biogeography, of colonic microbes have been observed in human inflammatory bowel disease (IBD) and mouse models of IBD. We have developed a mouse model of IBD that occurs spontaneously and consistently in the absence of adaptive immunity. Mice expressing tumor necrosis factor-induced protein 3 (TNFAIP3) in intestinal epithelial cells (villin-TNFAIP3) develop colitis when interbred with Recombination Activating 1-deficient mice (RAG1^-/-). The colitis in villin-TNFAIP3 × RAG1^-/- (TRAG) mice is prevented by antibiotics, indicating a role for microbes in this innate colitis. We therefore explored the biogeography of microbes and responses to antibiotics in TRAG colitis. Laser capture microdissection and 16S rRNA sequencing revealed altered microbial populations across the transverse axis of the colon as the inner mucus layer of TRAG, but not RAG1^-/-, mice was infiltrated by microbes, which included increased abundance of the classes Gammaproteobacteria and Actinobacteria. Along the longitudinal axis differences in the efficacy of antibiotics to prevent colitis were evident. Neomycin was most effective for prevention of inflammation in the cecum, while ampicillin was most effective in the proximal and distal colon. RAG1^-/-, but not TRAG, mice exhibited a structured pattern of bacterial abundance with decreased Firmicutes and Proteobacteria but increased Bacteroidetes along the proximal to distal axis of the gut. TRAG mice exhibited increased relative abundance of potential pathobionts including <i>Bifidobacterium animalis</i> along the longitudinal axis of the gut whereas others, like <i>Helicobacter hepaticus</i> were increased only in the cecum. Potential beneficial organisms including <i>Roseburia</i> were decreased in the proximal regions of the TRAG colon, while <i>Bifidobacterium pseudolongulum</i> was decreased in the TRAG distal colon. Thus, the innate immune system maintains a structured, spatially organized, gut microbiome along the transverse and longitudinal axis of the gut, and disruption of this biogeography is a feature of innate immune colitis.

Gastrokine-1, an anti-amyloidogenic protein secreted by the stomach, regulates diet-induced obesity.

Obesity and its sequelae have a major impact on human health. The stomach contributes to obesity in ways that extend beyond its role in digestion, including through effects on the microbiome. Gastrokine-1 (GKN1) is an anti-amyloidogenic protein abundantly and specifically secreted into the stomach lumen. We examined whether GKN1 plays a role in the development of obesity and regulation of the gut microbiome. Gkn1-/- mice were resistant to diet-induced obesity and hepatic steatosis (high fat diet (HFD) fat mass (g) = 10.4 ± 3.0 (WT) versus 2.9 ± 2.3 (Gkn1-/-) p < 0.005; HFD liver mass (g) = 1.3 ± 0.11 (WT) versus 1.1 ± 0.07 (Gkn1-/-) p < 0.05). Gkn1-/- mice also exhibited increased expression of the lipid-regulating hormone ANGPTL4 in the small bowel. The microbiome of Gkn1-/- mice exhibited reduced populations of microbes implicated in obesity, namely Firmicutes of the class Erysipelotrichia. Altered metabolism consistent with use of fat as an energy source was evident in Gkn1-/- mice during the sleep period. GKN1 may contribute to the effects of the stomach on the microbiome and obesity. Inhibition of GKN1 may be a means to prevent obesity.

Temporal Dynamics of Chronic Inflammation on the Cecal Microbiota in IL-10-/- Mice.

The intestinal microbiota is a critical component of mucosal health as evidenced by the fact that alterations in the taxonomic composition of the gastrointestinal microbiota are associated with inflammatory bowel diseases. To better understand how the progression of inflammation impacts the composition of the gastrointestinal microbiota, we used culture independent taxonomic profiling to identify temporal changes in the cecal microbiota of C3Bir IL-10-/- mice concomitantly with the onset and progression of colitis. This analysis revealed that IL-10-/- mice displayed a biphasic progression in disease severity, as evidenced by histopathological scores and cytokine production. Beginning at 4 weeks of age, pro-inflammatory cytokines including TNF-α, IFN-γ, IL-6, G-CSF, and IL-1α as well as chemokines including RANTES and MIP-1α were elevated in the serum of IL-10-/- mice. By 19 weeks of age, the mice developed clinical signs of disease as evidenced by weight loss, which was accompanied by a significant increase in serum levels of KC and IL-17. While the overall diversity of the microbiota of both wild type and IL-10-/- were similar in young mice, the latter failed to increase in complexity as the mice matured and experienced changes in abundance of specific bacterial taxa that are associated with inflammatory bowel disease in humans. Collectively, these results reveal that there is a critical time in young mice between four to six weeks of age when inflammation and the associated immune responses adversely affect maturation of the microbiota.

Resveratrol Represses Pokemon Expression in Human Glioma Cells.

POK erythroid myeloid ontogenic factor (Pokemon), an important proto-oncoprotein, is a transcriptional repressor that regulates the expression of many genes and plays an important role in tumorigenesis. Resveratrol (RSV), a natural polyphenolic compound, has many beneficial biological effects on health. In this study, we investigated the role of Pokemon in RSV-induced biological effects and the effect of RSV on the expression of Pokemon in glioma cells. We found that overexpression of Pokemon decreased RSV-induced cell apoptosis, senescence, and anti-proliferative effects. Moreover, we showed that RSV could efficiently decrease the activity of the Pokemon promoter and the expression of Pokemon. Meanwhile, RSV also inhibited Sp1 DNA binding activity to the Pokemon promoter; whereas, it did not influence the expression and nuclear translocation of Sp1. In addition, we found that RSV could increase the recruitment of HDAC1, but decreased p300 to the Pokemon promoter. Taken together, all these results extended our understanding on the anti-cancer mechanism of RSV in glioma cells.

Orally administered extract from Prunella vulgaris attenuates spontaneous colitis in mdr1a(-/-) mice.

AIM: To investigate the ability of a Prunella vulgaris (P. vulgaris) ethanolic extract to attenuate spontaneous typhlocolitis in mdr1a(-/-) mice. METHODS: Vehicle (5% ethanol) or P. vulgaris ethanolic extract (2.4 mg/d) were administered daily by oral gavage to mdr1a(-/-) or wild type FVB(WT) mice from 6 wk of age up to 20 wk of age. Clinical signs of disease were noted by monitoring weight loss. Mice experiencing weight loss in excess of 15% were removed from the study. At the time mice were removed from the study, blood and colon tissue were collected for analyses that included histological evaluation of lesions, inflammatory cytokine levels, and myeloperoxidase activity. RESULTS: Administration of P. vulgaris extracts to mdr1a(-/-) mice delayed onset of colitis and reduced severity of mucosal inflammation when compared to vehicle-treated mdr1a(-/-) mice. Oral administration of the P. vulgaris extract resulted in reduced (P < 0.05) serum levels of IL-10 (4.6 ± 2 vs 19.4 ± 4), CXCL9 (1319.0 ± 277 vs 3901.0 ± 858), and TNFα (9.9 ± 3 vs 14.8 ± 1) as well as reduced gene expression by more than two-fold for Ccl2, Ccl20, Cxcl1, Cxcl9, IL-1α, Mmp10, VCAM-1, ICAM, IL-2, and TNFα in the colonic mucosa of mdr1a(-/-) mice compared to vehicle-treated mdr1a(-/-) mice. Histologically, several microscopic parameters were reduced (P < 0.05) in P. vulgaris-treated mdr1a(-/-) mice, as was myeloperoxidase activity in the colon (2.49 ± 0.16 vs 3.36 ± 0.06, P < 0.05). The numbers of CD4(+) T cells (2031.9 ± 412.1 vs 5054.5 ± 809.5) and germinal center B cells (2749.6 ± 473.7 vs 4934.0 ± 645.9) observed in the cecal tonsils of P. vulgaris-treated mdr1a(-/-) were significantly reduced (P < 0.05) from vehicle-treated mdr1a(-/-) mice. Vehicle-treated mdr1a(-/-) mice were found to produce serum antibodies to antigens derived from members of the intestinal microbiota, indicative of severe colitis and a loss of adaptive tolerance to the members of the microbiota. These serum antibodies were greatly reduced or absent in P. vulgaris-treated mdr1a(-/-) mice. CONCLUSION: The anti-inflammatory activity of P. vulgaris ethanolic extract effectively attenuated the severity of intestinal inflammation in mdr1a(-/-) mice.

The Altered Schaedler Flora: Continued Applications of a Defined Murine Microbial Community.

The gastrointestinal (GI) microbiota forms a mutualistic relationship with the host through complex and dynamic interactions. Because of the complexity and interindividual variation of the GI microbiota, investigating how members of the microbiota interact with each other, as well as with the host, is daunting. The altered Schaedler flora (ASF) is a model community of eight microorganisms that was developed by R.P. Orcutt and has been in use since the late 1970s. The eight microorganisms composing the ASF were all derived from mice, can be cultured in vitro, and are stably passed through multiple generations (at least 15 years or more by the authors) in gnotobiotic mice continually bred in isolator facilities. With the limitations associated with conventional, mono- or biassociated, and germfree mice, use of mice colonized with a consortium of known bacteria that naturally inhabit the murine gut offers a powerful system to investigate mechanisms governing host-microbiota relationships, and how members of the GI microbiota interact with one another. The ASF community offers significant advantages to study homeostatic as well as disease-related interactions by taking advantage of a well-defined, limited community of microorganisms. For example, quantification and spatial distribution of individual members, microbial genetic manipulation, genomic-scale analysis, and identification of microorganism-specific host immune responses are all achievable using the ASF model. This review compiles highlights associated with the 37-year history of the ASF, including descriptions of its continued use in biomedical research to elucidate the complexities of host-microbiome interactions in health and disease.

Combined inhibition of EGFR and c-ABL suppresses the growth of triple-negative breast cancer growth through inhibition of HOTAIR.

Triple-negative breast cancer (TNBC) does not express conventional therapeutic targets and is the only type of malignant breast cancer for which no designated FDA-approved targeted therapy is available. Although overexpression of epidermal growth factor receptor (EGFR) is frequently found in TNBC, the therapeutic effect of EGFR inhibitors in TNBC has been underwhelming. Here we show that co-treatment with clinically validated inhibitors of c-ABL (imatinib) and EGFR (lapatinib) results in synergistic growth inhibition in TNBC cells. The dual treatment leads to synergistic repression of the long non-coding RNA (lncRNA) HOTAIR (HOX Antisense Intergenic RNA). HOTAIR has been known to induce tumor growth and metastasis in breast cancer. Depleting HOTAIR alone phenocopies the dual treatment in growth suppression. We show that expression of HOTAIR is regulated by ß-catenin through a LEF1/TCF4-binding site. The dual treatment blocks nuclear expression of ß-catenin and prevents its recruitment to the HOTAIR promoter. Consistently, forced expression of ß-catenin rescued HOTAIR expression and cell viability in the presence of both drugs. Upregulation of HOTAIR is associated with TNBC in cell lines and a cohort of primary tumors. This study elucidates a previously unidentified mechanism in TNBC linking signaling with lncRNA regulation which may be exploited for therapeutic gain.