Mae J. Ciancio

Exercise prevents weight gain and alters the gut microbiota in a mouse model of high fat diet-induced obesity.

Background Diet-induced obesity (DIO) is a significant health concern which has been linked to structural and functional changes in the gut microbiota. Exercise (Ex) is effective in preventing obesity, but whether Ex alters the gut microbiota during development with high fat (HF) feeding is unknown. Objective Determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO. Methods Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation. Results HF diet resulted in significantly greater body weight and adiposity as well as decreased glucose tolerance that were prevented by voluntary Ex (p<0.05). Visualization of Unifrac distance data with principal coordinates analysis indicated clustering by both diet and Ex at week 12. Sequencing demonstrated Ex-induced changes in the percentage of major bacterial phyla at 12 weeks. A correlation between total Ex distance and the ΔCt Bacteroidetes: ΔCt Firmicutes ratio from qPCR demonstrated a significant inverse correlation (r2 = 0.35, p = 0.043). Conclusion Ex induces a unique shift in the gut microbiota that is different from dietary effects. Microbiota changes may play a role in Ex prevention of HF-DIO.

β -Catenin activity negatively regulates bacteria-induced inflammation

Wild-type (WT) Salmonella typhimurium causes acute intestinal inflammation by activating the nuclear factor kappa B (NF-κB) pathway. Interestingly, WT Salmonella infection also causes degradation of β-catenin, a regulator of cellular proliferation. Regulation of β-catenin and the inhibitor of NF-κB, IκBα, is strikingly similar, involving phosphorylation at identical sites, ubiquitination by the same E3 ligase, and subsequent proteasomal degradation. However, how β-catenin directly regulates the NF-κB pathway during bacteria-induced inflammation in vivo is unknown. Using streptomycin-pretreated mice challenged with Salmonella, we demonstrated that WT Salmonella stimulated β-catenin degradation and decreased the physical association between NF-κB and β-catenin. Accordingly, WT Salmonella infection decreased the expression of c-myc, a β-catenin-regulated target gene, and increased the levels of IL-6 and TNF-α, the NF-κB-regulated target genes. Bacterial infection directly stimulated phosphorylation of β-catenin, both in vivo and in vitro. Closer examination revealed that glycogen synthase kinase 3β (GSK-3β) kinase activity was increased in response to WT Salmonella, whereas non-virulent Salmonella had no effect. siRNA of GSK-3β was able to stabilize IκBα in response to WT Salmonella. Pretreatment for 24 h with LiCl, an inhibitor of GSK-3β, reduced WT Salmonella induced IL-8 secretion. Additionally, cells expressing constitutively active β-catenin showed IκBα stabilization and inhibition of NF-κB activity not only after WT Salmonella infection but also after commensal bacteria (Escherichia coli F18) and TNF-α treatment. This study suggests a new role for β-catenin as a negative regulator of inflammation.

Erythropoietin Protects Intestinal Epithelial Barrier Function and Lowers the Incidence of Experimental Neonatal Necrotizing Enterocolitis

The impermeant nature of the intestinal barrier is maintained by tight junctions (TJs) formed between adjacent intestinal epithelial cells. Disruption of TJs and loss of barrier function are associated with a number of gastrointestinal diseases, including neonatal necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal diseases in preterm infants. Human milk is protective against NEC, and the human milk factor erythropoietin (Epo) has been shown to protect endothelial cell-cell and blood-brain barriers. We hypothesized that Epo may also protect intestinal epithelial barriers, thereby lowering the incidence of NEC. Our data demonstrate that Epo protects enterocyte barrier function by supporting expression of the TJ protein ZO-1. As immaturity is a key factor in NEC, Epo regulation of ZO-1 in the human fetal immature H4 intestinal epithelial cell line was examined and demonstrated Epo-stimulated ZO-1 expression in a dose-dependent manner through the PI3K/Akt pathway. In a rat NEC model, oral administration of Epo lowered the incidence of NEC from 45 to 23% with statistical significance. In addition, Epo treatment protected intestinal barrier function and prevented loss of ZO-1 at the TJs in vivo. These effects were associated with elevated Akt phosphorylation in the intestine. This study reveals a novel role of Epo in the regulation of intestinal epithelial TJs and barrier function and suggests the possible use of enteral Epo as a therapeutic agent for gut diseases.

Epithelial Secretory Response to Inflammation

As suggested by this and previous reviews, the neuroimmunoregulation of intestinal secretion is a complex series of endocrine, neurocrine, paracrine and autocrine interactions between the underlying cells in the mucosa and submucosa and the intestinal enterocyte. Under normal conditions, the balance of each of these systems is delicately controlled, thus allowing for normal, consistent intestinal function. However, when this finely-tuned system is altered, such as in a diseased state, the resultant effect is an amplification of the host defense response. Initially thought to be protective against further insult, this local immune response, if allowed to continue uncontrollably, can exacerbate the disease process.

Inducible heat shock protein 70 prevents multifocal flat dysplastic lesions and invasive tumors in an inflammatory model of colon cancer

BACKGROUND Heat shock protein 70 (Hsp70) regulates protein biosynthesis and refolding of denatured proteins. Since Hsp70 participates in recovery from stress injury, we examined the effect of Hsp70 genetic deletion in the azoxymethane (AOM)/dextran sulfate sodium (DSS) model of inflammation and colon cancer. METHODS Hsp70 mutant mice (Hsp70.1(-/-)/70.3(-/-)) and wild-type (WT) littermates received AOM and three cycles of DSS and were killed 24 weeks later. Tumors were graded for histology and immunostained for p53, adenomatous polyposis coli, beta-catenin, cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNOS) and sequenced for p53 mutations. RESULTS Elevated adenomas developed in 4/10 WT mice with no dysplasia in adjacent mucosa. In contrast, 7/8 Hsp70 knock out (KO) mice developed chronic mucosal inflammation and multifocal areas of flat dysplasia and 4/8 progressed to invasive carcinomas arising in a background of flat dysplastic mucosa. These differences in the incidence of flat dysplasia and invasive cancers were significant (P < 0.05). Nuclear p53 was stronger in Hsp70 KO tumors compared with WT tumors, and sequencing confirmed p53 mutations in 2/5 tumors from Hsp70(-/-) versus 0/5 in WT mice. In Hsp70 WT tumors, beta-catenin was predominantly nuclear, compared with membranous beta-catenin in Hsp70(-/-) tumors, suggesting that Hsp70 regulates beta-catenin in colonic tumorigenesis. Cox-2 and iNOS levels were increased in tumors from Hsp70(-/-) mice compared with Hsp70 WT tumors. CONCLUSIONS Hsp70-deleted mice treated with AOM/DSS develop flat invasive colonic tumors that mimic many histological and molecular features of ulcerative colitis colon cancer. This model will be useful to dissect the role of Hsp70 in inflammatory bowel disease colon cancer.

Flagellin is required for salmonella-induced expression of heat shock protein Hsp25 in intestinal epithelium

Flagellin is a bacterial protein responsible for activation of Toll-like receptor 5 (TLR5), which we hypothesize is involved in Salmonellas induction of cytoprotective heat shock proteins in intestinal epithelial cells. Flagellin induces the cytoprotective heat shock protein Hsp25 in different intestinal epithelial cell lines and in mouse intestine. Flagellin induces Hsp25 expression in a time-dependent manner in vitro. This effect is transcriptional, as confirmed by luciferase reporter assays and actinomycin D treatment. In addition, Hsp25 induction requires p38 MAPK activation and is only observed when flagellin is added to the basolateral side of polarized intestinal epithelial cells, consistent with the known location of TLR5. Flagellin-mediated Hsp25 induction is associated with increased protective effects against oxidant stress, an effect that is at least partially mediated by p38 MAPK. Use of small interfering RNA against Hsp25 demonstrates that flagellin-mediated protection against oxidant stress is to some degree mediated through Hsp25 induction. This suggests that, by protecting against oxidant injury, the induction of Hsp25 expression by flagellin may contribute to intestinal homeostasis. In a coculture cell model and in a mouse model of Salmonella enterica Serovar Typhimurium infection, not only does infection with wild-type and a flagellin-deletion mutant strain of Salmonella show that flagellin induces Hsp25 in vivo, but it also demonstrates that in the case of live Salmonella infection, flagellin serves as a major stimulus for the induction of Hsp25 expression. These data provide evidence that flagellin is required for Salmonella-mediated induction of Hsp25 expression in intestinal epithelium.