The Winnie-APCmin as an innovative tool to investigate the axis between inflammation and colorectal cancer

2017 - 2018During these last three yeas of PhD we aimed to create a new murine model of spontaneous inflammatory-induced colorectal cancer a combining the genetic susceptibility of the APCMin model and the chronic intestinal inflammation of the Winnie mice. Differently from the DSS induced colitis, Winnie are murine models of spontaneous, mild and progressive ulcerative colitis that require several months to show histological sign of disease. During these years, we characterized the disease progression in Winnie. 5-week old mice show reduced body weight, watery diarrhea, but now rectal bleeding or prolapse. Importantly, histologically no distinctive sign of UC is present in the colon, but a specific molecular pathway characterized by upregulated inflammatory cytokine transcript is present. In light of the mild inflammatory response observed in 5-wee old Winnie, it was surprising to realize that the colon of age matched Winnie-APCMin mice was rich of dysplastic ACFs along the all the colon length with a gradually increase in incidence and multiplicity moving from the proximal to the distal colon tract. The Winnie-APCMin molecular pathway underlines the unique molecular feature resulting from the combination of genetic predisposition and chronic inflammation. This still preliminary observation has been used to submit an experimental protocol aiming to prevent the upregulation of some of the Winnie-APCMin specific genes using nutritional strategies that suppress the intestinal inflammation and/or prevent dysbiosis. Indeed, nutritional based strategies to suppress or mitigate intestinal inflammation has been one of the most important research topic of our group. For this reason, we used BMDCs as a paradigm of cells potentially affected by polyphenol exposure. Dendritic cells respond to quercetin exposure producing secretory leukocyte protease inhibitor (SLPI). SLPI is an antimicrobial protein that is also involved in tissue repair and possess the ability to block NFkB nuclear translocation with the ultimate result to suppress inflammation. Using Slpi-KO DCs we demonstrated that Slpi induction was a necessary step following quercetin administration to suppress inflammatory cytokine secretion. Using an easy dose response experiment, we demonstrated that the administration of inorganic iron was able to block quercetin in a dose dependent manner. As quercetin is a strong iron chelating agent, we proposed that quercetin-iron chelation may result in DCs cytoplasmic loss of iron reservoir and consequent switch to an inflammatory-impaired phenotype. At the same time, nutritional intake of quercetin may contribute to sequestrate iron from the intestinal lumen, suppressing bacterial growth. This aspect was partially explored using polyphenol enriched diet administered to WT or Winnie mice. Dysbiosis is partially recovered after two weeks of 1% enriched diet. The Winnie-APCMin model will be crucial to evaluate the efficiency of the polyphenol-enriched nutritional strategies and, potentially, many different pharmaceutical approaches. [edited by Author]XXXI cicl

Plant Polyphenols-Biofortified Foods as a Novel Tool for the Prevention of Human Gut Diseases

Plant food biofortification is recently receiving remarkable attention, as it aims to increase the intake of minerals, vitamins, or antioxidants, crucial for their contribution to the general human health status and disease prevention. In this context, the study of the plant’s secondary metabolites, such as polyphenols, plays a pivotal role for the development of a new generation of plant crops, compensating, at least in part, the low nutritional quality of Western diets with a higher quality of dietary sources. Due to the prevalent immunomodulatory activity at the intestinal level, polyphenols represent a nutritionally relevant class of plant secondary metabolites. In this review, we focus on the antioxidant and anti-inflammatory properties of different classes of polyphenols with a specific attention to their potential in the prevention of intestinal pathological processes. We also discuss the latest biotechnology strategies and new advances of genomic techniques as a helpful tool for polyphenols biofortification and the development of novel, healthy dietary alternatives that can contribute to the prevention of inflammatory bowel diseases

Neglected and Underutilized Plant Species (NUS) from the Apulia Region Worthy of Being Rescued and Re-Included in Daily Diet

Neglected and underutilized species (NUS) are cultivated, semi-domesticated, or wild plant species, not included in the group of the major staple crops, since, in most cases, they do not meet the global market requirements. As they often represent resilient species and valuable sources of vitamins, micronutrients, and other phytochemicals, a wider use of NUS would enhance sustainability of agro-systems and a choice of nutritious foods with a strategic role for addressing the nutritional security challenge across Europe. In this review, we focused on some examples of NUS from the Apulia Region (Southern Italy), either cultivated or spontaneously growing species, showing interesting adaptative, nutritional, and economical potential that can be exploited and properly enhanced in future programs

Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement

Cells lining the gastrointestinal tract serve as both a barrier to and a pathway for infectious agent entry. Dendritic cells (DCs) present in the lamina propria under the columnar villus epithelium of the small bowel extend processes across this epithelium and capture bacteria, but previous studies provided limited information on the nature of the stimuli, receptors, and signaling events involved in promoting this phenomenon. Here, we use immunohistochemical as well as dynamic explant and intravital two-photon imaging to investigate this issue. Analysis of CD11c–enhanced green fluorescent protein (EGFP) or major histocompatibility complex CII-EGFP mice revealed that the number of trans-epithelial DC extensions, many with an unusual “balloon” shape, varies along the length of the small bowel. High numbers of such extensions were found in the proximal jejunum, but only a few were present in the terminal ileum. The extensions in the terminal ileum markedly increased upon the introduction of invasive or noninvasive Salmonella organisms, and chimeric mouse studies revealed the key role of MyD88-dependent Toll-like receptor (TLR) signaling by nonhematopoietic (epithelial) elements in the DC extension response. Collectively, these findings support a model in which epithelial cell TLR signaling upon exposure to microbial stimuli induces active DC sampling of the gut lumen at sites distant from organized lymphoid tissues

Altered miRNAs Expression Correlates With Gastroenteropancreatic Neuroendocrine Tumors Grades

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and heterogeneous tumors that present a wide spectrum of different clinical and biological characteristics. Currently, tumor grading, determined by Ki-67 staining and mitotic counts, represents the most reliable predictor of prognosis. This time-consuming approach fails to reach high reproducibility standards thus requiring novel approaches to support histological evaluation and prognosis. In this study, starting from a microarray analysis of paraffin-embedded tissue specimens, we defined the miRNAs signature for poorly differentiated NETs (G3) compared to well-differentiated NETs (G1 and G2) consisting of 56 deregulated miRNAs. We identified 8 miRNAs that were expressed in all GEP-NETs grades but at different level. Among these miRNAs, miR-96-5p expression level was progressively higher from grade 1 to grade 3; inversely, its target FoxO1 expression decreased from grade 1 to grade 3. Our results reveal that the miRNAs expression profile of GEP-NET is correlated with the tumor grade, showing a potential advantage of miRNA quantification that could aid clinicians in the classification of common GEP-NETs subtypes. These findings could reliably support the histological evaluation of GEP-NETs paving the way toward personalized treatment approaches

miR-369-3p modulates inducible nitric oxide synthase and is involved in regulation of chronic inflammatory response

Dendritic cells are the most important antigen-presenting cells that link the innate and acquired immune system. In our previous study, we identified that the upregulation of miR-369-3p suppresses the LPS-induced inflammatory response, reducing C/EBP-β, TNFα and IL-6 production. With the aim of gaining further insight into the biological function of miR-369-3p during acute inflammatory response, in the present study we identified novel gene targets of miR-369-3p and demonstrated the suppressive ability of these genes on the inflammatory dendritic cells. Bioinformatic analyses revealed that iNOS is a potential target of miR-369-3p. We demonstrated that the ectopic induction of miR-369-3p markedly reduced iNOS mRNA and protein as well as NO production. Moreover, we found that the upregulation of miR-369-3p decreased the release of TNFα, IL-6, IL-12, IL-1α, IL-1β in response to LPS, and increased the production of anti-inflammatory cytokines such as IL-10 and IL-1RA. In addition, LPS-induced nuclear translocation of NF-kB was inhibited by miR-369-3p. Levels of miR-369-3p were decreased in human inflamed regions of human intestine obtained from IBD patients. Our results provide novel additional information on miR-369-3p as a potential core of the signaling regulating the inflammatory response. These findings suggest that miR-369-3p should be considered as a potential target for the future development of new molecular therapeutic approaches

Extra Virgin Olive Oil Extracts Modulate the Inflammatory Ability of Murine Dendritic Cells Based on Their Polyphenols Pattern: Correlation between Chemical Composition and Biological Function

Extra virgin olive oil (EVOO) represents one of the most important health-promoting foods whose antioxidant and anti-inflammatory activities are mainly associated to its polyphenols content. To date, studies exploring the effect of EVOO polyphenols on dendritic cells (DCs), acting as a crosstalk between the innate and the adaptive immune response, are scanty. Therefore, we studied the ability of three EVOO extracts (cv. Coratina, Cima di Mola/Coratina, and Casaliva), characterized by different polyphenols amount, to regulate DCs maturation in resting conditions or after an inflammatory stimulus. Cima di Mola/Coratina and Casaliva extracts were demonstrated to be the most effective in modulating DCs toward an anti-inflammatory profile by reduction of TNF and IL-6 secretion and CD86 expression, along with a down-modulation of Il-1β and iNOS expression. From factorial analysis results, 9 polyphenols were tentatively established to play a synergistic role in modulating DCs inflammatory ability, thus reducing the risk of chronic inflammation

Secretory Leukoprotease Inhibitor (Slpi) Expression Is Required for Educating Murine Dendritic Cells Inflammatory Response Following Quercetin Exposure

Dendritic cells’ (DCs) ability to present antigens and initiate the adaptive immune response confers them a pivotal role in immunological defense against hostile infection and, at the same time, immunological tolerance towards harmless components of the microbiota. Food products can modulate the inflammatory status of intestinal DCs. Among nutritionally-derived products, we investigated the ability of quercetin to suppress inflammatory cytokines secretion, antigen presentation, and DCs migration towards the draining lymph nodes. We recently identified the Slpi expression as a crucial checkpoint required for the quercetin-induced inflammatory suppression. Here we demonstrate that Slpi-KO DCs secrete a unique panel of cytokines and chemokines following quercetin exposure. In vivo, quercetin-enriched food is able to induce Slpi expression in the ileum, while little effects are detectable in the duodenum. Furthermore, Slpi expressing cells are more frequent at the tip compared to the base of the intestinal villi, suggesting that quercetin exposure could be more efficient for DCs projecting periscopes in the intestinal lumen. These data suggest that quercetin-enriched nutritional regimes may be efficient for suppressing inflammatory syndromes affecting the ileo-colonic tract

Quercetin exposure suppresses the inflammatory pathway in intestinal organoids from winnie mice

Inflammatory bowel diseases (IBDs) are chronic and relapsing immune disorders that result, or possibly originate, from epithelial barrier defects. Intestinal organoids are a new reliable tool to investigate epithelial response in models of chronic inflammation. We produced organoids from the ulcerative colitis murine model Winnie to explore if the chronic inflammatory features observed in the parental intestine were preserved by the organoids. Furthermore, we investigated if quercetin administration to in vitro cultured organoids could suppress LPS-induced inflammation in wild-type organoids (WT-organoids) and spontaneous inflammation in ulcerative colitis organoids (UC-organoids). Our data demonstrate that small intestinal organoids obtained from Winnie mice retain the chronic intestinal inflammatory features characteristic of the parental tissue. Quercetin administration was able to suppress inflammation both in UC-organoids and in LPS-treated WT-organoids. Altogether, our data demonstrate that UC-organoids are a reliable experimental system for investigating chronic intestinal inflammation and pharmacological responses