Progress 06/01/23 to 05/31/24
Outputs
Target Audience:The target audiences for this research include clinicians, dieticians, nutritionists, researchers, and trainees with a focus on how diet impacts inflammation in the intestines. Generally, these audiences are reached through journal publications, conference presentations, and conversations with colleagues. Trainees are reached through classroom and laboratory instruction, as well as research training and mentoring. Audience also includes the public, as many people with gut inflammation are interested in using diet to reduce disease symptoms, and are reached through open-access journal publications, public talks, news and media articles, and social media content. ? Changes/Problems:The gut chip system we propose using in Objective 2.1 uses healthy intestinal cells, and is expensive to modify to create new versions. Thus, we modified the method for this Objective to use cultured cells instead of the gut chip system. Culturing cells is much cheaper and more flexible to experimental design changes, we have the equipment needed for this, and it does not require proprietary machines or consumables. We also modified the method for this Objective so it would better align with our goals: we obtained commercially available colon cells from someone with ulcerative colitis which was not controlled by medication at the time of biopsy. Using these cells instead of healthy ones which are included in the gut chip system allows us to better study the impact of broccoli exosomes on inflammatory bowel disease. This change was successfully implemented in summer 2024 and is continuing into the fall of 2024. What opportunities for training and professional development has the project provided?Students and postdocs have been involved in all aspects of the project: Wet-lab and related data analysis: chemical extraction for metabolite analysis, isolation and quantification of nanoparticles from diet, aerobic culturing and streak plating, gram staining and reading microscopy slides, aseptic technique and lab biosafety procedures, culture media making, anaerobic culturing and running an anaerobic chamber, optical density readings and using a spectrophotometer, using a robot liquid handling pipette, describing bacterial colonies, quantitative polymerase chain reaction (qPCR) for gene counts, cultuing human colon cells, co-culturing bacteria with human colon cells, cytokine analysis, cell growth quantification, western blot. Presentation: students make their own presentation slides and posters, and have presented in their courses, to the lab, to student audiences, and at university, regional, national, and international level conferences. Collaboration: The students work together, and coordinate activities across weeks, requiring excellent communication and organizational skills. How have the results been disseminated to communities of interest?Results and information related to the process of how diet affects microbial communities and inflammation in the gut have been disseminated via social media and scientific conference presentations. In the next reporting period, we will reach our communities of interest via journal publications, conference presentations, and social media posts. What do you plan to do during the next reporting period to accomplish the goals?The remaining portions of Objective 2will be completed in the next reporting period, and at least 2 manuscripts will be prepared for submission to a scientific journal for peer-review. At least 2 presentations are planned at scientific conferences.
Impacts
What was accomplished under these goals?
Objective 1: To assess the targeted delivery of BSDExo to inflammatory colon cells and tissues. 1.1 To assess the stability and release of dietary bioactives from BSDExo in simulated biorelevant GI fluids. We have isolated and purified Broccoli Sprout-Derived Exosomes (BSDExo) using a sequential ultracentrifugation method and characterized the size distribution. In order to reach the target inflammation sites in the colon, bioactives, for example sulforaphane, need to remain intact during the long transit in the stomach and small intestine. The stomach is a reservoir of a strong acid with an average pH of 1-2, and it takes up to 6 hours for things to move through the stomach and small intestine. We incubated BSDExo in a pH 1.5 buffer. The particle size remained constant after 4 hours, supporting the stability of BSDExo in the stomach environment. To test if BSDExo can protect the encapsulated bioactives, for example sulforaphane, from early release in the upper gastrointestinal tract to allow more of those to reach the lower gastrointestinal tract, we incubated exosomes in buffers with pH 1.5 or 7.4 at 37°C, and measure the amounts of SFN released over time. Our preliminary data showed that there was slower sulforaphane release at the stomach pH than at the neutral pH, suggesting that BSDExo may protect sulforaphane in the stomach to allow more of those to reach the lower gastrointestinal tract. 1.2 To investigate the cellular uptake mechanisms of BSDExo in colon cells. We labeled exosomes with ExoGlow fluorescent and performed a cellular uptake assay. CCD841 colon cells and Caco-2 colon cancer cells were pretreated with 1 ug/mL lipopolysaccharide (LPS) or dextran sodium sulfate (DSS) for 24 hours to stimulate inflammation via immune activation or chemical disturbance, respectively. This was followed by exosome treatment for 48 hours. We observed increased uptake of fluorescent-labeled BSDExo in inflamed cells, and more uptake was observed with more severe inflammation. We also observed more colon cell growth when cells were treated with BSDExo. We also noticed higher uptake of exosomes than microparticles across the board, regardless of the concentrations of LPS or DSS. The mechanisms are not clear, but we suspect that the small size of the exosome, its lipid bilayer structure, and surface marker proteins might all contribute to the preferable accumulation in the inflamed cells. The surface protein composition of exosomes is considered to be crucial to their function. We did a preliminary analysis of the BSDExo surface proteins using a protein microarray which has antibodies for mammalian cell-derived exosome markers, in order to identify some of those mammalian exosome markers on BSDExo. These proteins are important for communication and adhesion between mammalian cell-derived exosomes and recipient cells, and therefore the markers we have identified on BSDExo may be involved in the possible interactions between BSDExo and mammalian cells. 1.3 To determine the targeting and retention of BSDExo in inflamed GI tissues in a chemically induced colitis mouse model. We have tested the distribution of fluorescent-labeled BSDExo in the gastrointestinal tract of healthy mice and are examining its inflammation-targeting and retention in a colitis mouse model. We used 3% of DSS in drinking water to induce acute colitis in specific pathogen free B6 mice, in which inflammation and other symptoms occur most concentrated in the colon. We gave the mice one dose of BSDExo, at 50 ug/100 ul. We collected gastrointestinal tract samples at 8 hours and 24 hours post-dosing, and measured the fluorescent signal along the gastrointestinal tract. We found successful uptake in the stomach of healthy mice at 8 and 24 hours, and in their colon at 8 hours only. We found successful uptake in the stomach and the colon of mice receiving DSS at 8 and 24 hours, and a much higher concentration was found in the colon at 8 hours compared to other samples. This implies that the disturbance caused by DSS induced greater uptake at the site of inflammation. We plan to confirm this result with more mice per group and multiple doses in the next study. Objective 2: To evaluate the therapeutic effects of BSDExo against colitis. 2.1 To examine the anti-inflammatory effect of BSDExo in a human colitis-on-a-chip model. In Objective 2.1 of our research, we initially proposed using a gut chip system with healthy intestinal cells. However, due to the complexity involved in modifying the gut chip system for new versions, we decided to shift to using cultured cells. Culturing cells is significantly more economical and adaptable to changes in experimental design. Additionally, this approach does not require proprietary equipment or consumables, making it a more practical choice given our available resources. To align our methodology more closely with our research goals, we acquired commercially available colon cells from a patient with ulcerative colitis, whose condition was not controlled by medication at the time of the biopsy. This modification allows us to study the effects of broccoli exosomes on inflammatory bowel disease (IBD) more directly and effectively than if we had used healthy cells. This change was successfully implemented in the summer of 2024 and continues into the fall of 2024. We tested three different concentrations of broccoli exosomes, benchmarked against three concentrations of sulforaphane, in the ulcerative colitis colon cells at three different time intervals. Our analyses focus on measuring cytokine production, tight junction protein concentrations, and cell count and survival. Currently, data analysis is ongoing as we evaluate the impact of these treatments on the inflammatory processes and cellular integrity associated with IBD. 2.2 To evaluate the efficacy of BSDExo in a chemically induced colitis mouse model. We will run the colitis mouse study as proposed for this objective the following year.
Publications
|