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Abstract or Project Description

Ulcerative colitis (UC) is a chronic inflammatory disorder that affects the colon. There are multiple types of cells that drive inflammation and injury to the lining of the colon. Vedolizumab (VDZ) is a medication that blocks circulating immune cells from gaining access to the colon, but there are conflicting data about the primary cell types affected by this treatment. To identify the inflammatory cells that contribute to colitis and respond to VDZ, we performed a comprehensive single-cell transcriptomic and proteomic analysis of peripheral blood and colonic biopsies of patients with and without UC. We identified inflammatory cells called mononuclear phagocytes (MNPs) as a primary target of VDZ. We then used four new, complementary methods to look at where these cells are located in fixed tissue sections, and we discovered increased density and proximity of MNP and fibroblast subsets in UC biopsies when compared to HC, with inhibition by VDZ. We now want to confirm and extend these findings by looking in more detail at these cell types in tissue sections before and after therapy. We also want to culture epithelial cells from patients with MNPs and fibroblasts to see how these cells interact when grown in the lab.

Abstract or Project Description

The intestinal immune system must maintain a delicate balance between harmless bacteria in the gut and immunity against dangerous pathogens. Dysregulation of this balance can lead to Inflammatory Bowel Diseases (IBD) such as ulcerative colitis (UC) and Crohn’s disease. Many treatments for IBD focus on blocking inflammation and fail to repair damaged tissue and restore the integrity of the tissue. We have identified a long non-coding RNA that is only expressed in the colon. When this RNA is present the tissue is healthy and functional. In patients with ulcerative colitis however, the levels of this lncRNA decrease. To model this state, we generated mice lacking this lncRNA and like patients with UC, these mice develope colitis. The lncRNA localizes to the mitochondria in cells of the healthy colon. When the lncRNA is lost, the mitochondria in the cells in the colon are damaged, produce reactive oxygen species and damage colonic tissue. These findings provide new insights into how a lncRNA can help mitochondria work well to maintain homeostasis in the gut. These studies suggest new opportunities to restore tissue health by controlling the levels of this lncRNA.

Abstract or Project Description

Diet is thought to be a major environmental factor contributing to Inflammatory Bowel Disease (IBD) and represents a non-invasive path to improve this debilitating condition. To leverage diet to treat IBD the precise ways that dietary components influence development or healing of inflammation need to be determined. We discovered that different dietary protein sources have variable effects on intestinal inflammation and these effects are dependent on the community of microorganisms (microbiome) that inhabit the human colon. In particular, we discovered that soy protein promotes production of a beneficial metabolite called isobutyrate that reduces inflammation in a mouse model of IBD that we developed to mimic low fiber diet- and microbiome-driven inflammation. Isobutyrate is only produced by some bacteria and is derived from the dietary amino acid L-valine via at least two different metabolic pathways. Because soy protein specifically promotes isobutyrate, our model is that it is better at delivering L-valine-containing peptides to the colon after digestion, leading to bacterial isobutyrate production. The proposed experiments will uncover the specific component(s) of soy protein that promotes isobutyrate as well as the bacteria that produce this metabolite so that dietary protein can be optimized in the context of patients’ microbiomes to reduce inflammation.

Abstract or Project Description

Mental health profoundly impacts inflammatory diseases of the body. This is particularly apparent in Inflammatory Bowel Disease (IBD), where stressful events in a patient’s life often enhance the severity of subsequent disease flares. Psychological stress is also strongly linked to bowel motility problems in IBD patients. How the state of the brain influences inflammation in the gut is a fascinating question that remains poorly understood.

In this study, we will explore the mechanisms of brain-gut communication that link psychological stress to IBD severity. Using several different mouse models of IBD, we have recently discovered a pathway that links stress perception by the brain to the enteric nervous system (ENS), and in particular glia cells that reside in the gastrointestinal tract. We will explore how this neuronal brain-gut connection exacerbates the inflammatory state in the gut. We will also determine whether the same pathway is active in IBD patients in the context of stressful life events.

Collectively, this work will improve our understanding of how the brain and the gut communicate during psychological stress. We hope that an improved understanding of how stress influences intestinal inflammation will ultimately facilitate harnessing the power of brain-derived signals in the treatment of IBD.

Abstract or Project Description

Knowledge of the intestinal microbiota has transformed medical science, but not the treatment of Inflammatory Bowel Disease (IBD). The microbiota is difficult to use clinically because current analytical methods provide outputs that are too complex to provide useful data during an acute IBD flare. To solve these issues, we have developed a novel methodology, ‘MicFly’ that allows us to ‘count’ each type of bacteria present in the microbiota. Our method provides rapid, accurate, quantitative and simple measurements of the bacteria present in the microbiota and has the added benefit of being capable of identifying what genes are being expressed by each bacteria.

Clostridiodes difficile infection (Cdiff;CDI) is common in IBD patients because the pre-conditions for CDI, low microbiome diversity and immunosuppression, are common outcomes of treatment. Discriminating CDI from an IBD flare can be difficult because Cdiff is commonly found in IBD patients but is only dangerous when it expresses a toxin. As an example of the efficacy of MicFly, we propose to demonstrate that the technique can identify the number of Cdiff bacteria and their toxin expression. We will also measure whether a Fecal Microbiota Transplant for Cdiff has been successful in reducing Cdiff toxin levels and restoring microbiota diversity.

Abstract or Project Description

Inflammatory Bowel Disease (IBD) comprises incurable chronic diseases of the gut which oscillate between periods of disease worsening (i.e. relapse) and relatively normal health (i.e. remission). Whilst current medicines often reduce this see-sawing problem, their effectiveness eventually wanes to the point where a large percentage of IBD patients still need hospitalisation and major surgery during their lifetime. Indeed, our current inability to cure IBD denotes that existing medicines do not tackle the root cause of IBD.

In this context, we have discovered that gut cells in IBD patients are much more prone to dying than gut cells in healthy patients. Remarkably, this shift towards exaggerated gut cell death also occurs in IBD patients who are in remission and experiencing a period of “normal health” – leading us to predict that excessive gut cell death is a root cause of IBD. In this project, we will aim to understand why gut cells in IBD patients are primed to die, and test whether correcting this molecular defect can achieve long-lasting remission.

Abstract or Project Description

Our gut is constantly exposed to environmental harms, such as bacteria and dietary products, among others. The epithelium layer of our intestinal tract, which is the most exposed to the external world, contains a variety of immune cells, which likely play an important role in patrolling and defending our intestine. However, how these cells protect epithelial cells and the host from pathogens and other insults is still poorly understood. In this grant application, we aim to better understand how a specific subset of intraepithelial immune cells, known as gd T cells, contributes to gut homeostasis. We found that these cells are highly decreased in Crohn’s disease (CD) patients, suggesting that they exert protective roles during disease. In addition, we found that they are heterogeneous and can crosstalk with either epithelial cells or other immune cells located deeper in the tissue. Herein, we plan to design functional experiments to better understand their role in the context of gut immune responses and how they affect surrounding epithelial or other immune cell types. In addition, we aim to identify if they derive from a common precursor or if they are highly diverse and whether this scenario is altered in CD.

Abstract or Project Description

Microbiota small molecules correlate with Inflammatory Bowel Disease (IBD) pathogenesis, yet the molecular mechanisms behind remain elusive. We recently developed gene manipulation tools in multiple gut Clostridia commensals, and by mutating the responsible gene and mono-colonizing germ-free mice with the mutant, we can switch specific microbiota metabolites on/off in vivo and found that metabolites derived from Clostridia amino acid transamination, like branched short-chain fatty acids (BSCFAs) suppress intestine IgA plasma cells. Many microbiota-associated metabolites, including BSCFAs, are derived via microbial transamination of dietary amino acids and are highly abundant in the host. Multiple studies have shown that this group of microbial metabolites is significantly different between IBD patients and healthy humans. We hypothesize that microbiota metabolites from amino acid transamination mediate intestine immunity and inflammation. Herein we will apply Clostridium CRISPR-based toolsets to modulate these amino acid-derived metabolites in vitro and in vivo, investigate their effects on intestinal immunity and inflammation in germ-free and colitis mouse models, and look into clinical samples to determine if their levels are positively correlated with disease severity in IBD patients. Our findings will uncover previously unknown molecular mechanisms behind microbiome-IBD correlation and facilitate the identification of new therapeutic targets and new biomarkers for IBD prevention and treatment.

Abstract or Project Description

Crohn’s disease is a chronic inflammatory disease of the intestinal tract. It has been increasingly appreciated that two factors are involved beyond the genetic setup of individuals: 1) Intestinal microbial dysbiosis and 2) immune system dysregulation. Intestinal microbial dysbiosis refers to changes in the billions of bacteria that are growing in our gut. In healthy people these bacteria come from a certain core set of species. In Crohn’s disease patients these healthy microbes are perturbed. Immune system dysregulation refers to changes in the body’s capacity to deal with foreign particles (such as bacteria) but also your own body tissues. However, it is incompletely understood why and how the gut microbes and the immune system are perturbed in IBD.

Abstract or Project Description

We have discovered a novel single-gene defect in patients with pediatric IBD, setting the stage for new molecular and cellular discoveries to dissect biology stemming from human disease. Here, we will pursue two aims that will open new doors by leveraging the strength of preclinical models to uncover mechanisms by which the gene we identified in patients orchestrates immune responses and inflammation in the gastrointestinal tract. Specifically, we will assess roles for this gene in regulating production of the inflammatory IL-1 cytokine and begin to dissect interplay between immune cells lacking this gene and bacteria in the intestine. Since our work begins with patients, our findings will be directly translatable between humans and mice and will advance our unbiased gene discovery to mechanistic insights more broadly relevant for mapping immune interactions relevant for the larger IBD community. Together, these aims address innovative new hypotheses and will advance the mission of the Kenneth Rainin Foundation by enabling transformative IBD discoveries rooted in human biology and advanced with powerful modeling approaches.

Abstract or Project Description

Gut inflammation alters the bacterial communities in the gut (the microbiome), and these disruptions are thought to make inflammation even worse. Understanding and derailing this cycle could provide new avenues for IBD therapy. Although we know that inflammation can change “who’s there” in the gut microbiome, this project will determine whether harder-to-measure changes within the microbiome also impact the cycle of inflammation and microbiome disruption. Specifically, we will study whether episodes of inflammation cause these bacteria to change by altering their genetic instructions or how those instructions are read out. If successful, the study will uncover new measurements that we need to collect in order to understand how an individual’s microbiome responds to flares of inflammation and remission in IBD. In the future, this understanding will help us design microbiome-based therapies equipped to persist in the inflamed gut and break the cycle of microbiome disruption and inflammation that characterizes IBD.