Inflammatory bowel diseases (IBDs) cause chronic inflammation in the gastrointestinal (GI) tract, leading to symptoms like abdominal pain, fatigue, and diarrhea. The enteric nervous system (ENS) regulates GI function, and we aim to study how IBD affects its structure and function in a chronic mouse model. We hypothesize that IBD alters the ENS's cellular architecture and connectivity, impacting smooth muscle activity and causing GI dysfunction. Additionally, we plan to develop a drug-screening paradigm to evaluate pharmacotherapeutics' effects on ENS structure and smooth muscle activity.

Celiac disease is a prevalent condition in which dietary intolerance to gluten provokes the immune system to attack the intestine, causing significant medical complications. Here, we grow intestinal biopsies from celiac disease patients as 3-dimensional “organoids” in incubators, to dissect how dietary gluten induces immune responses and intestinal damage, and to achieve improved understanding and therapy of this disease.

In inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), B cells can harm the body by producing harmful antibodies and failing to produce enough anti-inflammatory IL-10. This research aims to understand these issues better to find new treatments, particularly for children. We will investigate whether B cells in UC express harmful antibodies early on due to ineffective elimination processes. Additionally, we will explore how high levels of CXCL4 in IBD patients may inhibit IL-10 production by interfering with the TLR9 receptor in B cells. Our goal is to identify new treatment options for IBD.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting children and adults. Nearly 30% of children in remission experience persistent abdominal pain, known as functional abdominal pain disorder (FAPD). This study aims to explore the gut microbiome's role in ongoing symptoms in pediatric UC using the non-invasive CapScan device to collect intestinal samples. Researchers will analyze these samples alongside stool, saliva, and blood to identify bacterial patterns and biochemical changes linked to pain and relapse. Findings may lead to new biomarkers and microbiome-based therapies, improving treatment and quality of life for affected children.

Ulcerative colitis is a chronic gut inflammation affecting millions, including children. Current treatments often have limited success and serious side effects, highlighting the need for safer options. Our research focuses on GSB-D341, a modified genistein derived from soy, designed to reduce inflammation and support gut health. We will test its effects on gut tissue samples from children with colitis and compare it to existing treatments. Additionally, we will evaluate GSB-D341 in mice to assess symptom relief and its impact on gut bacteria. This research aims to determine if GSB-D341 could be a safe and effective treatment for pediatric ulcerative colitis.

IBD therapies would greatly benefit from bioengineering approaches that would reduce drug off-target concerns and the need for multiple painful injections by enabling drugs to be delivered locally within the affected bowel in a reservoir that can protect their bioactivity, limit their off-target effects, and offer tunable release kinetics that can match the therapeutic window.  To that end, we have developed a catheter-injectable gel crosslinked with drug-eluting nanoparticles (NP gel) that can carry a reservoir of budesonide and infliximab for local delivery of sustained IBD therapy.

The intestine in young children undergoes rapid expansion that is characterized by higher metabolic demand and decreased metabolic reserve, which further compounds the damage induced by local inflammation encountered in IBD.  Recently, we identified the regenerative phenotype of MSCs based on their source and found umbilical cord tissue derived MSCs (UC-MSCs) have genomic and proteomic regenerative signatures which can address the inflammation encountered in IBD.   Our lab has been developing workflows (i.e. using ultrasound) to enhance the therapeutic cargo of  extracellular vesicles released by UC-MSCs (pUC-EVs) which can then be used as a cell-free therapy.  In addition, we have been investigating precision delivery approaches to help ensure optimal delivery of therapy to the bowel in a small animal translational model.

The parthogenesis of Crohn's disease involves the interplay between a dysregulated immune response and an altered gut microiome. Recent research has demonstrated that the total colonic microbial abundance is lower in patients with Crohns disease than in healthy controls, and the absolute abundance of the Bacteroidetes phylum is significantly higher in patients with Crohn's disease. The role of colonic protein availability in contributing to the alterations observed in the gut microbiome of Chron's disease patients is unclear. To adress the issue, our collaborateive team aims to (aim 1) characterize protein disgestion dynamics in vivo across strains and conditions, (aim 2) determine whether amino acid utilization pathways are enriched in CD payeints vs health controls, and (aim 3) estabish a mathematical model to estimate peptide digestion dynamics within the human large intestine. The results of this study will identify protein bioavailability in the colon as a potential mechanism by which protein-metabolizing colonic bacteria can drive a feed-forward cycle of inflammation. This project aims to understad the mechanism underlying Crohn's disease and develop new therapeutic approaches by targeting protein-utilizing microbes. it will enable a formal collaboration between pIs at Stanford, establish Dr. Cremers early career investigator group, and help the Bhatt lab extendgquantitative approaches for Crohn's disease and appications to follow on fundng from NIDDK, the Crohn's and Colitis Foundation and the Rainin Foundatio.

Cells lining the gut, called the epithelium, form a physical and chemical barrier keeping microbes and food from crossing into the body and triggering inflammation. However, this barrier is disrupted in Crohn’s disease (CD). The intestinal epithelium contains numerous cell types, including rare sensory cells called tuft cells. Surprisingly tuft cells share may properties with taste cells in the tongue and they use “taste” to detect microbes and communicate with neighboring epithelial and immune cells. Interestingly, parasite infections, which are far more prevalent in populations with lower incidence of CD, increase tuft cell numbers. Conversely, tuft cells decrease in both adult and pediatric patients with CD. In addition, stimulating tuft cells in a mouse CD model reduced epithelial damage and inflammation. Therefore, we hypothesize that tuft cells can be utilized to shape the intestinal immune response and restore epithelial barrier function in CD patients. Human tuft cells in CD remains poorly characterized and almost nothing is known about the role for these cells in pediatric disease. In particular, the tuft cell receptors that sense microbes and the tuft cell effector molecules that influence neighboring epithelial cells remain undefined. We propose to examine the gene expression of tuft cells and their neighboring epithelial cells from pediatric CD patients and non-CD patients using a technique called spatial transcriptomics. This approach allows us to examine a neighborhood of cells in native tissue to see what they’re doing and their potential interactions with neighboring cells. Tuft cells respond to commensal protists living in the intestines of both humans and mice. These protists are relatively common in non-industrialized countries but almost entirely absent in high-income countries where pediatric CD is most prevalent. We propose to examine how these protists affect CD severity under a variety of diets by using a mouse model of this disease.

Pediatric inflammatory bowel disease (IBD) is a severe condition where the intestines of children are attack by their own immune systems, resulting in smptoms such as diarrhea, weight loss, bleed and infection. Scientific research into pediatric IBD is limited because researchers lack appropriate tools. Our lab has developed a method to grow intestinal biopsy tissue from pediatric IBD patients in incubators outside of the body, as small three-dimensional aggregates of cells termed "organoids". Our pediatric IBD organoids grown in petri dishes are very advanced in that they ot only contain the intestinal lining cells, but also preserve he attacking immune cells. In this proposal, we add an additional component, the bacteria that populate the intestine, which is called the microbiome. Since the microbiome has bee hypothesized to be crucial for the development of IBD, we will test if adding components of the microbiome can stimulate the attacking immune responses within the IBD organoid cultures. This will help prove that the microbiome can promote the development of IBD, and create a more complete system in which potential pediatric IBD drugs can be tested.

Crohn disease (CD) is a chronic illness due to disease-causing (“autoimmune”) interactions between our immune systems and the cells lining the gastrointestinal (GI) tract. CD is probably triggered by abnormal stimulation of the immune system by environmental factors like the intestinal bacteria that normally help maintain our health. In some patients, CD has been shown to be caused by mutations in genes that control how the immune system reacts to the environment. These patients can be cured of their CD by a transplant of the blood-forming (“hematopoietic”) stem cells in the bone marrow from a healthy donor to provide the patient with a new immune system that won’t cause CD. Hundreds of such patients have been successfully treated and cured of their CD. There are also other patients with CD whose disease began at an early age and who frequently don’t improve with the usual medications to control CD. These patients are also thought to have inherited abnormalities of their immune system, but genetic mutations haven’t been found so there is uncertainty as to whether the benefits of transplant outweigh potential risks. Therefore only a few such patients have been treated by transplant. We have developed a safer way to do stem cell transplants that can shift the balance of this equation for certain patients with CD towards this treatment approach. We have used these “alpha-beta depleted stem cell transplants” combined with a safer way to prepare a patient’s body to accept the donor’s stem cells to cure other patients with autoimmune disorders. We have come together as a team of gastroenterology, stem cell transplant, genetics and infection doctors to develop a clinical trial to test this potentially curative approach for some patients with CD whose disease began at an early age and is not responding well to treatment.

Inflammatory Bowel Disease (IBD) has emerged as a global disease with increasing incidence of pediatric and adult cases worldwide. Only about a third of patients respond to biologics and a significant portion lose response over time. Clinicians are limited by IBD heterogeneity, an unclear understanding of mechanism of biologic therapy failure, and lack of tools to formulate data-driven and personalized therapies. While it is appreciated that IBD underlies a loss of tolerance to commensal gut bacteria and self-antigens, the adaptive(TCR and BCR) immune repertoire that mediate disease and clinical outcomes in IBD has not been characterized. Recent advanced in single-cell genomics have revealed complex cellular heterogeneity in T and B lymphocytes across inflamed and healthy states in IBD. Our group has revealed specific alterations in T cell and B cell immunophenotypes in IBD. Given that genes involved in antigen presentation in T cells and immunoglobulin genes in B cells are altered in active disease in IBD, we hypothesize that TCR and BCR signatures may predict future anti-TNF therapy response and that longitudinal changes in immunophenotypes mediate anti-TNF resistance. This proposal represents a unique collaboration where physician-scientists and adult and pediatric gastroenterologists and an expert bioinformatician/machine learning scientist will team up to identify immune repertoire biomarkers that predict anti-TNF response in a propective cohort of pediatric patients with IBD using machine learning, characterize longitudinal changes in peripheral immunophenotypes with anti-TNF therapy, and characterize colonic tissue single cell transcriptomics in anti-TNF responders versus non-responders. Together, these studies will result in novel biomarkers to predict anti-TNF response in pediatrice IBD, provide valuable insights into the mechanisms of anti-TNF resistance, and develop rich immune repertoire datasets for antigen discovery and a framework for precision medicine in pediatric patients with ID

Ulcerative colitis (UC) is a lifelong illness that affects approximately 3 million people in the United States and dramatically affects quality of life, especially in younger populations. Despite advances in medical management, up to 30% of patients eventually require major surgery with staged or total colectomy. Ulcerative colitis is typically defined by continuous mucosal inflammation; however, on endoscopy, peri-appendiceal inflammation in the cecum has been associated with left sided colitis, in up to 75% of cases. The clinical significance of this finding is unclear, but it could reflect a distinct clinical subtype of UC with a different course, and it could reflect inflammation in the appendix, an organ that has been implicated in the development of UC, particularly in children. Although the appendix is considered a vestigial organ, studies suggest that its removal can be beneficial for the course of UC especially in the young. One hypothesis is that the peri-appendiceal mucosa serves as an immune regulatory site that triggers colonic inflammation; another hypothesis is that the appendix serves as a reservoir of bacteria that seeds the colon. Neither hypothesis has been adequately tested. We propose to examine the microbiome, local and systemic immune responses, and histology of the peri-appendiceal mucosa in children with UC. We describe a prospective single-center pilot study of treatment naïve-children and young adults with UC, and children and young adults with Crohn’s disease or with normal colonic tissues as the comparison groups. We will characterize the microbiota and local immune responses as a function of distance to the appendiceal orifice, and correlate these features with the course of disease. Our longer-term goal is to identify features of the peri-appendiceal microbiota and mucosa that predict treatment response and clinical outcome in pediatric UC patients, and to identify those who may benefit from individualized therapy including appendectomy.

Very early onset IBD (VEO-IBD)  is a life-threatening disease of the gut, characterized by an excessive and chronic inflammation resulting from an uncontrollable and self-sustaining immune response to environmental triggers. The objective of this research is to develop a humanized mouse model of  VEO-IBD amenable for testing of gene and cell therapies for pediatric populations. The long-term goal is to develop type 1 regulatory T (Tr1) cell-based therapies to cure VEO-IBD in the absence of general immunosuppression.   The central hypothesis of this project is that immune reconstitution of mice transplanted with human IL10-deficient hematopoietic stem cells will result in the development of a novel mouse model suitable for testing therapeutics for VEO-IBD. 

In this project, we will develop the first ingestible pill technology that can track metabolite biomarkers of P-IBD - in vivo, continuously, and in real-time. To this end, we will leverage our expertise in aptamer probes and integrate them into a miniaturized opto-electronic device in an ingestible pill format. The success of this project holds the potential to dramatically improve understanding, assessment and treatment of P-IBD.

"Inflammatory Bowel Disease (IBD) is a chronic illness in the intestines. In people and especially children with IBD, the immune system does not work properly and can attack the intestines, making it difficult to absorb food. This inflammation can also cause complications like surgery and hospitalization, and be a source of pain and suffering for so many children with IBD. T regulatory cells (Tregs) are a part of the immune system that help dampen the problems caused by the dysfunctional immune system in IBD. This innovative proposal aims to use Tregs to heal the inflammation in the intestine, achieving independence from traditional medicines for IBD like immunosuppressants.

First, we will develop Tregs from patients with IBD using a very small amount of blood through a routine blood draw. Next, we will test the efficacy of these Tregs in a special cell culture called an organoid. An organoid is a collection of cells taken from a routine endoscopic or surgical procedure that can be grown indefinitely in a precision health model. The organoid allows us to non-invasively test the Tregs and to study several parts of intestinal immune function, so we can be certain that the Tregs can help achieve mucosal healing, the goal standard for care of IBD patients. With successful completion of these two aims, we could consider using Tregs cell therapies in future studies to definitively heal IBD patients."

How the microbiome impacts patients with Crohn’s Disease is a decades-old question that has presented many challenges to both scientists and physicians. The microbiome is complex and dynamic, containing 100s of species that exhibit enormous interindividual variability. While scientists know that the Crohn’s microbiome is altered showing a loss of strictly anaerobic bacteria, our understanding of how this altered microbial community worsens patient’s symptoms is unclear. Recently, our group and others have shown that one of the key ways the healthy microbiome promotes gut health is through the production of drug-like small molecules. Increasingly, we are realizing what is critical about the microbiome isn’t ‘who is there’ but rather, ‘what are they doing.’ We hypothesize that loss of microbes that produce beneficial small molecules in Crohn’s patients disrupts the normal balance in the gut, driving disease progression. If true, this presents an exciting new possibility: that by measuring small molecules in Crohn’s patients, we could predict disease outcomes. In the current application, we will measure microbiome-produced small molecules in the blood of patients in the largest clinical study of pediatric Crohn’s disease. We have the following objectives: 1) identify patterns of small molecules that could aid in diagnosis of Crohn’s disease, and 2) characterize whether microbiome-derived small molecules can be used to predict the development of severe disease. Our work would have two important implications: (1) it would provide fundamental new insights into how the microbiome’s functions change in Crohn’s patients, and (2) it would enable the development of new non-invasive tests to predict disease progression. Ultimately, this work could guide physician’s use of therapies to improve clinical outcomes in pediatric patients with newly diagnosed Crohn’s disease

Pediatric inflammatory bowel disease (IBD) is a severe chronic inflammatory disorder affecting over 70,000 children in the United States. Although extensive research over the past two decades has revealed the central role of immune cell-derived cytokines in the pathophysiology of pediatric IBD, a high percentage of patients still fail to respond to current immunosuppressive treatment modalities, highlighting the urgent need for new treatment options. Recently, the homeostatic cytokine interleukin-22 (IL-22) has garnered clinical interest for its ability to promote intestinal regeneration and enhance mucosal barrier integrity during inflammation. However, IL-22 is highly pleiotropic, and can also induce pro-inflammatory side effects and exacerbate inflammatory disease in other contexts, limiting its therapeutic utility. In an attempt to overcome this limitation and address this unmet therapeutic need, we have engineered IL-22 analogs with altered receptor-binding properties that display functionally selective signaling responses in colonic epithelial cells, driving expression of tissue protective genes without inducing the systemic inflammatory markers associated with wild type (WT) IL-22. These engineered IL-22 analogs therefore represent a novel therapeutic approach for protecting tissues from inflammatory damage without altering immune function. This proposal represents a unique collaboration where a physician-scientist gastroenterologist, and cytokine engineer post-doctoral fellow in the Garcia lab, will team up to characterize the therapeutic efficacy, tolerability, and mechanism of action of these engineered IL-22 analogs in two distinct genetic mouse models of IBD: (1) the IL10-/- model, in which homozygous deletion of the IL10 gene results in the spontaneous development of colitis, and (2) the SAMP1 model of spontaneous ileitis, which resembles the pathology of human Crohn’s Disease. Using these two models, we will assess the impact of WT IL-22 and our biased IL-22 analogs on mucosal healing, epithelial integrity, intestinal stem cell function, immune cell recruitment, and systemic inflammation. The results of the in vivo studies will guide optimization of the IL-22 analogs for therapeutic efficacy in a process we refer to as “cytokine tuning.” This is possible due to the synergy of the combined expertise of the Garcia lab members who will execute these experiments. Together, these studies will provide valuable insights into the therapeutic potential of engineered IL-22 receptor agonists for the treatment of pediatric IBD, facilitating the future development of these compounds for human clinical trials.

The prevalence of Crohn’s disease in pediatric patients continues to climb every year. Despite numerous medical advances in the treatment of this disease, up to 15% of these kids requires some form of abdominal surgery within the first five years from their diagnosis. Usually, this is due to stricture formation where the bowel remodels and scars from inflammation. Our goal in this project is to prevent stricture formation in pediatric Crohn’s disease patients. A poorly understood feature of Crohn’s disease is the development of what is known as “creeping fat.” Surgeons have long recognized the fat around the bowel has an abnormal shape in Crohn’s disease patients. It grows and starts to invade the space around the intestine. This growth has been associated with increased inflammation and stricture formation. Recent research has shown that fat has many biochemical activities that can interact with the surrounding tissue to increase inflammation. Our lab aims to look at how this “creeping fat” can play a role in changing the architecture of the intestine. We have developed a unique animal model that produces the creeping fat seen in Crohn’s disease. Our goal is the use the latest techniques to look at the exact process for how this occurs at the genetic level. We have the ability to map out genes in a three-dimensional way that has the promise of unlocking the exact cells involved in this process. We believe that we can find and track the genes and cells that cause stricture formation in Crohn’s disease and by that information, find different targets for possible therapeutics to alleviate this burden on Crohn’s disease patients. Despite our background in surgery, our goal is that one day, patients who suffer from Crohn’s disease will rarely, ever need surgery again due to stricture formation.

Celiac Disease (CeD) is characterized by an autoimmune destruction of the duodenal intestinal epithelium induced by gluten peptides, typically gliadin, which can cause nutrient malabsorption, abdominal pain, diarrhea, and failure to thrive. While it is known that the pathogenesis of CeD is initiated by gluten-specific CD4+ T cell activation and expansion, mechanistic insights have been substantially hindered by lack of experimental models. Our team has created an in vitro CeD organoid model from duodenal endoscopic biopsies that preserves both the syngeneic intestinal epithelium and a broad spectrum of native infiltrating intestinal immune cells without reconstitution. Importantly, our CeD organoid model exhibits signature hallmarks of CeD pathophysiology including T cell expansion and activation, plasma B cell anti-tTG2 autoantibody production, and epithelial IL-15 upregulation in response to in vitro gliadin challenge, with consequent epithelial cell death. Thus, we propose in Aim 1 to use pediatric CeD organoids to gain new insights into the acute progression crosstalk between immune and epithelial components underlying CeD pathogenesis, using unbiased single cell and multi-omic approaches. Aim 2 performs targeted T and B cell depletion experiments in pediatric CeD organoids using novel antibody-toxin conjugates, as well as manipulation of IL-15 levels and IEL/epithelium blocking assays, to experimentally dissect the immune cell requirements that culminates in death of epithelium in CeD. These studies exploit this robust in vitro human model of pediatric celiac disease to derive significant mechanistic insights into pathogenesis, validating this system for future downstream applications including diagnosis and therapeutics.

Celiac disease (CD) is a gluten-triggered enteropathy affecting ~1% children and adults around the world, with an apparently increasing incidence. The only effective treatment is gluten-free diet, although non-responsive individuals have been reported. Given disease comorbidity and the similar symptoms and clinical indications between CD and other intestinal disorders, CD diagnosis remains challenging, even with a combinatorial method of serology and endoscopic and histological examination of the duodenal mucosa. Understanding the cellular/molecular pathophysiology that links gluten digestion to enteropathy is essential to both research and clinical applications. CD association to dietary gluten and susceptibility to a genetic factor, HLA-DQ2 (up to 95%), strongly suggest unique characteristics of DQ2-expressing cells that “present” gliadin peptides to immune effector cells (T cells). The presenting cells (APCs) thus play their essential roles in connecting the gluten trigger with T cell-mediated small intestine damage. To effectively target these APCs, we and others have developed a few antibody-based reagents that specifically recognize gluten-derived peptides (i.e., gliadin- αI or -αII) bound to DQ2 proteins on the surface of APCs. These reagents recapitulate the binding characteristics of T cell receptors (TCRs) to gliadin/DQ2, but with greatly improved affinity and specificity. Here, we propose to utilize a specially designed engineering pipeline (a yeast display system) to further optimize these TCR-like (TCRL) reagents to allow the potential use of these TCRL in downstream targeting, staining, identification and isolation of gliadin-presenting APCs. A panel of APC-specific reagents including TCRL will enable multiplexed single-cell analysis (cutting edge techniques such as CyTOF, CODEX) to identify and localize gliadin-presenting APC in the small intestinal tissue, for investigation of CD pathogenesis. Reciprocally, understanding specific cell types presenting gliadin to T cells will guide in vitro (organoid models) or in vivo (animal models, in the future) evaluation of TCRL as adjuncts to diagnosis or therapy.