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.

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.

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.

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.

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.

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.

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. 

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.

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

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.

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.