Isolation and characterization of chemical species implicated in celiac disease - toward understanding pathogenesis in patients

Celiac disease (CeD) develops as the result of an inappropriate immune response to dietary gluten-derived peptides from wheat, rye, and barley. It affects approximately 1% of the population, and in the past 50 years, the prevalence has increased 4 times. Both the environmental and genetic drivers have been identified; specific HLA genes and consumption of gluten are necessary for the development of the CeD. Unlike most dietary proteins, gluten molecules are resistant to gastric and intestinal digestion, resulting in intact, partially digested gluten peptide fragments. These partially digested gluten peptides can cross the epithelial barrier of the gastrointestinal tract and be taken up by immune cells called antigen presenting cells (APCs). Gluten peptides can be displayed on human leukocyte antigen (HLA)-DQ2 or -DQ8 proteins on surfaces of APCs, which present these peptide:HLA complexes to T cells. This T cell activation initiates a TH1-type immune response and ultimately leads to mucosal damage. The binding affinity between gluten peptides and HLA proteins can be enhanced through a deamidation reaction by the enzyme transglutaminase 2 (TG2), the autoantigen in CeD; therefore, TG2 is thought to be central to the disease pathogenesis. Interestingly, TG2 is catalytically inactive under normal physiological conditions, but it can be activated by the protein thioredoxin-1 (TRX), a protein found in inflammatory environment. The only effective treatment for CeD is adherence to a gluten-free diet (GFD). As a result of the difficulties from a strict adherence to GFD, the burden of this treatment is similar to that of diabetes. Currently, both CeD serology testing and duodenal biopsy sampling through esophagogastroduodenoscopy (EGD) are required for the diagnosis of celiac disease in adults; both are invasive. In particular, EGD is also expensive and time consuming. In clinical management, detection of abnormally high autoantibody levels in blood is the primary tool to monitor a patient's disease status but has limited utility in the management of this lifelong disease, because most CeD patients become seronegative within a year after their initial diagnosis. These facts illustrate the need for the development of more advanced diagnostic, monitoring, and treatment modalities. However, efforts have been challenged by the lack of fundamental understanding of CeD molecular pathogenesis. The goals of this thesis are to develop tools and strategies to isolate and characterize various molecular species essential to CeD pathogenesis. Chapter 1 provides a more detailed background of CeD pathogenesis and highlights gaps in our current understanding of disease development from the perspective of molecular species and chemical transformations. In Chapter 2, we used NP161, a small molecular inhibitor of extracellular TRX, as a tool to identify extracellular substrates of TRX beyond TG2. In this chapter, we show that interleukin (IL)-4 is post-translationally regulated by TRX-mediated reduction. Through biochemical kinetic assays, in vitro cellular experiments, and an in vivo mouse model, we dissected the chemical mechanisms underlying this redox inactivation. Because IL-4 drives a TH2-type immune response by tuning macrophage polarization, the work in this chapter uncovers a potential secondary role of TRX. Namely, TRX may be secreted to terminate the path toward a TH2-type immune response. In Chapter 3, we further explored the role of extracellular TRX in macrophage polarization. Our results suggest that TRX is involved in initiating, enhancing, and maintaining an inflammatory phenotype in macrophages, which orchestrate TH1-type immune response. Taken together, Chapter 2 and 3 describe the role of TRX in tuning immune response -- initiating TH1 immune response and preventing the opposing TH2 counterpart. A number of gluten-derived immunotoxic peptide sequences have been identified through in vitro analyses. Previous studies suggested the presence of gluten peptides in urine, but the identities of these peptides were not investigated. Despite the key role that these peptides play in CeD pathogenesis, there is little knowledge of their absorption, metabolism, and excretion characteristics. Furthermore, not a single chemically-defined gluten peptide has ever been identified in vivo in the human circulatory system. Therefore, there is a need to identify pathophysiologically relevant peptides. Chapter 4 and 5 detailed our efforts to develop a non-invasive, quantitative technique to survey the prevalence of these immunoactive peptides in the population to further our understanding of CeD. To find the relevant structures produced from dietary gluten in CeD, we used liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to search in an unbiased manner. Chapter 4 focuses on the development of a novel sample preparation and LC-MS/MS method that excludes interfering molecules. With the optimized workflow, we recruited participants into an exploratory clinical study to test the utility of the new method in Chapter 5. Using mass spectrometric methods and untargeted sequence database analysis, we have unambiguously identified multiple gluten-derived peptides in both healthy controls and CeD patients. Our results provide the first examples of specific peptides from dietary gluten that enter systemic circulation in humans. The peptidomic investigation revealed an unexpected result -- the most prevalent peptides do not contain motifs recognized by antibodies commonly used in clinical assays for gluten detection. Consequently, we developed an immunoassay to detect gluten peptides with the most common chemical motif in Chapter 6. This immunoassay not only provides quantification of the most prevalent gluten peptides identified through the LC-MS/MS result, but also has potential for monitoring disease status of patients with CeD. Through these chapters, beyond developing non-invasive, quantitative diagnostic tools, our methods also provide a new path toward answering fundamental questions about CeD pathogenesis. Both gluten consumption and genetic susceptibility determinants are necessary but not sufficient to cause CeD. Our work may have revealed additional factors that contribute to the transition from healthy to disease state, and these molecules and their chemical transformations provide exciting new opportunities for diagnostics and treatment

Keywords: Chemistry.
Advisor: Khosla Chaitan
Burns Noah
Du Bois Justin