Abstract: Infection with Helicobacter pylori (H. pylori) and autoimmune gastritis both cause chronic inflammation and increase the risk of gastric cancer. Metaplasia in various tissues (lungs, pancreas, stomach) is associated with chronic inflammation and indicative of disease and an increased risk for developing cancer. In the stomach, metaplastic changes are induced in gastric epithelial cells, and >95% of all gastric cancers are epithelial cell derived adenocarcinomas. There is a critical gap in our understanding how individual immune cells and the inflammatory mediators they secrete (e.g. cytokines) regulate gastric metaplasia and gastric carcinogenesis. This application investigates the importance of a cytokine (IL-13) and immune cell type (mast cells) in inducing gastric metaplasia and promoting the development of gastric tumors. Experiments include a variety of genetically modified mouse models of gastritis, single cell transcriptomics of mouse and human cells, tumorigenesis studies, and analyses of human biopsies to develop mechanistic insight into how mast cells and IL-13 signaling regulate gastric metaplasia and tumorigenesis. Understanding how inflammation induces metaplasia and tumor development could improve the ability to identify individuals at an increased risk of disease progression, and new immune based strategies to prevent and treat preneoplastic lesions associated with gastric diseases, including gastric cancer. Work performed during this R56 funding period generate additional preliminary data to address some of the critiques from recent reviews of our application.
SPECIFIC AIMS: Metaplasia is associated with inflammation and indicative of disease and an increased risk for developing cancer. In the stomach, Helicobacter pylori (H. pylori) infection and autoimmune gastritis both cause chronic inflammation, metaplasia, and increase the risk of gastric cancer. Metaplastic changes are induced in gastric epithelial cells, and >95% of all gastric cancers are epithelial cell-derived adenocarcinomas. How specific components of inflammatory responses induce and regulate gastric metaplasia and gastric carcinogenesis is poorly understood. Determining the immune cells and cytokines that induce metaplasia and support its progression to cancer (adenocarcinoma) are essential in defining the pathophysiology of this disease process and could reveal new immune-based strategies to prevent and treat disease.
We recently discovered that mice lacking the IL-4RA receptors (Il4ra-/-) failed to develop metaplasia in a model of chronic gastritis and metaplasia. The IL-4RA is a component of receptors for two cytokines, IL-4 and IL-13. While IL-4 levels were nearly absent, IL-13 was secreted at relatively high levels in the gastric mucosa in two different mouse models of gastritis and gastric epithelium express IL-13 receptors. Surprisingly, most of the IL- 13 secreting cells were mast cells. Recent data in mouse models have associated IL-13 production with gastritis and gastric metaplasia, but the critical cellular sources and mechanism(s) of action are unknown. Our preliminary data demonstrate that IL-13 acts directly on gastric epithelium to induce growth, survival, and induce transcriptional changes associated with metaplasia. These data lead us to hypothesize that IL-13 plays a critical role in the development of gastric metaplasia, in part by acting directly on gastric epithelium, and that mast cells are a previously unrecognized critical source of IL-13 in chronic gastritis. The following specific aims will test these hypotheses:
Aim 1: Test whether IL-13 signaling in immune and/or epithelial cells is required for gastric metaplasia. IL-4RA and IL-13 have pleotropic effects on both immune and epithelial cells. To test whether IL-13 is necessary to induce gastric metaplasia, mice will be treated with IL-13 neutralizing antibodies. To test whether IL-13 is sufficient to induce gastric metaplasia, mice will be treated with IL-13 using mini-osmotic pumps and effects on metaplasia will be determined. Finally, to test the contributions of IL-4RA signaling in immune vs. epithelial cells we will assess gastric metaplasia in mice lacking IL-4RA in hematopoietic (immune) vs. non- hematopoietic (epithelial) cells. During R56 period additional data will be generated to identify the specific IL-13 (and IL-4) secreting immune cell subsets in both the H. pylori and the autoimmune disease models.
Aim 2: Determine the gastric epithelial cell responses to signaling through IL-4RA. We hypothesize that an important function of IL-4RA in metaplasia is to signal directly into mucous neck cells and/or chief cells to initiate transcriptional changes associated with metaplasia. Experiments will compare transcriptional profiles of gastric epithelial cells from a chronic gastritis setting in control and IL-4RA-/- mice, and mouse and human gastric epithelial cells cultured with IL-13. Experiments during this R56 period will generate stronger preliminary data on effects of IL-13 on transcriptional changes in gastric epithelium and will perform a more in-depth analyses of IL-4RA expression and address IL-13RA2 decoy receptor expression by both immune and cells and gastric epithelium in both mice and preliminary studies in humans.
Aim 3: Determine the role of mast cells in metaplasia using chronic mouse models and human tissue arrays. The goals of this aim are to identify the signals that induce gastric mast cells to secrete cytokines (including IL-13), to use mast cell-deficient Cpa3Cre/+ mice to define the requirement for mast cells in gastric metaplasia, to determine the requirement for mast cells for metaplasia to advance to invasive adenocarcinoma, and to extend our findings into human biopsies. During this R56 period of funding we will complete crosses of TxA23x Cpa3Cre/+ mice and characterize the extent of mast cell deficiency. We also plan to isolate and expand mast cell from stomachs, co-culture mast cells with gastric organoids to determine how activated mast cells from the gastric mucosa of mice with chronic gastritis affect organoid features (growth, viability, metaplasia associated gene expression). We will generate preliminary data with human tissue (healthy, and metaplastic caused by autoimmune and H. pylori induced gastritis).
This proposal explores a new cell type (mast cells) and a molecular mechanism for understanding how IL-13 drives gastric metaplasia and influences the development of gastric adenocarcinomas. Completion of these related yet independent aims is expected to provide essential mechanistic insight into the pathogenesis of gastric carcinogenesis which could help diagnose high risk individuals and develop