Allergic diseases are increasing in prevalence worldwide. Existing allergy increases the risk for developing another allergy in the same individual. Approximately 80% of children with atopic dermatitis develop asthma or allergic rhinitis later in life in a process known as the "atopic march". Immunoglobulin E (IgE)-mediated allergic inflammation is dependent on mast cell and basophil activation and release of mediators. Mast cells are long-lived tissue-resident hematopoietic cells that reside in nearly all tissues. Moreover, mast cells and their mediators have been shown to be particularly important for allergic responses and disease progression.

While mast cells and basophils are able to respond to several common stimuli, I hypothesized that each cell type would have unique activation-induced transcriptional signatures. Using mouse bone-marrow derived mast cells (BMMCs) and basophils (BMBs), I compared IgE- and non-IgE-mediated activation phenotypes. Interleukin-33 (IL-33) is a cytokine linked to the development of several allergic diseases, and IL-33 has recently been shown to potently activate mast cells and basophils. By stimulating mast cells with IgE/antigen and IL-33, I demonstrated that mast cells have activation-specific transcriptional signatures. Furthermore, by performing the same analysis in basophils, I was able to characterize heterogeneity in basophil and mast cell responses to the same activating signals. The limited homology that we observe between these cell types after activation with the same stimuli suggests that these cells play unique roles in the immune response. From these analyses, I found that mast cells induce enhanced Il33 gene expression upon IgE-mediated activation, but not with IL-33 stimulation itself. We hypothesized that IL-33 does not act in an autocrine fashion. The activation-induced signature in IL-33-deficient and ST2 (IL-33 receptor)-deficient was not different from wild-type mast cells suggesting that IL-33 does not act in an autocrine manner. Lastly, since mast cells become activated in tissues where there is a limited oxygen supply, we asked if hypoxia effects the IgE/antigen-induced transcriptome. We demonstrated that the mast cell activation-induced transcriptome is not altered if the activation occurs in hypoxic culture conditions.

From these transcriptional analyses, I found that IL-33-activated basophils selectively upregulate neutrophil-attracting chemokines, and I hypothesized that basophils help orchestrate allergic inflammation through this pathway. Using a model of mast cell-driven tissue inflammation, I demonstrated that mast cell-derived IL-33 acts on ST2-expressing basophils to promote neutrophil infiltration into murine ear tissue. Both mast cell-derived IL-33 and ST2-expressing basophils were both necessary and sufficient for antigen-driven tissue inflammation in vivo. These data define a mechanism for how mast cells via the release of IL-33 help to orchestrate a basophil-driven late phase inflammatory response during IgE-mediated inflammation.

In addition to investigating these mast cell activation phenotypes, I explored the role of mast cells in the setting of chronic allergic inflammation. By developing an in vitro protocol for the repeated activation of mast cells, I demonstrated that stimulation of mast cells results in transcriptional and epigenetic reprogramming akin to what can occur with other innate immune cells. These repeatedly activated mast cells become high expressers of IL-33 and take on an "trained" phenotype. Using a mouse model of the atopic march, I demonstrated that mast cell-derived IL-33 contributes to the progression of the atopic march by enhancing the severity of subsequently induced allergic airway inflammation.

Lastly, I explored the regulation of the mast cell activation phenotype by Siglec-8 (sialic acid-binding immunoglobulin-like lectin-8) an ITIM-containing inhibitory receptor. Siglec-8 is expressed on human, but not on mouse mast cells. Working in collaboration with the Zhu lab at Yale University, I characterized SIGLEC8Mc, a new knock-in strain designed for the investigation of Siglec-8 in mice. Crossing a Mcpt5(connective tissue mast cell protease)-Cre mouse with a mouse that transgenically expresses human SIGLEC8-fl/fl resulted in a mouse selectively expressing Siglec-8 in connective tissue mast cells. In this mouse, I showed that mast cell number is unaffected by the transgene. Furthermore, I demonstrated that Siglec-8 is highly and specifically expressed on mast cells and not on eosinophils or other cells. Thus, this SIGLEC8Mc is a novel and unique tool to understand how Siglec-8 regulates the mast cell activation phenotype which will be explored in future studies.

In conclusion, this thesis document compiles a body of evidence that shows the remarkable diversity and plasticity of the mouse and human mast cell in homeostasis and allergy.