Simon P. Hogan - US grants

DESCRIPTION (provided by applicant): Food allergy affects approximately 5% of individuals in the USA. The most severe form of food allergy, food-induced anaphylaxis causes approximately 100 deaths per year. An essential question in the food allergy field is to understand the molecular mechanisms that predispose to food allergy and anaphylaxis. In preliminary analysis, employing IL-9 deficient mice, intestinal IL-9 transgenic mice and experimental models of food allergy (intestinal anaphylaxis) we have demonstrated that IL-9 profoundly increases intestinal mast cells, intestinal permeability and predisposes to food allergy. The objective of this application is to define the molecular mechanisms involved in IL-9-mediated predisposition to intestinal anaphylaxis. The working hypothesis of this proposal is that IL-9 is a stimulus and required for the induction of experimental intestinal anaphylaxis. The central hypothesis will be addressed in three aims. In Aim 1, we will employ IL-9-deficient, IL-9R-deficient and WT mice and our experimental model of intestinal anaphylaxis to define the role of IL-9 in the immunopathology of intestinal anaphylaxis. In Aim 2, we will delineate the relationship between IL-9 overexpression in the gastrointestinal tract, intestinal permeability and predisposition to oral antigen sensitization. In Aim 3, we will define the role of mast cells and platelet activating factor in IL-9-induced altered intestinal permeability. We expect to find that IL-9 is central to the regulation of different components of the intestinal inflammatory response and the predisposition to oral antigen-induced intestinal anaphylaxis. Such results will have an important positive impact on human health, as identification of the molecular processes involved in food allergen sensitization and anaphylaxis will provide new and innovative approaches for the specific prevention and treatment of these diseases.Narrative: Food allergy and anaphylaxis are of increasing importance in the western world. Our hypothesis is that the cytokine IL-9 selectively stimulates mast cell- dependent pathways and promotes oral antigen sensitization and the onset of food allergy. Defining the biological processes involved in food allergy susceptibility will advance our understanding of these complex and poorly understood clinical events, and be applicable to the prevention/treatment of other allergic disorders such as eczema and asthma.

DESCRIPTION (provided by applicant): Corticosteriods (CS) remains the mainstay of therapy for pediatric UC; however, 20% of patients fail to respond to CS therapy and require secondary-line therapies and escalation of medical management or colectomy4. In recent patient-based studies, we demonstrated that CCL11 levels correlated with tissue eosinophil numbers, which, in turn, correlated with the UC Histologic Index of Severity (UCHIS) in pediatric UC. In preliminary studies, we determined that high rectosigmoid eosinophil levels at the time of pediatric UC diagnosis is linked with reduced likelihood of achieving steroid-free remission (SFR). Notably, high eosinophil levels positively correlated with levels of CCL11 and a low molecular weight intracellular calcium binding protein called calprotectin. In experimental studies, we have identified a link between calprotectin, M¿-derived CCL11, and eosinophils in experimental colitis. Importantly, we show that colonic M¿s express the calprotectin receptor (RAGE); and calprotectin stimulates p65 activation and CCL11 expression in M¿s in vitro. The object of this application is to further understand the relative contribution of calprotectin to inflammatory M¿-derived CCL11 and eosinophilic inflammation in experimental colitis and pediatric UC and treatment responses. Our central hypothesis is that calprotectin-induced activation of M¿s regulates CCL11-dependent colonic eosinophilic inflammation in experimental colitis and pediatric UC and that this pathway drives refractory disease. We will test this hypothesis by examining the relationship between calprotectin, M¿s, CCL11, and eosinophils in pediatric UC at diagnosis and the relationship of this pathway to refractory disease. We will employ S100A9-/-, RAGE-/-, CCR2-/-, CCR2-/-/Il10-/- mice; use DSS- and NSAID-induced Il10-/- models of colitis; generate bone marrow-derived M¿-specific chimeric mice and perform eosinophil chemotaxis assays to define M¿-derived CCL11 in eosinophil recruitment and histopathology in experimental colitis and the sensitivity of this pathway to CS treatment. With respect to expected outcomes, the studies proposed in Aim I are expected to define the association between calprotecin, CCL11+ M¿s, and eosinophils in pediatric UC; the sensitivity of this pathway to CS; and the predictive value of CCL11 and eosinophils disease as an indicator of resistance to first-line therapies. Aim II is expected to delineate colonic eosinophilc inflammation and histopathology dependency on calprotectin/RAGE-induced M¿-derived CCL11 in experimental colitis. Aim III is expected to define the capacity of calprotectin/RAGE to induce M¿-derived CCL11 and eosinophil chemotaxis and sensitivity to CS-induced inhibition. Demonstration of calprotectin/M¿/CCL11/eosinophil axis involvement in resistance to first-line therapies in pediatric UC will have important clinical implications for both the usage of CCL11 and eosinophils as a prognostic indicator for resistance to first-line therapies at diagnosis and the possible usage of therapeutic agents targeting eosinophils and eosinophil regulatory molecules as an approach for improved treatment of pediatric UC and refractory UC. PUBLIC HEALTH RELEVANCE: Pediatric ulcerative colitis (UC) has a high rate of corticosteroid (CS) dependency and frequent relapse requiring more aggressive therapy including surgery. We have identified a link between the level of the white blood cell, eosinophil at diagnosis in pediatric UC, and unresponsiveness to CS. In this application, we will unravel the mechanisms involved in the regulation of eosinophil movement and function in UC and identify if eosinophil levels can be used as a potential biomarker for CS-responsiveness in pediatric UC.

? DESCRIPTION (provided by applicant): Food allergy is a serious and common disorder within the U.S. and is thought to impact 1 in 4 families. The main approved therapy for food allergy is food avoidance; however, with the use of milk, eggs, peanuts and tree nuts in many foods in which their presence might not normally be anticipated, accidental and dangerous exposures are common. Recently, we identified a new mechanism of intestinal luminal soluble antigen sampling, which was mediated by goblet cell (GC) antigen passages (GAPs) 10. In preliminary studies, we identified a new pathway in the regulation of GAP formation and an important role for this pathway in atopic susceptible mice in both the development of clinical reactivity to foods and onset of an acute food allergic reaction. Our central hypothesis is that GAP-mediated intestinal antigen delivery primes for the sensitized food allergic state and stimulates clinical reactivity to foods. The Specific Aims outlined in this proposal will directly est the involvement of GAP formation in the facilitation of food antigen presentation, development of the sensitized food allergic state and clinical reactivity to foods. With respect to the expected outcomes, the studies proposed are expected to demonstrate that: Aim I) GAPs are the primary pathway of SI food antigen delivery to the immune system; Aim II) that cholinergic-induced GAPs promote the sensitized food allergic state in atopic susceptible individual by enhancing the delivery of food antigens; and Aim III) MC-derived IL- 13 in close proximity to GCs induces GAP formation, thereby augmenting antigen delivery and onset of a food- induced anaphylactic reaction. Successful completion of the proposed studies will provide a new and substantive departure from our current understanding of the underlying mechanisms of intestinal luminal antigen sampling and development of clinical reactivity to foods and identify GAPs as an attractive target for the development of therapeutic intervention to prevent development of new food allergies.

Project Summary Food allergy is rapidly increasing in prevalence and is the most common cause of anaphylaxis. While significant progress has been made in our understanding of the underlying immunologic pathways involved in dendritic cell (DC) presentation of food allergens, the development of the CD4+ Th2 repertoire, and the IgE-MC?dependent effector processes, there has been little attention to the processes underlying the passage of food allergens across the GI epithelium and presentation of these allergens to the immune compartment. We have recently reported 1) that goblet cells (GCs) in the small intestine (SI) of naïve mice can act as a conduit and permit the passage of food allergens across the intestinal epithelial (IE) layer and presentation to the SI lamina propria immune compartment (termed Goblet cell antigen passages; GAPs)13; 2) that food allergic mice utilize a different IE transport mechanism involving SI villus and crypt GCs, enteroendocrine cells, and Paneth cells that passage food allergens across the intestinal epithelial layer (termed secretory epithelial antigen passages; SAPs)24 and 3) a critical role for IE cell-derived pro-Type 2 cytokines such as TSLP, IL-25 and IL-33 in the development of food-induced anaphylaxis in mice14. In a series of preliminary studies, we interconnect these observations demonstrating that induction of SAPs and uptake of food allergens in murine intestinal epithelial cells leads to expression of the pro-allergic cytokine, IL- 33 and employing a human intestinal organoid (HIO) transplant model system demonstrate that the molecular processes, GAPs and SAPs are conserved in humans. The current gap in knowledge is the specificity of food allergen uptake by secretory intestinal epithelial cell lineages; the potential existence of environmental trigger programming of IEs which leads to modified antigen passage patterning (GAPs vs SAPs); and the relationship between food allergen uptake by SAPs and expression of pro-Type 2 cytokines in human tissue. We hypothesize that SAPs are a mechanism by which food allergens are channeled across the intestinal epithelium, promote the production of pro-Type 2 cytokines and whose composition and function are modulated by environmental triggers. In Aim I we will define the SI secretory cell lineages involved in food allergen passages and the impact of environmental triggers on food allergen passage patterning and Aim II, define the transcriptional inflammatory signature of human intestinal epithelial cells following food allergen uptake. With respect to the expected outcomes, the studies proposed in Aim I are expected to identify the involvement of distinct SI epithelial-specific food allergen transport processes and how they are influenced by environmental triggers, and those in Aim II are expected to reveal a link between pro-Th2 cytokine production and food allergen uptake by SI intestinal epithelial cells and define the pro-allergic transcriptional inflammatory signature of antigen passages. Collectively, these studies will illuminate new IE-restricted processes by which food allergens are sampled by the human SI compartment; link food allergen uptake with pro-Type 2 cytokine production and identify divergent pathways of SI IEs food allergen uptake that are influenced by FA status.

Project Summary Eosinophilic esophagitis (EoE) is an increasingly prevalent chronic inflammatory disease of the esophagus, mediated by dietary food antigens and clinically characterized by upper gastrointestinal (GI) symptoms including dysphagia and food impaction. Recently, a confounding esophageal disorder, termed proton-pump inhibitor (PPI)?responsive esophageal eosinophilia (PPI-REE), has emerged; PPI-REE is indistinguishable from EoE by clinical, endoscopic or histologic features or by gene profiles. The current clinical conundrums are whether PPI- REE represents a GERD-related phenomenon, a subtype of EoE or a completely new entity and why PPI-REE and EoE respond differently to PPI. RNA sequencing (RNA-Seq) analyses of esophageal biopsy samples from patients with active EoE disease revealed dysregulation of gene networks associated with regulating intracellular [pH]i and acid protection and that the most upregulated transmembrane transporter activity gene was SLC9A3, which encodes for the sodium-hydrogen exchanger family member 3 (NHE3). Recently, we have demonstrated 1) increased expression of SLC9A3 within the basal layer of ESSE biopsies from patients with EoE and that expression positively correlated with disease severity (eosinophils/HPF) and DIS; 2) IL-13 induced SLC9A3 expression and function in ESSE cells and that SLC9A3 activity positively correlating with DIS formation and 3) SLC9A3-mediated Na+-dependent proton secretion is the primary intracellular acid protective mechanism within IL-13?stimulated ESSE cells and blockade of this pathway abrogated DIS formation45. In new preliminary studies we have made several transformative observations: 1) IL-13 induced expression of the transcription factor aryl hydrocarbon receptor (AhR) and AhR-responsive genes in ESSE cells and EoE biopsies; 2) stimulating ESSE cells with AhR ligands, suppressed AhR-responsive gene expression including SLC9A3 and 3) a divergent effect of PPI therapy on SLC9A3 expression in ESSE biopsy samples from individuals with EoE and PPI-REE, suggesting an opposing impact of PPI on SLC9A3 transcriptional regulation between EoE and PPI-REE. Collectively, these observations underlie our central hypothesis that SLC9A3 activity promotes DIS formation in EoE subtypes and that this pathway is divergently responsive to PPI therapy via AhR-dependent signaling. The specific Aims outlined in this proposal will 1) Aim 1. Determine the relationship between SLC9A3 expression and function, disease severity and DIS formation in EE subtypes; 2) Define the requirement of SLC9A3 in ESSE DIS formation and 3) Define the involvement of PPI-induced AhR signaling in Type-2 cytokine-induced SLC9A3 expression and function in ESSE cells. With respect to the expected outcomes, the studies proposed in Aim I are expected to establish the contribution of SLC9A3 to histopathologic features of EoE and PPI-REE and responsiveness of this pathway to PPI trial; Aim II are expected to determine the necessity of SLC9A3 to ESSE acid transport and DIS and Aim III is expected to determine the interaction between IL-13? and PPI-induced AhR signaling in SLC9A3 expression and function in ESSE cells and DIS formation. Successfully completing the proposed studies will provide a new and substantive departure from our current understanding of the underlying molecular mechanisms underpinning the development of PPI-REE and EoE and provide an explanation for their differential responsiveness to PPI therapy, thereby directing the development of new and pre-existing therapeutics for treating esophageal eosinophilia?related disorders.