CARBOXYPEPTIDASE A3 IS ELEVATED IN MAST CELLS NEARBY BASAL EPITHELIAL CELLS AND DRIVES LOSS OF EPITHELIAL BARRIER INTEGRITY IN EOE

Background: Eosinophilic Esophagitis (EoE) is one of the most predominant causes of chronic esophageal symptoms in children. Pathological changes of the esophageal epithelium in EoE are characterized by basal cell hyperplasia (BCH), infiltration of eosinophils and elevated levels of cytokines (e.g. IL-4 and IL-13). Our knowledge of EoE pathogenesis has mostly relied on adult animal models, despite unique clinical phenotypes of pediatric EoE. Accumulation of the extracellular matrix protein tenascin-C (TNC) is associated with asthma; however, its role in EoE pathogenesis has not been investigated. Using proteomics, our preliminary data show that TNC is dramatically increased in IL-13 treated human esophageal basal cells (EPC2 cells). We hypothesize that IL-13 induced pediatric EoE progression is dependent on TNC which modulates BCH. Methods: We adapted our previously established transgenic mouse model to generate Krt5-rtTA; tetO-IL13 mice pups. To examine the role of TNC deletion in our pre-weaning mouse model of pediatric EoE, we generated Krt5-rtTA; tetO-IL-13; TNC^-/- pups to determine the effects on the development of EoE. We also investigated the effect of TNC knockdown in IL-13 treated EPC2 cells using siRNA. Furthermore, we examined human esophageal biopsies from EoE patients and healthy controls to evaluate TNC and IL-13 levels, BCH and esophageal eosinophilia and their correlation with EoE disease stage. Results: IL-13 overexpression was detected in hyperplastic basal cells of Krt5-rtTA; tetO-IL13 transgenic pre-weaning mice upon doxycycline water feeding. Keratin 5 (Krt-5), TNC and major basic protein (MBP) immunostaining confirmed TNC expression in hyperproliferative basal cells in addition to eosinophils in the esophageal epithelium of these pre-weaning mice. These mice weighed less than controls; resembling a failure to thrive phenotype as seen in human infants with EoE as well as skin lesions similar to atopic dermatitis. Deletion of TNC in the transgenic pre-weaning mouse esophagus reduces the thickness of basal cell layers (p63⁺ Krt5⁺), proliferation (p63⁺ Ki67⁺) and differentiation (Krt13⁺) of basal cells. TNC knockdown reduces the number of proliferating (p63⁺ Ki67⁺) EPC2 cells. Consistently, TNC expression is increased in esophageal biopsy specimens from EoE patients as compared to healthy controls. Conclusions: For the first time, we demonstrate that our novel transgenic pre-weaning EoE mouse model shows that TNC is not only critical but also plays a conserved role in epithelial remodeling in the early stages of EoE pathogenesis. Future directions include whether the mechanism involving TNC may be also used in other allergen associated diseases including atopic dermatitis. Keywords: eosinophilic esophagitis, IL-13, basal cell hyperplasia, Tenascin-C, pediatric mouse model

Introduction: Eosinophilic Esophagitis (EoE) involves epithelial barrier dysfunction characterized by basal zone hyperplasia (BZH). Mast cells (MC) are increased in EoE. MC activation leads to release of pre-formed proteases such as carboxypeptidase A3 (CPA3), and translocation of LAMP3 (CD63) to the cell surface. CPA3 has restricted expression in MCs and little is known regarding the role of CPA3, a zinc-dependent metalloproteinase, on epithelial barrier function. We hypothesized that activated MCs in EoE release CPA3 which drives mucosal barrier dysfunction through loss of barrier proteins.
Methods: Flow cytometry was performed on single cell suspensions of esophageal biopsies to detect activated MC and correlated to BZH . CPA3 ELISA was performed on biopsy lysates from patients with active and inactive EoE. IF was performed for CPA3 and SOX2 on biopsies from EoE patients and controls with varying degrees of BZH. Total intraepithelial CPA3+ MCs were counted along with CPA3+ MCs nearby and far from SOX2+ cells. Single cell RNA-Sequencing of esophageal biopsies was performed to assess protease expression among MC subsets between patients with varying degrees of BZH along with DSG1 among epithelial cell subsets. Air-liquid interface (ALI) culture of human esophageal epithelial cells (EPC2-hTERT) was generated and recombinant, mature CPA3 was exposed to the basal ALI layer for 48 hours with or without a carboxypeptidase inhibitor (PCI). Transepithelial electrical resistance (TEER) was measured at baseline and 48 hours after CPA3. Morphologic assessment of H&E-stained sections was performed along with DSG1 IHC.
Results: Increased CD63+ MCs were present in active compared to inactive EoE (p<0.01) and moderately correlated with BZH (p<0.01) severity. Patients with active EoE had significantly more CPA3 protein in biopsy lysates compared to inactive EoE (p<0.01). CPA3+ MCs were increased among patients with severe BZH (p<0.01), and spatially located near SOX2+ basal epithelial cells (p<0.01). MCs from patients with severe BZH and furrows had the highest CPA3 RNA (p<0.0001). Transient-type MCs, found only in patients with severe BZH, had the greatest amount of CPA3 compared to resident MCs (p<0.0001). DSG1 gene expression was significantly reduced only among transitioning epithelium (p<0.001). CPA3 significantly reduced TEER (p<0.001) and was reversible with a carboxypeptidase-specific inhibitor (p<0.05). CPA3-treated ALI showed increased basal cell layers with loss of squamous cells and DSG1.
Conclusion: Activated MCs are present in EoE and correlate with BZH severity. CPA3 is increased in transient-type MCs in EoE, and CPA3+ MCs are located near basal epithelial cells. CPA3 is sufficient to induce epithelial barrier disruption and BZH associated with loss of DSG1 in transitioning epithelial cells, and is reversible with a carboxypeptidase inhibitor.