424:
Deletion of SPINK7 by CRISPR/Cas9 Elicits Pro-Inflammatory and Impaired Epithelial Barrier Responses in Esophageal Epithelial Cells
Sunday, March 4, 2018
South Hall A2 (Convention Center)
Ayushi Jain, BS, Nurit P. Azouz, PhD, Julie M Caldwell, PhD, Marc E Rothenberg, MD PhD FAAAAI
RATIONALE: Eosinophilic esophagitis (EoE) is an inflammatory disorder characterized by an impaired epithelial barrier. SPINK7, a protease inhibitor and modulator of uPA, is depleted in EoE patients. Herein, we aimed to investigate if SPINK7 deficiency recapitulates predominant EoE features.

METHODS: CRISPR/Cas-9-editing was applied for knockout of SPINK7 in human esophageal epithelial cells. Control and SPINK7 KO cells were cultured at the air-liquid interface or in high calcium-high confluency conditions to promote squamous cell differentiation. SPINK7 protein expression was assessed by western-blot, barrier integrity by trans-epithelial electrical resistance, and filaggrin mRNA expression by qPCR. Trypsin-like proteolytic activity and uPA activity were determined through functional assays. Additionally, pro-inflammatory cytokine release of TSLP and IL-8 were assessed.

RESULTS: CRISPR/Cas-9-mediated knockout of SPINK7 was verified by undetectable SPINK7 protein expression in knockout cells. Functionally, SPINK7 KO cells produced 1.7-fold lower trans-epithelial resistance (p<.0001) during differentiation and exhibited 4-fold decrease in filaggrin expression (p=.0002), indicating impaired epithelial barrier function compared to control cells. Loss of SPINK7 unleashed proteolytic activity observed by enhanced trypsin-like (16-fold, p<.0001) and uPA activities (7-fold, p=.0005) of SPINK7 KO in comparison to control cells. Further, a pro-inflammatory response was triggered with increased release of cytokines IL-8 (2-fold, p=.0008) and TSLP (3-fold, p=.015) by SPINK7 KO compared to control cells.

CONCLUSIONS: SPINK7 loss induces an EoE-like phenotype with impaired esophageal barrier function, unhindered proteolytic activity and increased pro-inflammatory cytokine release, signifying its role in the inflammatory cascade of an allergic response. In addition, validated SPINK7 loss in CRISPR/Cas9-edited cells indicates potential as a drug screening model.