Herein we introduce a high-throughput method, INTERFACE, to reveal the capacity of contiguous RNA nucleotides to establish in vivo intermolecular RNA interactions for the purpose of functional characterization of intracellular RNA. INTERFACE enables simultaneous accessibility interrogation of an unlimited number of regions by coupling regional hybridization detection to transcription elongation outputs measurable by RNA-seq. We profile over 900 RNA interfaces in 71 validated, but largely mechanistically under-characterized, Escherichia coli sRNAs in the presence and absence of a global regulator, Hfq, and find that two-thirds of tested sRNAs feature Hfq-dependent regions. Further, we identify in vivo hybridization patterns that hallmark functional regions to uncover mRNA targets. In this way, we biochemically validate 25 mRNA targets, many of which are not captured by typically tested, top-ranked computational predictions. We additionally discover direct mRNA binding activity within the GlmY terminator, highlighting the information value of high-throughput RNA accessibility data.

Eosinophilic esophagitis (EoE) is an increasingly recognized allergic disease associated with dysphagia and esophageal fibrosis. We aimed to determine expression patterns of specific eosinophil integrins that promote eosinophilic infiltration of the esophageal epithelium, and to determine how key EoE-related cytokines influence eosinophil activation and survival. Esophageal and peripheral eosinophils were isolated from 20 adult subjects with EoE for immunophenotyping and integrin profiling using multicolor flow cytometry and immunohistochemistry. Expression signatures of eosinophil integrins were further assessed by immunohistochemistry using serial sections of esophageal biopsy specimens. Purified eosinophils were used to assess the effect of EoE-relevant cytokines and recombinant periostin on expression of known eosinophil integrins and eosinophil survival and activation. We found that resting eosinophils express high levels of the β2-pairing integrins αL and αM, and lower levels of α4, α6 and α4β7. The migration of peripheral eosinophils to the esophagus is characterized by the specific induction of αM, and a significant increase in the proportion of αM in high-activity conformation. Periostin, a secreted extracellular matrix protein that is significantly overexpressed in EoE, enhances eosinophil survival, and this effect is mediated by αM interaction. Integrin αM is a specific marker of activated tissue eosinophils in EoE, and promotes eosinophil survival through interactions with periostin. The ability of αMβ2 to mediate eosinophil tissue residency via periostin represents a key mechanism for disease development and a potential therapeutic target in EoE.

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Eosinophilic gastrointestinal diseases (EGIDs) are a rare set of conditions characterized by the pathologic accumulation of eosinophils in the gastrointestinal tract. While eosinophils have been strongly associated with EGIDs, localized mast cells are also elevated in eosinophilic esophagitis (EoE) and gastroenteritis (EGE). Despite evidence of mast cells being an important component of EGIDs, the mechanism by which they contribute to disease pathogenesis has yet to be established in human tissue. METHODS: Single-cell suspensions were prepared by enzymatic digestion of fresh esophageal biopsies from patients with EoE and nondiseased esophageal tissue. Flow cytometry was performed to quantify and phenotype immune cells. Mast cells were FACS-sorted from tissue and incubated overnight with PMA/ionomycin to induce mediator production. RESULTS: EoE biopsies had significantly increased numbers of mast cells and eosinophils compared to non-diseased esophageal tissue. Eosinophils and mast cells from EoE biopsies had elevated expression of activation markers, indicative of an activated and degranulating state. Mast cells from EoE biopsies also displayed increased expression of surfacebound IgE and FceRI. Lastly, upon PMA stimulation, FACS-sorted mast cells from EoE biopsies produced significant quantities of cytokines and chemokines, including IL-5, GM-CSF, TNFa, CCL2, and CCL4. CONCLUSIONS: Elevated and activated mast cells are found in patients with EoE. These mast cells produce abundant cytokines and chemokines that can induce inflammation and recruit other immune cells, such as eosinophils and T cells. EGIDs are chronic inflammatory diseases that are driven in part by mast cells. Therefore, targeting both eosinophils and mast cells may be needed to significantly reduce inflammation.