Proteomic profile of extracellular vesicles in anaphylaxis and their role in vascular permeability

Abstract

Background: Anaphylaxis is the most severe manifestation of allergic disorders. Diagnostic and understanding of molecular mechanisms need to improve. Extracellular vesicles (EVs) play a key role in cellular communication offering new possibilities to unravel patient-based particularities. The aim of this study is to analyze the protein profile of anaphylaxis-derived EVs providing a resource of potential markers for anaphylactic reactions, and to characterize their molecular mechanisms. Methods: EVs were isolated from 86 plasma samples (collected from 43 patients) during the acute phase of anaphylaxis (AnEVs) and at their baseline (BEVs). For comparison, EVs were characterized and their protein patterns were analyzed by mass spectrometry-based quantitative proteomics (LC-MS/MS). System Biology Analysis (SBA) was applied to identify main canonical pathways and molecules involved. In addition, in vitro permeability assays based on EVs-endothelial cells (ECs) were performed. Results: Differential proteomic analysis performed in 10 EVs paired patients’ samples identified 1206 proteins of which 99 were modulated in the AnEVs signature. CDC42, Ficolin-2 and S100A9 enrichment was confirmed in a larger cohort of patients. SBA revealed diverse group of immune proteins as the main canonical pathways altered in AnEVs. Thus, leukocyte extravasation and granulocyte adhesion-diapedesis processes stand out. In addition, marked-EVs from anaphylactic patients were captured by ECs decreasing the resistance of human endothelial monolayers. Conclusion: Our findings identify for the first time a differential EVs pattern signature in anaphylaxis revealing a source of potential biomarkers. Furthermore, these vesicles could participate in altered immune molecular mechanisms and present a role increasing vascular permeability.

Increased miR‐21‐3p and miR‐487b‐3p serum levels during anaphylactic reaction in food allergic children

“…Due to a recent study from our group that demonstrated an active participation of extracellular vesicles interacting with ECs in anaphylaxis, 16 an in vitro serum‐EC system was carried out to evaluate the functional role of miR‐21‐3p and miR‐487b‐3p in this event. In order to do so, intracellular levels of both miRNAs were measured after incubation with anaphylactic conditions: cocktail of mediators and serum from patients (acute and basal phases).…”

“…Human dermal microvascular ECs (HMVEC‐D) were acquired from Lonza CC‐2543 and maintained in EGM™‐2MV BulletKit™ as previously described 16,17 . EC monolayers were incubated for 2 hours with: EGM‐2 medium (negative control), a cocktail of anaphylaxis mediators, or serum samples (acute and basal) from 5 patients.…”

Anaphylaxis: Mediators, Biomarkers, and Microenvironments

“…Currently, the only study carried out demonstrated the existence of a differential proteomic profile in patients' EVs obtained during the anaphylactic reaction. Among these proteins, an increase in Ficolin-2, CDC42 and alarmin S100A9 levels was demonstrated [188].…”

“…as key players in cell-cell communication during the anaphylactic reaction [181,188] (FIGURE 4). Specifically, miR-155 regulates MC degranulation by modulating their calcium concentration through PI3K levels [179].…”

“…In addition, miR-21-3p could participate in this process by its action on Rac signaling, a protein involved in MC degranulation [181,224,225]. In turn, analysis of EVs obtained during anaphylaxis revealed that the main function of their differential proteins was neutrophil degranulation [188]. Specifically, elevation of circulating levels of miR-451a has been associated with increased degranulation of the effector cells of the reaction [88].…”