DISCUSSION
Anaphylaxis includes a variety of hypersensitivity reactions mainly classified accordingly to the clinical features. However, their underlying cellular and molecular mechanisms required to be fully deciphered. The fact why some reactions spread from local to systemic might be based on the importance of intercellular communication taking place in anaphylactic microenvironments and established between different cellular compartments. Therefore, circulating EVs might participate in the development of these pathophysiological processes [29]. Our studies show a different composition of plasma derived EVs in patients undergoing an anaphylactic reaction. This unique material has allowed us to identify a signature protein panel, which to our knowledge has not been previously characterized. Furthermore, our research reveals a close enrichment of immune canonical pathways related to leukocyte extravasation, agranulocytes adhesion and diapedesis. In addition, our data support a role of EVs from anaphylactic patients in vascular permeability.
The EVs proteome is increasingly considered as a rich source of biomarkers for various disease states [23]. We have identified 1206 plasma EVs-associated proteins using mass spectrometry-based quantification. A total of 83 proteins were exclusively detected in the acute phase, favoring their potential role as diagnostic markers and proposing them as candidates’ biomarkers. However, future studies are necessary to determine their possible diagnostic utility since only a few proteins found in this panel have been previously related with human anaphylaxis. In addition, 2% of the proteins contained in the panel were not already included in comprehensive EVs resources (Vesiclepedia).
CDC42, Ficolin-2 and S100A9 were selected in our study to confirm the AnEVs profile in a bigger cohort of sample patients, supporting a robust anaphylactic EV-protein signature identified. CDC42 is one of the most enriched proteins in AnEVs. It belongs to small GTPases Rho family, and it is one of the main endothelial barrier-stabilizer proteins [30], playing a crucial role in vascular injury recovery [31]. The cytoskeletal reorganization is essential for the secretion of EVs and CDC42 has been previously involved in this process [32]. Alterations of this molecule have been reported in chronic asthma in mice [33]. Ficolin-2 is a protein involved in the activation of the lectin complement pathway and it is closely related to immune processes. It induces opsonization by stimulating M1 polarization through the TLR4/MyD88/MAPK/NF-κβ signaling pathway in macrophages and the consequent release of several inflammatory mediators such as IFNγ, IL-6, TNFα and nitric oxide [34;35]. Interestingly, some of these molecules have been remarked as biomarkers in anaphylaxis [24;36]. Moreover, Ficolin-2 deficiency associates with allergic disorders in children [37]. Phagocytic S100 family proteins are activated under cell stress situations and participate in the regulation of inflammatory processes. From the proteomic studies performed here, it has been found S100A7, S100A8 and S100A9 increased in AnEVs. S100A8 and S100A9 form a heterodimer named calprotectin, modulating different functions such as leukocyte traffic, rearrangement of the cytoskeleton and neutrophil activation. Surprisingly, these are the main biological processes significantly altered in AnEVs, pointing to a calprotectin role in anaphylaxis. Even more, alarmins participate in endothelium activation increasing permeability and facilitating immune cell recruitment [38;39]. Specifically, the S100A8/A9 heterodimer complexes bind to human ECs [40]. In the allergy field, controversial roles have been pointed for calprotectin in asthma [41], however, it is not here the first time that increased levels of S100A9 in human anaphylaxis has been observed as well as the consequent activation of neutrophils [42;43]. These facts point to a possible role for alarmins in AnEVs.
The signaling pathways involved in anaphylaxis are multiple and thepicture including phenotypes, endotypes and mediators (potential biomarkers for diagnosis) is enormous [8]. Granulocytes and phagocytic cells (mainly mast cells and basophils) have been described as the key cell players, which release multiple mediators to the extracellular space during the acute phase of the anaphylactic reactions [44;45]. However, increasing evidences point to a role for other immune cells like macrophages and neutrophils [46-48]. Accordingly, our results show that around 25% of the AnEVs protein signature participates in leukocyte trans-endothelial migration and neutrophil degranulation. Some of these proteins (Anexin-1, CD14, S100A7/8/9 and Platelet Factor-4) are not only dysregulated, but they are also considered part of canonical pathways supporting the activation of monocytes and neutrophils. In addition, we have detected an important group of keratinocytes-derived proteins, which are addressed as related with glucocorticoid receptor pathway representing around of 12% of the AnVEs profile. The multisystem nature of anaphylaxis and in particular its relationship with epithelial and skin damage points to an interesting role for keratinocytes, considered contaminants in extracellular proteomics for a long time but supported as a systemically source of interleukins even in the allergy field [49].
Our knowledge regarding circulating EVs has increased exponentially in the last decade, and we know they can modify cellular functions due to the action of molecules they contain [50]. EVs participate in the destabilization of the endothelial barrier in inflammatory diseases such as sepsis or cancer [23]. This is related to a key pathophysiological characteristic of anaphylactic reactions, such as the increase in vascular permeability, which give rise to breakdown of the endothelial monolayer [51]. Our data show how ECs uptake AnEVs that are preferentially located in the perinuclear area. These results are consistent with previous publications [52], supporting a potential role in altering intracellular trafficking or even gene expression of the recipient cell, in this case the endothelial niche. In addition, AnEVs induce a greater leakage than BEVs by measuring the resistance values of the endothelial monolayer through an EndOhm device. However, further research needs to be conducted in order to clarify EVs role in endothelial damage and the differential effect of AnEVs.
We show here a challenging study of samples from human anaphylactic patients classified by allergology’s experts in the field. The EVs usage as disease biomarker is a matter of intense research [23] that could improve our knowledge and management of anaphylaxis. We have identified for the first time an anaphylactic EVs signature, and we performed pilot studies on the validity of some of these proteins as potential biomarkers. Nevertheless, based on the results of our work, analysis of EV-associated proteins in larger patient cohorts is well-warranted. Even more, our study supports a functional role for the AnEV signature in key processes related with the disease onset and progression. The functional involvement of EVs in activation of neutrophils and endothelial cells needs further clarification, but our results suggest a relevant underlying mechanism connecting these cell types with the bases of the anaphylactic reactions. Even considering our findings exploratory in nature, their potential diagnostic and prognostic value might be crucial for new therapeutic directions and the proteomic profiling of these plasma derived AnEV is a great resource for the allergy community.