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.