Single-Cell Analyses of Human Eosinophils at High Resolution to Understand
Compartmentalization and Vesicular Trafficking of Interferon-Gamma
#MMPMID30038615
Carmo LAS
; Bonjour K
; Spencer LA
; Weller PF
; Melo RCN
Front Immunol
2018[]; 9
(?): 1542
PMID30038615
show ga
Human eosinophils release numerous cytokines that are pre-synthesized and stored
within their cytoplasmic-specific (secretory) granules. For example, high levels
of interferon-gamma (IFN-?) are constitutively expressed in these cells, but the
intracellular compartments involved in the transport and release of this cytokine
remain to be established. In this work, we used a single-cell approach to
investigate the subcellular localization of IFN-? in human eosinophils stimulated
or not with tumor necrosis factor alpha (TNF-?) or CC-chemokine ligand 11 CCL11
(eotaxin-1), inflammatory mediators that induce eosinophil activation and
secretion. A pre-embedding immunonanogold transmission electron microscopy (TEM)
technique that combines optimal epitope preservation and access to membrane
microdomains was applied to detect precise localization of IFN-? in combination
with computational quantitative analyses. In parallel, degranulation processes
and formation of eosinophil sombrero vesicles (EoSVs), large transport carriers
involved in the transport of granule-derived cytokines, were investigated.
Quantitative TEM revealed that both CCL11 and TNF-?-activated eosinophils
significantly increased the total number of EoSVs compared to the unstimulated
group, indicating that this vesicular system is actively formed in response to
cell activation. Ultrastructural immunolabeling identified a robust pool of IFN-?
on secretory granules in both unstimulated and stimulated cells. Moreover, EoSVs
carrying IFN-? were seen around or/and in contact with secretory granules and
also distributed in the cytoplasm. Labeling was clearly associated with EoSV
membranes. The total number of IFN-?-positive EoSVs was significantly higher in
stimulated compared to unstimulated cells, and these labeled vesicles had a
differential distribution in the cytoplasm of activated cells, being
significantly higher in the cell periphery compared with the inner cell, thus
revealing intracellular IFN-? mobilization for release. IFN-? extracellular
labeling was found at the cell surface, including on extracellular vesicles. Our
results provide direct evidence that human eosinophils compartmentalize IFN-?
within secretory granules and identify, for the first time, a vesicular
trafficking of IFN-? associated with large transport carriers. This is important
to understand how IFN-? is trafficked and secreted during inflammatory responses.
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