Antigen mediators such as leukotrienes and prostaglandins as well

(Ag) –mediated crosslinking of the high-affinity immunoglobulin E (IgE)
receptor (Fc?RI) on mast cells results in the degranulation and the release of
pre-stored granular mediators, followed by the production of many allergic and
inflammatory cytokines and chemokines, which are key effectors in allergic
disorders, such as asthma and anaphylaxis. Previous studies have demonstrated
that ELKS, an active zone protein, involves in the neurotransmitter release in
neuronal cells as well as exocytotic release in rat basophilic leukemia
(RBL-2H3) cells. In this study, we generated conditional knockout(KO) mice for
ELKS to delete ELKS specifically in mast cells and showed that peritoneal
cell-derived mast cells (PCMCs) lacking ELKS exhibited significantly less
degranulation in vitro while cytokine
and chemokine production was slightly affected. Our finding suggests that ELKS
is a positive regulator for mast cell degranulation.



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prevalence of allergic diseases has been increasing continuously in the
developed countries over the past decades and approximately one third of the
population worldwide is affected by allergic diseases such as asthma, allergic
rhinitis and dermatitis (ref).


having a role in innate and adaptive defense against pathogens, mast cells have
long been considered as the central effectors in allergic inflammation. Mast
cells are granulated cells derived from the bone marrow and they localise at
tissues that are exposed to the external environment such as the skin and lung (ref).
Mast cells express the high-affinity IgE receptor Fc?RI on their surface and
multivalent antigen binds to Fc?RI-bound IgE causes receptor aggregation and
thereby mast cell activation. Activated mast cells degranulate within seconds
to minutes after its exposure to antigen and release an array of pre-formed,
granule-stored mediators including histamine and ?-hexosaminidase (ref). Several
hours after activation, mast cells also produce newly synthesized of lipid
mediators such as leukotrienes and prostaglandins as well as de novo synthesis
and secretion of cytokines and chemokines (for example interleukin (IL)-6,
IL-4, IL-13, MCP-1) driven by transcription factors including Nuclear factor
kappa B (NF-?B) (ref).


The NF-?B
family is a group of evolutionarily conserved transcription factors that play
an important role in cell survival, immunity and inflammatory responses. In
unstimulated cells, the most abundant NF-?B dimer, p50/p65, is bound by inhibitors
of ?B (I?Bs) and therefore retains in the cytoplasm and remains inactive (ref).
The NF-?B pathway can be activated by a wide range of stimuli such as pathway lipopolysaccharide
(LPS), tumour necrosis factor (TNF) and IL-1. After these inducers bind to
their corresponding receptors, the IKK complex that contains IKK?, IKK? and IKK?/NEMO
is activated, leading to the phosphorylation, ubiquitination and degradation of
I?Bs. As a result, the p50/p65 dimer enters into the nucleus, causing the
transcription of many target genes that involve in inflammatory and immune
response as well as cell differentiation and survival (ref). Apart from IKK?,
IKK? and IKK?/NEMO, ELKS has been identified as a regulatory subunit within the
IKK complex (ref).


exocytotic machinery in mast cell degranulation and neurotransmitter release in
neuronal cells share some similarities and both require the SNARE (soluble N-ethylmaleimide-sensitive factor
attachment protein receptors) proteins (ref). In neuronal cells, ELKS, together
with several cytomatrix-at-the-active –zone (CAZ) -associated structural
protein (CAST) family members including Rab3 interacting molecule 1 (RIM1),
Bassoon and Piccolo have been reported to be involved in the Ca2+ dependent
exocytosis of neurotransmitters (ref). In addition, a study has demonstrated
that using siRNA to silence ELKS in rat basophilic leukemia (RBL-2H3) cells has
led to a decrease in mast cell degranulation, suggesting that ELKS also has a
role in regulating the exocytosis of granular contents in mast cells (ref).


Base on
the above, we would like to explore the role of ELKS in mast cell degranulation
through the use of animal model and to decipher the role of ELKS in other mast
cell functions.


the aims for this project are:

To generate the mast cell specific ELKS
knockout mouse – Mcpt5-Cre ELKS Strain

To study the role of ELKS in mast cell
degranulation in vitro

To study the role of ELKS in de novo
synthesis of cytokines and chemokines in mast cells in vitro

To investigate if ELKS have a role in early intracellular
signaling in mast cells

To examine the localization of ELKS in mast

To study the role of ELKS in mast cell
degranulation in vivo



Materials And Methods


Mast Cells Harvesting and Culture


marrow cells were isolated from femurs and tibias of mice and cultured with
RPMI-1640 (Hyclone) plus 10% FBS (Gibco), 5% non-essential amino acids (Gibco),
5% penicillin/streptomycin (Gibco), 10ng/mL IL-3 (Miltenyi Biotec.) and 10ng/mL
stem cell factor (SCF) (Miltenyi Biotec). Medium was changed every 4 days with
fresh medium with cytokines. After 6 weeks of culture, purity of BMMCs (cKit
and Fc?RI expression) was confirmed by flow cytometry.

For PCMCs,
8mL of PBS was injected into the mouse peritoneal cavity using 19G needle.
Massage the abdomen for 30sec and collect the fluid with the 19G needle.
Centrifuge the cells (300g, 4°C, 10min)
for 2 times and resuspend the cells in 5mL RPMI-1640 10% FBS (Gibco), 5%
non-essential amino acids (Gibco), 5% penicillin/streptomycin (Gibco), 30ng/mL
IL-3 (Miltenyi Biotec.) and 30ng/mL stem cell factor (SCF) (Miltenyi Biotec) and
culture for 15-21 days. Change medium every 3 days for the first 9 days and
supplement the culture with 1mL RPMI plus 30ng/mL IL-3 and 30ng/mL SCF
thereafter. The purity of PCMCs (cKit and Fc?RI expression) was confirmed by
flow cytometry.


Flow Cytometry


To determine
the purity of mast cells, 2×105 BMMCs, PCMCs or peritoneal lavage
cells were ruspended in 100?L PBS and incubated with 1?LPE-anti-mouse CD117/Kit (BD Bioscieces) and 1?L APC-anti-mouse
Fc?RI (eBioscience) for 20min on ice. The cells were washed with PBS and
resuspended in 200?L PBS for analysis using BD LSRII flow cytometer (BD

evaluate degranulation of PCMCs, surface expression of CD107a was measured
using flow cytometry. IgE-sensitised PCMCs were stimulated with 10ng/mL DNP-BSA
for 30min. Cells were washed with PBS and incubated with CD107a (1:100) and Fc?RI (1:100)
for 20min on ice. The cells were washed with PBS and resuspended in 200?L PBS
for analysis using BD LSRII flow cytometer (BD Biosciences.)


RT-PCR Analysis


Total RNA was
isolated from 1x 106 harvested mast cells with Trizol (Invitrogen)
and purified with column using QIAGEN RNeasy Mini Kit. 1?g of the isolated RNA
was used for cDNA synthesis with the Maxima First Strand cDNA Synthesis Kit
(ThermoFisher). RT-qPCR was then performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad)
and was run on the CFX96tm Real-Time System (Bio-Rad). Experiments were
performed in duplicate for each sample and the mRNA expression was normalized
to the ?-Actin RNA.


SDS-PAGE and Western Blot


1x 106
BMMCs or PCMCs were harvested and lysed with Totex Buffer (20mM HEPES at pH
7.9, 0.35M NaCl, 20% glycerol, 1% NP-40, 1mM MgCl2, 0.5mM EDTA,
0.1mM EGTA, 50mM NaF and 0.3mM NaVO3, protease inhibitor cocktail)
to obtain whole-cell extracts. The protein concentration was quantified using
Bradford. Proteins were run in 4-12% Bis-Tris SDS-PAGE gel and transferred to
PVDF membrane (Bio-Rad). Membrane was probed with the following antibodies: …..


?-hexosaminidase Assay


PCMCs were
sensitized with 0.5?g/mL IgE anti-DNP (Sigma-Aldrich) overnight at 37°C. The IgE-sensitised PCMCs were
washed with Tyrode’s Buffer ( ) and stimulated with 10ng/mL DNP-BSA for 1 hour
at 37°C. Cells were centrifuged and supernatant was collected
and the cell was lysed with Triton
X-100. The supernatant and cell lysate were incubated in Tyrode’s buffer with
p-nitrophenyl-N-acetyl-?-D-glucosaminide for 1 hour at 37°C. The reaction was stopped by adding glycine. Absorbance was recorded at 405nm and the
percentage degranulation = absorbance of culture supernatant at 405nm X100 /
absorbance of total cell lysate at 405nm



Generation of mast cell specific ELKS knockout
mice (ELKS Mcpt5-Cre Mice)


Since we
would like to study the specific role of ELKS in mast cell and whole body
knockout of ELKS in mouse has resulted in embryonic lethality (Liu et al.,
2014; Wu et al., 2010), ELKS conditional knockout mice were generated using
Cre-LoxP system. Mice with their ELKS alleles floxed with LoxP sequence (ELKS
f/f) was first crossed with Mcpt5-Cre mice that express Cre recombinase
selectively in connective tissue mast cells (Ref.). Then, ELKS f/f mice was
crossed with ELKS f/f Mcpt5-Cre mice and the number of ELKS f/f and ELKS f/f
Mcpt5-Cre pups in F2 progeny was similar, which matched the expected Mendelian
ratio (Table 1).


Cells were
extracted from the peritoneal cavity of wild-type (WT) and ELKS Mcpt-Cre
knockout (KO) mice and there are similar population of mast cells in the
peritoneal lavage cells between WT and KO mice (Fig. ). These cells are then cultured
for 21 days in the presence of interleukin (IL)-3 and stem cell factor (SCF).
The surface expression levels of mast cell-specific markers Fc?RI and c-Kit on
KO PCMCs were similar to that of WT PCMCs (Fig. ). Similarly, the generation of
BMMCs in the presence of IL-3 and SCF was not affected by ELKS deficiency as
both WT and ELKS KO BMMCs had comparable levels of Fc?RI
and c-Kit surface expression (Fig. ). Therefore, ELKS is not required for mast
cell development.


Next, the
mRNA and protein level expression of ELKS in PMMCs and BMMCs from ELKS f/f mice
(WT) and ELKS f/f Mcpt5-Cre mice (KO) were quantified at mRNA and protein
levels using real-time PCR and Western blot respectively. Deletion of ELKS in
PMMCs at mRNA and protein levels were confirmed as shown in Fig. . However, as stated
in previous literature that the efficacy of Cre/Lox recombination in BMMCs for
Mcpt-Cre strain is not 100%, the deletion of ELKS in BMMCs from ELKS f/f
Mcpt5-Cre mice was not complete (Fig. ). Therefore, we only used PCMCs from
these mice for later experiments.


ELKS regulates mast cell degranulation in vitro


Mast cells
degranulate rapidly after being stimulated through the Fc?RI.
To determine if ELKS plays a role in such mast cell function, WT and ELKS KO PCMCs
were first sensitised with anti-DNP-IgE antibodyand then stimulated with
DNP-BSA and the release of granular-stored enzyme, ?-hexosaminidase was
measured. Release of ?-hexosaminidase
was optimal at a dose of antigen at 10ng/mL in WT PCMCs (Fig. ) and
ELKS-deficient PCMCs had significantly lower release of ?-hexosaminidase
compared to WT PMMCs upon Fc?RI activation (Fig. ). Likewise, less surface
exposure of CD107a was detected in KO PCMCs than WT PCMCs following IgE/Ag stimulation
(Fig. ). Hence, these data indicated that ELKS-deficient mast cells have a
deficit in their capacity to degranulate in


ELKS is required for cytokine production from
mast cells


of the Fc?RI receptor can also result in de
novo synthesis of various cytokines and chemokines that charaterises the
late-phase pro-inflammatory response. Therefore, we analysed gene expression of
a selection of pro-inflammatory and Th2-related cytokines and chemokines
including TNF?, IL-6, CCL1, IL-1?, IL-33, GM-CSF, MCP-1 and
IL-13. WT and KO PCMCs were sensitised with anti – DNP IgE overnight and
stimulated with DNP-BSA for 1.5h. Real-time PCR analysis demonstrated that ELKS-deficient
mast cells have slight increase in mRNA expressions for TNF?,
IL-6, CCL1, IL-1? and IL-33 compared to WT mast cells. Collectively, these
results suggest that ELKS is playing an additional role in Fc?RI-mediated
cytokine and chemokine synthesis in mast cells besides degranulation.


ELKS is not required for early signaling


NF-?B and
MAP kinase cascades orchestrate the production of cytokines from mast cells
following Ag-induced IgE-Fc?RI aggregation and ELKS is part of the NF-?B signaling
pathway. Hence, we next examined
whether ELKS is required for early signaling pathways in mast cells. WT and KO
mast cells were again sensitised with anti-DNP IgE and then stimulated with
DNP-BSA. KO mast cells have reduced I?B? mRNA expression compare to WT mast
cells upon IgE-Ag stimulation (Fig. ). There was no difference in p-pERK and
p-p38 between WT and KO mast cells (Fig. ).





In the
present study, we have generated conditional knockout mice for ELKS in
connective tissue mast cells and have demonstrated that ELKS deletion in mast
cells causes reduced degranulation. Mast cells from KO mice also produced more
inflammatory cytokines and chemokines upon IgE-induced activation compare to
those from WT mice. We have also shown that loss of ELKS has resulted in less I?B?.
Collectively, our data has reconfirmed the role of ELKS in positive regulation
of exocytosis.


studies have implicated the involvement of different IKK complex subunits
within the NF-?B signaling pathway in mast cell functions. I?B kinase ? (IKK?)
was shown to be critical for mast cell degranulation as fetal liver-derived
mast cells from IKK?-deficient mice had impaired degranulation upon IgE-Ag
stimulation (ref.). However, another study by Peschke et al. (2014) found that there
was unaffected degranulation but impaired production of cytokine in peritoneal mast
cells generated from mice with connective tissue mast cell-specific IKK? deletion.
In the same study by Peschke et al. (2014), they have also reported that activated
peritoneal NEMO/IKK? KO mast cells had impaired cytokine production.


addition, several lines of evidence suggest that ELKS, a regulatory subunit of
the IKK complex, is a positive regulator for exocytosis. A study by Inoue et
at. (2006) has shown that ELKS regulates Ca2+ dependent exocytosis
in PC12 cells (ref.) while another study by Ohara-Imaizumi et al. (2005) has
demonstrated that there was a decrease in insulin exocytosis after silencing
ELKS with RNA interference (RNAi) in MIN6? cells (ref). In addition, another
study has demonstrated that knockdown and overexpression of ELKS in RBL-2H3
cells have resulted in a decrease and increase in their exocytotic activity
respectively (ref.). Therefore, our data showing less ?-hexosaminidase release
from KO PCMCs than WT PCMCs after stiumation (Fig. ) further supported the role
of ELKS in positively regulating degranulation in mast cells through the use of
animal model generated by the Cre/LoxP system.


considered to be an essential regulatory subunit within the IKK complex as knocking
down ELKS by RNAi inhibited expression of I?B? (ref.). Here, we have shown that
KO mast cells have less I?B? mRNA expression. Furthermore, we have demonstrated
that the gene expression for some pro-inflammatory cytokines and chemokines are
higher in activated KO mast cells than in activated WT mast cells (Fig. ),
suggesting that ELKS might have an additional role in cytokine and chemokine
production in mast cells. However, more biological repeats are needed to
confirm this result and secreted cytokines and chemokines should also be
measured in the future experiments.


Taken together,
components within the IKK complex, including ELKS, could contribute to
different mast cell functions and our work will provide further insight into
how ELKS regulate mast cell functions and thereby extend our understanding in
the molecular mechanisms for allergic and anaphylactic disorders and to
identify potential therapeutic targets for allergic inflammation.