Antigen
(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.

 

Introduction

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The
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).

 

Besides
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).

 

The
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.

 

Therefore,
the aims for this project are:

1.    
To generate the mast cell specific ELKS
knockout mouse – Mcpt5-Cre ELKS Strain

2.    
To study the role of ELKS in mast cell
degranulation in vitro

3.    
To study the role of ELKS in de novo
synthesis of cytokines and chemokines in mast cells in vitro

4.    
To investigate if ELKS have a role in early intracellular
signaling in mast cells

5.    
To examine the localization of ELKS in mast
cells

6.    
To study the role of ELKS in mast cell
degranulation in vivoGeneration 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
vitro. ELKS is required for cytokine production from
mast cells Engagement
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. ).  Discussion 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. Previous
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.  In
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. ELKS is
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.