Antigen production was enhanced. Our findings suggest that ELKS

Antigen
(Ag) –mediated crosslinking of the high-affinity immunoglobulin E (IgE)
receptor (Fc?RI) on mast cells results in degranulation and the production of
many inflammatory cytokines and chemokines, which are key effectors in allergic
disorders including asthma, atopic dermatitis and food allergies such as peanut
allergy. Previous in vitro studies
have demonstrated that ELKS, an active zone protein in presynaptic neurons, is involved
in the neurotransmitter release as well as in the exocytosis process in rat
basophilic leukemia (RBL-2H3) cells. Here, to understand the in vivo roles of ELKS, we generated mast
cell-specific ELKS knockout (KO) mice and showed that ELKS-deficient peritoneal
mast cells (PCMCs) exhibited significantly less degranulation while inflammatory
cytokine and chemokine production was enhanced. Our findings suggest that ELKS differentially
regulates mast cell degranulation and cytokine production.

 

 

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 fourth of the population worldwide
is affected by allergic diseases such as asthma, allergic rhinitis and atopic dermatitis
(1).

Besides having a role
in innate and adaptive defense against pathogens, mast cells have long been
considered as the central effectors in allergic inflammation (2). 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 (2). Mast
cells express the high-affinity IgE receptor Fc?RI on their surface and binding
of multivalent antigen to Fc?RI-bound IgE causes receptor aggregation and
thereby mast cell activation (3, 4). 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 (3, 5). Mast
cells also de novo synthesise lipid
mediators of inflammation such as leukotrienes and prostaglandins, as well as 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) (3, 5, 6).

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 (7, 8). The NF-?B
pathway can be activated by a wide range of stimuli such as lipopolysaccharide
(LPS), tumour necrosis factor (TNF) and IL-1. After these ligands 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, initiating the
transcription of many target genes involved in inflammatory and immune response
as well as cell differentiation and survival (7, 8). Apart from IKK?, IKK? and
IKK?/NEMO, ELKS (a protein riched in glutamic acid (E), leucine (L), lysine
(K), and serine (S)) has also been identified as a regulatory subunit within
the IKK complex (9). ELKS was originally identified as part of a translocation
fusion protein fused with the receptor tyrosine kinase (RET) in papillary
thyroid carcinoma (10). ELKS was proposed to be an essential regulatory subunit
within the IKK complex as TNF?-induced phosphorylation and degradation of I?B?
was lost and delayed after silencing of ELKS with siELKS, indicating that ELKS
regulates the function of IKK complex by recruiting I?B? to the IKK complex (9).

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 (11-13). 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 (14-15). In
addition, the study by Nomura et al.
(2009) 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 RBL cells (16).

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

Therefore,
the aims of 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 has a role in early intracellular
signaling in mast cells.

5.    
To examine the localisation of ELKS in mast
cells.

6.    
To measure exocytosis of wild-type (WT) and ELKS
KO mast cells at single-cell level with patch clamp studies.

7.    
To confirm the role of ELKS in mast cell
degranulation in vivo with passive
cutaneous anaphylaxis model.

 

 

Materials And Methods

 

Mast Cells Isolation and Culture

Bone 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 supplemented with cytokines. After 6 weeks of culture, purity of bone
marrow-derived mast cells (BMMCs) was confirmed by flow cytometry (cKit+,
Fc?RI+).

For isolation of PCMCs,
8mL of sterile PBS was injected into the mouse peritoneal cavity using 19G
needle. The abdomen was massaged for 30sec and the fluid was collected. Cells
were then centrifuged at 300g, 4°C, 10min
and resuspended in 5mL RPMI-1640 containing 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 cultured for 18
days. The purity of PCMCs was confirmed by flow cytometry (cKit+ ,
Fc?RI+).

 

Flow Cytometry

To determine the
purity of mast cells, 2×105 BMMCs, PCMCs or peritoneal lavage cells
were resuspended in 100?L PBS and incubated with 1?L PE-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 Biosciences).

To evaluate
degranulation of PCMCs, surface expression of LAMP1 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 LAMP1 –APC (Miltenyi Biotec)
(1:100) and Fc?RI-FITC (Miltenyi Biotec) (1:100) for 20min on ice. The cells
were washed with PBS and resuspended in 200?L of 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 normalised to the ?-Actin mRNA.

 

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: ELKS
(Santa Cruz; Sc-47877), p-p38 (Thr180/Tyr182) (Cell Signaling; #9215S),
p-p44/42 (Thr202/Tyr204) (Cell Signaling; # 9101), HSP90 (BD Bioscience; 610419)

 

?-hexosaminidase Assay

PCMCs were sensitised
with 0.5?g/mL IgE anti-DNP (Sigma-Aldrich) overnight at 37°C. The IgE-sensitised PCMCs were
washed with Tyrode’s Buffer (10mM HEPES, 129mM NaCl, 5mM KCl, 1.4mM CaCl2,
1mM MgCl2, 8.4mM D-glucose, 0.1%BSA at pH 7.4) and stimulated with
10ng/mL DNP-BSA for 1 hour at 37°C. Cells
were centrifuged and supernatant was collected and cells were lysed with 0.5% Triton
X-100. The supernatant and cell lysate were incubated in substrate buffer
(155mM Na2HPO4 and 88mM citiric aicd, pH 4.5) with
p-nitrophenyl-N-acetyl-?-D-glucosaminide for 1 hour at 37°C. The reaction was stopped by adding 0.2 M glycine.
Absorbance was recorded at 405nm and the percentage degranulation = absorbance
of culture supernatant at 405nm X100 / absorbance of total cell lysate at 405nm
was calculated.

 

 

Results

 

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 cells and whole body knockout of
ELKS in mouse has been reported to result in embryonic lethality (17, 18), ELKS
conditional knockout mice were generated using Cre-LoxP system. Mice with ELKS
alleles floxed with LoxP sequence (ELKS f/f) were first crossed with Mcpt5-Cre mice
that express Cre recombinase selectively in connective tissue mast cells (19). Then,
ELKS f/f mice were crossed with ELKS f/f Mcpt5-Cre mice (Fig. 1). The number of
ELKS f/f and ELKS f/f Mcpt5-Cre pups in F2 progeny was similar, in line with
the expected Mendelian ratio (Table 1).

To understand if ELKS
regulates the population of resident mast cells in peritoneal cavity, cells were
extracted from the peritoneal cavity of WT and ELKS Mcpt-Cre KO mice. Flow
cytometric analysis suggested similar population of mast cells in the
peritoneal lavage of WT and ELKS KO mice (Fig. 2a). Furthermore, these cells
are then cultured for 21 days in the presence of IL-3 and 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. 2b). Similarly, the generation of bone
marrow-derived mast cells (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. 2c).
Therefore, ELKS is not required for mast cell development in vivo and in vitro.

Next, the mRNA and
protein levels of ELKS in PCMCs and BMMCs from WT and ELKS KO mice were quantified
using real-time PCR and Western blot respectively. Absence of ELKS mRNA and
protein in PCMCs were confirmed as shown in Fig. 2d. However, as stated in
previous literature that the efficacy of Cre/Lox recombination in BMMCs for
Mcpt-Cre strain is not 100% (20), the deletion of ELKS in BMMCs from ELKS f/f
Mcpt5-Cre mice was not complete (Fig. 2e). Therefore, we only used PCMCs from
these mice for later experiments which allows 100% deletion of ELKS in PCMCs.

 

ELKS positively regulates mast cell
degranulation

Mast cells rapidly degranulate
after being activated through Fc?RI. To determine
if ELKS plays a role in IgE-mediated mast cell activation and degranulation, WT
and ELKS KO PCMCs were first sensitised with anti-DNP-IgE antibody and then
stimulated with DNP-BSA and the release of granule-stored enzyme, ?-hexosaminidase was
measured. Release of ?-hexosaminidase
was optimal at a dose of antigen at 10ng/mL in WT PCMCs (Fig. 3a) and
ELKS-deficient PCMCs had significantly lower release of ?-hexosaminidase
compared to WT PCMCs upon Fc?RI activation (Fig. 3b). Likewise, less surface
exposure of LAMP1, a marker for exocytosis of granules, was detected in ELKS KO
PCMCs compared to WT PCMCs following IgE/Ag stimulation (Fig. 3c). Hence, these
data indicated that ELKS-deficient mast cells are impaired in their capacity to
degranulate in vitro.

 

ELKS negatively regulates cytokine production
from mast cells

Engagement of the Fc?RI
receptor by IgE and specific antigen also results in de novo synthesis of various cytokines and chemokines that characterises
the late-phase pro-inflammatory response. Therefore, we analysed gene
expression of a selection of pro-inflammatory cytokines and chemokines
including TNF?, IL-6, CCL1, IL-1?, IL-33, GM-CSF,
MCP-1 and IL-13. To this end, WT and ELKS KO PCMCs were sensitised with anti –
DNP IgE overnight and stimulated with DNP-BSA for 1.5h. Interestingly, real-time
PCR analysis demonstrated that ELKS-deficient mast cells have augmented mRNA
expressions for TNF?, IL-6,
CCL1, IL-1? and IL-33 compared to WT mast cells upon stimulation (Fig. 4a).
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 essential for early signal
transduction of IgE-activated mast cells

Next, we examined
whether ELKS is required for the Fc?RI – induced early intracellular signalling
pathways in mast cells. To this end, WT and ELKS KO mast cells were again
sensitised with anti-DNP IgE and then stimulated with DNP-BSA. However, there
was no difference in p-pERK and p-p38 between WT and ELKS KO mast cells (Fig. 4b),
suggesting that ELKS does not play an essential role in ERK and p38 signalling
after mast cell activation.

 

 

Discussion

 

In the present study,
we generated conditional knockout mice for ELKS in connective tissue mast cells
and demonstrated that ELKS deletion in mast cells causes reduced degranulation
but enhanced cytokine synthesis. Collectively, our data has confirmed the role
of ELKS in positive regulation of exocytosis and has identified a negative
regulatory role of ELKS in cytokine transcription.

Previous studies have
implicated the involvement of different IKK complex subunits within the NF-?B signalling
pathway in mast cell functions. I?B kinase ? (IKK?) was shown to be critical
for mast cell degranulation as Suzuki et
al. (2008) has demonstrated that fetal liver-derived mast cells from IKK?-deficient
mice had impaired degranulation upon IgE-Ag stimulation (21). However, another
study by Peschke et al. (2014) had found
that there was unaffected degranulation but impaired
production of cytokines in peritoneal mast cells generated from mice with
connective tissue mast cell-specific IKK? deletion (20). In the same study by
Peschke et al. (2014), they have also
reported that activated peritoneal NEMO/IKK? KO mast cells had reduced cytokine
production (20).

In addition, several
lines of evidence suggested that ELKS, a regulatory subunit of the IKK complex,
is a positive regulator of exocytosis. The study by Inoue et al. (2006) has shown that ELKS regulates Ca2+ dependent
exocytosis in PC12 cells (22) 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 (23). Moreover, another study by Nomura et al. (2009) has demonstrated that
knockdown and overexpression of ELKS in RBL-2H3 cells have resulted in reduced
and enhanced exocytotic activity respectively (16). Therefore, our data showing
less ?-hexosaminidase release from ELKS KO PCMCs than WT PCMCs after stiumation
(Fig. 3b) further supported the role of ELKS in positively regulating
degranulation in mast cells.

Furthermore, we
demonstrated that the gene expression for some pro-inflammatory cytokines and
chemokines are higher in activated ELKS KO mast cells than in activated WT mast
cells (Fig. 4a), suggesting that ELKS might have an additional role in cytokine
and chemokine production in mast cells. Since previous study by
Kandere-Grzybowska et al. (2003) has
demonstrated that IL-1 stimulated mast cells can release IL-6 without
degranulation (24) and another study by Foger et al. (2011) has found that Coro1a KO BMMCs displayed enhanced degranulation
but diminished cytokine secretion (25), the trafficking for cytokine/chemokine in
mast cells probably employs a different secretory lysosomal pathway to those
pre-formed mediators within the secretory granules. However, secreted cytokines
and chemokines should be measured in our future experiments in order to verify
that cytokine and chemokine secretion is upregulated in ELKS-deficient PCMCs.

 

 

Conclusion and Future Directions

 

Taken together, components
within the IKK complex, including ELKS, could contribute to different mast cell
functions. The present findings strengthen the idea that ELKS is a positive
regulator of mast cell degranulation and ELKS might also negatively regulate
cytokine and chemokine production in mast cells. We will also study the localisation
of ELKS in mast cells during unstimulated and IgE-Ag stimulated conditions and
confirm its role in mast cell degranulation in
vitro at single-cell level through patch clamp studies and in vivo through the passive cutaneous anaphylaxis
model. Therefore, our work will provide further insight into how ELKS regulate
mast cell functions, allowing us to identify potential therapeutic targets for
allergic inflammation.