Antigen ELKS differentially regulates mast cell degranulation and

Antigen(Ag) –mediated crosslinking of the high-affinity immunoglobulin E (IgE)receptor (Fc?RI) on mast cells results in degranulation and the production ofmany inflammatory cytokines and chemokines, which are key effectors in allergicdisorders including asthma, atopic dermatitis and food allergies such as peanutallergy. Previous in vitro studieshave demonstrated that ELKS, an active zone protein in presynaptic neurons, is involvedin the neurotransmitter release as well as in the exocytosis process in ratbasophilic leukemia (RBL-2H3) cells. Here, to understand the in vivo roles of ELKS, we generated mastcell-specific ELKS knockout (KO) mice and showed that ELKS-deficient peritonealmast cells (PCMCs) exhibited significantly less degranulation while inflammatorycytokine and chemokine production was enhanced. Our findings suggest that ELKS differentiallyregulates mast cell degranulation and cytokine production.   Introduction The prevalence ofallergic diseases has been increasing continuously in the developed countriesover the past decades and approximately one fourth of the population worldwideis affected by allergic diseases such as asthma, allergic rhinitis and atopic dermatitis(1). Besides having a rolein innate and adaptive defense against pathogens, mast cells have long beenconsidered as the central effectors in allergic inflammation (2).

Mast cells aregranulated cells derived from the bone marrow and they localise at tissues thatare exposed to the external environment such as the skin and lung (2). Mastcells express the high-affinity IgE receptor Fc?RI on their surface and bindingof multivalent antigen to Fc?RI-bound IgE causes receptor aggregation andthereby mast cell activation (3, 4). Activated mast cells degranulate within secondsto minutes after its exposure to antigen and release an array of pre-formed,granule-stored mediators including histamine and ?-hexosaminidase (3, 5). Mastcells also de novo synthesise lipidmediators of inflammation such as leukotrienes and prostaglandins, as well as cytokinesand chemokines (for example interleukin (IL)-6, IL-4, IL-13, MCP-1) driven bytranscription factors including Nuclear factor kappa B (NF-?B) (3, 5, 6). The NF-?B family is agroup of evolutionarily conserved transcription factors that play an importantrole 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) andtherefore retains in the cytoplasm and remains inactive (7, 8). The NF-?Bpathway 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 theircorresponding receptors, the IKK complex that contains IKK?, IKK? and IKK?/NEMOis activated, leading to the phosphorylation, ubiquitination and degradation ofI?Bs.

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As a result, the p50/p65 dimer enters into the nucleus, initiating thetranscription of many target genes involved in inflammatory and immune responseas well as cell differentiation and survival (7, 8). Apart from IKK?, IKK? andIKK?/NEMO, ELKS (a protein riched in glutamic acid (E), leucine (L), lysine(K), and serine (S)) has also been identified as a regulatory subunit withinthe IKK complex (9). ELKS was originally identified as part of a translocationfusion protein fused with the receptor tyrosine kinase (RET) in papillarythyroid carcinoma (10). ELKS was proposed to be an essential regulatory subunitwithin the IKK complex as TNF?-induced phosphorylation and degradation of I?B?was lost and delayed after silencing of ELKS with siELKS, indicating that ELKSregulates the function of IKK complex by recruiting I?B? to the IKK complex (9).The exocytotic machineryin mast cell degranulation and neurotransmitter release in neuronal cells sharesome 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 Rab3interacting molecule 1 (RIM1), Bassoon and Piccolo have been reported to be involvedin the Ca2+ dependent exocytosis of neurotransmitters (14-15). Inaddition, the study by Nomura et al.(2009) has demonstrated that using siRNA to silence ELKS in rat basophilicleukemia (RBL-2H3) cells has led to a decrease in mast cell degranulation,suggesting that ELKS also has a role in regulating the exocytosis of granularcontents in RBL cells (16).

Based on the above, wewould like to explore the role of ELKS in mast cell degranulation through theuse of animal models and to decipher the role of ELKS in other mast cellfunctions.Therefore,the aims of this project are:1.    To generate the mast cell specific ELKSknockout mouse – Mcpt5-Cre ELKS Strain.

2.    To study the role of ELKS in mast celldegranulation in vitro.3.    To study the role of ELKS in de novo synthesis of cytokines andchemokines in mast cells in vitro.4.    To investigate if ELKS has a role in early intracellularsignaling in mast cells.5.    To examine the localisation of ELKS in mastcells.

6.    To measure exocytosis of wild-type (WT) and ELKSKO mast cells at single-cell level with patch clamp studies.7.    To confirm the role of ELKS in mast celldegranulation in vivo with passivecutaneous anaphylaxis model.  Materials And Methods Mast Cells Isolation and CultureBone marrow cellswere 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/mLstem cell factor (SCF) (Miltenyi Biotec).

Medium was changed every 4 days withfresh medium supplemented with cytokines. After 6 weeks of culture, purity of bonemarrow-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 19Gneedle. The abdomen was massaged for 30sec and the fluid was collected. Cellswere then centrifuged at 300g, 4°C, 10minand resuspended in 5mL RPMI-1640 containing 10% FBS (Gibco), 5% non-essentialamino acids (Gibco), 5% penicillin/streptomycin (Gibco), 30ng/mL IL-3 (MiltenyiBiotec) and 30ng/mL stem cell factor (SCF) (Miltenyi Biotec) and cultured for 18days. The purity of PCMCs was confirmed by flow cytometry (cKit+ ,Fc?RI+). Flow CytometryTo determine thepurity of mast cells, 2×105 BMMCs, PCMCs or peritoneal lavage cellswere 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 usingBD LSRII flow cytometer (BD Biosciences).

To evaluatedegranulation of PCMCs, surface expression of LAMP1 was measured using flowcytometry. 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 cellswere washed with PBS and resuspended in 200?L of PBS for analysis using BDLSRII flow cytometer (BD Biosciences). RT-PCR AnalysisTotal RNA wasisolated from 1x 106 harvested mast cells with Trizol (Invitrogen)and purified with column using QIAGEN RNeasy Mini Kit. 1?g of the isolated RNAwas used for cDNA synthesis with the Maxima First Strand cDNA Synthesis Kit(ThermoFisher). RT-qPCR was then performed using SsoAdvanced Universal SYBRGreen 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 expressionwas normalised to the ?-Actin mRNA. SDS-PAGE and Western Blot1x 106 BMMCsor 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, 0.1mMEGTA, 50mM NaF and 0.3mM NaVO3, protease inhibitor cocktail) toobtain whole-cell extracts. The protein concentration was quantified usingBradford. Proteins were run in 4-12% Bis-Tris SDS-PAGE gel and transferred toPVDF 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 AssayPCMCs were sensitisedwith 0.

5?g/mL IgE anti-DNP (Sigma-Aldrich) overnight at 37°C. The IgE-sensitised PCMCs werewashed 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 with10ng/mL DNP-BSA for 1 hour at 37°C. Cellswere centrifuged and supernatant was collected and cells were lysed with 0.

5% TritonX-100. The supernatant and cell lysate were incubated in substrate buffer(155mM Na2HPO4 and 88mM citiric aicd, pH 4.5) withp-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 = absorbanceof culture supernatant at 405nm X100 / absorbance of total cell lysate at 405nmwas calculated.  Results Generation of mast cell-specific ELKS knockoutmice (ELKS Mcpt5-Cre Mice)Since we would liketo study the specific role of ELKS in mast cells and whole body knockout ofELKS in mouse has been reported to result in embryonic lethality (17, 18), ELKSconditional knockout mice were generated using Cre-LoxP system.

Mice with ELKSalleles floxed with LoxP sequence (ELKS f/f) were first crossed with Mcpt5-Cre micethat 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 ofELKS f/f and ELKS f/f Mcpt5-Cre pups in F2 progeny was similar, in line withthe expected Mendelian ratio (Table 1).To understand if ELKSregulates the population of resident mast cells in peritoneal cavity, cells wereextracted from the peritoneal cavity of WT and ELKS Mcpt-Cre KO mice. Flowcytometric analysis suggested similar population of mast cells in theperitoneal lavage of WT and ELKS KO mice (Fig. 2a). Furthermore, these cellsare then cultured for 21 days in the presence of IL-3 and SCF.

The surfaceexpression levels of mast cell-specific markers Fc?RI and c-Kit on KO PCMCswere similar to that of WT PCMCs (Fig. 2b). Similarly, the generation of bonemarrow-derived mast cells (BMMCs) in the presence of IL-3 and SCF was notaffected by ELKS deficiency as both WT and ELKS KO BMMCs had comparable levelsof 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 andprotein levels of ELKS in PCMCs and BMMCs from WT and ELKS KO mice were quantifiedusing real-time PCR and Western blot respectively. Absence of ELKS mRNA andprotein in PCMCs were confirmed as shown in Fig. 2d. However, as stated inprevious literature that the efficacy of Cre/Lox recombination in BMMCs forMcpt-Cre strain is not 100% (20), the deletion of ELKS in BMMCs from ELKS f/fMcpt5-Cre mice was not complete (Fig. 2e). Therefore, we only used PCMCs fromthese mice for later experiments which allows 100% deletion of ELKS in PCMCs. ELKS positively regulates mast celldegranulation Mast cells rapidly degranulateafter being activated through Fc?RI.

To determineif ELKS plays a role in IgE-mediated mast cell activation and degranulation, WTand ELKS KO PCMCs were first sensitised with anti-DNP-IgE antibody and thenstimulated with DNP-BSA and the release of granule-stored enzyme, ?-hexosaminidase wasmeasured. Release of ?-hexosaminidasewas optimal at a dose of antigen at 10ng/mL in WT PCMCs (Fig. 3a) andELKS-deficient PCMCs had significantly lower release of ?-hexosaminidasecompared to WT PCMCs upon Fc?RI activation (Fig. 3b). Likewise, less surfaceexposure of LAMP1, a marker for exocytosis of granules, was detected in ELKS KOPCMCs compared to WT PCMCs following IgE/Ag stimulation (Fig. 3c).

Hence, thesedata indicated that ELKS-deficient mast cells are impaired in their capacity todegranulate in vitro. ELKS negatively regulates cytokine productionfrom mast cellsEngagement of the Fc?RIreceptor by IgE and specific antigen also results in de novo synthesis of various cytokines and chemokines that characterisesthe late-phase pro-inflammatory response. Therefore, we analysed geneexpression of a selection of pro-inflammatory cytokines and chemokinesincluding 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-timePCR analysis demonstrated that ELKS-deficient mast cells have augmented mRNAexpressions 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-mediatedcytokine and chemokine synthesis in mast cells besides degranulation.  ELKS is not essential for early signaltransduction of IgE-activated mast cellsNext, we examinedwhether ELKS is required for the Fc?RI – induced early intracellular signallingpathways in mast cells.

To this end, WT and ELKS KO mast cells were againsensitised with anti-DNP IgE and then stimulated with DNP-BSA. However, therewas 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 signallingafter mast cell activation.

  Discussion In the present study,we generated conditional knockout mice for ELKS in connective tissue mast cellsand demonstrated that ELKS deletion in mast cells causes reduced degranulationbut enhanced cytokine synthesis. Collectively, our data has confirmed the roleof ELKS in positive regulation of exocytosis and has identified a negativeregulatory role of ELKS in cytokine transcription.Previous studies haveimplicated the involvement of different IKK complex subunits within the NF-?B signallingpathway in mast cell functions. I?B kinase ? (IKK?) was shown to be criticalfor mast cell degranulation as Suzuki etal. (2008) has demonstrated that fetal liver-derived mast cells from IKK?-deficientmice had impaired degranulation upon IgE-Ag stimulation (21). However, anotherstudy by Peschke et al. (2014) had foundthat there was unaffected degranulation but impairedproduction of cytokines in peritoneal mast cells generated from mice withconnective tissue mast cell-specific IKK? deletion (20). In the same study byPeschke et al.

(2014), they have alsoreported that activated peritoneal NEMO/IKK? KO mast cells had reduced cytokineproduction (20). In addition, severallines 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+ dependentexocytosis in PC12 cells (22) while another study by Ohara-Imaizumi et al. (2005) has demonstrated thatthere was a decrease in insulin exocytosis after silencing ELKS with RNAinterference (RNAi) in MIN6? cells (23).

Moreover, another study by Nomura et al. (2009) has demonstrated thatknockdown and overexpression of ELKS in RBL-2H3 cells have resulted in reducedand enhanced exocytotic activity respectively (16). Therefore, our data showingless ?-hexosaminidase release from ELKS KO PCMCs than WT PCMCs after stiumation(Fig.

3b) further supported the role of ELKS in positively regulatingdegranulation in mast cells.Furthermore, wedemonstrated that the gene expression for some pro-inflammatory cytokines andchemokines are higher in activated ELKS KO mast cells than in activated WT mastcells (Fig. 4a), suggesting that ELKS might have an additional role in cytokineand chemokine production in mast cells. Since previous study byKandere-Grzybowska et al.

(2003) hasdemonstrated that IL-1 stimulated mast cells can release IL-6 withoutdegranulation (24) and another study by Foger et al. (2011) has found that Coro1a KO BMMCs displayed enhanced degranulationbut diminished cytokine secretion (25), the trafficking for cytokine/chemokine inmast cells probably employs a different secretory lysosomal pathway to thosepre-formed mediators within the secretory granules. However, secreted cytokinesand chemokines should be measured in our future experiments in order to verifythat cytokine and chemokine secretion is upregulated in ELKS-deficient PCMCs.   Conclusion and Future Directions Taken together, componentswithin the IKK complex, including ELKS, could contribute to different mast cellfunctions. The present findings strengthen the idea that ELKS is a positiveregulator of mast cell degranulation and ELKS might also negatively regulatecytokine and chemokine production in mast cells.

We will also study the localisationof ELKS in mast cells during unstimulated and IgE-Ag stimulated conditions andconfirm its role in mast cell degranulation invitro at single-cell level through patch clamp studies and in vivo through the passive cutaneous anaphylaxismodel. Therefore, our work will provide further insight into how ELKS regulatemast cell functions, allowing us to identify potential therapeutic targets forallergic inflammation. 



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