LAT is essential for the mast cell stabilising effect of tHGA in IgE-mediated mast cell activation

Accepted Manuscript LAT is Essential for the Mast Cell Stabilising Effect of tHGA in IgE-Mediated Mast Cell Activation Ji Wei Tan, Daud Ahmad Israf, Nur Fariesha Md Hashim, Yoke Kqueen Cheah, Hanis Hazeera Harith, Khozirah Shaari, Chau Ling Tham PII: DOI: Reference:

S0006-2952(17)30537-3 http://dx.doi.org/10.1016/j.bcp.2017.08.010 BCP 12896

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

15 June 2017 10 August 2017

Please cite this article as: J.W. Tan, D.A. Israf, N.F. Md Hashim, Y. Kqueen Cheah, H.H. Harith, K. Shaari, C.L. Tham, LAT is Essential for the Mast Cell Stabilising Effect of tHGA in IgE-Mediated Mast Cell Activation, Biochemical Pharmacology (2017), doi: http://dx.doi.org/10.1016/j.bcp.2017.08.010

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

LAT is Essential for the Mast Cell Stabilising Effect of tHGA in IgE-Mediated Mast Cell Activation Ji Wei Tan1, Daud Ahmad Israf1, Nur Fariesha Md Hashim1, Yoke Kqueen Cheah1, Hanis Hazeera Harith1, Khozirah Shaari2, Chau Ling Tham1* 1

Department of Biomedical Science, Faculty of Medicine and Health Sciences,

Universiti Putra Malaysia, Serdang 43300, Malaysia. 2

Faculty of Science, Universiti Putra Malaysia, Serdang 43300, Malaysia.

* Corresponding Author’s Email: [email protected]

Abstract Mast cells play a central role in the pathogenesis of allergic reaction. Activation of mast cells by antigens is strictly dependent on the influx of extracellular calcium that involves a complex interaction between signalling molecules located within the cells. We have previously reported that tHGA, an active compound originally isolated from a local shrub known as Melicope ptelefolia, prevented IgE-mediated mast cell activation and passive systemic anaphylaxis by suppressing the release of interleukin4 (IL-4) and tumour necrosis factor (TNF)-α from activated rat basophilic leukaemia (RBL)-2H3 cells. However, the mechanism of action (MOA) as well as the molecular target underlying the mast cell stabilising effect of tHGA has not been previously investigated. In this study, DNP-IgE-sensitised RBL-2H3 cells were pre-treated with tHGA before challenged with DNP-BSA. To dissect the MOA of tHGA in IgEmediated mast cell activation, the effect of tHGA on the transcription of IL-4 and TNF-α mRNA was determined using Real Time-Polymerase Chain Reaction (qPCR)

1

followed by Calcium Influx Assay to confirm the involvement of calcium in the activation of mast cells. The protein lysates were analysed by using Western Blot to determine the effect of tHGA on various important signalling molecules in the LATPLCγ-MAPK and PI3K-NFκB pathways. In order to identify the molecular target of tHGA in IgE-mediated mast cell activation, the LAT and LAT2 genes in RBL-2H3 cells were knocked-down by using RNA interference to establish a LAT/LAT2 competition model. The results showed that tHGA inhibited the transcription of IL-4 and TNF-α as a result of the suppression of calcium influx in activated RBL-2H3 cells. The results from Western Blot results revealed that tHGA primarily inhibited the LAT-PLCγ-MAPK pathway with partial inhibition on the PI3K-p65 pathway without affecting Syk. The results from RNAi further demonstrated that tHGA failed to inhibit the release of mediators associated with mast cell degranulation under the LAT/LAT2 competition model in the absence of LAT. Collectively, this study concluded that the molecular target of tHGA could be LAT and may provide a basis for the development of a mast cell stabiliser which targets LAT.

Keyword: tHGA, RBL-2H3, IgE-mediated mast cell activation, LAT, LAT 2, Melicope ptelefolia.

1. Introduction Allergy is a hypersensitivity disorder mediated by immunological mechanisms which can cause tissue damage and life-threatening reactions, including atopic dermatitis, asthma, and anaphylactic shock [1,2]. One of the key characteristics of allergy is the excessive activation of mast cells by an antigen specific immunoglobulin E (IgE) antibody which results in extreme inflammatory responses [1,3,4]. Mast cells are the 2

major effector cells of allergic inflammation, where their roles in innate and adaptive immune responses have been increasingly recognised within the last few years [5]. The binding of antigen-specific IgE to FcεRI sensitizes mast cells, enabling them to release mediators in response to subsequent encounter with that specific antigen [3,4,6]. The rat basophilic leukaemia cell line (RBL-2H3), which is known to be a mast cell analogue, has been widely used to determine the signal transduction of FcεRI as these cells express abundant FcεRI receptors on their cell surfaces [7,8,9]. RBL-2H3 cells release preformed and newly synthesised mediators of immune allergic response following crosslinking of IgE-bound FcεRI with multivalent allergens [7,10,11,12]. In addition, activated RBL-2H3 cells produce various cytokines, chemokines, prostaglandins and leukotrienes that play important roles in the infiltration of inflammatory cells and the induction of the late-phase reactions [4,13]. The effect of these mediators on surrounding cells and tissues is what causes the symptoms and severity of an allergic reaction [6]. Signalling pathways leading to the degranulation of mast cells after engagement of the IgE on the FcεRI receptor have been well studied and extensively characterised [14,15,16]. Following FcεRI aggregation, a complex series of intracellular signalling processes within mast cells will be initiated [17]. Although the immediate receptorproximal signalling events seem to be common for the release of all categories of mediators, the receptor-distal signalling events must diverge to regulate the different mechanisms by which these mediators are released [15]. Several lines of evidence have shown that the LAT-PLCγ axis as well as the PI3K-NFκB axis pathways play vital roles in coordinating the IgE-dependant mast cell degranulation [15,18,19]. Both pathways are activated by a tyrosine kinase known as Spleen tyrosine kinase (Syk) 3

through the phosphorylation of adapter molecules such as linker for activation of T cells (LAT) and Non-T cell activation linker (LAT2) [20]. This will lead to the downstream cascade effects which cause the activation of various key enzymes or molecules along the signalling pathways such as phosphoinositide 3-kinase (PI3K), NFκB p65, phospholipase Cγ (PLCγ), mitogen-activated protein kinase (MAPK) and arachidonic acid-associated enzymes [14,15]. Eventually, this will result in the massive influx of Ca2+ from the surrounding environment into the cell which is considered an important event that triggers mast cell degranulation [20,21]. As mast cell degranulation has been shown to play an important role in allergic inflammation, coagulation cascades, host defence, and tissue remodelling, increased understanding of mast cell degranulation and the mechanisms involved will lead to the discovery of effective therapy for diseases associated with mast cell degranulation. [22]. Until now, the treatment of allergic diseases relies on clinically-prescribed drug classes of mast cell stabilisers or H1 antagonists such as ketotifen fumarate, olopatadine and cromolyn [6]. These mast cell stabilisers have been reported to attenuate the release of allergic mediators such as histamine, leukotrienes C4 and PAF, as well as playing a part in the signalling pathway that is associated with Ca2+ influx inhibition during mast cell activation [23]. For example, cromolyn has been reported to inhibit NF-κB nuclear translocation and the action of phosphorylated MAPK proteins during mast cell activation [24,25]. At the same time, unwanted side effects such as drowsiness, upset stomach, chest congestion and dry mouth have been associated with the use of these mast cell stabilisers [26]. As such, there are ongoing researches into potential mast cell stabilising agents derived from natural sources, however their mechanisms of action and safety are yet to be established [6].

4

2,4,6-trihydroxy-3-geranylacetophenone (tHGA) is an active compound originated from a local shrub, namely Melicope ptelefolia [27]. It has been traditionally used by locals to treat a wide range of diseases, including itches [28]. Scientific evidence on the anti-allergic properties of tHGA has been previously reported where the oral and systemic treatment of tHGA prevented the ovalbumin-induced allergic airway inflammation in a murine model of allergic asthma [29,30]. Despite the important roles of mast cells as the main immune effector cells involved in the pathogenesis of allergic diseases, there was no report on the mast cell stabilising effect of tHGA until recently, when our research group demonstrated that tHGA inhibited IgE-mediated mast cell degranulation and passive systemic anaphylaxis [31]. However, the mechanism underlying its protective effects is still poorly understood. Therefore, the purpose of this study was to investigate the underlying mechanism of the inhibitory effects of tHGA in a cellular model of IgE-mediated mast cell degranulation.

2. Material and methods 2.1. Compound synthesis and preparation tHGA, with its chemical structure previously reported [30], was synthesised according to a previously described method [30] and the stock solution (20 mM) was prepared according to a previous study [31]. Prior to experiments, the activity of several batches of synthetic tHGA was tested in vitro using the β-hexosaminidase release assay. There was minimal variation between different batches of tHGA (<3% variation).

2.2. Antibodies and reagents 5

Mouse anti-dinitrophenol (DNP) monoclonal IgE and 4-Nitrophenyl N-acetyl-β-Dglucosaminide (PNAG) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). DNP-conjugated bovine serum albumin (DNP-BSA) was purchased from Calbiochem (San Diego, CA, USA). The enzyme-linked immunoassay (EIA) kits for histamine, prostaglandins D2 (PGD2) and leukotrienes C4 (LTC4) were purchased from Cayman Chemicals (Ann Arbor, MI, USA). The ELISA kits for interleukin 4 (IL-4) and tumour necrosis factor-alpha (TNF-α) were purchased from R&D systems (Minneapolis, MN, USA). Ketotifen fumarate was purchased from MP Biomedicals (Santa Ana, California, USA). Eagle’s minimum essential medium (EMEM), foetal bovine serum, penicillin and streptomycin were purchased from Life Technologies Inc. (Waltham, MA, USA). Silencer® Select siRNAs (LAT, LAT2, GAPDH and negative control), Opti-MEM® I-reduced serum media and Lipofectamine® RNAiMAX transfection reagent were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). FLUOFORTE® Calcium assay kit was purchased from Enzo Life Sciences (Farmingdale, NY, USA). The QuantiNova™ SYBR® Green PCR kit and RNeasy® Plus Mini kit were purchased from QIAGEN Inc. (Valencia, CA, USA). Rabbit polyclonal primary antibodies against 5-LOX, COX-2, IKK, PI3K, PLCγ1, p38, JNK, ERK, p65, Syk, LAT, β-Actin, TFIIB, phospho-Syk, phospho-PLCγ1, phospho-p38, phospho-JNK, phospho-ERK, phospho-IKK, phospho-PI3K, phosphoLAT, HRP-conjugated goat anti-rabbit IgG secondary antibodies, HRP-conjugated mouse monoclonal IgG1 for β-actin and mouse monoclonal IgG2b for TFIIB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal primary antibodies against IκB, cPLA2, phospho-IκB and phospho-cPLA2 were purchased from Cell Signalling Technology (Danvers, MA, USA). SuperSignal West Pico Chemiluminescent Substrate was purchased from Pierce Biotechnology

6

(Rockford, IL, USA). Other Western Blotting chemical reagents were purchased from Amresco LLC (Solon, OH, USA). ADP-Glo™ Kinase assay and Syk Kinase Enzyme System assay kits were purchased from Promega Corporation (Madison, WI, USA).

2.3 Cell culture and activation RBL-2H3 cell line (rat basophilic leukaemia cell line) was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in EMEM medium containing 10% foetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin. RBL-2H3 cells were incubated at 37°C in a 5% CO2 humidified incubator and were subcultured to new T25 tissue culture flask (3x105 cells/flask) or used for assays when the confluence of cells reached 80%. Only RBL-2H3 cells of passage number ranging from 6 to 11 were used throughout this study.

2.4. Quantitative real-time PCR (qPCR) To determine the effect of tHGA on the gene expression of IL-4 and TNF-α, RBL2H3 cells (1×10 6 cells/well in 6-well plate) were seeded and sensitised with 1 µg/mL of DNP-IgE for 24 hrs. On the next day, the sensitised cells were rinsed with Tyrode’s buffer before pre-treatment with tHGA (1.25, 5, 20 µM) for 20 min. The pre-treated cells were then challenged with 1 µg/mL of DNP-BSA for 1 hr. At the end of DNPBSA challenge, the RNA extraction, reverse-transcription of the extracted RNA and quantitative real-time PCR were performed according to the manufacturer’s instructions. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Forward (F) and reverse (R) primers for the

cytokines

and

GAPDH

were 7

as

follows:

IL-4:

5’-

TGCACCGAGATGTTTGTACCAGA-3’

(F)

TTGCGAAGCACCCTGGAAG-3’

(R);

AACTCGAGTGACAAGCCCGTAG-3’

and

5’-

TNF-α:

5’-

and

5’-

GAPDH:

5’-

and

5’-

(F)

GTACCACCAGTTGGTTGTCTTTGA-3’

(R);

GGCACAGTCAAGGCTGAGAATG-3’

and (F)

ATGGTGGTGAAGACGCCAGTA-3’ (R). The cycling protocol was identical for all primer pairs, with an initial incubation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 5 s and 60°C for 10 s. The fold-changes in the transcription levels were calculated using the 2 −∆∆CT method where ∆∆Ct = (Cttarget

gene

− CtGAPDH)treated groups −

(Cttarget gene − CtGAPDH)Normal.

2.5 Measurement of intracellular Ca 2+ To

determine

the

effect

of

tHGA

on

intracellular

Ca2+

levels,

a

FLUOFORTE® Calcium assay kit was used according to the manufacturer’s protocol. Briefly, RBL-2H3 cells (4×104 cells/well in 96-well plate) were sensitised with 1 µg/mL of DNP-IgE for 24 hrs. On the next day, the sensitised cells were rinsed with Tyrode’s buffer before being pre-treated with tHGA (1.25, 5, 20 µM) for 20 min. Then, the cells were incubated with the Ca2+ reagent loading solution for 1 hr at room temperature (RT). The pre-treated cells were then challenged with 1 µg/mL of DNPBSA for 1 hr. At the end of DNP-BSA challenge, the fluorescence signal was measured using a fluorometric plate reader at an excitation wavelength of 494 nm and an emission wavelength of 516 nm (Tecan Infinite® M200, Männedorf, Switzerland).

2.6 Western blotting 8

To determine the effect of tHGA on the signalling molecules in IgE-mediated mast cell activation, RBL-2H3 cells (1×107 cells) were sensitised with 1 µg/mL of DNPIgE for 24 hrs. On the next day, the sensitised cells were rinsed with Tyrode’s buffer before pre-treatment with tHGA (1.25, 5, 20 µM) for 20 min. The pre-treated cells were then challenged with 1 µg/mL of DNP-BSA for 10 min (phospho-Syk, phosphoLAT, phospho-PLCγ1, phospho-p38, phospho-JNK, phospho-ERK, phospho-PI3K and phospho-cPLA2) or 15 min (5-LOX, COX-2, p65, phospho-IKK, phospho-IκB, phospho-cPLA2). At the end of DNP-BSA challenge, the cells were washed with cold PBS, resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1% protease and phosphatase inhibitors cocktail) and centrifuged to obtain whole cell lysates. For nuclear and cytoplasmic protein extracts, the cells were lysed using the NucBuster™ protein extraction kit according to the manufacturer’s protocols with slight modifications. Briefly, the cells were lysed with 300 µL of NucBuster reagent 1 for 5 min on ice. Then, the whole cell lysates were vortexed for 30 seconds at 8000 x g followed by 10 min of incubation on ice. The same steps were repeated twice. The cell suspension was then centrifuged for 5 min at 16000 x g and 4°C and the supernatant was stored as a cytosolic fraction. The remaining cell pellet containing nuclei was further lysed with 150 µL of NucBuster reagent 2 containing protease inhibitor cocktail and dithiothreitol (DTT). The cells were vortexed for 30 seconds at 8000 x g followed by 10 min of incubation on ice. The same steps were repeated thrice. The nuclear debris was then spun down for 5 min at 16000 x g and 4°C and the supernatant was stored as a nuclear fraction. The protein samples were separated using 10% (w/v) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes, 9

blocked with 5% BSA for 1 hr, and subsequently incubated overnight at 4°C with primary antibody (1:1000) specific for 5-LOX, COX-2, Syk, p-Syk, LAT, p-LAT, PLCγ1, p-PLCγ1, p38, p-p38, JNK, p-JNK, ERK, p-ERK, cPLA2, p-cPLA2, PI3K, p65 NF-κB, IκBα, p-IκBα, IKKα/β, p-IKKα/β in Tris Buffered Saline-Tween (TBST) containing 5% BSA. On the next day, the membranes were incubated for 1 hr with goat anti-rabbit IgG secondary antibody (1:5000) diluted in 5% BSA at RT. Finally, the membranes were washed three times in TBST, incubated with chemiluminescent substrate solution for 1 min, and visualised using the Fusion FX gel documentation system (Vilber Lourmat, Eberhardzell, Germany).

2.7 Kinase assay To determine the effect of tHGA on Syk kinase activity, a Syk kinase enzyme system assay kit was used according to the manufacturer’s protocols. Briefly, 25µL of kinase reaction (50 ng Syk kinase, 10 µM ATP and 2 µg Poly E4Y1) containing tHGA (5 or 20 µM) in 1X kinase buffer was added to the wells of a 96-well solid white plate and incubated for 60 min at RT. Then, 25 µL of ADP-Glo™ reagent was added to the kinase reaction followed by further incubation of the plate for 40 min at RT. Finally, the plate was treated with 50 µL of kinase detection reagent and incubated for 30 min at RT, before the plate was taken to measure the luminescence signal using a Panomics luminometer (Santa Clara, CA, USA).

2.8 siRNA knockdown To perform transient knockdown of LAT or both LAT/LAT2 in RBL-2H3 cells, short interference RNA (siRNA) constructs around 21 nucleotide sequences that matched 10

rat LAT (siRNA1: 5’-CAU CAA ACC ACC UCA AAU ATT-3’ and siRNA2: 5’CUA UGA CAG UGC UUC CAC ATT-3’) and/or LAT2 (siRNA1: 5’-ACA AGU CAU UUG AGA GGA ATT-3’ and siRNA2: 5’- CCA GAA CUU CUA CAA AGG ATT-3’) were used. All transfection protocols were carried out according to the manufacturer’s instructions with slight modifications. Briefly, 7×105 cells/well in 6well plate were transfected with siRNA at a final concentration of 50 pmol/well using Lipofectamine RNAiMAX transfection reagent. Cells transfected with non-target siRNA or GAPDH siRNA were used as the negative and positive control respectively. Cells receiving Opti-MEM® I reduced serum media without any siRNAs served as a mock-transfected control. Twenty-four hours after the transfection, the release of key mediators during IgE-antigen complex-stimulation of siRNA-treated or non-treated cells, including the negative and positive controls as well as the mock transfection control, were measured as explained in sections 2.9 and 2.10. Results of the siRNA treatment groups were presented as follow: (1) GAPDH siRNA positive control (positive control) (2) Scramble siRNA negative control (negative control) (3) Mock transfection control (mock control) (4) Normal RBL-2H3 cells (normal cells) (5) IgE-antigen complex-stimulated cells (challenged cells) (6) LAT-deficient IgE-antigen complex-stimulated cells (LAT-deficient challenged cells) (7) LAT2-deficient IgE-antigen complex-stimulated cells (LAT2-deficient challenged cells)

11

(8) LAT/LAT2-deficient IgE-antigen complex-stimulated cells (LAT/LAT2-deficient challenged cells) (9) LAT-deficient IgE-antigen complex-stimulated cells pre-treated with tHGA (tHGA pre-treated LAT-deficient challenged cells) (10) LAT2-deficient IgE-antigen complex-stimulated cells pre-treated with tHGA (tHGA pre-treated LAT2-deficient challenged cells) (11) LAT/LAT2-deficient IgE-antigen complex-stimulated cells pre-treated with tHGA (tHGA pre-treated LAT/LAT2-deficient challenged cells)

2.9 Analysis of histamine release To determine the effect of tHGA on the release of preformed mediators in LATdeficient RBL-2H3 cells, the release of histamine upon treatment with tHGA was examined. LAT-deficient RBL-2H3 cells were sensitised with 1 µg/mL of DNP-IgE for 24 hrs. On the next day, the sensitised cells were washed with Tyrode's buffer before pre-treatment with tHGA (5 and 20 µM) for 20 min. The pre-treated cells were then challenged with 1 µg/mL of DNP-BSA for 1 hr. At the end of DNP-BSA challenge, the culture supernatant was centrifuged (17,000 x g, 10 min) at 4°C. The level of histamine in the culture supernatant was measured by using a Histamine EIA kit according to the manufacturer’s instructions. The same protocol was also applied to determine the release of histamine in LAT/LAT2-deficient RBL-2H3 cells.

2.10 Analysis of TNF-α, IL-4, PGD2 and LTC4

12

To determine the effect of tHGA on the release of de novo mediators in LAT-deficient RBL-2H3 cells, the cells (2×10 5 cells/well) were sensitised with 1 µg/mL of DNP-IgE for 24 hrs. On the next day, the sensitised cells were rinsed with Tyrode's buffer before pre-treatment with tHGA (5 and 20 µM) for 20 min. The pre-treated cells were then challenged with 1 µg/mL of DNP-BSA for 6 hrs. At the end of DNP-BSA challenge, the culture supernatant was centrifuged (17,000 g, 10 min) at 4°C. The levels of PGD2 and LTC4 were measured by using PGD2 and LTC4 EIA kits, whereas the levels of IL-4 and TNF-α were measured by using IL-4 and TNF-α ELISA assay kits according to the manufacturer’s instructions. The same protocol was also applied to determine the release of de novo mediators in LAT/LAT2-deficient RBL-2H3 cells.

2.11 Statistical analysis All experiments described were performed three times. The results were expressed as means ± S.E.M. Statistical analyses were performed using SPSS 19.0 (Chicago, IL, USA). One-way analysis of variance (ANOVA) followed by Tukey's test was used to compare the results from different treatment groups. P values of less than 0.05 was considered as statistically significant.

3. Results 3.1 tHGA inhibits IL-4 and TNF-α mRNA expression in IgE-antigen complexstimulated RBL-2H3 cells Our previous study demonstrated that tHGA was able to inhibit mast cell degranulation through the attenuation of mediator release, including IL-4 and TNF-α

13

from IgE-antigen complex-stimulated RBL-2H3 cells [31]. In order to understand the mechanism of action of tHGA on IgE-antigen complex-stimulated RBL-2H3 cells, we continued to determine the effect of tHGA on the gene expression of IL-4 and TNF-α using Real Time-PCR. As shown in Figs. 1A and 1B, DNP-BSA challenge for 6 hrs significantly increased the mRNA levels of IL-4 and TNF-α in IgE-antigen complexstimulated cells by approximately 16.7- and 20.6-fold, respectively. On the other hand, at 5 and 20 µM, tHGA significantly and concentration-dependently reduced the mRNA levels of IL-4 and TNF-α by approximately 26-50% in comparison to the IgEantigen complex-stimulated cells. However, tHGA failed to significantly decrease the mRNA levels of both IL-4 and TNF-α at the concentration of 1.25 µM.

3.2 tHGA suppresses the level of intracellular Ca2+ in IgE-antigen complexstimulated RBL-2H3 cells An increase of the intracellular Ca2+ concentration is important in causing mast cell activation followed by degranulation [32]. Thus, the inhibitory effect of tHGA on the level of intracellular Ca2+ was examined. As shown in Fig. 1C, DNP-BSA challenge for 1 hour significantly increased the level of intracellular Ca2+ by 15.9-fold in IgEantigen complex-stimulated cells. However, the intracellular Ca2+ level was significantly reduced in the presence of tHGA. Pre-treatment of 5 and 20 µM tHGA significantly reduced the intracellular Ca2+ by 32% and 47%, respectively, in comparison to the IgE-antigen complex-stimulated cells. Apparently, no significant inhibition was observed at 1.25 µM of tHGA.

14

3.3 tHGA inhibits the phosphorylation of LAT-PLCγ pathway in IgE-antigen complex-stimulated RBL-2H3 cells It is well known that the increase of intracellular Ca2+ influx will cause activation of the LAT-PLCγ pathway in IgE-antigen complex-stimulated RBL-2H3 cells [5]. Figs. 2A and 2B show that DNP-BSA significantly up-regulated the phosphorylation of PLCγ and LAT in IgE-antigen complex-stimulated RBL-2H3 cells by approximately 6.7-fold. On the other hand, pre-treatment with 5 and 20 µM tHGA significantly attenuated the phosphorylation of these two signalling proteins by approximately 23% to 45%. However, tHGA at the concentration of 1.25 µM was too low to cause a significant reduction on the phosphorylation of both PLCγ and LAT in comparison to the IgE-antigen complex-stimulated RBL-2H3 cells.

3.4 tHGA inhibits the phosphorylation of cPLA2 and enzymes associated with the arachidonate cascade pathway in IgE-antigen complex-stimulated RBL-2H3 cells Our previous study reported that tHGA inhibited the release of PGD2 and LTC4 in IgE-mediated mast cell degranulation [31] whereby the production of PGD2 and LTC4 is mediated by the arachidonate cascade pathway and involves enzymes such as cPLA2, 5-LO and COX-2 [33,34]. Figs. 3A to 3C show that DNP-BSA significantly induced the phosphorylation of cPLA2 as well as the levels of both 5-LO and COX-2 proteins in IgE-antigen complex-stimulated cells by approximately 6.2- to 6.6-fold. However, these adverse effects were significantly reduced in a concentrationdependent manner in the presence of tHGA. Pre-treatment with 5 and 20 µM tHGA significantly reduced the phosphorylation of cPLA2 by approximately 23.9% and 41.4%, respectively, in comparison to the IgE-antigen complex-stimulated cells.

15

Furthermore, the expression levels of both 5-LO and COX-2 were also reduced by approximately 17% to 24% (5 µM tHGA) and 24% to 48% (20 µM tHGA) with respect to the IgE-antigen complex-stimulated RBL-2H3 cells. Conversely, at the concentration of 1.25 µM, tHGA failed to down-regulate the levels of all of the enzymes associated with the arachidonate cascade pathway.

3.5 tHGA inhibits the activation of MAPK proteins in IgE-antigen complexstimulated RBL-2H3 cells The activated LAT-PLCγ pathway in IgE-antigen complex stimulated RBL-2H3 cells is reported to be associated with the activation of MAPK proteins including ERK 1/2, p38 and JNK [33, 34]. Figs. 3D to 3F show that DNP-BSA significantly induced the phosphorylation of all MAPK proteins in IgE-antigen complex-stimulated cells by approximately 5.6- to 6.0-fold. However, the phosphorylation or protein expression levels of MAPK proteins were significantly reduced in a concentration-dependent manner in the presence of tHGA. Pre-treatment with 5 and 20 µM tHGA significantly reduced the phosphorylation of all MAPK proteins by approximately 15-20% and 3235%, respectively, in comparison to the IgE-antigen complex-stimulated cells. Apparently, consistent with other results so far, 1.25 µM of tHGA failed to inhibit the phosphorylation of all of the MAPK proteins.

3.6 tHGA partially inhibits activation of the PI3K-NFκB pathway in IgE-antigen complex-stimulated RBL-2H3 cells Previous reports demonstrated crosstalk between the LAT-PLCγ pathway and the PI3K-NFκB pathway through MAPK proteins, which collectively leads to the 16

production of cytokines from IgE-antigen complex-stimulated mast cells [33,35]. In the present study, Fig. 4A to 4D demonstrated that DNP-BSA significantly induced, by approximately 5.0- to 8.7-fold, the expression of PI3K, phosphorylated IKKα/β and phosphorylated IκBα, as well as the translocation of p65 into the cell nucleus of IgE-antigen complex-stimulated cells. However, in the presence of 20 µM tHGA, the phosphorylation of IκBα and nuclear translocation of NFκB p65 were significantly reduced by 13.2% and 13.7%, respectively. There was no inhibition of these two signalling proteins in IgE-sensitised RBL cells pre-treated with 1.25 and 5 µM of tHGA. Interestingly, the inhibitory effect of tHGA was not significant, even at 20 µM, on the expression of PI3K and phosphorylation of IKK, which are located upstream of IκBα and NFκB p65.

3.7 tHGA does not inhibit the phosphorylation of Syk and its kinase activity in IgE-antigen complex-stimulated RBL-2H3 cells As Syk is the upstream molecule of both LAT-PLCγ and PI3K-NFκB pathways, the significant role of Syk, especially in IgE-dependent mast cell activation, has been previously shown by various studies [36,37]. According to Fig. 5A, DNP-BSA significantly induced the phosphorylation of Syk in IgE-antigen complex-stimulated cells by approximately 6.6-fold. Surprisingly, the presence of tHGA was not able to reduce the phosphorylation of Syk. In order to exclude the possibility that tHGA might inhibit Syk kinase activity without inhibiting its phosphorylation, the effect of tHGA on Syk kinase activity was examined and negative results were obtained. None of the three concentrations of tHGA used (1.25, 5 and 20 µM) were able to show any inhibitory effect on Syk kinase activity (Fig. 5B). Nonetheless, ketotifen fumarate,

17

which served as the drug control in this study, was able to significantly reduce the Syk kinase activity by approximately 22% without affecting Syk phosphorylation.

3.8 The effects of LAT, LAT2, and LAT/LAT2 deficiency on the release of mediators from IgE-antigen complex-stimulated RBL-2H3 cells Thus far, the results in this present study have shown that tHGA does not inhibit the upstream signalling of the PI3K-NFκB pathway. At the same time, the inhibitory effect of tHGA on the LAT-PLCγ-MAPK axis pathway stops at LAT without further interfering the phosphorylation and kinase activity of its upstream molecule, Syk. Collectively, all these findings indicated that LAT could be the molecular target of tHGA in IgE-mediated mast cell degranulation. In order to confirm whether LAT is the molecular target of tHGA in this study, cellular models of LAT, LAT2 and LAT/LAT2 gene knockdown were developed by using transient transfection. The efficacy of siRNA-mediated down-regulation of LAT, LAT2 and LAT/LAT2 protein expressions were examined by performing Western Blot. Two siRNA (siRNA1 and siRNA2) were used to knock down the expression of both LAT and LAT2. As shown in Fig. 6A, after 24 hrs of transfection, siRNA1 and siRNA2 significantly down-regulated the protein expression of LAT by 70.1% and 71.9% respectively. When the transfection period was prolonged to 48 hrs, the protein expressions of LAT by cells transfected by siRNA1 and siRNA2 were downregulated by approximately 70.3% and 70.5% respectively. Similarly, Fig 6B shows that the proteins expressions of LAT2 were significantly down regulated by siRNA1 and siRNA2 by 72.7% and 73.3% respectively after 24 hrs of transfection. The efficacy did not increase significantly when the cells were transfected with siRNA1

18

and siRNA2 of LAT2 up to 48 hrs. As the efficacy of all siRNA used to downregulate LAT and LAT2 protein expressions were similar, siRNA1 of LAT and LAT2 were selected to knock down the protein expressions of LAT and LAT2 simultaneously. Fig. 6C shows that the proteins expressions of both LAT and LAT2 were significantly down- regulated by approximately 70.3% and 71.8% respectively, after 24 hrs of siRNAs transfection. Consistent with the earlier findings in Figs. 6A and 6B, the efficacy of siRNA-mediated down-regulation of LAT and LAT2 did not increase significantly after 48 hrs of transfection. Hence, subsequent experiments in this study were conducted by transfecting the cells with siRNA for 24 hrs. For the positive control, the protein expressions of GAPDH in all the experiments were being down regulated consistently by approximately 88.7% to 90.1% when compared to the normal cells. In order to confirm that these siRNAs specifically silence the LAT and LAT2 genes without interfering the phophorylation of upstream signalling molecules, the phosphorylation level of Syk was also examined in these siRNAs treated RBL2H3 cells. The Western Blot results confirmed that the silencing of both or either one of the adapter protein did not affect the upstream signalling event of IgE-mediated mast cell activation (data not shown). The effects of silencing LAT, LAT2 and LAT/LAT2 in IgE-antigen complexstimulated RBL-2H3 cells were determined by measuring the release of histamine, which is a preformed mediator released during the early stage of mast cell activation [38]. According to Fig. 7A, under normal condition, histamine release was significantly increased by 21.3-fold following 1 hour of DNP-BSA incubation in challenged cells compared to normal cells. However, LAT deficiency alone or both LAT/LAT2 deficiency led to only 14.1 and 6.2-fold increase respectively. This indicates that histamine release in LAT or LAT/LAT2 deficient challenged cells was 19

suppressed respectively by 34% and 71% compared to challenged cells. By contrast, LAT2 deficiency alone led to 27.6-fold increase, indicating that histamine release by LAT2-deficient challenged cells was greater by 30% compared to challenged cells. Apart from the release of preformed mediators in the early phase of mast cell degranulation, prolonged exposure of IgE-antigen complex-stimulated RBL-2H3 cells to antigen results in the late phase reaction and production of de novo inflammatory mediators including PGD2, LTC4, IL-4 and TNF-α [39,40]. As shown in Fig. 7B to 7E, under normal condition, PGD2, LTC4, IL-4 and TNF-α release were significantly increased by approximately 13- to 21-fold following 6 hrs of DNP-BSA incubation in challenged cells compared to normal cells. However, LAT deficiency alone or both LAT/LAT2 deficiency led to only 9- to 13-fold and 4- to 6-fold increase respectively. This indicates that the de novo mediators’ release in LAT or LAT/LAT2 deficient challenged cells was suppressed respectively by 33-46% and 71-75% compared to challenged cells. In contrast, LAT2-deficient challenged cells demonstrated an increase in the release of de novo mediators by approximately 16- to 28-fold in comparison to normal cells, which was 22% to 35% higher than challenged cells.

3.9 tHGA does not inhibit the release of key mediators associated with mast cell degranulation by LAT-deficient IgE-antigen complex-stimulated RBL-2H3 cells In order to confirm that tHGA inhibits mast cell activation by specifically targeting LAT, the ability of tHGA to inhibit the release of key mediators associated with mast cell degranulation in LAT-deficient challenged cells were examined. Consistent with the results from Figure 7, one hour of DNP-BSA challenge significantly up-regulated the release of histamine by 17.9-fold in the challenged cells, 11.2-fold in LAT20

deficient challenged cells, 23-fold in LAT2-deficient challenged cells and 6.2-fold in LAT/LAT2-deficient challenged cells in comparison to the normal cells (Figure 8A). However, pre-treatment with tHGA did not down-regulate the release of histamine, which is an indicator of mast cell degranulation, in both LAT-deficient and LAT/LAT2-deficient challenged cells, even at the concentration of 20 µM. On the other hand, 20 µM of tHGA demonstrated a significant decrease on the release of histamine by 71.20% in LAT2-deficient challenged cells. Figs. 8B to 8E also show that, after 6 hrs of DNP-BSA challenge, the levels of all de novo mediators released by challenged cells, LAT-deficient challenged cells, LAT2deficient challenged

cells and

LAT/LAT2-deficient challenged

cells were

significantly up-regulated by approximately 19- to 28-fold, 11- to 19-fold, 25- to 42fold and 4- to 6-fold, respectively. However, both LAT-deficient as well as LAT/LAT2-deficient challenged cells pre-treated with 20 µM tHGA did not show a significant difference in the release of all de novo mediators. In consistent with the release of histamine, 20 µM of tHGA significantly reduced the release of all de novo mediators by approximately 60% to 67% in LAT2-deficient challenged cells.

4. Discussion tHGA, a geranyl acetophenone and also a phloroglucinol compound, has been previously reported to contain mast cell stabilising properties by preserving the mast cell structure and attenuating the release of pro-inflammatory mediators in the in vitro model of IgE-mediated mast cell degranulation as well as in an animal model of systemic anaphylaxis [31]. Allergic diseases have been considered to be highly associated with mast cell activation and the release of various inflammatory mediators

21

such as histamine, PGD2, LTC4, IL-4 and TNF-α. Over the years, therapeutic approaches in allergic medicine attempt to target mast cell activation as well as the release of mediators associated with mast cell activation [20,41]; for example, ketotifen fumarate, one of the second-generation antihistamine drugs which is widely used to ameliorate many allergic disorders in individuals such as allergic conjunctivitis and chronic rhinitis by suppressing many pro-inflammatory signalling pathways [42,43,44]. Studies reported that ketotifen fumarate inhibits the phosphorylation of various important signalling molecules during mast cell activation such as Syk, MAPKs, PI3K and NFκB [6,25,45,46]. However, the use of such drugs on allergic patients is always associated with unwanted effects such as irritation, headache, runny nose, increased sensitivity to light and even a decrease in drug tolerability [26]. Recent studies and development have been heading towards the discovery of substances isolated from natural sources as the next generation mast cell stabilisers [6]; it is important to understand the mechanism of action (MOA) of these substances before they can be safely entered into clinical trials. Our research group has previously reported that tHGA is able to prevent mast cell degranulation and attenuate the release of preformed mediators such as histamine and the synthesis of de novo pro-inflammatory cytokines such as IL-4 and TNF-α. However, the underlying mechanism by which tHGA exerts its mast cell stabilizing effects is currently unclear. The present study dissected the underlying mechanisms by determining whether tHGA inhibits the mRNA expression of IL-4 and TNF-α. Upon confirmation of that, the two major signalling pathways in IgE-mediated mast cell activation, the LATPLCγ-MAPK and PI3K-NFκB pathways, were studied. Lastly, the possible molecular

22

targets located along the LAT-PLCγ-MAPK or PI3K-NFκB pathways in IgEmediated mast cell degranulation. In line with the ELISA results in our previous study, which demonstrated the inhibitory effect of tHGA on the release of IL-4 and TNF-α in IgE-mediated mast cell degranulation [31], the results from real-time PCR show that tHGA inhibited the mRNA expression of IL-4 and TNF-α. This finding has proven that tHGA inhibits the synthesis of IL-4 and TNF-α in IgE-mediated mast cell degranulation, not only at the cellular level but also at the gene transcriptional level. As the transcriptional process of IL-4 and TNF-α genes is regulated by the transcription factors and signalling molecules along the major signalling pathways in IgE-mediated mast cell activation, such as the LAT-PLCγ-MAPK and PI3K-NFκB pathways [15], we continued the study by examining the effect of tHGA on the signalling molecules involved in LATPLCγ-MAPK and PI3K-NFκB pathways. Previous studies have demonstrated that Ca2+ is highly associated with the LATPLCγ-MAPK pathway in mast cell degranulation [15,16]. An increase of intracellular Ca2+, as a result of massive entry from the extracellular surrounding, is necessary for the activation of various transcription factors which are crucial for the transcription of many cytokine genes including IL-4, TNF-α, and IL-13 [47]. The results show that tHGA was able to attenuate the level of intracellular Ca2+ in IgE-antigen complexstimulated mast cells. This finding also indicates that Ca2+ flux, which is one of the hallmarks in response to FcεRI aggregation, is an unequivocal essential feature of the inhibitory effect of tHGA in IgE-mediated mast cell activation. As LAT and PLCγ are the two signalling molecules which cause the influx of Ca2+ in an activated mast cell [48,49,50], we continued to study the effect of tHGA on LAT and PLCγ by using Western Blot. The results show that 20 µM of tHGA inhibited the phosphorylation of 23

both LAT and PLCγ by as much as 50%, with the inhibitory effect comparable to 300 µM of ketotifen fumarate, which is a clinically used mast cell stabiliser in allergic reactions. This finding once again confirmed that tHGA exerts its mast cell-stabilising effect primarily via the LAT-PLCγ pathway. Interestingly, while tHGA inhibited the activation of most signalling molecules involved in the LAT-PLCγ pathway, including LAT, PLCγ and Ca2+ influx, it did not inhibit the phosphorylation of Syk. Syk is located upstream of LAT and PLCγ and is responsible for activation of the LAT-PLCγ pathway. As Syk is a tyrosine kinase, the effect of tHGA on Syk kinase activity was studied by performing a kinase assay before Syk was ruled out as the possible molecular target of tHGA. This finding is interesting as Syk has been known as the common molecular target for most of the mast cell stabilisers, including ketotifen fumarate. Our results show that ketotifen fumarate significantly inhibited the kinase activity of Syk without inhibiting its phosphorylation, which is consistent with previous studies [46,51]. Activated Syk phosphorylates a variety of substrates including LAT, which orchestrates downstream signalling resulting in degranulation and cytokine gene transcription [6]. However, mast cell stabilisers with Syk as the molecular target could be associated with unwanted side effects as Syk is required for the development and function of various tissues. Syk −/− mice have also been reported to have high rates of perinatal lethality, and exhibit cardiovascular, immune, and hematopoietic defects [36]. After confirming that tHGA did not inhibit both phosphorylation and kinase activity of Syk, we speculate that tHGA may be able to inhibit other signalling cascades that are related to the LAT axis pathway, such as the LAT-MAPK pathway [17,48]. The present study demonstrates that tHGA attenuated the phosphorylation of all MAPK enzymes including ERK1/2, JNK, and p38. This is expected because we have 24

demonstrated earlier that tHGA inhibited LAT activation. Since LAT is known to positively regulate MAPKs activation, tHGA’s inhibitory effect on LAT activation subsequently attenuated MAPK activation. MAPKs have been shown to mediate the activation of several enzymes associated with arachidonic acid, including cPLA2, 5LOX and COX-2 [15,33]. These enzymes are important for the production of key mediators such as LTC4 and PGD2 in IgE-mediated mast cell degranulation [33,52]. As expected, tHGA inhibited the phosphorylation of cPLA2 and downregulated 5LOX and COX-2 protein expression, effects which are likely attributed to the inhibitory effect of tHGA on MAPKs activation. These findings explain the mechanisms underlying tHGA’s inhibitory effect on the release of PGD2, LTC4, IL-4 and TNF-α in IgE-mediated mast cell degranulation as reported in our previous study [31]. Thus far, we have demonstrated that tHGA exerts its mast cell-stabilising effect via the LAT-PLCγ pathway without affecting Syk. The inhibitory effect of LAT-PLCγ pathway was further confirmed by the evidence that tHGA inhibited the phosphorylation of MAPKs, which play vital roles in the LAT axis signalling pathway. However, it is still questionable whether tHGA also exerts its mast cell stabilising effect via another major pathway in IgE-mediated mast cell activation, the PI3K-NFκB pathway. PI3K and NFκB are both important signalling molecules in mast cell biology. Both of these molecules, together with the adapter protein LAT2, are responsible for forming the complementary activation pathway which can also govern the release of mediators in IgE-mediated mast cell degranulation [15]. The results from this complementary pathway show that only 20 µM of tHGA was able to subtly inhibit (~13%) the phosphorylation of IκBα and nuclear translocation of NFκB p65 in IgE-antigen complex-stimulated mast cells. However, tHGA did not show any 25

inhibitory effect on the phosphorylation of IκB kinase (IKK) and PI3K along the PI3K-NFκB pathway. Notably, the inhibitory effects of tHGA on IκB and NFκB were not as strong as those of signalling molecules located in the LAT axis pathway. The partial inhibitory effect of tHGA on the PI3K-NFκB pathway can be explained by several recent studies which reported about the presence of crosstalk between the LAT-MAPK and PI3K-NFκB signalling pathways, specifically in between MAPKs and NFκB p65 [33,53]. This may explain why tHGA inhibited NFκB p65 and IκBα as well as their downstream molecules, but not the signalling molecules located upstream of NFκB p65 and IκBα. Up to this point, our results strongly demonstrate that tHGA primarily exerts its mast cell-stabilising effect via the LAT-PLCγ pathway. Although tHGA also inhibited some of the signalling molecules from the PI3K-NFκB pathway, it could have resulted from the crosstalk between MAPKs and NFκB. Collectively, our findings indicate that it is highly probable that tHGA exerts its mast cell-stabilising effect in IgE-mediated mast cell degranulation by targeting the adapter molecule LAT. In order to confirm that LAT is the molecular target of tHGA in IgE-mediated mast cell degranulation, the RNAi method was performed to establish LAT, LAT2 and LAT/LAT2-deficient RBL-2H3 cells. The rationale for the use of LAT or LAT2 knockdown strategy in this study is, the competition between LAT and LAT2 which eventually lead to the release of mediators in the event of mast cell activation allows the identification of the molecular target of tHGA by examining the release of mediators when LAT or LAT2 gene is silenced. For example, if the molecular target of tHGA is LAT, tHGA pretreatment will not affect the release of mediators when only the LAT gene is silenced. Interestingly, silencing LAT gene will not impede the cells from undergoing mast cell activation and release all the relevant mediators as 26

LAT2 will take over the dominant role of LAT when the expression of LAT is low. On the other hand, if tHGA targets signalling molecules other than LAT, the release of mediators will be inhibited when the challenged cells are pretreated with tHGA. When this happens, it is important to confirm whether tHGA also targets LAT2 as LAT2 is a homolog to LAT. Therefore, in order to determine the molecular target of tHGA in a IgE-mediated mast cell activation model, it is extremely important to establish the LAT/LAT2 competition model. The relationship and interaction between LAT and LAT2 has been suggested by Orr and McVicar (2010) [54], by using RNA interference (RNAi) method in primary cells such as bone marrow mast cells and human mast cells [55,56,57]. However, this is the first time that LAT/LAT2 competition model was demonstrated in RBL-2H3 cells. LAT and LAT2 are both important adaptor molecules that can be found on the lipid raft of mast cells [58]. These adaptor molecules are substrates of the tyrosine kinase Syk following their engagement by an activated FcεRI receptor. The main function of LAT and LAT2 is to serve as a scaffold protein for the recruitment of other signalling molecules such as PI3K and PLCγ in order to propagate down the activation signal [58,59]. Previous studies have demonstrated that antigen-mediated degranulation, Ca2+ mobilisation, cytokine production and the activation of signalling molecules such as MAPKs and PLCγ were all inhibited partially in LAT-/- mast cells, further decreased in LAT-/-/LAT2 -/- mast cells, and surprisingly, increased in LAT2 -/- mast cells [55,56,57]. Therefore, the effects of tHGA on the release of mediators from LAT, LAT2 and LAT/LAT2-deficient RBL-2H3 cells will be utilised in this study to determine whether LAT is the specific molecular target of tHGA in IgE-antigen complex-stimulated RBL-2H3 cells.

27

Consistent with previous studies [56,57], similar trend was observed in challenged cells following knockdown of either LAT or LAT2 alone or both, whereby the release of mediators was partially decreased in LAT-deficient challenged cells, further decreased in LAT/LAT2-deficient challenged cells and finally increased in LAT2deficient challenged cells. According to the LAT/LAT2 competitive model proposed by Orr and McVicar (2010), the competition between LAT and LAT2 in the localisation to lipid rafts and the availability of binding proteins may account for such observations [54]. Therefore, the LAT2, through its complementary PI3K-NFκB signalling cascade, acts as a negative regulator of LAT signalling in wild type (WT) mast cells but may also deliver weaker degranulation signals when expressed in LATdeficient mast cells [56,57,60] (Fig. 9). Once the validity of the LAT/LAT2 competitive model was demonstrated in IgEantigen complex-stimulated RBL-2H3 cells, the LAT-deficient challenged RBL-2H3 cells were then pre-treated with tHGA to observe the release of key mediators in IgEmediated mast cell activation. It is hypothesised that there will not be significant decrease in the release of mediators by the challenged cells in the deficiency of LAT if tHGA specifically targets LAT without interfering with LAT2 or other signalling molecules associated with mast cell activation. Interestingly, the results show that the release of key mediators such as IL-4, TNF-α, PGD2 and LTC4 were not affected by tHGA in LAT-deficient challenged cells. Several studies have suggested that, when LAT is being lowly expressed in a mast cell, LAT2 will take over the dominant role of LAT in regulating the release of mediators in the event of mast cell activation [54,55,56]. This may explain the inability of tHGA pre-treatment to decrease the release of mediators in LAT-deficient challenged cells despite the fact that there were approximately 30% of LAT remaining in the cells. 28

Such results confirmed that tHGA indeed exerts its mast cell stabilising effect via the LAT-PLCγ pathway, with LAT as the possible molecular target. Up to this point, it is important to address the following two questions: (i) does tHGA inhibit the expression of LAT? or (ii) does tHGA influence the adaptor function of LAT? We excluded the possibilities that tHGA could inhibit the expression of LAT or LAT2, as demonstrated by our results (data not shown). Thus, we strongly believe that tHGA exerts it mast cell stabilising effect by affecting the adaptor function of LAT. LAT is an adaptor protein which plays a critical role in FcεRI-mediated signalling in mast cells by acting as a signalling hub for the binding of various important downstream messenger proteins such as PLCγ [61,62]. It is possible that tHGA may interfere with the phosphorylation site of the LAT residues, resulting in the destabilisation and deformation of the LAT-based signalling complexes that causes the attenuation of mast cell activation [61]. However, due to difficulty in crystallising the membrane proteins, the complete crystallised structure of LAT is still unknown [60]. Therefore, it remains a question on how tHGA acts upon LAT to cause a loss of function. More in-depth studies should be carried out before any decisive conclusion can be drawn. At this current stage, it can only be concluded that LAT is important in the mast cell stabilising effect of tHGA. To the best of our knowledge, there is currently no compound or small molecule that attenuates IgE-mediated mast cell degranulation through the specific inhibition of LAT. Many of the pharmacological studies reported compounds which target other signalling molecules such as Syk and PI3K [63,64,65], or soluble mediators such as histamine, lipid mediators, cytokines and chemokines [66,67]. However, as discussed earlier, there are some drawbacks and side effects associated with these compounds. Apart from that, wide range selectivity of current drug treatment in allergy increases 29

potential side effects and adverse drug reactions [66]. In comparison to Syk which is required for the development and function of various tissues, LAT is central to FcεRImediated signalling and effector function in mast cells, while not having any obvious role in mast cell development [68]. This suggests that inhibiting IgE-mediated mast cell degranulation through LAT might be one of the potential options for drug treatment in the future. However, whether tHGA might target other cell types such as basophils or T cells is still a mystery and should be addressed in the future. In summary, the present study demonstrated that tHGA attenuated IgE-antigen complex-stimulated RBL-2H3 degranulation primarily via the LAT-PLCγ pathway. The inhibition of LAT enables tHGA to partially inhibit the PI3K-NFκB pathway via the crosstalk between MAPK and NFκB, resulting in an overall attenuation of the IgE-mediated mast cell activation. This study also validated the LAT/LAT2 competitive model in IgE-antigen complex-stimulated RBL-2H3 cells via the establishment of LAT, LAT2 and LAT/LAT2-deficient RBL-2H3 cells. The LAT/LAT2 competitive model successfully proved that the possible molecular target of tHGA in IgE-mediated mast cell degranulation could be LAT. However, more studies should be conducted in the future to fully understand the interaction between tHGA and LAT in attenuating IgE-mediated mast cell degranulation.

5. Acknowledgements This study was supported by Exploratory Research Grant Scheme (ERGS) 2013 from Ministry of Higher Education of Malaysia (No. 5527166) and Putra Grant - Putra Graduate Initiative 2017 from Universiti Putra Malaysia (No. 9512400). Ji Wei Tan is a recipient of MyPhD scholarship under MyBrain15 programme.

30

6. Conflict of Interest The authors declare that there are no conflicts of interest.

7. References [1] Jutel M, Akdis CA. Immunological mechanisms of allergen-specific immunotherapy. Allergy 2011;66:725–732. [2] Kaplan DH, Igýartó BZ, Gaspari AA. Early immune events in the induction of allergic contact dermatitis. Nat Rev Immunol 2012;12:114–124. [3] Galli SJ, Tsai M, Piliponsky AM. The development of allergic inflammation. Nature 2008;454:445–454. [4] Schroeder JT. Basophils: emerging roles in the pathogenesis of allergic disease. Immunol Rev 2011;242:144–160. [5] Lu, Y., Li, Y., Jin, M., Yang, J.H., Li, X., Chao, G.H., et al. Inula japonica extract inhibits mast cell-mediated allergic reaction and mast cell activation. J Ethnopharmacol 2012;143:151-157. [6] Finn DF, Walsh JJ. Twenty-first century mast cell stabilizers. Br J Pharmacol 2013;170:23-37. [7] Passante E, Frankish N. The RBL-2H3 cell line: its provenance and suitability as a model for the mast cell. Inflamm Res 2009;58:737-745. [8] Moriya K, Rivera J, Odom S, Sakuma Y, Muramato K, Yoshiuchi T, et al. ER27319, an acridine-related compound, inhibits release of antigen-induced allergic 31

mediators from mast cells by selective inhibition of Fcε receptor I-mediated activation of Syk. Proc Natl Acad Sci USA 1997;94:12539-44. [9] Choi OH, Adelstein RS, Beaven MA. Secretion from rat basophilic RBL-2H3 cells is associated with diphosphorylation of myosin light chains by myosin light chain kinase as well as phosphorylation by protein kinase C. J Biol Chem 1994;269:536-541. [10] Ng NM, Jiang SP, Zhang W. 2-Aminoethoxydiphenyl borate reduces degranulation and release of cytokines in a rat mast cell line. Eur Rev Med Pharmacol Sci 2012;16:1017-21. [11] Tang JM, Liu J, Wu W. Studies on the Degranulation of RBL-2H3 Cells Induced by Traditional Chinese Medicine Injections. Chin Med 2012;3:200-208. [12] Barsumian EL, Isersky MG, Petrino MG, Siraganian RP. IgE-induced histamine release from rat basophilic leukemia cell lines: isolation of releasing and nonreleasing clones. Eur J Immunol 1987;11:317-322. [13] Aung G, Niyonsaba F, Ushio H, Kajiwara N, Saito H, Ikeda S, et al. Catestatin, a neuroendocrine antimicrobial peptide, induces human mast cell migration, degranulation and production of cytokines and chemokines. Immunology 2011;13:527–539. [14] Metcalfe DD, Peavy RD, Gilfillan AM. Mechanisms of mast cell signalling in anaphylaxis. J Allergy Clin Immunol 2009;124:639-646. [15] Gilfillan AM, Tkaczyk C. Integrated signalling pathways for mast-cell activation. Nat Rev Immunol 2006;6:218-230.

32

[16] Sibilano R, Frossi B, Pucillo CE. Mast cell activation: a complex interplay of positive and negative signalling pathways. Eur J Immunol 2014;44:2558-66. [17] Rivera J, Gilfillan AM. Molecular regulation of mast cell activation. J Allergy Clin Immunol 2006;117:1214–25. [18] Gilfillan AM, Beaven MA. Regulation of mast cell responses in health and disease. Crit Rev Immunol 2011;31:475-529. [19] Kim MS, Rådinger M, Gilfillan AM. The multiple roles of phosphoinositide 3kinase in mast cell biology. Trends Immunol 2008;29:493-501. [20] Galli SJ, Tsai M. IgE and mast cells in allergic disease. Nat Med 2012;18:693704. [21] Beaven MA, Rogers J, Moore JP, Hesketh TR, Smith GA, Metcalfe JC. The mechanism of the calcium signal and correlation with histamine release in 2H3 cells. J Biol Chem 1984;259:7129-36. [22] Amin K. The role of mast cells in allergic inflammation. Respir Med 2012;106:9–14. [23] Zhang T, Finn DF, Barlow JW, Walsh JJ. Mast cell stabilisers. Eur J Pharmacol 2016;778:158-168. [24] Weng Z, Zhang B, Asadi S, Sismanopoulos N, Butcher A, Fu X, et al. Quercetin is more effective than cromolyn in blocking human mast cell cytokine release and inhibits contact dermatitis and photosensitivity in humans. PLoS One 2012;7:e33805.

33

[25] Zhang A, Chi X, Luo G, Hei Z, Xia H, Luo, C, et al. Mast cell stabilization alleviates acute lung injury after orthotopic autologous liver transplantation in rats by downregulating inflammation. PLoS One. 2013;8:e75262. [26] Oppenheimer JJ, Casale TB. Next generation antihistamines: therapeutic rationale, accomplishments and advances. Expert Opin Investig Drugs 2002;11:807–817. [27] Shaari K, Suppaiah V, Wai LK, Stanslas J, Tejo BA, Israf DA, et al. Bioassayguided identification of an anti-inflammatory prenylated acylphloroglucinol from Melicope ptelefolia and molecular insights into its interaction with 5lipoxygenase. Bioorg. Med. Chem. 2011;19:6340-7. [28] Shaari K, Safri S, Abas F, Lajis NH, Israf DA. A geranylacetophenone from the leaves of Melicope ptelefolia. Nat Prod Lett 2006;20:415–419. [29] Lee YZ, Shaari K, Cheema MS, Tham CL, Sulaiman MR, Israf DA. An orally active geranyl acetophenone attenuates airway remodeling in a murine model of chronic asthma. Eur J Pharmacol 2017;797:53-64. [30] Ismail N, Jambari NN, Zareen S, Akhtar MN, Shaari K, Zamri-Saad M, et al. A geranyl acetophenone targeting cysteinyl leukotriene synthesis prevents allergic airway inflammation in ovalbumin-sensitized mice. Toxicol Appl Pharmacol 2012;259:257-262. [31] Tan JW, Israf DA, Harith HH, Md Hashim NF, Ng CH, Shaari K, et al. Antiallergic

activity

of

2,4,6-trihydroxy-3-geranylacetophenone

(tHGA)

via

attenuation of IgE-mediated mast cell activation and inhibition of passive systemic anaphylaxis. Toxicol Appl Pharmacol 2017;319:47-58.

34

[32] Huang F, Yamaki K, Tong X, Fu L, Zhang R, Cai Y, et al. Inhibition of the antigen-induced

activation

of

RBL-2H3

cells

by

sinomenine.

Int

Immunopharmacol 2008;8:502–507. [33] Lian Q, Cheng Y, Zhong C, Wang F. Inhibition of the IgE-mediated activation of RBL-2H3 cells by TIPP, a novel thymic immunosuppressive pentapeptide. Int J Mol Sci. 2015;16:2252-68. [34] Kwon JH, Lee JH, Kim, KS, Chung YW, Kim IY. Regulation of cytosolic phospholipase A2 phosphorylation by proteolytic cleavage of annexin A1 in activated mast cells. J Immunol 2012;188:5665-73. [35] Valim CX, da Silva EZ, Assis MA, Fernandes FF, Coelho PS, Oliver C, et al. rPbPga1

from Paracoccidioides brasiliensis Activates Mast Cells and

Macrophages via NFkB. PLoS Negl Trop Dis 2015;9:e0004032. [36] Siraganian RP, de Castro RO, Barbu EA, Zhang J. Mast cell signalling: the role of protein tyrosine kinase Syk, its activation and screening methods for new pathway participants. FEBS Lett 2010;584:4933-40. [37] Denyer J, Patel V. Syk kinase inhibitors in allergic diseases. Drug News Perspect 2009;22:146-150. [38] Han EH, Hwang YP, Kim HG, Choi JH, Im JH, Yang JH, et al. Inhibitory effect of Pleurotus eryngii extracts on the activities of allergic mediators in antigenstimulated mast cells. Food Chem Toxicol 2011;49:1416-25. [39] Wang W, Zhou Q, Liu L, Zou, K. Anti-allergic activity of emodin on IgEmediated activation in RBL-2H3 cells. Pharmacol Rep. 2012;64:1216-22.

35

[40] Theoharides TC, Kalogeromitros D. The critical role of mast cells in allergy and inflammation. Ann N Y Acad Sci 2006;1088:78-99. [41] Holgate ST. Pathophysiology of asthma: what has our current understanding taught us about new therapeutic approaches? J Allergy Clin Immunol 2011;128:495–505. [42] Barnes PJ. Glucocorticosteroids: current and future directions. Br J Pharmacol 2011;163:29–43. [43] Kato A, Chustz RT, Ogasawara T, Kulka M., Saito H, Schleimer RP, et al. Dexamethasone and FK506 inhibit expression of distinct subsets of chemokines in human mast cells. J Immunol 2009;182:7233–43. [44] Matsuda K, Piliponsky AM, Iikura M, Nakae S, Wang EW, Dutta SM, et al. Monomeric IgE enhances human mast cell chemokine production: IL-4 augments and dexamethasone suppresses the response. J Allergy Clin Immunol 2005;116:1357–63. [45] Sinniah A, Yazid S, Perretti M, Solito E, Flower RJ. The role of the AnnexinA1/FPR2 system in the regulation of mast cell degranulation provoked by compound

48/80

and

in

the

inhibitory

action

of

nedocromil.

Int

Immunopharmacol 2016;32:87-95. [46] Hisamichi H, Naito R, Toyoshima A, Kawano N, Ichikawa A, Orita A, et al. Synthetic studies on novel Syk inhibitors. Part 1: Synthesis and structure-activity relationships of pyrimidine-5-carboxamide derivatives. Bioorg Med Chem Lett 2005;13:4936-51.

36

[47] Baba Y, Nishida K, Fujii Y, Hirano T, Hikida M, Kurosaki T. Essential function for the calcium sensor STIM1 in mast cell activation and anaphylactic responses. Nat Immunol 2008;9:81–88. [48] Blank U, Madera-Salcedo IK, Danelli L, Claver J, Tiwari N, Sánchez-Miranda E, et al. Vesicular trafficking and signaling for cytokine and chemokine secretion in mast cells. Front Immunol 2014;22:5:453. [49] Wernersson S, Pejler G. Mast cell secretory granules: armed for battle. Nat Rev Immunol 2014;14:478-494. [50] Han SY, Bae JY, Park SH, Kim YH, Park JH, Kang YH. Resveratrol Inhibits IgE-Mediated Basophilic Mast Cell Degranulation and Passive Cutaneous Anaphylaxis in Mice. J Nutr 2013;143:632-639. [51] Singh R, Masuda ES. Chapter 24 Spleen Tyrosine Kinase (Syk) Biology, Inhibitors and Therapeutic Applications. Annu. Rep. Med Chem 2007;42:379391. [52] Huang L, Li T, Zhou H, Qiu P, Wu J, Liu L. Sinomenine potentiates degranulation of RBL-2H3 basophils via up-regulation of phospholipase A2 phosphorylation by Annexin A1 cleavage and ERK phosphorylation without influencing on calcium mobilization. Int Immunopharmacol 2015;28:945-951. [53] Matsui T, Ito C, Furukawa H, Okada T, Itoigawa M. Lansiumamide B and SB204900 isolated from Clausena lansium inhibit histamine and TNF-a release from RBL-2H3 cells. Inflamm Res 2013;62:333-341. [54] Orr SJ, McVicar DW. LAB/NTAL/Lat2: a force to be reckoned with in all leukocytes? J. Leukoc Biol 2011;89:11-19.

37

[55] Tkaczyk C, Horejsi V, Iwaki S, Draber P, Samelson LE, Satterthwaite AB, et al. NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and FcεRI aggregation. Blood 2004;104:207–214. [56] Volna P, Lebduska P, Draberova L, Simova S, Heneberg P, Boubelik M, et al. Negative regulation of mast cell signaling and function by the adaptor LAB/NTAL. J Exp Med 2004;200:1001–13. [57] Zhu M, Liu Y, Koonpaew S, Granillo O, Zhang W. Positive and negative regulation of FcεRI-mediated signalling by the adaptor protein LAB/NTAL. J Exp Med 2004;200:991–1000. [58] Fuller DM, Zhu M, Ou-Yang C, Sullivan SA, Zhang W. A Tale of Two TRAPs: LAT and LAB in the Regulation of Lymphocyte Development, Activation, and Autoimmunity. Immunologic research 2011;49:97-108. [59] Rivera J. NTAL/LAB and LAT: a balancing act in mast-cell activation and function. Trends Immunol 2005;26:119-122. [60] Moran LA, Horton RA, Scrimgeour G, Perry M. 2012. Principles of Biochemistry. 5th ed. New York: Pearson Education. [61] Balagopalan L, Kortum RL, Coussens NP, Barr VA, Samelson LE. The linker for activation of T cells (LAT) signalling hub: from signalling complexes to microclusters. J Biol Chem 2015;290:26422-29. [62] Balagopalan L, Coussens NP, Sherman E, Samelson LE, Sommers CL. The LAT story: a tale of cooperativity, coordination, and choreography. Cold Spring Harb Perspect Biol. 2010;2:a005512.

38

[63] Braselmann S, Taylor V, Zhao H, Wang S, Sylvain C, Baluom M, et al. R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation. J Pharmacol Exp Ther 2006;319:998-1008. [64] Norman P. Selective PI3Kd inhibitors, a review of the patent literature. Expert Opin Ther Pat 2011;21:1773-90. [65] Simmons DL. Targeting kinases: a new approach to treating inflammatory rheumatic diseases. Curr Opin Pharmacol 2013;13:426-434. [66] Harvima IT, Levi-Schaffer F, Draber P, Friedman S, Polakovicova I, Gibbs BF, et al. Molecular targets on mast cells and basophils for novel therapies. J. Allergy Clin. Immunol. 2014;134:530-544. [67] Krause K, Spohr A, Zuberbier T, Church MK, Maurer M. Up-dosing with bilastine results in improved effectiveness in cold contact urticaria. Allergy 2013;68:921-928. [68] Saitoh S, Arudchandran R, Manetz TS, Zhang W, Sommers CL, Love PE, et al. LAT Is Essential for FcεRI-Mediated Mast Cell Activation. Immunity 2000;12:525–535.

Figure legends Fig.1. The effects of tHGA on the levels of (A) IL-4 mRNA, (B) TNF-α mRNA and (C) intracellular Ca 2+ in IgE-antigen complex-stimulated RBL-2H3 cells. IgE-sensitised RBL-2H3 cells were pre-treated with tHGA (1.25, 5 and 20 µM) for 20 min, followed by DNP-BSA challenge (1 µg/mL) for 6 hrs. The mRNA levels of IL-4 39

and TNF-α were determined by performing qPCR whereas the intracellular Ca2+ level was measured by using a fluorometric kit according to the manufacturer’s instructions. Results are expressed as the mean ± S.E.M values of three independent experiments. ***P < 0.005 and **P < 0.01 versus IgE-antigen complex-stimulated RBL-2H3 group (black coloured bar).

Fig.2. The effects of tHGA on the protein expression of (A) p-PLCγ and (B) pLAT in IgE-antigen complex-stimulated RBL-2H3 cells. IgE-sensitised RBL-2H3 cells were pre-treated with tHGA (1.25, 5 and 20 µM) for 20 min, followed by DNPBSA challenge (1 µg/mL) for 30 min. Whole cell protein extracts were assayed by using Western Blot to determine the expression level of the proteins of interest. The phosphorylated forms of PLCγ1 and LAT were normalised with their respective native proteins in the experiment. β-actin protein (1:5000) was used as an internal control for the experiment. Results are expressed as the mean ± S.E.M values of three independent experiments. ***P < 0.005 and **P < 0.01 versus IgE-antigen complexstimulated RBL-2H3 group (black coloured bar).

Fig.3. The effects of tHGA on the expression of (A) 5-LO, (B) COX-2, and phosphorylation of (C) cPLA2, (D) ERK1/2, (E) p38 and (F) JNK in IgE-antigen complex-stimulated RBL-2H3 cells. IgE-sensitised RBL-2H3 cells were pre-treated with tHGA (1.25, 5 and 20 µM) for 20 min, followed by DNP-BSA challenge (1 µg/mL) for 30 min. Whole cell protein extracts were assayed by using Western Blot to determine the expression level of the proteins of interest. The phosphorylated forms of ERK1/2, p38, JNK, and cPLA2 were normalised with their respective native

40

proteins in the experiment whereas 5-LOX and COX-2 were normalised with β-actin. β-actin protein (1:5000) was used as an internal control for the experiment. Results are expressed as the mean ± S.E.M values of three independent experiments. ***P < 0.005 and *P < 0.05 versus IgE-antigen complex-stimulated RBL-2H3 group (black coloured bar).

Fig.4. The effects of tHGA on the expression levels of (A) p-IκB, (B) IκB, (C) nuclear p65, (D) cytosolic p65, (E) IKK α/β, and (F) PI3K in IgE-antigen complex-stimulated RBL-2H3 cells. IgE-sensitised RBL-2H3 cells were pre-treated with tHGA (1.25, 5 and 20 µM) for 20 min, and followed by DNP-BSA challenge (1 µg/mL) for 30 min. Whole cell protein extracts were assayed by using Western Blot to determine the expression levels of p-IκB, IκB, IKK α/β, and PI3K. Nuclear and cytosolic fraction were used to determine the levels of nuclear p65 and cytosolic p65 respectively. The phosphorylated forms IKK α/β were normalised with its native proteins in the experiment. The internal control used for p-IκB, IκB, cytosolic p65 and PI3K was β-actin whereas for nuclear p65 was TFIIB. Results are expressed as the mean ± S.E.M values of three independent experiments. ***P < 0.005, **P < 0.01 and *P < 0.05 versus IgE-antigen complex-stimulated RBL-2H3 group (black coloured bar).

Fig.5. The effects of tHGA on the (A) phosphorylation and (B) kinase activity of Syk in IgE-antigen complex-stimulated RBL-2H3 cells. IgE-sensitised RBL-2H3 cells were pre-treated with tHGA (1.25, 5 and 20 µM) for 20 min, followed by DNPBSA challenge (1 µg/mL) for 30 min. Whole cell protein extracts were assayed by

41

using Western Blot to determine the phosphorylation level of Syk. The phosphorylated form of Syk was normalised with its respective native protein in the experiment. β-actin (1:5000) was used as an internal control for the experiment. Syk kinase activity assay was determined using the ADP-Glo™ Syk kinase enzyme luminescence kit according to the manufacturer’s instructions. Results are expressed as the mean ± S.E.M values of three independent experiments. ***P < 0.005 and *P < 0.05 versus IgE-antigen complex-stimulated RBL-2H3 group (black coloured bar).

Fig. 6. Optimal transfection conditions for gene silencing experimental of (A) LAT, (B) LAT2 or (C) LAT/LAT2 in RBL-2H3 cells. RBL-2H3 cells were transfected with siRNAs specific for LAT, LAT2 or both for (i) 24 hr or (ii) 48 hrs incubation. Whole cell protein extracts were assayed by using Western Blot to determine the expression level of the proteins of interest. β-actin protein (1:5000) was used as an internal control for the experiment. Results are expressed as the mean ± S.E.M values of three independent experiments. ***P < 0.005 versus normal cell group (black coloured bar).

Fig. 7. The effects of gene silencing on the levels of (A) histamine, (B) PGD2, (C) LTC4, (D) IL-4 and (E) TNF-α released by LAT, LAT 2 and LAT/LAT2deficient challenged RBL-2H3 cells. RBL-2H3 cells were treated with siRNAs for GAPDH, scramble, LAT, LAT2 or both. After 24 hrs of incubation, the siRNAs treated cells and the normal cells were sensitized with 1 µg/mL of DNP-IgE for another 24 hrs, followed by DNP-BSA challenge (1 µg/mL) for 1 or 6 hrs. The levels of histamine, PGD2, LTC4, IL-4 and TNF-α released were determined using EIA and

42

ELISA kits according to the manufacturer’s instructions. (1) Positive control, (2) Negative control, (3) Mock control, (4) Normal cells, (5) Challenged cells, (6) LATdeficient challenged cells, (7) LAT2-deficient challenged cells, (8) LAT/LAT2deficient challenged cells. Results are expressed as the mean ± S.E.M values of three independent experiments. ***P < 0.005, **P < 0.01 and *P < 0.05 versus mock control group (black coloured bar). ###P < 0.005 versus LAT-deficient challenged cells group.

Fig. 8. The effects of tHGA on the levels of (A) histamine, (B) PGD2, (C) LTC4, (D) IL-4 and (E) TNF-α released by LAT-deficient or LAT/LAT2-deficient challenged RBL-2H3 cells. LAT siRNA or LAT/LAT2 siRNAs treated RBL-2H3 cells were pre-treated with 20 µM tHGA for 20 min, followed by DNP-BSA challenge (1 µg/mL) for 1 or 6 hrs. The levels of histamine, PGD2, LTC4, IL-4 and TNF-α were determined using EIA and ELISA kits according to the manufacturer’s instructions. (1) Positive control, (2) Negative control, (3) Mock control, (4) Normal cells, (5) Challenged cells, (6) LAT-deficient challenged cells, (7) LAT-deficient challenged cells pre-treated with tHGA, (8) LAT2-deficient challenged cells, (9) LAT2-deficient challenged cells pre-treated with tHGA, (10) LAT/LAT2-deficient challenged cells, (11) LAT/LAT2-deficient challenged cells pre-treated with tHGA. Results are expressed as the mean ± S.E.M values of three independent experiments. ***P < 0.005 and **P < 0.01 versus mock control group (black coloured bar), 0.005 versus LAT2-deficient challenged cells group

43

###

P<

Fig. 9. The LAT/LAT2 competition model. (A) In IgE-mediated mast cell degranulation, aggregation of FcεRI receptor will result in the mediators’ release mainly through activation of LAT in normal mast cells as LAT2 can only activate mast cell weakly. (B) LAT2 deficient mast cells display an enhanced mediators’ release as a result of increased accumulation of highly active LAT in lipid rafts in the absence of the relatively poorly activating LAT2. (C) In the absence of LAT, LAT2 can also cause mast cell activation, but with a more relatively lower in mediators’ release. (D) In the absence of both adaptors, mast cell activation is largely ablated.

Fig. 10. Possible inhibitory mechanisms of tHGA on FcεRI-induced activation of mast cells. Engagement of FcεRI with cognate antigen triggers the activation of Syk, a receptor-proximal tyrosine kinase. Activated Syk phosphorylates LAT, resulting in the formation of a macromolecular signalling complex that allows the diversification of downstream signalling, such as PLCγ and MAPK proteins. Activated PLCγ is essential for Ca2+ influx responses, which are pivotal for the release of histamine from activated mast cells. The MAPKs-mediated pathway is crucial for optimal activation of cPLA2 and 5-LO, leading to eicosanoid generation such as PGD2 and LTC4. Activated Syk also regulates the PI3K pathway through the adaptor protein LAT2, which plays a key role in NFκB-mediated expression of COX-2 and mRNA generation for the pro-inflammatory cytokines.

44

45

46

47

48

49

50

52

53

54

55

LAT/LAT2 competition model in RBL-2H3 cells demonstrated that tHGA exerts mast cell stabilising activity via specific interaction with LAT without interfering with LAT2 and upstream signalling molecule Syk

56