Mast cells Dectin-1 in innate anti-fungal immunity

Dectin-1 recognizes β-glucan and plays important roles for the anti-fungal immunity through the activation of Syk in dendritic cells (DCs) or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1-specific agonist Curdlan induced tyrosine phosphorylation of cellular proteins and the interaction of Dectin-1 with the Src homology 2 (SH2) domain of Syk. These responses depended on tyrosine phosphorylation of hemi-immunoreceptor tyrosine-based activation motif (hemITAM) in cytoplasmic tail of Dectin-1, whereas those were independent of γ-subunit of high-affinity IgE receptor (FcεRIγ). DNA microarray and real-time PCR analyses showed that Dectin-1-mediated signaling stimulated gene expression of transcription factor Nfkbiz and inflammatory cytokines, such as monocyte chemoattractant protein-1 (MCP-1), IL-3, IL-4, IL-13 and tumor necrosis factor (TNF)-α. The response was abrogated by pretreatment with Syk inhibitor R406. These results suggest that Syk is critical for Dectin-1-mediated activation of mast cells, although the signaling differs from that triggered by FcεRI activation. In addition, these gene expressions induced by Curdlan stimulation were specifically observed in mast cells, suggesting that Dectin-1-mediated signaling of mast cells offers new insight into the anti-fungal immunity. by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Mast cells Dectin-1 in innate anti-fungal immunity 3 INTRODUCTION Fungal infections are major health threat and clinical problems due to increasing numbers of immuno-compromised host because of the increase in immunosuppressive diseases such as AIDS and immunosuppressive therapies against chronic inflammatory diseases, autoimmune diseases, cancers and transplant rejections. Anti-fungal immunity, thus grows increasingly important, is initiated by recognition of fungal pathogens with innate immune receptors. Pattern recognition receptors, such as Toll-like receptors 5 (TLRs) and C-type lectin receptors (CLRs), play important roles in the innate anti-microbial immunity by recognition of pathogen-associated molecular patterns (PAMPs), including carbohydrates, lipids, nucleic acids, and proteins. β-Glucan, a major carbohydrate component of fungal cell wall along with mannans and chitin, are known as a PAMP recognized by Dectin-1. Dectin-1 is a type II transmembrane receptor which recognizes β-glucan. Dectin-1 was first identified by Ariizumi, et al. and originally thought to be a DC-specific receptor, from which its name ‘dendritic-cell-associated C-type lectin-1’ was derived (1). However the receptor is now known to be expressed by many other cell types, including macrophages, monocytes, neutrophils and T cells (2, 3). Especially in DCs and macrophages, Dectin-1-mediated mechanisms of anti-fungal immunity have been studied. The following shows the facts brought out by prior studies in DCs and macrophages. Dectin-1 is composed of four domains, carbohydrate recognition domain (CRD), stalk domain, transmembrane domain, and cytoplasmic domain which possesses hemITAM. Alternative splicing produces two major isoforms, which vary by the inclusion, or exclusion, of the stalk region in rat (4), mouse (5) and human (6) (although Dectin-1 in human has eight splicing variants in total). The CRD of Dectin-1 specifically recognizes soluble and particle β(1→3)and β(1→6)-linked glucan (2). In contrast to classic Ca 2+ -dependent CLRs, CRD of Dectin-1 can recognize carbohydrate by Ca 2+ -independent manner (2). Dectin-1 also recognizes impure particulate β-glucan zymosan, a stimulatory cell-wall extract of Saccharomyces cerevisiae that is composed mainly of β-glucan but also mannan, chitin, protein and lipid (7). A large number of receptors have been implicated in the recognition of zymosan, including mannose receptor (8), complement receptor 3 (9, 10), Dectin-1 (2) and TLR2 (11). Thereby, analysis using zymosan does not reflect the independent molecular mechanisms of Dectin-1, whereas zymosan acts as an ideal model of a complex microorganism displaying several PAMPs. Curdlan consists of purified β(1→3)-glucan polymer from Alcaligenes faecalis, therefore Curdlan was utilized as a specific agonist of Dectin-1 (12), in order to investigate independent molecular mechanism of Dectin-1 in this study. Upon ligand binding, hemITAM of Dectin-1 is phosphorylated by Src family by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Mast cells Dectin-1 in innate anti-fungal immunity 4 protein-tyrosine kinases and recruits spleen tyrosine kinase (Syk) (10), which initiates a signaling cascade leading to nuclear factor-κB (NF-κB) (13, 14), nuclear factor of activated T-cells (NFAT) (15, 16), and MAPK activation (17-19). Traditional ITAM sequences, found in such as Fc receptors, consist of a tandem repeat of YXXI/L sequences (where X is any amino acid) which, on ligand binding and receptor clustering, become tyrosine-phosphorylated by Src kinases. In contrast to this, Dectin-1 has a single ITAM motif termed as hemITAM and phosphorylation of this hemITAM sequence is sufficient to mediate the interaction with Syk (which normally requires two phospho-tyrosines for binding) through an unknown mechanism (10, 20, 21). Syk kinase has two SH2 domains in tandem, which bind to specific phosphorylated tyrosine residues in protein and result in the assembly of signaling complexes (22). In the previous study used recombinant N-terminal (Syk-SH2(N)), C-terminal (Syk-SH2(C)), and tandem SH2 (Syk-SH2(NC)) domains of Syk to precipitate C-type lectin-like receptor 2 (CLEC2) both SH2 domains of Syk are required for binding and signaling downstream of CLEC2. This suggests that the mechanism of the binding of Syk to Dectin-1 is similar to CLEC2, because CLEC2 is a member of CLRs and has hemITAM as same as Dectin-1 (23). Through above, Dectin-1 activates a number of cellular responses, including phagocytosis (21) and reactive oxygen species (ROS) production (21, 24) and the production of various cytokines (IL-1, IL-2, IL-6, IL-10, IL-12, IL-22, TNF-α) and chemokines (CCL17, CCL22) (25), leading to anti-fungal immunity. Mast cells are now known to be critical effectors of not only allergic disease but also host defense (26, 27). Recently, it has been reported that Dectin-1 is expressed in mouse and human mast cells and its activation elicits leukotriene release, ROS production and Dectin-1 expression, indicating the relationship between mast cells and anti-fungal immunity (24, 28, 29). However, the signaling pathway and physiological roles of Dectin-1 in mast cells are still largely unknown. The purpose of this study is to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells in order to analyze how Dectin-1 in mast cells contributes to innate anti-fungal immunity. by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Mast cells Dectin-1 in innate anti-fungal immunity 5 EXPERIMENTAL PROCEDURES Antibodies and Materials―Anti-phosphotyrosine (pY) (clone 4G10) and anti-GAPDH mAbs, and anti-phosphotyrosine (pY) (clone 4G10) agarose conjugate were purchased from Millipore (Bedford, MA, USA). Anti-dinitrophenyl (DNP) IgE mAb (clone SPE-7) was obtained from Sigma (St. Louis, MO, USA). Anti-mouse Dectin-1/CLEC7A polyclonal antibody was from R&D systems (Minneapolis, MN, USA). anti-phosphoERK, Anti-ERK polyclonal antibodies were from Cell signaling Technology (Danvers, MA, USA). Syk polyclonal antibody was raised against rat Syk-specific peptide (EPTGGAWGPDRGLC), as previously described (30). Anti-phospholipase C (PLC)γ2 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein A-agarose was from Sigma. Curdlan was from Wako (Osaka, Japan). Antigen DNP-BSA (30 mol of DNP/1 mol of BSA) was from LSL (Tokyo, Japan). Syk inhibitor R406 was from Selleck chemicals (Houston, TX, USA). BAY61-3606, PD98059, BAY11-7082, Cyclosporin A were from Wako. Curdlan was prepared as previously described (12). Curdlan is insoluble at neutral pH, thereby it was once solubilized with 0.15M NaOH solution at 10 mg/ml and then added to the culture medium to neutralize pH by diluting more than 100-fold. cDNAs―The mouse Dectin-1A cDNA was obtained as follows. Total RNA from RAW 264.7 cells was isolated by using RNeasy mini kit (Qiagen, Valencia, CA, USA) and the first-strand cDNA was generated by using superscript III (Life technologies, Carlsbad, CA, USA) with oligo(dT) primers, according to the manufacturer’s instructions. The mouse Dectin-1A cDNA was subsequently amplified by PCR using following primers, 5’-CAAGTGCTCTGCCTACCTAGGGCCCTGT3’ (forward) and 5’-CACCATCTTTATATTCTCACATACATTTAC AGTTCCTT-3’ (reverse). The PCR product was subcloned into pGEM-T easy vector (Promega, Madison, WI, USA), and DNA sequence was verified. The cDNA fragment was inserted into pcDNA3.1(-) myc-His expression vector (Life technologies) to add epitope tag at the C-terminal site. The cDNA fragment encoding myc-His tagged Dectin-1A was transferred into pSVL expression vector (GE Healthcare, Buckinghamshire, UK). Substitution of Tyr 15 to Phe (Y15F) by a point mutation of Dectin-1A cDNA was generated by QuickChange Lightning Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) using two primers, 5’-GAGAATCFTGGATGAAGATGGATTTACTC AATTAGACTTCAGCAC-3’ (forward) and 5’-GTGCTGAAGTCTAATTGAGTAAATCCATC TTCATCCAGATTCTC-3’ (reverse). The resulted point mutation was confirmed by the DNA sequencing. The mutant cDNA (Dectin-1AY15F) was then transferred into the pSVL vector. Cell culture and transfections―Rat basophilic leukemia RBL-2H3 cells were by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Mast cells Dectin-1 in innate anti-fungal immunity 6 maintained as monolayer cultures in DMEM with 100 U/ml of penicillin and 10% (v/v) heat-inactivated FCS. For stable transfection, 20 μg of linearized expression constructs and 2 μg of pSV2-neo vector were cotransfected into 5×10 6 RBL-2H3 cells by electroporation (950 microfarads, 310 V) using GenePulserXcell (Bio-Rad, Hercules, CA, USA) as described (31). Stably transfected cell lines were selected with 0.4 mg/ml active G418 (Nacalai tesque, Kyoto, Japan). Cell lines were screened by the level of protein expression by the immunoblotting of detergent-soluble lysates with anti-Dectin-1 and anti-FcεRIβ antibodies (a gift from Dr. Reuben P. Siraganian (National Institutes of Health, MD, USA)). Preparation of cell lysates, immunoprecipitation and immunoblotting―10 6 cells were incubated without or with 100 μg/ml of Curdlan in the medium for the indicated periods of time, or cultured overnight with anti-DNP IgE mAb (1:5000) for sensitization and stimulated with 300 ng/ml of DNP-BSA for 10 min at 37°C. In some experiments, cells were pretreated with Syk inhibitor R406, prior to the stimulation. After the stimulation, cells were washed with ice-cold PBS twice and solubilized in the lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na3VO4, 1 mM PMSF and 2 μg/ml aprotinin) containing 1% Triton X-100. Preparation of detergent-soluble cell lysates, immunoprecipitation, and immunoblotting were performed as previously described (32-34). FACS analysis―Parental or Dectin-1-expressing RBL-2H3 cells were reacted with anti-Dectin-1 antibody or goat anti-mouse IgG antibody (Jackson immunoresearch, West Grove, PA, USA) as a negative control for 30 min at 4°C. After washing with PBS, cells were stained with Alexa Flour 488 F(ab')2 fragment of rabbit anti-goat IgG (Life technologies). Data from stained cells were acquired by FACScantII (Beckton Dickinson, Franklin Lakes, NJ, USA), and analyzed with FlowJo software (FlowJo, LLC, Ashland, OR, USA). Pull-down assay―The GST-rat Syk-SH2 (both Nand C-terminal SH2 domains) expression construct was a gift from Dr. Reuben P. Siraganian. 5×10 6 cells were incubated without or with 100 μg/ml Curdlan in medium, and were solubilized with the lysis buffer containing 1% Triton X-100. In vitro binding experiments were performed as previously described (32-34). Microarray analysis―10 6 cells were pretreated without or with R406 (2 μM) for 5min, and subsequently stimulated without or with 100 μg/ml of Curdlan for 2 h. Total RNAs were isolated and the qualities were evaluated by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The sense-strand cDNAs were generated using Ambion WT Expression kit (Life technologies), and the synthesized cDNAs were subjected to fragmentation and labeling by GeneChip WT Terminal labeling kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Hybridization with by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Mast cells Dectin-1 in innate anti-fungal immunity 7 GeneChip Rat Gene 1.0 ST Array, scanning and generation of probe cell intensity data were carried out using an Affymetrix fluidics station 450 and GeneChip Scanner 3000 7G using Affymetrix GeneChip Command Console Software (Affymetrix). The data were imported into Subio platform version 1.16 (Subio, Kagoshima, Japan) for database management and quality control. All samples were assayed in two different biological replicates. Raw data were published in GEO database (GSE56246). Quantitative Real-time PCR―10 6 cells were seeded in 35-mm dishes and cultured overnight. Cells were pretreated without or with any of following, R406, PD98059, BAY11-7082, or Cyclosporin A and subsequently stimulated without or with 100 μg/ml of Curdlan for 2 h at 37°C. Total RNA was extracted using High Pure RNA Isolation Kit (Roche, Mannheim, Germany) and the first-strand cDNAs were prepared using the superscript III with random primers. Real-time PCR was performed using KAPA SYBR FAST Universal qPCR Kit (KAPA Biosystems, Wilmington, MA, USA) according to the manufacturer’s instructions. The primers used in this study were as follows: IL-3 (forward: 5’-ACAATGGTTCTTGCCAGCTCTAC-3’; reverse: 5’-AGGAGCGGGAGCAGCAT-3’), IL-4 (forward: 5’-CAGGGTGCTTCGCAAATTTTAC-3’: reverse: 5’-ACCGAGAACCCCAGACTTGTT-3’), IL-13 (forward: 5’-AGGAGCTGAGCAACATCACAC-3’: reverse:5’-CCATAGCGGAAAAGTTGCTT-3’), TNF-α (forward: 5’-GTAGCCCACGTCGTAGCAA-3’; reverse: AAATGGCAAATCGGCTGAC-3’), MCP-1 (forward: CGGCTGGAGAACTACAAGAGA-3’; reverse: 5’-CTCTTGAGCTTGGTGACAAATACT-3’), Nfkbiz (forward: 5’-TGCTCCAGGCAATCCAGAAG-3’; reverse: GTTGCCTCCAGATCCACAAAC-3’), GAPDH (forward: 5’-TTCACCACCATGGAGAAGGC-3’; reverse: 5’-GGCATGGACTGTGGTCATGA-3’). The expression of the housekeeping gene GAPDH was used as a reference for normalization. The data were developed by using the StepOne Software version 2.1 (Life technologies) and the analyzed results were finally expressed as relative units. Cytokine production―10 6 cells were seeded in 35-mm dishes and cultured overnight without or with (for DNP-BSA) anti-DNP IgE mAb (1:5000) for sensitization. Cells were pretreated without or with R406, then stimulated with 100 μg/ml of Curdlan or 30 ng/ml of DNP-BSA for 6 h at 37°C. Concentrations of MCP-1, IL-4, and TNF-α secreted into cell culture supernatants of untreated and stimulated cells were analyzed with plate-bound ELISA-kits (Quantikine ELISA, R&D systems) according to the manufacture’s recommendations. Statistical analysis―Significant differences were evaluated by the paired t-test; *P<0.05, **P<0.01, ***P<0.001 were considered by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Mast cells Dectin-1 in innate anti-fungal immunity