Download Protein Secretion in Human Macrophages Autophagy

Dectin-1 Pathway Activates Robust
Autophagy-Dependent Unconventional
Protein Secretion in Human Macrophages
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of June 16, 2017.
Tiina Öhman, Laura Teirilä, Anna-Maria
Lahesmaa-Korpinen, Wojciech Cypryk, Ville Veckman,
Shinobu Saijo, Henrik Wolff, Sampsa Hautaniemi, Tuula A.
Nyman and Sampsa Matikainen
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Copyright © 2014 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2014; 192:5952-5962; Prepublished online 7 May
2014;
doi: 10.4049/jimmunol.1303213
http://www.jimmunol.org/content/192/12/5952
The Journal of Immunology
Dectin-1 Pathway Activates Robust Autophagy-Dependent
Unconventional Protein Secretion in Human Macrophages
Tiina Öhman,*,1 Laura Teirilä,†,1 Anna-Maria Lahesmaa-Korpinen,‡ Wojciech Cypryk,*
Ville Veckman,† Shinobu Saijo,x,{ Henrik Wolff,† Sampsa Hautaniemi,‡ Tuula A. Nyman,*,2
and Sampsa Matikainen†,2
F
ungi (e.g., yeast, filamentous forms) are heterotrophic
eukaryotes that are associated with a wide spectrum of
diseases in humans and animals, such as respiratory allergy
and skin diseases. b-Glucans are naturally occurring carbohydrates that are the major structural components of the fungal cell
wall and therefore represent potentially important immunostimulatory components of fungi. Particulate but not soluble b-glucans
are potent activators of dendritic cells and macrophages (1). The
*Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland;
†
Finnish Institute of Occupational Health, 00250 Helsinki, Finland; ‡Computational
Systems Biology Laboratory, Institute of Biomedicine, Genome-Scale Biology Research Program, Faculty of Medicine, University of Helsinki, 00014 Helsinki,
Finland; xDepartment of Molecular Immunology, Medical Mycology Research Center, Chiba University, Chiba 260-8673, Japan; and {Presto, Japan Science and Technology Agency, Saitama 332-0012, Japan
1
T.Ö. and L.T. contributed equally to this work.
2
T.A.N and S.M. contributed equally to this work.
Received for publication December 6, 2013. Accepted for publication April 2, 2014.
This work was supported by Academy of Finland Grants 125826, 135628, and
140950, The Finnish Work Environment Fund, the Sigrid Jusélius Foundation, the
Graduate School in Environmental Health ‘SYTYKE’ (to L.T.), the Helsinki Biomedical Graduate School (to A.-M.L.-K.), the Viikki Doctoral Programme in Molecular Biosciences (to W.C.), the Nummela Foundation, and the Finnish Medical
Society (Finska läkaresällskapet).
The microarray data presented in this article have been submitted to the Gene
Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number
GSE32282. The mass spectrometry data in this article have been submitted to the
ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) under
dataset identifier PXD000574.
Address correspondence and reprint requests to Dr. Sampsa Matikainen, Finnish
Institute of Occupational Health, Topeliuksenkatu 41b, 00250 Helsinki, Finland.
E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: ASC, apoptosis-associated speck-like protein,
which contains a caspase recruitment domain; BMDC, bone marrow–derived dendritic cell; DAMP, danger-associated molecular pattern molecule; ER, endoplasmic
reticulum; GBY, baker’s yeast; iTRAQ, isobaric tag for relative and absolute quantification; LDH, lactose dehydrogenase; 3-MA, 3-methyladenine; MS, mass spectrometry; PRR, pattern recognition receptor; siRNA, small-interfering RNA.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303213
major pattern recognition receptor (PRR) for b-glucans is dectin-1,
a transmembrane C-type lectin receptor, which is predominantly expressed by the myeloid cells such as macrophages and
dendritic cells (2–4). Receptor binding by b-glucans, including
curdlan and glucan from baker’s yeast (GBY), triggers a variety of
protective cellular responses via the Syk kinase-signaling pathway. These responses include phagocytosis and killing, which
is mediated through the respiratory burst, and the production of
numerous cytokines and chemokines (5). In addition to dectin-1,
the NLRP3 inflammasome is a PRR that participates in antifungal
defense. The NLRP3 inflammasome is a caspase-1–activating
molecular platform that regulates proteolytic processing of proinflammatory cytokines IL-1b and IL-18 (6, 7).
The “secretome” is generally referred to as the complex set of
proteins secreted from living cells at a given time and under defined conditions. These proteins can be released through various
mechanisms, including classical secretion and nonclassical, vesiclemediated mechanisms such as exosome- and lysosome-mediated
release of proteins (8). The secreted proteins can control and
coordinate many biological activities in multicellular organism,
such as growth, differentiation, apoptosis, and immune response,
and they hold additional therapeutic importance as either targets
for pharmacologic intervention in disease or cancer biomarkers.
Studies using quantitative mass spectrometry (MS)-based proteome analysis combined with bioinformatics have highlighted
the benefits of this method for elucidating which proteins are
secreted in response to various stimuli (9–12). We have shown
previously that b-glucans activate the membrane-associated
dectin-1 and the cytoplasmic NLRP3 inflammasome in human
macrophages, resulting in IL-1b gene transcription and IL-1b
protein secretion, respectively (13), but the global protein secretion pattern and the associated intracellular signaling pathways after b-glucan stimulation have not been characterized. In
this study, we have used MS-based quantitative proteomics—that
is, the iTRAQ (isobaric tag for relative and absolute quantification)
technique—to conduct a secretome analysis with the data being
combined with transcriptomics, bioinformatics, and functional studies
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Dectin-1 is a membrane-bound pattern recognition receptor for b-glucans, which are the main constituents of fungal cell walls.
Detection of b-glucans by dectin-1 triggers an effective innate immune response. In this study, we have used a systems biology
approach to provide the first comprehensive characterization of the secretome and associated intracellular signaling pathways
involved in activation of dectin-1/Syk in human macrophages. Transcriptome and secretome analysis revealed that the dectin-1
pathway induced significant gene expression changes and robust protein secretion in macrophages. The enhanced protein secretion correlated only partly with increased gene expression. Bioinformatics combined with functional studies revealed that the
dectin-1/Syk pathway activates both conventional and unconventional, vesicle-mediated, protein secretion. The unconventional
protein secretion triggered by the dectin-1 pathway is dependent on inflammasome activity and an active autophagic process. In
conclusion, our results reveal that unconventional protein secretion has an important role in the innate immune response against
fungal infections. The Journal of Immunology, 2014, 192: 5952–5962.
The Journal of Immunology
to characterize the global innate immune response of human primary
macrophages stimulated through the dectin-1 pathway.
Materials and Methods
Cell culture and stimulations
Mouse bone marrow-derived dendritic cells
Mouse bone marrow–derived dendritic cells (BMDCs) were derived from
bone marrow cells isolated from the femurs of wild type C57BL/6 (NOVASCB AB) and dectin-1 knockout mice (15). The animal study was approved by the Health Services of State Provincial Office of Southern
Finland.
iTRAQ labeling and mass spectrometry
Before stimulation, the cells were washed and the culture media was
changed to RPMI 1640. After stimulation, cell culture media were collected
and concentrated, and the proteins were precipitated with 2-D Clean-Up Kit
(GE Healthcare), followed by protein alkylation, trypsin digestion, and
iTRAQ labeling (16) of the resulting peptides according to the manufacturer’s instructions (Applied Biosystems). The control sample was labeled with 114, LPS-stimulated was labeled with 115, Curdlan-stimulated
was labeled with 116, and GBY-stimulated was labeled with 117 isobaric
tag. After labeling, the samples were pooled and dried, and the peptides
were fractionated by strong cation exchange chromatography using
an Ettan HPLC system (Amersham Biosciences) connected to a PolySULFOETHYL A column. Each SCX-fraction containing the labeled
peptides was analyzed twice with nano-liquid chromatography-tandem
mass spectrometry using Ultimate 3000 nano-liquid chromatograph
(Dionex) and QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems) with nano-ESI ionization as described
previously (11). MS data were acquired automatically using Analyst QS
2.0 software.
Gene expression microarray
Microarray experiments were performed at Biomedicum Genomics
(Helsinki, Finland) using an Agilent Whole Human Genome 4 3 44K
1-Color Array (Agilent Technologies). Integrity and purity of the RNA
were verified with Agilent 2100 Bioanalyzer (Agilent Technologies).
Three independent experiments were performed.
Cell death assays
APOPercentage apoptosis assay was performed according to the manufacturer’s guidelines (Biocolor Life Science Assays). Photographs were
taken with an Olympus DP70 Digital microscope camera, connected to an
Olympus IX71 light microscope, and using software in DP Controller
(version 2.2.1.227) and DP Manager (2.2.1.195; Center Valley, PA). The
lactose dehydrogenase (LDH) assay was performed according to the
manufacturer‘s instructions (Roche Diagnostics). Cell viability was determined by measuring intracellular ATP using the CellTiter-Glo Luminescent Cell Viability Assay (Promega).
Data files from both technical replicates of an iTRAQ sample set were
processed together. Database searching was done against UniProt human
database (version 2008-01-28 with 20330 human sequences) and ‘decoy’
database (the reverse amino acid sequence for false discovery rate estimation). The search criteria were: cysteine alkylation with MMTS, trypsin
digestion, biological modifications allowed, thorough search, and detected
protein threshold of 95% confidence (Unused ProtScore . 1.3). Importantly, no automatic bias correction was applied in the quantitation. The
false discovery rates were calculated as described previously (18) and were
1.4% and 2% for the two biological replicates.
The secreted proteins identified were classified based on their Gene
Ontology annotations using GeneTrail (http://genetrail.bioinf.uni-sb.de/)
(19) and GoMiner (http://discover.nci.nih.gov/gominer/index.jsp) using
Uniprot as data source and Homo sapiens as the organism. Human diseases
were left out of KEGG pathway results. SignalP 4.0 (http://www.cbs.dtu.
dk/services/SignalP/) was used to predict classically secreted proteins, and
ExoCarta version 3.2 database (20) (http://www.exocarta.org/) was used to
determine exosomal proteins.
The microarray expression values were preprocessed with LOWESS
using the Agilent Feature Extractor and then median-centered, followed by
reannotation of the probes using the Ensembl genomic database (version
60). Fold changes for genes were calculated for each stimulation versus the
control, and the three replicates were combined using the median value.
Gene Ontology enrichment was calculated with Fisher exact test using
a genome-wide reference set for human genes as the reference. The pathway
enrichment analysis was performed with SPIA (21), which uses the KEGG
pathway database (22). All data analyzes were performed with the freely
available Anduril framework (23).
Exosome enrichment
Cells were stimulated in RPMI 1640 without FCS, after which the cell
culture media were collected, and the cells and cell debris were removed
(500 3 g for 10 min and 3000 3 g for 30 min). Exosomal fractions were
enriched using centrifugal filter units (100 kDa cut-off), and the flowthrough was further concentrated with 10-kDa centrifugal filter units.
The enriched exosomal fractions were used directly for Western blotting,
or the fractions were diluted with PBS and ultracentrifuged twice at
100,000 3 g for 1 h. The purified vesicles were resuspended in PBS and
used for electron microscopy or Western blotting.
Electron microscopy
The exosome suspension was fixed with 1% paraformaldehyde, transferred
to Pioloform-carbon–coated copper grids, and allowed to absorb for 20
min. The grids were subsequently washed and contrasted with uranyl acetate to visualize proteins and viewed with a Jeol 1200 EX II transmission
electron microscope.
Immunoblotting
To analyze the secreted proteins, cell culture media were concentrated with
centrifugal filter units (cutoff, 10 kDa). The following Abs were used for
immunoblotting: cleaved caspase-3 (Asp175) Ab (Cell Signaling Technology), anti-Alix (1A12, Santa Cruz), anti- tsg 101 (C-2; Santa Cruz), antiannexin I (EH17a; Santa Cruz), anti-a/b-tubulin (Cell Signaling), anti–
apoptosis-associated speck-like protein, which contains a caspase recruitment
domain (ASC; TMS1; Millipore), anti-cathepsin B (Ab-3; Calbiochem), anticathepsin D (C-20; Santa Cruz), anti-Atg7 (D12B11; Cell Signaling), and
anti-optineurin (C-2; Santa Cruz). The mAb against b2-integrin
(R2E7B) was a gift from Prof. Gahmberg (Division of Biochemistry,
University of Helsinki). The IL-1b Ab has been described previously
(14).
To confirm equal loading and transfer of the protein, membranes were
stripped and stained with ready-to-use SYPRO Ruby Protein Blot Stain
(Sigma-Aldrich) or detected with anti-GAPDH ab (0411; Santa Cruz).
Luminex assay
The human cytokine Luminex Bio-Plex Pro immunoassay kit designed to
detect chemokines TNF, CCL2 and CCL5 was from Bio-Rad Laboratories.
The Luminex assay was performed according to the manufacturer’s
instructions using Bio-Plex 200 system hardware and version 4.1.1 of BioPlex 200 software.
ELISA and RT-PCR
Proteomics and transcriptomics data-analysis
Protein identification and relative quantitation were performed with the
Paragon search algorithm (17) using ProteinPilot 2.0 interface (AB Sciex).
Human IL-1b and mouse IL-1b ELISAs were purchased from Diaclone
and eBioscience, respectively. The experiments were performed three
times with similar results.
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PBMCs were isolated from healthy blood donors (Finnish Red Cross Blood
Transfusion Service) and differentiated into macrophages in the presence
Macrophage-SFM medium (Life Technologies) supplemented with GMCSF as described previously (14). The isolated cells were identified as
macrophages by their typical morphology and CD14 expression pattern
(14). Macrophages were untreated or stimulated with LPS (Escherichia
coli O111:B4) or 1,3-b-glucans curdlan or GBY (all purchased from
Sigma-Aldrich). The concentrations used in experiments were 1 mg for
LPS, 10 mg for curdlan, 100 mg for GBY, or as indicated in the figures.
Curdlan and GBY were suspended to PBS as a homogeneous dispersion. In
the secretome analysis, the cells were stimulated for 18 h and subjected to
gene expression microarray analysis for 6 h. Each macrophage sample
represents a pool of separately stimulated cells from at least two different
blood donors.
Caspase-1 inhibitor VI (Z-YVAD-FMK, 25 mM; Millipore), Syk tyrosine kinase inhibitor II (5 or 10 mM; Calbiochem), Src inhibitor PP2 (10
mM; Sigma), Src inhibitor-1 (20 mM; Sigma), and 3-methyladenine (3MA; 10 mM; Sigma) were added to human macrophages 1 h before curdlan stimulation. Brefeldin A (Sigma) was used at 100 ng/ml and was
added to cells 1 h after curdlan stimulation.
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DECTIN-1 AND VESICLE-MEDIATED PROTEIN SECRETION
Total cellular RNA was isolated using RNeasy Plus Mini Kit (Qiagen) and
were reverse transcribed using the High Capacity cDNA Reverse Transcription
kit (Applied Biosystems) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with an ABI PRISM 7500 Sequence
Detection System applying TaqMan chemistry and Predeveloped TaqMan
assay primers and probes (human IL-1b Hs01555410_m1 and mouse-IL-1b
Mm01336189_m1; Applied Biosystems) and PerfeCTa qPCR FastMix
(Quanta Biosciences). RT-PCR data were processed and quantified as
described previously (24). The results are expressed as relative units. The
experiments were performed three times with similar results.
Small-interfering RNA approach
Macrophages were transfected twice with 200 nM non-targeting control
small-interfering RNA (siRNA; AllStars Negative Control siRNA; Qiagen)
and with 100 nM of each of two different beclin-1 siRNAs (Hs_BECN1_1,
Hs_BECN1_3; Qiagen; final concentration being 200 nM) by using
HiPerFect Transfection Reagent (Qiagen) according to the manufacturer’s
instructions.
Accession codes
Results
b-Glucans induce significant gene expression changes and
activate robust protein secretion from human primary
macrophages
To characterize secretome upon dectin-1 activation in human
macrophages, we stimulated the cells with either curdlan or GBY
for 18 h, and subsequently the secreted proteins were analyzed by
4plex iTRAQ labeling combined with liquid chromatographytandem MS analysis. In addition, bacterial LPS, a well-known
inflammatory stimulus unrelated to the fungi activating TLR4
signaling, was used as a control. iTRAQ analysis was performed on
two independent biological experiments resulting in the identification and quantification of 1597 distinct proteins with highconfidence (Supplemental Table I). Activation of the dectin-1
pathway effectively induced protein secretion, because the average ratio of total secreted protein compared with untreated cells
was elevated by 2.7-fold when the cells were stimulated with
curdlan compared with the 3.7-fold increase obtained with GBY,
whereas the total protein secretion was only modestly (1.1 average
ratio) increased by LPS (Supplemental Table I). Curdlan and GBY
stimulation increased the secretion of 1258 and 1492 distinct
proteins (fold change . 2), respectively (Fig. 1A). From these
proteins, 1198 were common to both curdlan and GBY, evidence
of a similar response to both b-glucans. Gene ontology analysis
based on the known cellular locations for the identified proteins
revealed that most of the secreted proteins detected were intracellular, including nuclear and cytosolic proteins and proteins
from different cell organelles (Fig. 1A). Pathway analysis using
the KEGG database of the identified proteins identified 14 pathways as being significantly (p , 0.05) overrepresented after
b-glucan stimulation (Fig. 1A). Ten of these pathways are part of
the immune system, including the phagosome, chemokine signaling pathway, leukocyte transendothelial migration, and the
NOD-like receptor signaling pathway (marked as asterisk in Fig.
1A). To confirm that the robust secretion of intracellular proteins
seen after dectin-1 activation was not a result of cell death, we first
applied the APOPercentage apoptosis assay, in which we did not
detect any morphologic signs of cell death or activation of apoptosis in macrophages that had been activated through the dectin-1
pathway (Fig. 2A). In accordance with these findings, we did not
FIGURE 1. Secretome and transcriptome analysis of human primary
macrophages stimulated with b-glucans and LPS. (A) Secretome analysis.
Venn diagram of the identified proteins with relative quantity .2 in curdlan- and GBY-stimulated macrophages, respectively (upper left). GO
classification based on cellular localization of the identified proteins (upper
right) and the most significant KEGG pathways found in the b-glucan
secretome data (lower panel). Pathways associated with the immune system are marked by an asterisk. (B) Transcriptome analysis. Correlation
between curdlan and GBY induced genes (upper left panel), and LPS and
b-glucans induced genes (the median of curdlan and GBY samples; upper
right panel). Each dot represents a single gene, and its position is determined by its induction in response to stimulation. Data are combined from
three independent experiments. Overrepresented (+) and underrepresented
(2) KEGG pathways. ND = not determined (lower panel).
detect caspase-3 activation (Fig. 2B), which is a hallmark of apoptosis, in macrophages that had been activated through the
dectin-1 or TLR4 pathways. Furthermore, both LDH release (Fig.
2C) and CellTiter-Glo Luminescent Cell Viability (Fig. 2D) assays
demonstrated the lack of necrosis in these macrophages.
We then performed gene expression microarray experiments to
compare the global transcriptional response induced by b-glucans
and LPS with their secretomes. We identified 767, 1447, and 1683
genes with .2-fold increase or decrease in curdlan-, GBY-, or LPSstimulated macrophages, respectively (Supplemental Table II). The
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The Gene Expression Omnibus accession number for the microarray data
reported in this article is GSE32282 (http://www.ncbi.nlm.nih.gov/geo/).
The mass spectrometry data have been deposited to the ProteomeXchange
Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE
partner repository (25) with the dataset identifier PXD000574 (http://
proteomecentral.proteomexchange.org).
The Journal of Immunology
5955
Dectin-1 pathway activates both conventional and
unconventional, vesicle-mediated protein secretion
Inflammasome activity is essential for dectin-1–induced
unconventional protein secretion
To characterize the secretion mechanism activated by dectin-1
pathway in macrophages, the identified proteins were first classified using SignalP to determine the presence of signal peptide
sequence needed for conventional protein secretion. Only 26%
of curdlan-induced and 20% of GBY-induced secreted proteins
were confirmed as secretory proteins, with the presence of signal
sequences with a good prediction value and cleavage site position
The dectin-1 pathway activates NLRP3 inflammasome in human
macrophages (13). After activation, the NLRP3 binds the adaptor
protein ASC and procaspase-1 in the inflammasome multiprotein
complex. The inflammasome assembly will spontaneously activate caspase-1, which cleaves pro–IL-1b and pro–IL-18 triggering
a release of these cytokines. In line with previous study (13), our
secretome data showed enhanced IL-1b and IL-18 secretion after
FIGURE 2. Robust secretion of intracellular proteins after b-glucan
stimulation is not a result of cell death. (A) Human macrophages were left
untreated or stimulated with curdlan or cytosolic pI:C for 18 h, after which
the possible ongoing apoptosis was studied with the APOPercentage apoptosis assay. Apoptotic cells are stained purple. pI:C, a cytosolic viral
dsRNA analog, was used as a positive control. Original magnification 310.
(B) Western blot analysis with caspase-3 Ab from total cell lysates. Human
macrophages were stimulated with LPS, curdlan, or GBY or infected with
influenza A virus for 18 h, after which the cell lysates were analyzed with
anti–caspase-3 p19/17. Influenza A virus infection was used as a positive
control. (C) LDH cytotoxicity assay was used to quantify lactose dehydrogenase (LDH) release into cell culture media. (D) Cell viability was
determined by measuring intracellular ATP using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) after 18 h stimulation with LPS,
curdlan, or GBY or influenza A virus infection.
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high coefficient of determination (r2 = 0.5) and scatter plot of curdlanand GBY-stimulated genes demonstrated similar induction ratios
after both b-glucan treatments (Fig. 1B). In contrast, the transcriptional profile of LPS-stimulated macrophages did not correlate with
the transcriptional profile of b-glucan–stimulated macrophages (r2 =
0.28; Fig. 1B), indicating that distinctly different downstream signaling pathways are being triggered by LPS and b-glucans. The
differentially expressed genes were analyzed further for their pathway associations using the KEGG database (Fig. 1B). The main
significantly (p , 0.05) overrepresented pathways identified from
all three datasets were chemokine signaling pathway, cytokinecytokine receptor interaction, and MAPK signaling pathways,
whereas cytosolic DNA-sensing pathway, Jak-STAT signaling pathway, and NOD-like receptor signaling pathways, which were overrepresented after LPS but underrepresented after b-glucan stimulation.
(Fig. 3A, Supplemental Table IIIA). This result led us to hypothesize that mechanisms other than conventional, signal peptide
sequence–dependent secretion mechanisms were also being activated during b-glucan stimulation in human macrophages. The
cells secrete the proteins into the extracellular space through
membrane vesicles of endosomal and plasma membrane origin
called “exosomes” and “microvesicles,” respectively (26). Based
on the exosomal protein database ExoCarta, 49% of curdlan and
GBY-induced secreted proteins were previously reported as being
exosomal proteins (Fig. 3A, Supplemental Table IIIB). We then
classified the secreted proteins based on their gene ontology
annotations for “biological processes” (Fig. 3B). The exosomal
proteins identified in our secretome data included typical exosomal proteins, such as proteins involved in protein metabolic processes (e.g., proteosome subunits and ribosomal proteins) and in
vesicle-mediated trafficking (Rab protein family), signaling proteins (annexins and small GTPases), cell structure proteins (actin
cytoskeleton proteins), and cell adhesion and migration proteins
(intergrins, ICAMs). The classically secreted proteins are mainly
immune system proteins, such as chemokines and cytokines, and
unclassified proteins include proteins involved in gene expression
and RNA processing.
The exosomal nature of the protein secretion was confirmed in
b-glucan–induced macrophages by conducting an experiment in
which we enriched the fraction containing extracellular vesicles
from the growth media by ultracentrifugation. Electron microscopy analysis revealed membrane-bound particles with a characteristic exosomal size and shape (Fig. 3C). Western blot analysis
with Abs against known exosomal marker proteins including Alix,
tsg 101, tubulin, and annexin I demonstrated their increased secretion after b-glucan stimulation (Fig. 3D). In addition, protein
separation by SDS-PAGE and visualization with silver staining
were used to estimate the total protein amount in exosomal fractions (Fig. 3E), further confirming that activation of the dectin-1
pathway notably increases the secretion of exosomal proteins.
Our secretome analyzes revealed that dectin-1 pathway also
increased the secretion of several chemokines and cytokines, most
of which were being secreted through conventional signal peptide–mediated pathway (yellow boxes in Fig. 4A, Supplemental
Table IIIA). TNF and selected chemokines were quantified from
the growth media using the Luminex assay, with a high level secretion of TNF and chemokines after stimulation of macrophages
with b-glucans and LPS (Fig. 4B). To characterize further the
conventional protein secretion in macrophages, we used Brefeldin
A, which inhibits transport of proteins from endoplasmic reticulum (ER) to Golgi, thereby blocking conventional protein secretion. Macrophages were stimulated with curdlan for 1 h, after
which Brefeldin A was added. After 9 h of stimulation, the cell
culture supernatants were collected. Luminex assay revealed that
Brefeldin A-treatment of macrophages completely inhibited conventional protein secretion including TNF and chemokines CCL-2
and CCL-5 in macrophages that had been activated through the
dectin-1 pathway (Fig. 4C). However, Brefeldin A had no effect
on dectin-1–induced unconventional IL-1b secretion (Fig. 4D).
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DECTIN-1 AND VESICLE-MEDIATED PROTEIN SECRETION
b-glucan stimulation, and this was confirmed by Western blot
analysis (Supplemental Table I, Fig. 5A). Cathepsins are lysosomal
proteases that are produced as inactive preproenzymes, which need to
be cleaved into their active, mature forms (27). b-Glucan induced
NLRP3 inflammasome activation has been shown to be dependent on
cathepsin activity (13), and our current data indicate that there is a
major increase in the secretion of mature cathepsins upon b-glucan
stimulation (Fig. 5A). Interestingly, the mature form of cathepsin D
was seen in macrophage secretome already at 3 h after dectin-1 activation. To characterize the secretion mechanism of inflammasomecomponents and regulators after curdlan stimulation in more detail,
we enriched the extracellular vesicles from the cell culture media, and
IL-1b, IL-18, ASC, and cathepsin secretion was analyzed (Fig. 5B).
Mature forms of IL-1b and IL-18 cytokines, as well as ASC isoforms
p22 and p10, could be detected only in the flow-through fraction,
which indicates that these molecules are not secreted by vesicles.
Cathepsins are presumably released with secretory lysosomes, and as
expected, we detected intact cathepsins in the flow-through fraction,
which included also lysosomal proteins. Interestingly, the mature forms
of cathepsins were detected only in the fraction containing vesicles.
Caspase-1 has been implicated in the regulation of unconventional protein secretion in ultraviolet-activated human keratinocytes (28). We used a pharmacologic inhibitor of caspase-1 to
examine the possible role of inflammasome activation in the
dectin-1–induced protein secretion. Macrophages were stimulated through dectin-1 pathway in the presence or absence of the
caspase-1 inhibitor, and cell culture supernatants were collected
after 18 h of stimulation. Western blot analysis of cell culture
supernatants indicated that in addition to total blockade of IL-1b
secretion, the caspase-1 inhibitor also abolished the release of
two unconventionally secreted proteins, tubulin and annexin I
(Fig. 5C). However, the caspase-1 inhibitor had no effect on the
secretion of the mature form of cathepsin D, indicating that
cathepsins are activated upstream of the inflammasome. Luminex
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FIGURE 3. b-Glucans activate unconventional, vesicle-mediated protein secretion from human macrophages. (A) b-Glucan induced secreted proteins
were classified using SignalP to determine the presence of the signal peptide sequence needed for conventional protein secretion and ExoCarta database to
determine exosomal proteins. For analysis, SwissProt accesses were converted to gene names. (B) Exosomal, conventionally secreted, and unclassified
proteins were classified based on GO classification for biological functions. The p value shows the significance of process. (C) Human macrophages were
left untreated or stimulated with curdlan for 18 h, after which the cell culture was collected and extracellular vesicles were enriched using ultracentrifugation. Electron microscopy image reveals exosome-like vesicles in the macrophage supernatant. Scale bar, 200 nm. Original magnification 325,000. (D)
Exosomal fractions were analyzed by immune blotting with known exosomal proteins Alix, tsg 101, annexin 1, and tubulin. (E) Silver-stained SDS-PAGE
gel was used to estimate the total protein amount in exosomal fractions after curdlan stimulation.
The Journal of Immunology
5957
analysis of cell culture supernatants revealed that caspase-1 inhibition has no effect on dectin-1–induced TNF and chemokine
secretion (Fig. 5D). In conclusion, our results demonstrate that
dectin-1–induced inflammasome activation is required to trigger
unconventional protein secretion, but it is not essential for the
release of conventionally secreted proteins.
b-Glucan-activated protein secretion is dependent on
dectin-1/Syk pathway
Dectin-1 has an ITAM-motif within its cytoplasmic tail, and tyrosine
phosphorylation of this motif by Src kinase followed by Syk
recruitment is required for receptor activation and initiation of
downstream signaling events (29). To confirm the role of dectin-1 in
the b-glucan induced secretion of IL-1b and cathepsins, we used
primary BMDCs from a mouse strain that is deficient in the dectin-1
receptor (15). In general, mouse bone marrow-derived macrophages
are not responsive to b-glucans, and for this reason we used
BMDCs (31). Dendritic cells from dectin-12/2 mice were unable to
release the mature, biologically active form of IL-1b, and the secretion of mature forms of cathepsins were dectin-1 dependent
(Fig. 6A, 6B). In contrast, expression of IL-1b mRNA in response
to curdlan stimulation was only partially dependent on dectin-1
(Fig. 6C). These data indicate that other receptors, such as scavenger receptors CD36 and SCARF1 (30) or complement receptor
CR3 (31) may be involved in the activation of IL-1b gene transcription in response to curdlan stimulation. Dendritic cells prepared from dectin-12/2 mice did not express IL-1b after stimulation
with GBY, whereas dectin-1 deficiency had no effect on the LPS
response (Fig. 6C).
The role of Syk and Src kinase in curdlan-induced secretion of
inflammasome components and associated regulators was investigated using specific inhibitors for Syk (SykII) and Src (PP2 or Src
inhibitor I). SykII clearly decreased the secretion of the mature
forms of IL-1b, cathepsins B and D, and ASC p10 in macrophages
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FIGURE 4. b-Glucans activate effective secretion of chemokines and cytokines. (A) Heat map visualization of chemokine and cytokine secretion and
gene expression during LPS and b-glucan stimulation of human macrophages. Colors indicate the normalized FC-values. Proteins that were secreted by
classical signal peptide mediated mechanisms are marked with yellow boxes. (B) Human macrophages were stimulated with LPS, curdlan, and GBY for
18 h, after which TNF and selected chemokines were quantified from macrophage media using Luminex cytokine assay. The experiments were conducted
twice with similar results. (C) Human macrophages were stimulated with curdlan for 1 h, after which the cells were cultured in the presence and absence of
100 mg/ml Brefeldin A (BFA). After 9 h of stimulation, the cell culture supernatants were collected and secretion of TNF, CCL2, and CCL5 was measured
with the Luminex assay. The experiments were performed twice with similar results. (D) The effect on BFA for IL-1b secretion was analyzed by ELISA.
The experiments were performed three times with similar results.
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DECTIN-1 AND VESICLE-MEDIATED PROTEIN SECRETION
(Fig. 6D). In contrast, PP2 or Src inhibitor I treatment had no
effect on the secretion of these proteins (Fig. 6D, Supplemental
Fig. 1A). The effect of these inhibitors on the production of pro–
IL-1b was also studied: the inhibitors alone had only a marginal
effect on pro–IL-1b expression, but the combination of Syk and
Src inhibitors completely abolished the production of pro–IL-1b
(Fig. 6E). We also investigated the role of Syk and Src signaling in
the total protein secretion: SykII alone clearly reduced curdlaninduced protein secretion, whereas PP2 or Src inhibitor I on their
own had no effect on total protein secretion (Fig. 6F, Supplemental
Fig. 1A). Western blot analysis of annexin I and tubulin demonstrated that secretion of these proteins was solely dependent on Syk,
whereas the secretion of b2-integrin was inhibited only when both
Syk and Src signaling were blocked (Fig. 6F).
Inhibition of autophagy suppresses dectin-1–induced vesiclemediated protein secretion
Autophagy is a highly conserved biological process involved in
the degradation of defective organelles and long-lived proteins.
Previous studies have demonstrated that autophagy regulates
inflammasome activation and IL-1b secretion (32–35); however,
the results have been somewhat conflicting. Our secretome data
revealed that several autophagy proteins were secreted during
b-glucan stimulation (Fig. 7A). In addition, a recent report identified 94 autophagy-associated proteins (36), and 84 of them were
detected in our secretome data (Supplemental Table IIIC). This
finding strongly suggests that autophagy participates in the protein
secretion activated by dectin-1 pathway. To characterize the role
of autophagy in protein secretion from human macrophages in
response to b-glucan stimulation, we performed a Western blot
analysis of LC3, a well-known marker of autophagy activation.
This revealed that the conversion of the cytosolic LC3-I to the
lipid-associated LC-II, which is found on autophagy membranes,
was increased in response to curdlan already at the 3-h time point
(Fig. 7B). Next, the dectin-1–induced initiation of autophagy was
inhibited using 3-MA (Fig. 7C), which inhibits the activity of
class III phosphatidylinositol-3-OH kinase (PI(3)K) and the formation of autophagosomes. 3-MA treatment decreased significantly
curdlan-induced IL-1b secretion (Fig. 7D), but this effect was not
due to any reduction in IL-1b mRNA expression (Supplemental
Fig. 1B). The silver-stained SDS-PAGE gel showed that the dectin-1–induced total protein secretion was clearly suppressed after
3-MA treatment (Fig. 7E). Western blot analysis of exosomal,
inflammasome-associated and autophagy proteins indicated that
the secretion of unconventionally secreted proteins was suppressed. To determine the involvement of beclin-1, a key component of the class III PI(3)K complex, in dectin-1–induced
protein secretion we reduced beclin-1 protein expression with the
siRNA approach. Transfection of macrophages with beclin-1–
specific siRNA molecules reduced beclin-1 expression by 50%
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FIGURE 5. Inflammasome activity is essential for dectin-1–induced unconventional protein secretion. (A) Human macrophages were stimulated with
curdlan for the times indicated, and the secretion of IL-1b, IL-18, ASC, cathepsin B, and cathepsin D was analyzed from the concentrated cell culture
media by immunoblotting. (B) Human macrophages were left untreated or stimulated with curdlan for 18 h, after which the cell culture was collected and
exosomes were enriched using 100-kDa centrifugal filter units. Samples were analyzed with Western blotting. (C and D) Macrophages were stimulated with
curdlan in the presence or absence of caspase-1 inhibitor VI (Z-YVAD-FMK) and after 18 h of stimulation, the cell culture supernatants were collected. The
secretion of IL-1b, annexin 1, tubulin, ASC, galectin-3, and cathepsin D was analyzed from the concentrated cell culture media by immunoblotting (C). The
secretion of TNF, CCL-2, and CCL-5 was analyzed from cell culture supernatants with Luminex assay (D). ASC, apoptosis-associated specklike protein
containing a caspase recruitment domain.
The Journal of Immunology
5959
(Supplemental Fig. 1C), and the decreased beclin-1 expression
resulted in the inhibition of total protein secretion (Fig. 7F).
Similarly, Western blot analysis showed that silencing of beclin-1
leads to decreased release of annexin-I, tubulin, galectin-3, and the
mature form of cathepsin D in dectin-1–activated macrophages
(Fig. 7F). In agreement with these data, secretion of IL-1b was
decreased in beclin-1–silenced macrophages (Fig. 7G). In contrast, Luminex analysis of cell-cultured supernatants revealed that
silencing of beclin-1 did not inhibit the secretion of TNF, CCL2,
and CCL5 (Fig. 7H). In conclusion, our data suggest that an active
autophagy process is required for unconventional protein secretion
(Fig. 8), but not for conventional protein secretion, in macrophages that have been activated through dectin-1 pathway.
Discussion
Fungi are associated with a wide spectrum of diseases in humans
and animals. These diseases include acute, self-limiting pulmonary
manifestations and cutaneous lesions in immunologically competent individuals. In immunologically compromised patients,
fungi can cause severe, life-threatening infections. An increased
prevalence of cancer, chemotherapy, organ transplantation, and
autoimmune disease has been reported to be associated with fungal
infections in immunologically compromised patients. In this study,
we have characterized the global innate immune response of human
primary macrophages in response to dectin-1 activation using
a systems biology approach. We demonstrate that the dectin-1
pathway induces significant gene expression changes and robust
protein secretion in macrophages (summarized in Fig 8).
Many eukaryotic proteins are secreted through the conventional
ER-Golgi secretory pathway, which is primarily regulated at the
level of gene expression. These proteins contain N-terminal or
internal signal peptides that direct them to the ER. In our quantitative secretome analysis, we identified many classically secreted
proteins in cell culture supernatants of human macrophages in
response to dectin-1 activation. The classically secreted proteins
identified included many chemokines (e.g., CCL2-5, CXCL1-3,8)
and cytokines (IL-6, IL-23, and TNF-a) that contribute to the
innate immune response during microbial infections. In addition
to classical protein secretion, there is extensive protein secretion
through unconventional protein release was seen after dectin-1
activation. The unconventional protein secretion seen in macrophages activated through the dectin-1 pathway includes exosomelike vesicle-mediated release of proteins. This vesicle type of
secretion delivers more efficiently signaling molecules to adjacent
cells as compared with classically secreted proteins, which can
diffuse throughout the extracellular environment (37).
One major group of inflammatory proteins identified in our
secretome data were danger-associated molecular pattern molecules (DAMPs), which are preformed endogenous molecules,
usually with a well-defined intracellular function, released or exposed following an injury or a stress. Our secretome and transcriptome data reveal that both curdlan and GBY stimulation
clearly increased the secretion of several proteins that have been
demonstrated, or at least proposed to have a role as endogenous
danger signals (38, 39) (e.g., galectins, heat shock proteins,
HMGB-proteins, S100-proteins; Supplemental Table I), but the
gene expression levels of these molecules were not upregulated
during cell stimulation (Supplemental Table II). Interestingly, all
the identified DAMPs, except for HMGBs, are found in the
ExoCarta (Supplemental Table IIIB), evidence that DAMPs are
secreted through exosomes. Galectin-3 was one of the DAMPs
robustly secreted in response to dectin-1 activation; this is also
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FIGURE 6. Dectin-1 and Syk kinase are required for b-glucan–induced secretion of inflammasome components. (A) ELISA analysis of IL-1b secretion
from WT and dectin-12/2 BMDCs stimulated with curdlan for 6 h. (B) Western blot analysis of the secretion of active IL-1b (p17) and mature forms of
cathepsin B and D from WT and dectin-12/2 BMDCs after 18 h curdlan stimulation. (C) Quantitative RT-PCR analysis of IL-1b mRNA in WT and dectin12/2 BMDCs stimulated with curdlan, GBY, or LPS for 3 h. (D) Syk inhibitor (5 mM SykII) and Src inhibitor (10 mM PP2) were added to macrophages 1 h
before curdlan stimulation (18). The secretion IL-1b, ASC, cathepsin B, and cathepsin D was analyzed with Western blotting. (E) Quantitative RT-PCR
analysis of IL-1b mRNA expression from human macrophages that were pretreated with 5 mM SykII or 10 mM PP2, or both, for 1 h and then stimulated
with curdlan for 3 h (upper panel). Immunoblot analysis of intracellular pro–IL-1b (p31) expression that were pretreated with 5 mM SykII or 10 mM PP2,
or both, for 1 h and then stimulated with curdlan for 18 h (lower panel). (F) The effect of Syk and Src inhibitors on total protein secretion was studied. The
secreted proteins were separated by SDS-PAGE and visualized with silver-staining (upper panel) with the secretion of annexin I, tubulin, b2-integrin being
analyzed with Western blotting.
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DECTIN-1 AND VESICLE-MEDIATED PROTEIN SECRETION
a PRR that recognizes the carbohydrates uniquely present in the
cell walls of Candida albicans. Interestingly, it was recently
shown that galectin-3 is directly associated with dectin-1 and is
essential for pathogenic C. albicans–induced proinflammatory
response (40). It is likely that the robust release of galectin-3 from
human macrophages upon b-glucan stimulation results in the
enhanced activation of dectin-1/Syk signaling pathway and more
efficient antifungal defense.
One common feature of many types of vesicles is their expression
of adhesion molecules. Our analysis demonstrated that b-glucan
stimulation clearly induced the secretion of proteins involved in
leukocyte migration like integrins, intracellular adhesion molecules
and matrix metalloproteinases (Supplemental Table I). In addition,
proteins that regulate actin cytoskeleton reorganization were highly
upregulated in cell culture media. Leukocyte migration from the
blood into tissues is vital for immune surveillance and inflammation.
During migration, the leukocytes bind to endothelial cell adhesion
molecules and then migrate across the vascular endothelium. In this
process, intracellular signals trigger an actin cytoskeleton reorganization and endothelial cell junction dissociation. These molecules
are believed to mediate cell-to-cell communication by facilitating
the interaction of secreted vesicles with recipient cells (41). After
interacting with molecules on the recipient cell surface, the exosomes may fuse with the recipient plasma membrane, leading to the
incorporation of proteins from the exosomal membrane into the
membrane and the release of exosome contents into the cytoplasm of
the recipient cell.
b-Glucans are potent activators of NLRP3 inflammasome in
macrophages (13, 42); this is an essential part of the innate immune
response to fungal infection also in vivo (43). Inflammasome activation is associated with the secretion of its central components, and
active caspase-1 has been shown to be a regulator of unconventional
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FIGURE 7. Inhibition of autophagy suppresses unconventional protein secretion in macrophages that have been activated through the dectin-1 pathway.
(A) List of autophagy proteins that were identified in the secretomes of b-glucan–stimulated macrophages. (B) Human macrophages were stimulated with
curdlan for the times indicated. Next, cell lysates were prepared, and the expression LC3 was analyzed with immunoblotting. Numbers below lanes indicate
the ratio of LC3-II/LC3-I. (C–E) Human macrophages were untreated or treated with 10 mM 3-MA for 1 h before curdlan stimulation (18). Next, the
expression of LC3 was analyzed from cell lysates with Western blotting (C), and the expression of IL-1b was analyzed from cell culture supernatants by
ELISA and Western blotting (D), respectively. The secreted proteins were separated by SDS-PAGE and visualized with silver-staining and Western blotting
(E). (F) Human macrophages were transfected with control siRNA and beclin-1 specific siRNA molecules, after which they were left unstimulated or
stimulated with curdlan for 18 h. Subsequently, cell culture supernatants were collected and concentrated, and the secreted proteins were separated by SDSPAGE and visualized with silver-staining and Western blotting. (G) The IL-1b secretion was analyzed from cell culture supernatants with ELISA, and (H)
TNF, CCL2, and CCL5 secretion was analyzed with Luminex assay.
The Journal of Immunology
protein secretion (28, 44, 45). In line with these reports, our current
results demonstrate the secretion of core components of NLRP3
inflammasome, caspase-1 and ASC, and known inflammasome
regulators cathepsin B and D in dectin-1–activated macrophages. A
more detailed analysis indicates that the mature forms of cathepsins
were secreted in exosomes, which might facilitate the release of
these proteases to adjacent cells in their biologically active forms
and result in NLRP3 inflammasome activation also in these cells.
Furthermore, our current results show that inflammasome function
is essential for the activation of unconventional protein secretion in
response to the dectin-1 pathway.
Both Src and Syk kinases have been linked to dectin-1 signaling
and NLRP3 inflammasome activation (13, 29, 43, 46). Our present
experiments revealed that the secretion of IL-1b, cathepsin B, and
cathepsin D upon dectin-1 activation was completely dependent
on Syk kinase. In contrast, Src kinases had only a minor role
in this process. In general, Src-family kinases and Syk tend to
operate together in signaling, with the Src acting upstream of Syk.
However, our results show that Syk can act independently of Src.
Recently, it was reported that particulate b-glucans, including
curdlan, activate dectin-1 signaling by clustering the receptor into
synapse-like structures from which tyrosine phosphatases CD45
and CD148 are excluded resulting in the activation of Src and Syk
kinases (47). Our results show that the activation of this phagocytic synapse by b-glucans results in the robust activation of unconventional protein secretion. The phagocytic synapse formed in
response to fungal cell walls provides a novel mechanism by
which the innate immune system can distinguish direct microbial
contact, from the detection of microbes at a distance.
Autophagy is an evolutionary conserved lysosomal pathway
involved in the turnover of long-lived proteins, cytoplasm, and
whole organelles. In response to nutrient-limiting conditions,
autophagy is activated to form a double-membrane autophagosome
(48). Next, the autophagosomes mature by fusing with lysosomes
resulting in the degradation of the cargo. In addition to starvationinduced autophagy, selective autophagic degradation is activated
in response to several other cellular stressors. These include
aggregated and misfolded proteins, damaged organelles, and
cytosol-invasive bacteria and viruses (49). Autophagy is emerging
as a central component of antimicrobial defense against a variety
of bacterial infections; however, little is known about the role of
autophagy in antifungal defense. Our current data demonstrate
that dectin-1/Syk signaling pathway, the major pattern recognition
receptor pathway involved in the induction of antifungal defense,
activates autophagy. The conversion of the cytosolic LC3-I to the
lipid-associated LC-II, which is found on autophagy membranes,
was increased in response to curdlan stimulation. Interestingly, it
has been shown previously that lipid-associated LC-II is recruited
to phagosomes containing b-glucans (50); this points to a role for
LC3 in regulating phagocytosis, in addition to being a central
player in autophagy. Furthermore, autophagy was essential for IL1b release and total unconventional protein secretion, but not for
conventional protein secretion, in macrophages activated through
the dectin-1 pathway. In line with these results, it was recently
shown that when autophagy is induced by starvation, enhanced
secretion of IL-1b is seen in the cells that have been stimulated by
known inflammasome activators (i.e., silica, nigericin, b-amyloid
fibrils, and alum) (32). It has also been shown that autophagy
negatively regulates IL-1b production by targeting ubiquitylated
NLRP3 inflammasomes for degradation (35); this indicates a dual
role for autophagy in the regulation of inflammatory response.
First, it is required for the secretion of IL-1b and for other unconventionally secreted proteins. Second, autophagy restricts inflammation by targeting inflammasome complexes to degradation.
In conclusion, we provide the first comprehensive characterization of the secretome and the associated intracellular signaling
pathways involved in dectin-1/Syk signaling in human primary
macrophages. In addition, we demonstrate an important role for
unconventional, vesicle-mediated protein secretion in dectin-1–
activated innate immune response.
Disclosures
The authors have no financial conflicts of interest.
References
1. Qi, C., Y. Cai, L. Gunn, C. Ding, B. Li, G. Kloecker, K. Qian, J. Vasilakos,
S. Saijo, Y. Iwakura, et al. 2011. Differential pathways regulating innate and
adaptive antitumor immune responses by particulate and soluble yeast-derived
b-glucans. Blood 117: 6825–6836.
2. Brown, G. D., and S. Gordon. 2001. Immune recognition. A new receptor for
beta-glucans. Nature 413: 36–37.
3. Brown, G. D., P. R. Taylor, D. M. Reid, J. A. Willment, D. L. Williams,
L. Martinez-Pomares, S. Y. Wong, and S. Gordon. 2002. Dectin-1 is a major
beta-glucan receptor on macrophages. J. Exp. Med. 196: 407–412.
4. Taylor, P. R., S. V. Tsoni, J. A. Willment, K. M. Dennehy, M. Rosas, H. Findon,
K. Haynes, C. Steele, M. Botto, S. Gordon, and G. D. Brown. 2007. Dectin-1 is
required for beta-glucan recognition and control of fungal infection. Nat.
Immunol. 8: 31–38.
5. Romani, L. 2011. Immunity to fungal infections. Nat. Rev. Immunol. 11: 275–
288.
6. Franchi, L., T. Eigenbrod, R. Muñoz-Planillo, and G. Nuñez. 2009. The
inflammasome: a caspase-1-activation platform that regulates immune responses
and disease pathogenesis. Nat. Immunol. 10: 241–247.
7. Martinon, F., K. Burns, and J. Tschopp. 2002. The inflammasome: a molecular
platform triggering activation of inflammatory caspases and processing of proILbeta. Mol. Cell 10: 417–426.
8. Nickel, W., and C. Rabouille. 2009. Mechanisms of regulated unconventional
protein secretion. Nat. Rev. Mol. Cell Biol. 10: 148–155.
9. Meissner, F., R. A. Scheltema, H. J. Mollenkopf, and M. Mann. 2013. Direct
proteomic quantification of the secretome of activated immune cells. Science
340: 475–478.
10. K€uttner, V., C. Mack, K. T. Rigbolt, J. S. Kern, O. Schilling, H. Busch,
L. Bruckner-Tuderman, and J. Dengjel. 2013. Global remodelling of cellular
microenvironment due to loss of collagen VII. Mol. Syst. Biol. 9: 657.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 8. The global innate immune response activated by b-glucans
in human macrophages. Detection of b-glucans by dectin-1 triggers significant transcriptional changes and activates both conventional and unconventional, vesicle-mediated protein secretion in macrophages. The
unconventional protein secretion is dependent on inflammasome activity
and active autophagy process, and it has an important role in the innate
immune response activated by b-glucans. MPR, mannose-6–phosphate
receptor; MVB, multivesicular body.
5961
5962
31. Goodridge, H. S., A. J. Wolf, and D. M. Underhill. 2009. Beta-glucan recognition by the innate immune system. Immunol. Rev. 230: 38–50.
32. Dupont, N., S. Jiang, M. Pilli, W. Ornatowski, D. Bhattacharya, and V. Deretic.
2011. Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1b. EMBO J. 30: 4701–4711.
33. Harris, J., M. Hartman, C. Roche, S. G. Zeng, A. O’Shea, F. A. Sharp,
E. M. Lambe, E. M. Creagh, D. T. Golenbock, J. Tschopp, et al. 2011. Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation. J.
Biol. Chem. 286: 9587–9597.
34. Nakahira, K., J. A. Haspel, V. A. Rathinam, S. J. Lee, T. Dolinay, H. C. Lam,
J. A. Englert, M. Rabinovitch, M. Cernadas, H. P. Kim, et al. 2011. Autophagy
proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat. Immunol. 12: 222–
230.
35. Shi, C. S., K. Shenderov, N. N. Huang, J. Kabat, M. Abu-Asab, K. A. Fitzgerald,
A. Sher, and J. H. Kehrl. 2012. Activation of autophagy by inflammatory signals
limits IL-1b production by targeting ubiquitinated inflammasomes for destruction. Nat. Immunol. 13: 255–263.
36. Dengjel, J., M. Høyer-Hansen, M. O. Nielsen, T. Eisenberg, L. M. Harder,
S. Schandorff, T. Farkas, T. Kirkegaard, A. C. Becker, S. Schroeder, et al. 2012.
Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens. Mol. Cell. Proteomics. 11:
M111.014035.
37. Record, M., C. Subra, S. Silvente-Poirot, and M. Poirot. 2011. Exosomes as
intercellular signalosomes and pharmacological effectors. Biochem. Pharmacol.
81: 1171–1182.
38. Bianchi, M. E. 2007. DAMPs, PAMPs and alarmins: all we need to know about
danger. J. Leukoc. Biol. 81: 1–5.
39. Gallucci, S., and P. Matzinger. 2001. Danger signals: SOS to the immune system.
Curr. Opin. Immunol. 13: 114–119.
40. Esteban, A., M. W. Popp, V. K. Vyas, K. Strijbis, H. L. Ploegh, and G. R. Fink.
2011. Fungal recognition is mediated by the association of dectin-1 and galectin3 in macrophages. Proc. Natl. Acad. Sci. USA 108: 14270–14275.
41. Théry, C., M. Ostrowski, and E. Segura. 2009. Membrane vesicles as conveyors
of immune responses. Nat. Rev. Immunol. 9: 581–593.
42. Kumar, H., Y. Kumagai, T. Tsuchida, P. A. Koenig, T. Satoh, Z. Guo, M. H. Jang,
T. Saitoh, S. Akira, and T. Kawai. 2009. Involvement of the NLRP3 inflammasome in innate and humoral adaptive immune responses to fungal beta-glucan. J. Immunol. 183: 8061–8067.
43. Gross, O., H. Poeck, M. Bscheider, C. Dostert, N. Hannesschläger, S. Endres,
G. Hartmann, A. Tardivel, E. Schweighoffer, V. Tybulewicz, et al. 2009. Syk
kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence. Nature 459: 433–436.
44. Martinon, F., A. Mayor, and J. Tschopp. 2009. The inflammasomes: guardians of
the body. Annu. Rev. Immunol. 27: 229–265.
45. Qu, Y., L. Franchi, G. Nunez, and G. R. Dubyak. 2007. Nonclassical IL-1 beta
secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J. Immunol.
179: 1913–1925.
46. Shio, M. T., S. C. Eisenbarth, M. Savaria, A. F. Vinet, M. J. Bellemare,
K. W. Harder, F. S. Sutterwala, D. S. Bohle, A. Descoteaux, R. A. Flavell, and
M. Olivier. 2009. Malarial hemozoin activates the NLRP3 inflammasome
through Lyn and Syk kinases. PLoS Pathog. 5: e1000559.
47. Goodridge, H. S., C. N. Reyes, C. A. Becker, T. R. Katsumoto, J. Ma, A. J. Wolf,
N. Bose, A. S. Chan, A. S. Magee, M. E. Danielson, et al. 2011. Activation of the
innate immune receptor Dectin-1 upon formation of a ‘phagocytic synapse’.
Nature 472: 471–475.
48. Rubinsztein, D. C., T. Shpilka, and Z. Elazar. 2012. Mechanisms of autophagosome biogenesis. Curr. Biol. 22: R29–R34.
49. Kuballa, P., W. M. Nolte, A. B. Castoreno, and R. J. Xavier. 2012. Autophagy
and the immune system. Annu. Rev. Immunol. 30: 611–646.
50. Ma, J., C. Becker, C. A. Lowell, and D. M. Underhill. 2012. Dectin-1-triggered
recruitment of light chain 3 protein to phagosomes facilitates major histocompatibility complex class II presentation of fungal-derived antigens. J. Biol. Chem.
287: 34149–34156.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
11. Lietzén, N., T. Ohman, J. Rintahaka, I. Julkunen, T. Aittokallio, S. Matikainen,
and T. A. Nyman. 2011. Quantitative subcellular proteome and secretome profiling of influenza A virus-infected human primary macrophages. PLoS Pathog.
7: e1001340.
12. Miettinen, J. J., S. Matikainen, and T. A. Nyman. 2012. Global secretome
characterization of herpes simplex virus 1-infected human primary macrophages.
J. Virol. 86: 12770–12778.
13. Kankkunen, P., L. Teirilä, J. Rintahaka, H. Alenius, H. Wolff, and S. Matikainen.
2010. (1,3)-beta-glucans activate both dectin-1 and NLRP3 inflammasome in
human macrophages. J. Immunol. 184: 6335–6342.
14. Pirhonen, J., T. Sareneva, M. Kurimoto, I. Julkunen, and S. Matikainen. 1999.
Virus infection activates IL-1 beta and IL-18 production in human macrophages
by a caspase-1-dependent pathway. J. Immunol. 162: 7322–7329.
15. Saijo, S., N. Fujikado, T. Furuta, S. H. Chung, H. Kotaki, K. Seki, K. Sudo,
S. Akira, Y. Adachi, N. Ohno, et al. 2007. Dectin-1 is required for host defense
against Pneumocystis carinii but not against Candida albicans. Nat. Immunol. 8:
39–46.
16. Ross, P. L., Y. N. Huang, J. N. Marchese, B. Williamson, K. Parker, S. Hattan,
N. Khainovski, S. Pillai, S. Dey, S. Daniels, et al. 2004. Multiplexed protein
quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging
reagents. Mol. Cell. Proteomics 3: 1154–1169.
17. Shilov, I. V., S. L. Seymour, A. A. Patel, A. Loboda, W. H. Tang, S. P. Keating,
C. L. Hunter, L. M. Nuwaysir, and D. A. Schaeffer. 2007. The Paragon Algorithm, a next generation search engine that uses sequence temperature values and
feature probabilities to identify peptides from tandem mass spectra. Mol. Cell.
Proteomics 6: 1638–1655.
18. Elias, J. E., and S. P. Gygi. 2007. Target-decoy search strategy for increased
confidence in large-scale protein identifications by mass spectrometry. Nat.
Methods 4: 207–214.
19. Backes, C., A. Keller, J. Kuentzer, B. Kneissl, N. Comtesse, Y. A. Elnakady,
R. M€
uller, E. Meese, and H. P. Lenhof. 2007. GeneTrail—advanced gene set
enrichment analysis. Nucleic Acids Res. 35(Web Server issue): W186–W192.
20. Mathivanan, S., and R. J. Simpson. 2009. ExoCarta: A compendium of exosomal
proteins and RNA. Proteomics 9: 4997–5000.
21. Tarca, A. L., S. Draghici, P. Khatri, S. S. Hassan, P. Mittal, J. S. Kim, C. J. Kim,
J. P. Kusanovic, and R. Romero. 2009. A novel signaling pathway impact
analysis. Bioinformatics 25: 75–82.
22. Kanehisa, M., S. Goto, M. Furumichi, M. Tanabe, and M. Hirakawa. 2010.
KEGG for representation and analysis of molecular networks involving diseases
and drugs. Nucleic Acids Res. 38(Database issue): D355–D360.
23. Ovaska, K., M. Laakso, S. Haapa-Paananen, R. Louhimo, P. Chen, V. Aittomäki,
E. Valo, J. Núñez-Fontarnau, V. Rantanen, S. Karinen, et al. 2010. Large-scale
data integration framework provides a comprehensive view on glioblastoma
multiforme. Genome Med 2: 65.
24. Öhman, T., J. Rintahaka, N. Kalkkinen, S. Matikainen, and T. A. Nyman. 2009.
Actin and RIG-I/MAVS signaling components translocate to mitochondria upon
influenza A virus infection of human primary macrophages. J. Immunol. 182:
5682–5692.
25. Vizcaı́no, J. A., R. G. Côté, A. Csordas, J. A. Dianes, A. Fabregat, J. M. Foster,
J. Griss, E. Alpi, M. Birim, J. Contell, et al. 2013. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids
Res. 41(Database issue): D1063–D1069.
26. Raposo, G., and W. Stoorvogel. 2013. Extracellular vesicles: exosomes, microvesicles, and friends. J. Cell Biol. 200: 373–383.
27. Colbert, J. D., S. P. Matthews, G. Miller, and C. Watts. 2009. Diverse regulatory
roles for lysosomal proteases in the immune response. Eur. J. Immunol. 39:
2955–2965.
28. Keller, M., A. R€
uegg, S. Werner, and H. D. Beer. 2008. Active caspase-1 is
a regulator of unconventional protein secretion. Cell 132: 818–831.
29. Kerrigan, A. M., and G. D. Brown. 2010. Syk-coupled C-type lectin receptors
that mediate cellular activation via single tyrosine based activation motifs.
Immunol. Rev. 234: 335–352.
30. Means, T. K., E. Mylonakis, E. Tampakakis, R. A. Colvin, E. Seung, L. Puckett,
M. F. Tai, C. R. Stewart, R. Pukkila-Worley, S. E. Hickman, et al. 2009. Evolutionarily conserved recognition and innate immunity to fungal pathogens by
the scavenger receptors SCARF1 and CD36. J. Exp. Med. 206: 637–653.
DECTIN-1 AND VESICLE-MEDIATED PROTEIN SECRETION