Global Characterization of Protein Secretion from Human Macrophages Following Non-canonical Caspase-4/5 Inflammasome Activation
2017; Elsevier BV; Volume: 16; Issue: 4 Linguagem: Inglês
10.1074/mcp.m116.064840
ISSN1535-9484
AutoresMartina B. Lorey, K. Rossi, Kari K. Eklund, Tuula A. Nyman, Sampsa Matikainen,
Tópico(s)Immune Response and Inflammation
ResumoGram-negative bacteria are associated with a wide spectrum of infectious diseases in humans. Inflammasomes are cytosolic protein complexes that are assembled when the cell encounters pathogens or other harmful agents. The non-canonical caspase-4/5 inflammasome is activated by Gram-negative bacteria-derived lipopolysaccharide (LPS) and by endogenous oxidized phospholipids. Protein secretion is a critical component of the innate immune response. Here, we have used label-free quantitative proteomics to characterize global protein secretion in response to non-canonical inflammasome activation upon intracellular LPS recognition in human primary macrophages. Before proteomics, the total secretome was separated into two fractions, enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation. We identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction. From these, 640 were identified from both fractions suggesting that the non-canonical inflammasome activates multiple, partly overlapping protein secretion pathways. We identified several secreted proteins that have a critical role in host response against severe Gram-negative bacterial infection. The soluble secretome (RS fraction) was highly enriched with inflammation-associated proteins upon intracellular LPS recognition. Several ribosomal proteins were highly abundant in the EV fraction upon infection, and our data strongly suggest that secretion of translational machinery and concomitant inhibition of translation are important parts of host response against Gram-negative bacteria sensing caspase-4/5 inflammasome. Intracellular recognition of LPS resulted in the secretion of two metalloproteinases, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and MMP14, in the enriched EV fraction. ADAM10 release was associated with the secretion of TNF, a key inflammatory cytokine, and M-CSF, an important growth factor for myeloid cells probably through ADAM10-dependent membrane shedding of these cytokines. Caspase-4/5 inflammasome activation also resulted in secretion of danger-associated molecules S100A8 and prothymosin-α in the enriched EV fraction. Both S100A8 and prothymosin-α are ligands for toll-like receptor 4 recognizing extracellular LPS, and they may contribute to endotoxic shock during non-canonical inflammasome activation. Gram-negative bacteria are associated with a wide spectrum of infectious diseases in humans. Inflammasomes are cytosolic protein complexes that are assembled when the cell encounters pathogens or other harmful agents. The non-canonical caspase-4/5 inflammasome is activated by Gram-negative bacteria-derived lipopolysaccharide (LPS) and by endogenous oxidized phospholipids. Protein secretion is a critical component of the innate immune response. Here, we have used label-free quantitative proteomics to characterize global protein secretion in response to non-canonical inflammasome activation upon intracellular LPS recognition in human primary macrophages. Before proteomics, the total secretome was separated into two fractions, enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation. We identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction. From these, 640 were identified from both fractions suggesting that the non-canonical inflammasome activates multiple, partly overlapping protein secretion pathways. We identified several secreted proteins that have a critical role in host response against severe Gram-negative bacterial infection. The soluble secretome (RS fraction) was highly enriched with inflammation-associated proteins upon intracellular LPS recognition. Several ribosomal proteins were highly abundant in the EV fraction upon infection, and our data strongly suggest that secretion of translational machinery and concomitant inhibition of translation are important parts of host response against Gram-negative bacteria sensing caspase-4/5 inflammasome. Intracellular recognition of LPS resulted in the secretion of two metalloproteinases, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and MMP14, in the enriched EV fraction. ADAM10 release was associated with the secretion of TNF, a key inflammatory cytokine, and M-CSF, an important growth factor for myeloid cells probably through ADAM10-dependent membrane shedding of these cytokines. Caspase-4/5 inflammasome activation also resulted in secretion of danger-associated molecules S100A8 and prothymosin-α in the enriched EV fraction. Both S100A8 and prothymosin-α are ligands for toll-like receptor 4 recognizing extracellular LPS, and they may contribute to endotoxic shock during non-canonical inflammasome activation. Gram-negative bacteria are associated with a wide spectrum of infectious diseases in humans, including pneumonia, bloodstream infections, wound infections, meningitis, as well as several sexually transmitted diseases (1.Kaye K.S. Pogue J.M. Infections Caused by Resistant Gram-Negative Bacteria: Epidemiology and Management.Pharmacotherapy. 2015; 35 (doi:10.1002/phar.1636): 949-962Crossref PubMed Scopus (231) Google Scholar). Innate immunity is the first defense response against pathogens. Macrophages are central effector cells of innate immunity detecting the presence of Gram-negative bacteria with their pattern recognition receptors (2.Storek K.M. Monack D.M. Bacterial recognition pathways that lead to inflammasome activation.Immunol. Rev. 2015; 265: 112-129Crossref PubMed Scopus (89) Google Scholar). Gram-negative bacteria contain pathogen-associated molecular patterns, including the major cell wall component LPS, a potent activator of the innate immune response. Extracellular LPS is recognized by pattern recognition receptors called Toll-like receptor 4 (TLR4) (3.Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6440) Google Scholar), which activates transcription of genes encoding cytokines, chemokines, and co-stimulatory molecules in antigen-presenting cells (4.Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.Nature. 1997; 388: 394-397Crossref PubMed Scopus (4426) Google Scholar). This results in the activation of the anti-microbial defense and adaptive immune response. Infection with Gram-negative bacteria may lead to the life-threatening condition called endotoxic shock, which is one of the major causes of death in intensive care units (5.Angus D.C. van der Poll T. Severe sepsis and septic shock.N. Engl. J. Med. 2013; 369: 840-851Crossref PubMed Scopus (1860) Google Scholar). This condition develops due to a dysregulation of the immune response, and the mechanisms initially recruited to fight the infection produce life-threatening tissue damage. Inflammasomes are multimeric protein complexes of the innate immune system that are critical for both local and systemic inflammation (6.Schroder K. Tschopp J. The inflammasomes.Cell. 2010; 140: 821-832Abstract Full Text Full Text PDF PubMed Scopus (4137) Google Scholar). The most studied inflammasome structure is the canonical NLRP3 (NACHT, LRR, and PYD domain-containing protein 3) inflammasome, which is activated by several microbial stimuli as well as by endogenous danger signals, including ATP and monosodium urate (7.He Y. Hara H. Núñez G. Mechanism and regulation of NLRP3 inflammasome activation.Trends Biochem. Sci. 2016; 41: 1012-1021Abstract Full Text Full Text PDF PubMed Scopus (1484) Google Scholar). The NLRP3 inflammasome activates caspase-1, which in turn facilitates the proteolytic processing and secretion of pro-inflammatory cytokines IL-1β and IL-18. Recently, Kayagaki et al. (8.Kayagaki N. Warming S. Lamkanfi M. Vande Walle L. Louie S. Dong J. Newton K. Qu Y. Liu J. Heldens S. Zhang J. Lee W.P. Roose-Girma M. Dixit V.M. Non-canonical inflammasome activation targets caspase-11.Nature. 2011; 479: 117-121Crossref PubMed Scopus (1657) Google Scholar) showed that mouse caspase-11 is involved in the recognition of infections with Gram-negative bacteria. This led to the discovery of the non-canonical caspase-11 inflammasome, which activates pyroptosis, an inflammatory form of cell death, in response to infections with Gram-negative bacteria. Subsequently, it was shown that caspase-11 recognizes intracellular LPS independently of TLR4 and mediates endotoxic shock in mice (9.Hagar J.A. Powell D.A. Aachoui Y. Ernst R.K. Miao E.A. Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock.Science. 2013; 341: 1250-1253Crossref PubMed Scopus (821) Google Scholar, 10.Shi J. Zhao Y. Wang Y. Gao W. Ding J. Li P. Hu L. Shao F. Inflammatory caspases are innate immune receptors for intracellular LPS.Nature. 2014; 514: 187-192Crossref PubMed Scopus (47) Google Scholar). Very recently, also endogenous oxidized phospholipids were discovered to activate the caspase-11 inflammasome and IL-1 release without inducing pyroptosis (11.Zanoni I. Tan Y. Di Gioia M. Broggi A. Ruan J. Shi J. Donado C.A. Shao F. Wu H. Springstead J.R. Kagan J.C. An endogenous caspase-11 ligand elicits interleukin-1 release from living dendritic cells.Science. 2016; 352: 1232-1236Crossref PubMed Scopus (324) Google Scholar). Human caspase-4 and caspase-5 are homologs of mouse caspase-11, and it was demonstrated that these inflammatory caspases also directly bind to intracellular LPS resulting in their activation (8.Kayagaki N. Warming S. Lamkanfi M. Vande Walle L. Louie S. Dong J. Newton K. Qu Y. Liu J. Heldens S. Zhang J. Lee W.P. Roose-Girma M. Dixit V.M. Non-canonical inflammasome activation targets caspase-11.Nature. 2011; 479: 117-121Crossref PubMed Scopus (1657) Google Scholar, 9.Hagar J.A. Powell D.A. Aachoui Y. Ernst R.K. Miao E.A. Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock.Science. 2013; 341: 1250-1253Crossref PubMed Scopus (821) Google Scholar, 10.Shi J. Zhao Y. Wang Y. Gao W. Ding J. Li P. Hu L. Shao F. Inflammatory caspases are innate immune receptors for intracellular LPS.Nature. 2014; 514: 187-192Crossref PubMed Scopus (47) Google Scholar). Subsequent studies have shown that the human non-canonical caspase-4/5 inflammasome can activate the canonical NLRP3 inflammasome resulting in secretion of IL-1β and IL-18 (12.Yang J. Zhao Y. Shao F. Non-canonical activation of inflammatory caspases by cytosolic LPS in innate immunity.Curr. Opin. Immunol. 2015; 32: 78-83Crossref PubMed Scopus (170) Google Scholar). Protein secretion from cells is mediated through conventional and unconventional pathways. Conventionally secreted proteins have a signal peptide on their N terminus. The signal sequence directs them through the endoplasmic reticulum and Golgi apparatus to vesicles, which fuse with the plasma membrane and release their cargo into the extracellular space. Unconventionally secreted proteins lack the signal peptide and are secreted directly through the plasma membrane through vesicles, including exosomes derived from multivesicular bodies (13.Rabouille C. Malhotra V. Nickel W. Diversity in unconventional protein secretion.J. Cell Sci. 2012; 125: 5251-5255Crossref PubMed Scopus (192) Google Scholar, 14.Raposo G. Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends.J. Cell Biol. 2013; 200: 373-383Crossref PubMed Scopus (5199) Google Scholar). Recent system-level studies of protein secretion by activated immune cells, including macrophages, have highlighted the importance of different secretory pathways in innate immune response (15.Meissner F. Scheltema R.A. Mollenkopf H.J. Mann M. Direct proteomic quantification of the secretome of activated immune cells.Science. 2013; 340: 475-478Crossref PubMed Scopus (149) Google Scholar, 16.Öhman T. Teirilä L. Lahesmaa-Korpinen A.M. Cypryk W. Veckman V. Saijo S. Wolff H. Hautaniemi S. Nyman T.A. Matikainen S. Dectin-1 pathway activates robust autophagy-dependent unconventional protein secretion in human macrophages.J. Immunol. 2014; 192: 5952-5962Crossref PubMed Scopus (66) Google Scholar, 17.Cypryk W. Ohman T. Eskelinen E.-L. Matikainen S. Nyman T.A. Quantitative proteomics of extracellular vesicles released from human monocyte-derived macrophages upon β-glucan stimulation.J. Proteome Res. 2014; 13: 2468-2477Crossref PubMed Scopus (41) Google Scholar, 18.Cypryk W. Lorey M. Puustinen A. Nyman T.A. Matikainen S. Proteomic and bioinformatic characterization of extracellular vesicles released from human macrophages upon influenza A virus infection.J. Proteome Res. 2017; 16: 217-227Crossref PubMed Scopus (47) Google Scholar). We have shown that NLRP3 inflammasome activators ATP and crystallized monosodium urate induce robust protein secretion in human macrophages (19.Välimäki E. Miettinen J.J. Lietzén N. Matikainen S. Nyman T.A. Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages.Mol. Cell. Proteomics. 2013; 12: 749-763Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 20.Välimäki E. Cypryk W. Virkanen J. Nurmi K. Turunen P.M. Eklund K.K. Åkerman K.E. Nyman T.A. Matikainen S. Calpain activity is essential for ATP-driven unconventional vesicle-mediated protein secretion and inflammasome activation in human macrophages.J. Immunol. 2016; 197: 3315-3325Crossref PubMed Scopus (44) Google Scholar), but the effect of non-canonical inflammasome activation on global protein secretion has remained uncharacterized. Here, we studied protein secretion in human macrophages in response to non-canonical caspase-4/5 inflammasome activation using label-free quantitative proteomics combined with bioinformatics. We show that non-canonical inflammasome activation triggers robust protein secretion through multiple secretion pathways and that the secreted proteins have important roles in host response against severe Gram-negative bacterial infection. Primary human macrophages were derived from leukocyte-rich buffy coats from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). All human blood donors provided written informed consent. Monocytes from three donors per experiment were isolated and differentiated into macrophages as described previously (21.Pirhonen J. Sareneva T. Kurimoto M. Julkunen I. Matikainen S. Virus infection activates IL-1β and IL-18 production in human macrophages by a caspase-1-dependent pathway.J. Immunol. 1999; 162: 7322-7329PubMed Google Scholar). In total, 1.4 × 106 monocytes were seeded per well on 6-well plates. The monocytes were cultured in serum-free macrophage media (Macrophage-SFM, Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (ImmunoTools, Germany) and 50 units/ml penicillin/streptomycin (Lonza, Basel, Switzerland) at 37 °C and 5% CO2 for 6 days to polarize the monocytes into macrophages of the pro-inflammatory M1-phenotype. On day 6, the cells were washed with PBS, supplied with fresh RPMI 1640 medium (Gibco) supplemented with l-glutamate and antibiotics, and subsequently mock-transfected with Lipofectamine or transfected with Ultrapure LPS (Invivogen, Escherichia coli 0111:B4 1 mg/ml) using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific) for the times indicated. Caspase-4 inhibitor (Z-YVAD-fmk, 1The abbreviations used are: Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; DAMP, damage-associated molecular pattern; EV, enriched extracellular vesicle; IPA, Ingenuity Pathway Analysis; ITGAX, integrin α-X; KEGG, Kyoto Encyclopedia of Genes and Genomes; LDH, lactate dehydrogenase; M-CSF, macrophage colony-stimulating factor; RS, Rest-secretome; VAS, V-type proton ATPase subunit S1; FDR, false discovery rate. 25 μm) was purchased from R&D Systems (Minneapolis, MN), and when used, it was added to media 1 h before LPS transfection. Label-free quantitative proteomics was used to identify and quantify proteins secreted in response to LPS transfection. Total secretomes were fractionated into two fractions. The extracellular vesicles were enriched from equal supernatant volumes (40–60 ml of medium per condition) by size-exclusion filtration using Amicon 15-ml tubes with a 100-kDa cutoff. Subsequently, the flow-through was concentrated using Amicon 15-ml filters with a 10-kDa cutoff. The sample volumes were equalized using PBS, and equal volumes of the protein fractions were separated by SDS-PAGE and silver-stained (22.O'Connell K.L. Stults J.T. Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymatic digests.Electrophoresis. 1997; 18: 349-359Crossref PubMed Scopus (208) Google Scholar). Then the gel lanes were cut into 5–6 pieces each, and the proteins were in-gel digested with trypsin (Promega) overnight in 37 °C and eluted as described previously (23.Ohman T. Lietzén N. Välimäki E. Melchjorsen J. Matikainen S. Nyman T.A. Cytosolic RNA recognition pathway activates 14-3-3 protein mediated signaling and caspase-dependent disruption of cytokeratin network in human keratinocytes.J. Proteome Res. 2010; 9: 1549-1564Crossref PubMed Scopus (46) Google Scholar). Peptides were desalted and concentrated before mass spectrometry by the STAGE-TIP method using a C18 resin disk (3 M Empore). The peptides were eluted twice with 0.1% TFA, 50% ACN, dried, and solubilized in 7 μl of 0.1% TFA for mass spectrometry analysis. Each peptide mixture was analyzed on an Easy nLC1000 nano-LC system connected to a quadrupole Orbitrap mass spectrometer (QExactive, ThermoElectron, Bremen, Germany) equipped with a nanoelectrospray ion source (EasySpray/Thermo). For the liquid chromatography separation of the peptides, we employed an EasySpray column capillary of 25-cm bed length (C18, 2-μm beads, 100 Å, 75-μm inner diameter, Thermo). The flow rate was 300 nl/min, and the peptides were eluted with a 2–30% gradient of solvent B in 60 min. Solvent A was aqueous 0.1% formic acid, and solvent B was 100% acetonitrile, 0.1% formic acid. The data-dependent acquisition automatically switched between MS and MS/MS mode. Survey full scan MS spectra were acquired from a mass-to-charge ratio (m/z) of 400 to 1200 with the resolution R = 70,000 at m/z 200 after accumulation to a target of 3,000,000 ions in the quadruple. For MS/MS, the 10 most abundant multiple-charged ions were selected for fragmentation on the high energy collision dissociation (HCD) cell at a target value of 100,000 charges or maximum acquisition time of 100 ms. The MS/MS scans were collected at a resolution of 17,500. Target ions already selected for MS/MS were dynamically excluded for 30 s. The resulting MS raw files were submitted to the MaxQuant software version 1.5.3.8 for protein identification using the Andromeda search engine. Carbamidomethyl (C) was set as a fixed modification, and protein N-acetylation and methionine oxidation were set as variable modifications. First search peptide tolerance of 20 ppm and main search error of 4.5 ppm were used. Trypsin without proline restriction enzyme option was used, with two allowed miscleavages. The minimal unique + razor peptides number was set to 1, and the allowed FDR was 0.01 (1%) for peptide and protein identification. Label-free quantitation was employed with default settings. The SwissProt human database was used (August, 2016, with 154,660 entries) for the database searches. Known contaminants as provided by MaxQuant and identified in the samples were excluded from further analysis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (24) partner repository with the dataset identifier PXD005083 and to MS-Viewer (45) with Search Keys: zrbigvruog and vvsyumthop. The proteomic datasets were submitted to EnrichR (25.Chen E.Y. Tan C.M. Kou Y. Duan Q. Wang Z. Meirelles G.V. Clark N.R. Ma'ayan A. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool.BMC Bioinformatics. 2013; 14: 128Crossref PubMed Scopus (3073) Google Scholar). The output files of the enrichment analysis are tables that include p values, Benjamini-Hochberg adjusted p values, the z score of the deviation from the expected rank, as well as the "combined score," which is the combination of the p value with the z score by multiplying these two numbers as follows: c = ln(p)·z. In addition, the proteomic datasets were analyzed with Ingenuity Pathway Analysis software (IPA, Ingenuity Systems, Mountain View, CA, www.ingenuity.com) and STRING (http://string-db.org/ (26.Szklarczyk D. Franceschini A. Wyder S. Forslund K. Heller D. Huerta-Cepas J. Simonovic M. Roth A. Santos A. Tsafou K.P. Kuhn M. Bork P. Jensen L.J. von Mering C. STRING v10: protein-protein interaction networks, integrated over the tree of life.Nucleic Acids Res. 2015; 43: SD447-SD452Crossref Scopus (6757) Google Scholar)). The LDH release of cells was measured using the cytotoxicity detection kit (LDH) (Roche Diagnostics, Switzerland) according to the manufacturer's instructions. The human cytokine Luminex Bio-Plex Pro immunoassay kit designed to detect cytokines IL-1β, IL-18, and TNF were from Bio-Rad. The Luminex assay was performed according to the manufacturer's instructions. Protein samples were denatured at 95 °C for 10 min and separated on SDS-PAGE, transferred to PVDF transfer membranes (Trans-Blot Turbo Transfer System, Bio-Rad), blocked with 5% non-fat milk or 5% BSA in TBS/Tween (TBS-T), and incubated overnight at 4 °C with primary antibodies. The membranes were washed and incubated with appropriate HRP-conjugated secondary antibody for 1 h at room temperature, and unbound antibody was removed by washing with TBS-T. Proteins were visualized with Western Lightning ECL (PerkinElmer Life Sciences) on a ChemiDoc MP Imaging System (Bio-Rad). Antibodies against annexin-1 (sc-12740), galectin-3 (sc-56108), and Alix (sc-53540) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody against P-eIF2α (catalog no. 9721S) was purchased from Cell Signaling Technologies (Danvers, MA). The antibody against CD11c (ITGAX, ab52632) was purchased from Abcam PLC (Cambridge, UK). Secondary antibodies were purchased from Dako (Dako Denmark A/S). We analyzed three independent biological replicates with cells from two or three individual donors in each replicate with label-free quantitative proteomics. The allowed FDR was 0.01 (1%) for peptide and protein identification, and label-free quantitation was employed with default MaxQuant settings. The proteins with at least 2-fold increased secretion in all three biological replicates or in two out of three biological replicates with no opposing quantification values in the third replicate were considered to have increased secretion upon LPS transfection and were included for further bioinformatics analysis. Protein secretion is a critical component of the innate immune response. Here, we have characterized the global protein release during intracellular LPS recognition in human macrophages. Intracellular LPS that activates the non-canonical inflammasome in macrophages triggers pyroptosis, which is associated with LDH release from the cells (27.Rayamajhi M. Zhang Y. Miao E. Detection of pyroptosis by measuring released lactate dehydrogenase activity.Methods Mol. Biol. 2013; 1040: 85-90Crossref PubMed Scopus (107) Google Scholar). We first measured the kinetics of LDH release from macrophages upon intracellular LPS recognition. Macrophages were mock-transfected or transfected with LPS after which cell culture supernatants were collected, and the LDH assay was performed. LDH release increased in a time-dependent manner from human macrophages starting at 3 h after LPS transfection (Fig. 1A). Triggering non-canonical caspase-4/5 inflammasome results in the activation of the NLRP3 inflammasome and leads to secretion of the biologically active form of IL-18. To study the kinetics of IL-18 secretion, macrophages were transfected with LPS for different time periods after which cell culture supernatants were collected, and IL-18 secretion was studied by Luminex assay. IL-18 secretion started at 3 h after LPS transfection correlating with LDH release (Fig. 1B). Then, we examined how the intracellular LPS recognition pathway affects global protein secretion. For this, macrophages were mock-transfected or transfected with LPS for different time periods, and the cell culture supernatants were collected and concentrated. The total secretome was separated into two fractions as follows: enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation as described previously (16.Öhman T. Teirilä L. Lahesmaa-Korpinen A.M. Cypryk W. Veckman V. Saijo S. Wolff H. Hautaniemi S. Nyman T.A. Matikainen S. Dectin-1 pathway activates robust autophagy-dependent unconventional protein secretion in human macrophages.J. Immunol. 2014; 192: 5952-5962Crossref PubMed Scopus (66) Google Scholar). After this, the proteins in EV and RS fractions were separated by SDS-PAGE and visualized with silver staining. Enhanced protein secretion was seen in both EV and RS fractions already at 1.5 h after LPS transfection, and protein secretion clearly increased time-dependently after stimulation (supplemental Fig. 1). Protein secretion dramatically increased at 6 h after LPS transfection (supplemental Fig. 1), probably due to cell death, which was seen as high secretion of LDH at 6 h post-stimulation (Fig. 1A). This showed that intracellular LPS recognition pathway is a potent activator of protein secretion. To further characterize activation of EV secretion in human macrophages in response to LPS transfection, we studied the secretion kinetics of known EV marker proteins Alix, annexin-1, galectin-3, and ITGAX. In accordance with silver staining results, secretion of these proteins increased time-dependently in human macrophages in response to LPS transfection (Fig. 1C). We have previously shown that TLR4 activation by extracellular LPS does not induce EV-mediated protein secretion in human macrophages (16.Öhman T. Teirilä L. Lahesmaa-Korpinen A.M. Cypryk W. Veckman V. Saijo S. Wolff H. Hautaniemi S. Nyman T.A. Matikainen S. Dectin-1 pathway activates robust autophagy-dependent unconventional protein secretion in human macrophages.J. Immunol. 2014; 192: 5952-5962Crossref PubMed Scopus (66) Google Scholar). In contrast to this, the current results strongly suggest that intracellular LPS is a potent activator of EV-mediated protein secretion. Next, we wanted to verify that LDH release and IL-18 secretion induced by LPS transfection is dependent on caspase activity. For this, macrophages were pre-treated with caspase-4 inhibitor (Z-YVAD-fmk) before stimulation with intracellular LPS. Both LDH release (Fig. 2A) and IL-18 secretion (Fig. 2B) were completely suppressed by the inhibitor demonstrating that LPS transfection-induced LDH and IL-18 response is dependent on the non-canonical inflammasome. To demonstrate that intracellular LPS-induced EV-mediated protein secretion is dependent on the non-canonical inflammasome, macrophages were transfected with LPS for 3 h in the absence and presence of caspase-4 inhibitor. After this, the EV fraction was enriched, and the proteins were separated by SDS-PAGE and visualized with silver staining. The inhibitor clearly decreased EV-mediated protein secretion in response to LPS transfection (Fig. 2C). In accordance with this result, Western blotting analysis showed that caspase-4 inhibitor completely blocks secretion of EV marker proteins ITGAX and annexin-1 induced by LPS transfection (Fig. 2D). In conclusion, these results show that EV-mediated protein secretion following intracellular LPS recognition is dependent on non-canonical inflammasome activation. To characterize the effect of intracellular LPS stimulation to total protein secretion, we performed LC-MS/MS analysis using label-free quantification (Fig. 3). Based on the kinetic experiments, we isolated EV and RS fractions from mock-transfected and LPS-transfected macrophages at 1.5 h after stimulation for subsequent analysis. The proteomic analysis was performed for three independent biological replicates. Altogether, we identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction (Fig. 3 and supplemental Tables 1 and 2). From these, 640 were identified from both fractions. This overlap is in line with a recent study by Zhu et al. (28.Zhu Y. Chen X. Pan Q. Wang Y. Su S. Jiang C. Li Y. Xu N. Wu L. Lou X. Liu S. A comprehensive proteomics analysis reveals a secretory path- and status-dependent signature of exosomes released from tumor-associated macrophages.J. Proteome Res. 2015; 14: 4319-4331Crossref PubMed Scopus (57) Google Scholar), who characterized exosomes and exosome-free secretome fractions from tumor-associated macrophages. They postulate that the secretion of macrophage proteins follows multiple pathways, including vesicles only, conventional secretion only, and dual pathways. All the identified proteins were classified based on their cellular localization, biological processes, and molecular functions as well as canonical pathways to get an overview of the EV and RS proteomes (supplemental Tables 3 and 4). Classification based on cellular localization showed that extracellular vesicular exosome, cytosol, and focal adhesion are the main components in both fractions. The top canonical pathways in the EV fraction dataset include
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