Inflammasome-Derived Exosomes Activate NF-ÎºB Signaling in

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Inflammasome-derived exosomes activate NF-#B signaling in macrophages Yuehui Zhang, Fangbing Liu, Yanzhi Yuan, Chaozhi Jin, Cheng Chang, Yunping Zhu, Xiuyuan Zhang, Chunyan Tian, Fuchu He, and Jian Wang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00599 • Publication Date (Web): 29 Sep 2016 Downloaded from http://pubs.acs.org on September 30, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

Inflammasome-derived exosomes activate NF-κB signaling in macrophages Yuehui Zhang‡§, Fangbing Liuǁ§, Yanzhi Yuan‡, Chaozhi Jin‡, Cheng Chang‡, Yunping Zhu‡, Xiuyuan Zhang‡, Chunyan Tian‡, Fuchu He‡*, Jian Wangǁ* ǁ

Anhui Medical University, Hefei, Anhui 230032, China; State Key Laboratory of Proteomics,

Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China; National Center for Protein Sciences Beijing, Beijing 102206, China. ‡

State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of

Radiation Medicine, Beijing 102206, China; National Center for Protein Sciences Beijing, Beijing 102206, China.

ABSTRACT Exosomes are secreted small vesicles that mediate various biological processes, such as tumorigenesis and immune response. However, whether the inflammasome signaling leads to the change of constituent of exosomes and its roles in immune response remain to be determined. We isolated the exosomes from macrophages with treatment of mock, endotoxin or endotoxin/ nigericin. A label-free quantification method by MS/MS was used to identify the components of exosomes. Totally 2331 proteins were identified and 513 proteins were exclusively detected in exosomes with endotoxin and nigericin treatment. The differentially expressed proteins were classified by Gene Ontology and KEGG pathways. The immune response related proteins and signaling pathways were specifically enriched in inflammasome-derived exosomes. Moreover, we treated macrophages with the exosomes from different stimulation. We found that 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

inflammasome-derived exosomes directly activate NF-κB signaling pathway, while the control or endotoxin-derived exosomes have no effect. The inflammatory signaling was amplified in neighbor cells in an exosome-dependent way. The inflammsome-derived exosomes might be used to augment the immune response in diseases treatment and prevent of the transfer of these exosomes might ameliorate auto-immune diseases. KEYWORDS: exosomes, inflammatory signaling, inflammasome, proteomics

2


Page 2 of 32

Page 3 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Inflammasomes are multimeric protein complexes formed during innate immune response1-3. The pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs)4. The members of PRRs, such as NOD-like receptor (NLR) family, form inflammasome to activate caspase-1. Then, the cytokines, interleukin 1β (IL-1β) and IL-18 are processed by caspase-1. The activation of inflammsome leads to pyroptosis (a type of inflammatory cell death) and release of mature IL-1β and IL-18, which is mediated by gasdermin D (GSDMD)5, 6. NLRP3 is a most-studied member of NLR family7. The activation of NLRP3 inflammasome needs two distinct steps4, 8. First, the expression of NLRP3 and pro-IL-1β is induced by NF-κB pathway, such as endotoxin (lipopolysaccharide, LPS) binding to TLR4. Second signal triggers the assembly of NLRP3 inflammasome, which includes ATP, nigericin and monosodium urate (MSU) crystals etc. ASC (also named as PYCARD) has been known as an essential adaptor of different PRRs. After inflammasome activation, ASC forms a perinuclear large speck in cells9. ASC specks have been reported to be released by dying cells4, 9. The engulfment of ASC specks leads to activate caspase-1 in macrophages10. The inflammasomes are related to various inflammatory diseases, such as multiple sclerosis, Alzheimer’s disease, diabetes, rheumatoid arthritis and gout etc4, 11, 12. It has been reported that NLRP1 inflammasome is found in exosomes from brain and spinal cord injury patients13. Exosomes are secreted small vesicles with diameters of 30-150 nm from cells14-17. These vesicles transfer protein, mRNA and miRNA to recipient cells to mediate many biological processes, including tumorigenesis, metastasis, drug resistance and immune response etc16, 18-21. Dendritic cells-derived exosomes modulate the inflammatory response to endotoxin22. Another 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 32

study suggests that the secretion of IL-1β is correlated with exosome release23. Exosomes also transfer virulence factors into host to restrain innate immune responses24. The proteome of exosomes has been analyzed from cell lines, tumor cells and body fluid. The specific proteins of different exosomes are regarded as biomarkers for disease diagnosis19, 25. The macrophage migration inhibitory factor (MIF) has been identified in exosomes of pancreatic cancer and might be a prognostic maker for the liver metastasis26. Glypican-1 is identified as biomarker for the patients with early and late-stage pancreatic cancer27. Melanoma exosomes enhance metastasis to bones and lungs through Met28. The integrins of exosomes are associated with lung metastasis and liver metastasis of tumor29. Exosomes have potential uses in diseases treatment19.

Urinary

exosomes

inhibit

bacterial

growth30.

Mesenchymal

stem

cells

(MSCs)-derived exosomes induce angiogenesis through proteins of PDGF, EGF, FGF and NF-κB signaling pathways31. MSCs-derived exosomes inhibit macrophage activation through suppressing of TLR signaling pathway32. Reducing expression of MIF and Met in exosomes inhibits tumor metastasis26, 28. However, whether inflammasome signaling affects the secretion of exosomes and its functional roles remain to be determined. In this study, we challenged the murine macrophages with an agonist of NLRP3 inflammasome signaling, LPS/nigericin. More than 2300 exosomal proteins were obtained. The inflammasome-derived exosomes contain specific components that directly activate NF-κB signaling pathway.

4


Page 5 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EXPERIMENTAL SECTION Cell Culture For the generation of bone-marrow derived macrophages (BMDMs), 8-wk-old C57BL/6 mice were sacrificed, and the cells from tibial bone marrow were washed into a culture dish with phosphate-buffered saline (PBS). The cells were collected in a sterile 50 ml conical centrifuge tube on ice. Then, the cells were centrifuged 5 min at 200 g. The supernatant was removed and the cell pellet was resuspended with culture medium. The cells were plated onto a sterile plastic petri dish and cultured in RPMI 1640 with 50 ng/mL macrophage colony-stimulated factor (MCSF) and 10% FBS (which was depleted of endogenous exosomes by overnight centrifugation at 100,000g). Don't rock the cells and change culture medium after three days. The cells were cultured in a humidified incubator with 5% CO2 at 37°C for 7 days. After that, the culture supernatant was removed and discarded. Two surface antigens, CD11b and F4/80, were checked for the isolated BMDMs. More than 98% of the isolated BMDMs were CD11b+/F4/80+. Isolation of Exosomes To obtain exosomes, about 3×107 BMDMs were plated onto two 150 mm petri dishes. BMDMs were primed with 1,000 ng/ml LPS (Sigma) for 6 h or not, and then stimulated with 10 µM nigericin (InvivoGen) for 30 min. Exosomes from culture supernatants were isolated by differential centrifugation as described33. Briefly, the supernatants were centrifuged at 300 g for 10 min to remove whole cells and debris. Then, the supernatants were centrifuged at 2,000 g for 10 min and 10,000 g for 30 min to remove large membrane fragments. Finally, the supernatants were centrifuged at 100,000 g for 70 min to obtain exosomes. Next, the exosome-rich pellet was washed with phosphate-buffered saline (PBS) and centrifuged at 100,000 g for 70 min. The 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

isolated exosomes were fixed and examined by a Phillips CM120 BioTwin transmission electron microscopy (TEM). Western Blot The protein samples were separated by SDS-PAGE, followed by immunoblot analyses with appropriate antibodies and detected with the ECL substrate. Antibodies to NLRP3, and caspase-1 were from AdipoGen. Antibodies to TSG101 and β-actin were from Sigma. Antibody to IL-1β was from Abcam. All other antibodies were from Cell Signaling Technology. Proteomic Analysis of Exosomes by LC-MS/MS The protein samples were resolved by 10% SDS-PAGE, and stained with Coomassie Brilliant Blue R250 in parallel. The full lanes were cut into small bands, despite visible protein bands or not. These bands were digested with 100 ng of trypsin overnight, extracted twice with 100% acetonitrile, and dried in a Savant Speed-Vac. The peptides were then re-suspended in 0.1% Formic acid and analyzed using an ultra-performance LC-MS/MS platform. The LC separation was performed on an Easy nLC 1000 (Thermo Fisher Scientific) with an in-house packed capillary column (150 µm I.D.×12 cm) with 1.9 µm C18 reverse-phase fused-silica (Michrom Bioresources Inc.). The sample was eluted with a 78 min nonlinear gradient ramped from 5% to 30% mobile phase B (phase A: 0.1% formic acid in water, phase B: 0.1% FA in ACN) at a 0.6 µL/min flow rate. Eluted peptides were analyzed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific). The MS1 was analyzed over a mass range of 300-1400 Da with a resolution of 70000 at m/z 200. The isolation width was 3m/z for precursor ion selection. The automatic gain control (AGC) was set to 3×106, and the maximum injection time (MIT) was 60 ms. The MS2 was analyzed using data-dependent mode searching for the 20 most intense ions fragmented in the HCD. For each 6


Page 6 of 32

Page 7 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


scan with a resolution of 17500 at m/z 200, the AGC was set at 5×104 and the MIT was 80 ms. The dynamic exclusion was set at 18 s to suppress repeated detection of the same fragment ion peaks. The relative collision energy for MS2 was at 27% for HCD. Spectral data were then searched against Uniprot Swiss-Prot Human (v201503) database with Mascot v2.3.2 software. The peptide and protein false discovery rates (FDR) were set to 0.01. The cutoff of ion score from Mascot was set to 10. The SILVER software was used to get the intensity of each protein34. Only the proteins with more than one peptide were kept for further analysis. The raw MS data was deposited in iProx database with identifier IPX00079700 (http://www.iprox.org/index). Data Analysis Differential expressed proteins were obtained by using R packages, ProstaR and DAPAR. For each condition of the label-free quantification data, we required a protein to have non-zero intensity in two out of the three replicates. Then the data was logarithmic transformed and normalized by median centering. The missing values were replaced with two. Differentially expressed proteins were detected using a Limma moderated t-test. P-values were adjusted by Benjamini-Hochberg procedure. Proteins were regarded as significantly differentially expressed between two groups with P-value less than 0.01, and fold change < -2 or > 2. The Pearson’s correlation coefficients were measured for the normalized data. The functional classification of differentially expressed proteins with Gene Ontology was carried out by the DAVID webtool35 . The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways were analyzed by an R package, clusterProfiler 36. Fluorescent Labeling of Exosomes and Immunofluorescence Microscope PKH26 red fluorescent cell linker kit for general cell membrane labeling was purchased from 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Sigma. As described37, the exosomes were resuspended in 200 µL of Diluent C. Then, the resuspended exosomes were mixed with 200 µL of Diluent C containing PKH26 at the final concentration of 2 µM PKH dye. After 5 min incubation at 22°C, the reaction was stopped by addition of 400 µL PBS containing 5% BSA. After 1 min incubation, labeled exosomes were washed three times with PBS and resuspended in PBS. Fluorescently labeled exosomes were added to the culture medium of BMDMs. After 4 hours, the medium was replaced with fresh medium with Hoechst. After 30 min, the medium was removed and the cells were washed with PBS. Then, the cells were fixed with methanol and pictures were taken by confocal microscope. Cytotoxicity assay BMDMs were primed with exosomes for 6 hours, and then stimulated with 10 µM nigericin for 30 min. Cell death was measured by the LDH assay using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). ELISA BMDMs were primed with exosomes for 6 hours, and then stimulated with 10 µM nigericin for 30 min. The amounts of secreted IL-1β, IL-6 and TNF-α in culture supernatants were determined by using commercially available ELISA kits (Neobioscience Technology Company), and followed by analysis using a microplate reader. Statistical Analysis The statistical evaluation was performed using a two-tailed Student’s t-test. P

Inflammasome-Derived Exosomes Activate NF-ÎºB Signaling in

Recommend Documents