Deep Coverage Tissue and Cellular Proteomics ...

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Deep Coverage Tissue and Cellular Proteomics Revealed IL-1 β Can Independently Induce the Secretion of TNF-Associated Proteins from Human Synoviocytes Shengquan Tang, Suyuan Deng, Jiahui Guo, Xing Chen, Wanling Zhang, Yizhi Cui, Yanzhang Luo, Ziqi Yan, Qing-Yu He, Shan Shen and Tong Wang

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http://www.jimmunol.org/content/suppl/2017/12/01/jimmunol.170048 0.DCSupplemental

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This article cites 47 articles, 6 of which you can access for free at: http://www.jimmunol.org/content/200/2/821.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 2018; 200:821-833; Prepublished online 1 December 2017; doi: 10.4049/jimmunol.1700480 http://www.jimmunol.org/content/200/2/821

The Journal of Immunology

Deep Coverage Tissue and Cellular Proteomics Revealed IL-1b Can Independently Induce the Secretion of TNFAssociated Proteins from Human Synoviocytes Shengquan Tang,*,1 Suyuan Deng,*,1 Jiahui Guo,*,1 Xing Chen,* Wanling Zhang,* Yizhi Cui,* Yanzhang Luo,* Ziqi Yan,* Qing-Yu He,* Shan Shen,† and Tong Wang*

O

steoarthritis (OA) is one of the most common and debilitating joint diseases. Other than the typical manifestations, such as cartilage destruction and subchondral

*Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China; and †The First Affiliated Hospital, Jinan University, Guangzhou 510632, China 1

S.T., S.D., and J.G. contributed equally to this work.

ORCIDs: 0000-0001-7451-4301 (Y.L.); 0000-0001-7981-8224 (S.S.); 0000-00025980-3380 (T.W.). Received for publication April 3, 2017. Accepted for publication October 31, 2017. This work was supported by the National Basic Research Program 973 of China (Grant 2014CBA02000), the National Natural Science Foundation of China (Grant 81372135), the Key Project for Research and Development of Guangdong Province (Grant 2016B020238002), and the Key Special Project on the Integration of Industry, Education and Research of Guangzhou (Grant 201604020002) to T.W., the Fundamental Research Funds for the Central Universities of China (Grant 21615461), and the National Key Research and Development Program of China (Grants 2017YFA0505100 and 2017YFA0505000). The mass spectrometry data have been submitted to the iProX database (http://www. iprox.org/) under accession number IPX0000873000. Address correspondence and reprint requests to Prof. Tong Wang or Dr. Shan Shen, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, 601 Huangpu Avenue W, Room 916, The 2nd Science and Technology Building, Guangzhou 510632, China (T.W.) or The First Affiliated Hospital, Jinan University, 601 Huangpu Avenue W, Guangzhou 510632, China (S.S.). E-mail addresses: [email protected] (T.W.) or [email protected] (S.S.) The online version of this article contains supplemental material. Abbreviations used in this article: DAB, diaminobenzidine; DDA, data-dependent acquisition; DEP, differentially expressed protein; DIA, data-independent acquisition; ER, endoplasmic reticulum; FDR, false discovery rate; FLNc, filamin C; FSC, forward scatter; GO, gene ontology; HS, human synoviocyte; IB, immunoblotting; IHC, immunohistochemistry; IPA, Ingenuity Pathway Analysis; MS, mass spectrometry; OA, osteoarthritis; PCNA, proliferating cell nuclear Ag; PLGEM, power law global error mode. Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700480

bone remodeling, the synovium inflammation or synovitis has now been accepted as a source of proinflammatory mediators that are difficult to contain in progressive OA (1–3). In this regard, synoviocytes have autoimmune cell properties as they respond to certain stimuli, such as IL-1b and TNF-a, in a destructive manner to become activated and attack bystander healthy cells. Meanwhile, synovium will be abnormally thickened, which in turn harms joint structures and functions (4). It is known that synovitis in OA can be initiated by cartilage breakdown, with the subsequent phagocytosis of debris, and activation of synoviocytes, leading to the formation of an inflaming microenvironment and synovial fluid (5). Interestingly, Melin et al. (6) recently found with a quantitative secretome analysis that the cartilage itself represents a major inducer of complement proteins upon IL-1a stimulation. Indeed, monocytes/macrophages and dendritic cells are the primary producers of both IL-1 and TNF in the OA joint, but they have a classical self-containing mechanism with NO-induced apoptosis and/or anergy (7). Unfortunately, the excessive NO accumulation can selectively kill chondrocytes and induce cartilage degradation (8). In contrast, inflaming synovial cells tend not to be self-limited by the NO system, and the synovitis is rarely self-healing. All these findings implicate that a continuously homeostatic state of inflammation that lacks negative feedback signals exists in the OA synovium. However, the systems biological feature of such a state has not been fully understood. Specifically, although phosphoprotein changes in chondrocytes have been associated with the mechanism of OA inflammation (9), no phosphoproteome data in human OA synovitis has been published to date. Particularly, we previously found that the synovium tissue of OA and fractures had differential IL-1b persistence, whereas TNF remained the same (2). With a functional proteomics strategy, we have found that IL-1b can activate the TNF-downstream signals in synoviocytes, which requires no existence of TNF itself (2). Indeed, clinical trials of


Synovitis is a key contributor to the inflammatory environment in osteoarthritis (OA) joints. Currently, the biological therapy of OA is not satisfactory in multiple single-target trials on anti-TNF agents, or IL-1 antagonists. Systems biological understanding of the phosphorylation state in OA synovium is warranted to direct further therapeutic strategies. Therefore, in this study, we compared the human synovial phosphoproteome of the OA with the acute joint fracture subjects. We found that OA synovium had significantly more phosphoproteins, and 82 phosphoproteins could only be specifically found in all the OA samples. Differentially expressed proteins of the OA synovium were focusing on endoplasmic reticulum–/Golgi-associated secretion and negative regulation of cell proliferation, which was verified through an IL-1b–treated human synoviocyte (HS) in vitro model. With data-independent acquisition–based mass spectrometry, we found that IL-1b could induce HS to secrete proteins that were significantly associated with the endosomal/vacuolar pathway, endoplasmic reticulum/Golgi secretion, complement activation, and collagen degradation. Especially, we found that while specifically suppressing HS endocytosis, IL-1b could activate the secretion of 25 TNF-associated proteins, and the change of SERPINE2 and COL3A1 secretion was verified by immunoblotting. In conclusion, our results suggest that OA synovium has a polarized phosphoproteome to inhibit proliferation and maintain active secretion of HS, whereas IL-1b alone can transform HS to produce a synovitis-associated secretome, containing numerous TNF-associated secretory proteins in a TNF-independent mode. The Journal of Immunology, 2018, 200: 821–833.

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SYNOVIUM PHOSPHOPROTEOME AND SYNOVIOCYTE SECRETOME quantification. We employed the Scaffold PTM software version 3.0 (Proteome Software) to determine the confidence of phosphosites using the Ascore algorithm, and only the phosphopeptides with a localization probability .99% were considered confident identifications (12).

Determination of differentially expressed proteins We employed the power law global error mode (PLGEM) algorithm (14) to compare the abundance difference at the proteome level in both phosphoproteome and secretome analyses. We have found this method a useful tool for comparative proteomics per our published work, which addresses the detailed PLGEM method (15, 16). A protein was deemed a significantly differentially expressed protein (DEP) when p , 0.05 per PLGEM.

Gene ontology analysis Upregulated DEPs were subjected to the gene ontology (GO) and pathway term network analysis with CluGO + CluePedia (17, 18) in Cytoscape software version 3.4.0, as we previously described with minor modifications (12, 15). Parameters were set as follows: Pathways, Reactome pathway (data: 09.11.2016); evidence code, All_Experimental_(EXP, IDA, IPI, IMP, IGI, IEP); GO term fusion, applied; pathways p value cutoff, p # 0.01; p value correction algorithm, Benjamini–Hochberg; k Score $0.7; leading group term was based on percentage of genes per term.

Immunohistochemistry

Materials and Methods Human synovium tissue and fibroblast acquisition The scientific and ethics review committee of Jinan University approved this study, and written informed consent was obtained from all study participants. All methods were performed in accordance with the relevant guidelines and regulations. Synovium tissue samples from 13 subjects were obtained from The First Affiliated Hospital of Jinan University. Among them, seven subjects were diagnosed with knee joint OA and sampled upon synovectomy; the other six with acute knee fractures were included for synovial tissue acquisition during surgical operation. For the tissue phosphoproteome analysis, four OA and three fracture samples were used; the other three OA and three fracture samples were used for the immunohistochemistry (IHC) verification. Detailed clinical features and experimental information for each subject can be found in Table I. Specifically, all OA and fracture subjects had no prior history of corticosteroid injection or nonsteroidal anti-inflammatory drug use during the preceding 8 wk. Prepuce dermis samples from three healthy donors were acquired upon circumcision, and human primary fibroblasts were isolated and cultured as described previously (10).

Protein extraction from synovial tissues Synovial tissues were mixed with the 1% SDS lysis buffer, supplemented with 1 mM PMSF, PhosSTOP Phosphatase Inhibitor Mixture (Roche, Shanghai, China), and phosphatase inhibitor (Roche), followed by grounding in liquid nitrogen. Postincubation on ice for 30 min, tissue lysates were centrifuged at 17,000 3 g, for 30 min. Supernatants were collected, and the protein concentration was determined by a BCA kit (Thermo Fisher Scientific, Shanghai, China).

Protein digestion and phosphopeptide enrichment We next performed in-solution protein digestion following our published procedure (11, 12). The phosphopeptide enrichment was conducted as we previously reported (12). Briefly, Titansphere TiO particles (GL Sciences, Tokyo, Japan) were used to trap phosphopeptides, which were eluted by 5% NH3·H2O. Phosphopeptides were then fractionated by gradient elutions with 0, 2, 5, 8, 10, and 40% acetonitrile in 5% NH3·H2O. Mono Tip C18 Pipette Tips (GL Sciences) were employed for desalting.

Shotgun mass spectrometry analysis of tissue phosphoproteins Phosphopeptides were analyzed by a TripleTOF 5600 mass spectrometer (AB Sciex, Framingham, CA). The exact mass spectrometry (MS) parameters can be found in our published work (12). The raw MS data are publicly available through the iProX database (accession number IPX0000873000). The MS raw data were first searched with the Mascot server version 2.5.1 (Matrix Science, London, U.K.) against the Swiss-Prot HUMAN FASTA database (downloaded on Jan 4, 2016, 20,193 entries). Searching parameters were used as we previously described (12). Mascot search-result DAT files were then loaded into the Scaffold software version 4.5.0 (Proteome Software, Portland, OR) for controlling the protein level false discovery rate (FDR) to ,0.01. The normalized spectral abundance factor method (13), as provided by the Scaffold software, was used for phosphoprotein

IHC was performed as we previously described with minor modifications (2). In brief, sections were sequentially treated with the primary Ab rabbit anti-pFLNC(S2233) pAb (1:100; Biorbyt, San Francisco, CA), HRPconjugated goat anti-rabbit mAb (1:3000; Bioworld), the Vectastain ABC kit (Vector Laboratories, Burlingame, CA), and the ImmPACT Diaminobenzidine (DAB) Substrate (Vector Laboratories). ImageJ (19) and the IHC Image Analysis Toolbox (20) were used to quantify the grayscale of DAB staining in the synovium tissue. The thickness of the synovium was determined by the Measure tool provided by ImageJ.

Cell culture and treatment Primary HS were purchased from ScienCell (Carlsbad, CA; lot number 2070), and maintained in Synoviocyte Medium (ScienCell) consisting of 500 ml of basal medium, 10 ml of FBS, 5 ml of synoviocyte growth supplement, and 5 ml of penicillin/streptomycin solution (all from ScienCell), as we previously described (2). Per the manufacturer’s instructions, these primary HS were isolated from a healthy nonarthritic subject; they were CD90 and fibronectin positive, although negative for HIV type 1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. Only HS with ,5 population doublings were used. In addition, culture surfaces were treated with poly-L-lysine (ScienCell) at 2 mg per cm2. Either IL-1b or TNF-a (Sino Biological, Beijing, China) was used at 10 ng/ml per cell modeling needs.

Immunoblotting analysis The immunoblotting (IB) analysis was performed as we previously described (16). The primary Abs and their working dilution are listed as follows: mouse anti-human proliferating cell nuclear Ag (PCNA) mAb (1:5000; Thermo Fisher Scientific), rabbit anti-human p38 MAPK pAb (1:1000; Cell Signaling Technology, Shanghai, China), rabbit anti-human phospho-p38 MAPK (Thr180/Tyr182) mAb (1:1000; CST), rabbit antihuman JNK1/2 mAb (1:1000; CST), mouse anti-human phospho-SAPK/ JNK (Thr183/Tyr185) mAb, rabbit anti-human ERK1/2 (phospho-T202/ Y204) mAb (1:1000; CST), rabbit anti-human Collagen Type III (C-terminal) pAb (1:1000; Proteintech, Wuhan, China), rabbit anti-human SERPINE2 pAb (1:1000; Proteintech), and rabbit anti-human a-tubulin pAb (1:1000; CST). Secondary Abs were HRP-conjugated goat anti-rabbit mAb (1:2000; Bioworld) and HRP-conjugated goat anti-mouse mAb (1:2000; Bioworld).

WST-1 assay The proliferation of HS was analyzed by a WST-1 Assay Kit (Beyotime, Shanghai, China), following the manufacturer’s instructions. In detail, cells were cultured in 96-well plates at 2000 cells per well in 100 ml culture medium. Cells were treated with 10 ng/ml IL-1b for 24 or 48 h, followed by addition of 20 ml WST-1 solution and 2 h incubation. The light absorbance was measured at 450 and 630 nm, respectively.

Cytometric bead array analysis Cytometric bead array was used to measure the supernatant concentration of IL-8, IL-1b, IL-6, IL-10, TNF, and IL-12p70 by a Human Inflammatory Cytokines Kit (BD, Guangzhou, China), as we previously described


both anti-TNF and anti–IL-1b therapies on OA synovitis have shown certain efficacy; however, the evidence is not strong enough to draw generalized conclusions on whether such biological therapies are beneficial (5). As IL-1b can bypass TNF to function just like TNF in synoviocytes, OA synovitis tends to be a specialized inflammation and far more complex than a disease with single etiology, which can be solved by a single-target treatment. Hence, investigation of the systems biology feature of OA synovitis is warranted. As most signaling proteins are phosphoproteins, in this study we analyzed the phosphoproteome of the knee joint synovium tissues of subjects with OA and acute fractures. We recognized that the phosphoproteome of OA synovium had a unique pattern shift from the fracture synovial tissues, focusing on regulating the secretion and inhibiting cell proliferation. Accordingly, with an in vitro IL-1b–treated human synoviocyte (HS) model, we characterized the proliferation, secretome change, and endocytosis capacity of HS. We found that IL-1b can bypass TNF to induce the release of secretory proteins detrimental to cartilage, while reducing the endocytotic capacity of HS.

The Journal of Immunology (2, 15). Samples were analyzed by using a C6 flow cytometer (BD) and the FCAP Array software (version 3.0.1; BD).

Protein preparation for secretome analysis HS cells were cultured to reach 60–70% confluency, and washed four times with the serum- and phenol red–free DMEM medium (Invitrogen), and incubated in the same medium at 37˚C for 24 h. Supernatants were harvested, with addition of PMSF to reach a final concentration of 1 mM, followed by sequential centrifugations at 300 3 g, for 10 min, and 15,000 3 g, for 30 min, to remove debris. The supernatant was passed through a 0.22 mm filter, and concentrated to 500 ml using an Amicon Ultra 3K device (Merck Millipore, Guangzhou, China), followed by three washes with 50 mM triethylammonium bicarbonate for buffer change. Post– freeze-dry, secretory proteins were resuspended by the SDS lysis buffer (Beyotime), supplemented with 1 mM PMSF and protease inhibitor mixture (Roche). The protein samples were then digested with the same method as the phosphoproteome analysis described above.

Data-independent acquisition–based MS analysis

activation type, HCD; HCD collision energy, 30%; detector type, orbitrap; orbitrap resolution, 15,000; AGC target, 5.0 3 104; maximum injection time, 30 ms. Next, individual samples were analyzed in the data-independent acquisition (DIA) mode with the same instrument. The parameters were largely the same as the DDA analysis, except the following: MS scan range, 350–1200 m/z; MS/MS scan range, 200–2000 m/z; MS/MS maximum injection time, 50 ms.

Data processing for the DIA MS To build the spectral library, the DDA raw files were searched using the Sequest HT (v2.5) engine in the Proteome Discoverer software version 2.1 (Thermo Fisher Scientific) against a customized reference database incorporating the Swiss-Prot HUMAN FASTA database (downloaded on Nov 24, 2016, 20,120 entries) and the iRT standard peptides sequence in the FASTA format. Search parameters were set as follows: MS tolerance, 10 ppm; fragment mass tolerance, 0.02 Da; enzyme, trypsin; static modifications, carbamidomethyl (C); dynamic modifications, oxidation of methionine, deamination of Q and N, and acetyl of the N terminus. Peptides were filtered for high confidence (FDR , 1%), and the minimum peptide length was 9 aa. Confident protein identification should meet the following criteria: 1) protein level FDR , 1%; 2) unique peptides $2. The pdResult files of the DDA searches were exported from the PD software, and imported into the Spectronaut software version 10 (Biognosys) to build the ion spectral library. Next, we converted the DIA raw files into an htrm format by using the HTRMS Converter provided by the Spectronaut. Finally, the DIA htrm files, DDA raw files, DDA pdResult files, and the customized Swiss-Prot HUMAN database + iRT standard peptides FASTA file were collectively loaded into the Spectronaut, and processed with the BGS factory settings with minor modification by changing the enzyme from trypsin/P to trypsin. The proteins were inferred by the software, and the quantification information was acquired at the protein level by using the q-value ,0.01 criteria, which was used for subsequent analyses.

FIGURE 1. Phosphoproteome comparison of human synovium tissues from OA and fracture subjects. (A) Venn diagram comparison of phosphoprotein identifications. Numbers indicate the phosphoproteins identified across all samples within each group, respectively. (B) Comparison of the phosphoprotein numbers for MS analyses on individual subjects. As data did not follow normal distribution, the nonparameter Kolmogorov–Smirnov test, two-tailed, was used. All data are shown as mean 6 SEM, OA donor n = 4, and fracture donor n = 3. (C) PLGEM fitting of the abundance of the 219 phosphoproteins quantified across samples from all subjects. (D) Residuals versus rank of protein mean abundances. (E) Quantile-quantile (Q-Q) plot. (F) Venn diagram comparison of phosphoproteins identified across all OA subjects, but not detected in any subject in the fracture group.


HS secretome peptides were mixed with the retention time of standard peptides provided by the iRT-Kit (Biognosys, Schlieren, Switzerland) at a volume ratio of 9:1. To build the precursor ion library, a pooled sample was prepared by mixing the same amount of protein from each sample of the IL-1b untreated and treated groups. This pooled sample was analyzed by an Orbitrap Fusion Lumos (Thermo Fisher Scientific) in a datadependent acquisition (DDA) mode for four repetitive runs. The parameters for the precursor ion analysis (MS) were set as follows: ion source type, NSI; spray voltage, static; positive ion, 2000; negative ion, 600; ion transfer tube temp, 320˚C; detector type, orbitrap; orbitrap resolution, 60,000; mass range, normal; scan range, 400–1500 m/z; RF lens, 30%; AGC target, 4.0 3 105; maximum injection time, 50 ms; include charge state(s), 2–7; intensity threshold, 5.0 3 104; datadependent mode, top speed. For the product ion analysis (MS/MS), the parameters were as follows: MS/MS isolation mode, quadrupole;

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SYNOVIUM PHOSPHOPROTEOME AND SYNOVIOCYTE SECRETOME

Table I. Donor clinical features and sample applications Donor No.

Age

Gender

Diagnosis

Application

1 2 3 4 5 6 7 8 9 10 11 12 13

35 58 85 89 76 80 67 28 56 64 66 53 39

Male Female Male Male Male Male Male Male Male Male Female Female Male

Fracture Fracture Fracture OA OA OA OA Fracture Fracture OA OA OA Fracture

MS MS MS MS MS MS MS IHC IHC IHC IHC IHC IHC

Physical and chemical features of proteins

Ingenuity Pathway Analysis We performed Ingenuity Pathway Analysis (IPA; www.ingenuity.com, Qiagen, Shanghai, China) with the Core Analysis module to discern the bioprocess, disease, and upstream regulators, as we described previously (2, 12, 22). The Connect Tool provided by IPA was used to find any association between proteins per IPA knowledge base.

Network analysis The network analysis was performed in the Cytoscape environment with the ReactomeFIPlugIn (version 5.1.0.b) (23, 24), as we previously described, with minor modifications (15). The 2015 version of functional interactions was used.

Cellular internalization assay The fluorophore conjugated beads with sizes of 0.2 mm (fluorescent at 660/ 680; Thermo Fisher Scientific) and 0.5 mm (fluorescent at 505/515; Thermo Fisher Scientific) were used to treat the HS for 2 h prior to the flow cytometry analysis with a C6 flow cytometer (BD). Cells were gated based on forward scatter (FSC) and side scatter to exclude debris, and bead-untreated cells were used as the negative control for fluorescence determination.

Statistics The bootstrap comparison was performed with the MATLAB R2016b software (MathWorks). Intergroup difference was examined by either parameter

Results Phosphoproteome of human synovium tissues With liquid chromatography MS/MS analysis, a total of 951 phosphoproteins could be identified as a union set, i.e., a protein would be counted if it was identified from any of the seven samples (http://www.iprox.org/). Among them, 537 and 239 phosphoproteins were confidently identified and quantified across all OA samples, and all fracture samples, respectively (Fig. 1A; http:// www.iprox.org/). In overlap, 219 phosphoproteins were identified across all seven samples (Fig. 1A, Table I). Interestingly, when analyzing tissue samples at the individual donor level, the OA samples had significantly more phosphoproteins than the fracture samples (Fig. 1B). The abundance of the 219 overlapped phosphoproteins could be well fitted by the PLGEM model (r2 = 0. 984) (Fig. 1C), with no observed bias in terms of rank of mean (Fig. 1D). In addition, the residues were found to be normally distributed (Fig. 1E). With these features, we could only discern 12 significant DEPs (p , 0.05) per PLGEM (Table II). But we found that 82 phosphoproteins were consistently and exclusively expressed in the OA synovium tissues (Fig. 1F; http://www.iprox. org/), whereas there were 645 proteins that could be identified in one of the three fracture, but not the OA, samples (Fig. 1F). Hence, these 82 proteins were also treated as DEPs for the subsequent analyses. OA phosphoproteome focuses on secretion and proliferation With GO analysis of the DEPs, we found that the upregulated phosphoproteins of the OA groups were significantly enriched in the terms of Golgi-associated vesicle biogenesis and the downregulation of EGFR and MET (Fig. 2A). These bioprocesses correspond to secretion and compromised cellular proliferation. As a plasma membrane protein, filamin C (FLNc) is potentially associated with intracellular trafficking and secretory processes (25). Among the 12 DEPs shown in Table II, the only commercialized Ab available was against phosphorylated FLNc at Ser2233. We then obtained three additional pairs of clinical samples to perform IHC analysis (Fig. 2B). We found that the OA group had significantly more pFLNC (s2233) staining than the OA group (Fig. 2C), which was consistent with the MS quantitation. Furthermore, we found that the synovium was significantly thicker in the OA group (Fig. 2D).

Table II. Differentially expressed phosphoproteins of human synovium

Swiss-Prot ID

Q9UPQ0 P35606 Q63ZY3 Q14315 O75475 Q92954 Q8WVC0 P68871 Q969G5 Q08495 P02794 P24821

Protein Name

LIM and calponin homology domains-containing protein 1 Coatomer subunit b9 KN motif and ankyrin repeat domain-containing protein 2 Filamin-C PC4 and SFRS1-interacting protein Proteoglycan 4 RNA polymerase-associated protein LEO1 Hemoglobin subunit b Protein kinase C d-binding protein Dematin Ferritin H chain Tenascin

OA/Fracture Ratio

PLGEM p Value

4.08

1.374 3 1022 22

Mass (kDa)

Isoelectric Point

121.9

6.41

28.67

LIMCH1

Charge

HGNC Gene Name

3.23 2.82

2.899 3 10 4.468 3 1022

102.5 91.2

4.93 5.33

233.89 218.83

COPB2 KANK2

2.33 0.42 0.46 0.37

4.530 4.939 4.917 2.822

1022 1022 1022 1022

291.0 60.1 151.1 75.4

5.82 9.86 10.16 4.12

249.46 14.57 66.60 299.87

FLNC PSIP1 PRG4 LEO1

0.27 0.3

1.917 3 1022 1.851 3 1022

16.0 27.7

7.32 6.39

0.37 22.18

HBB PRKCDBP

0.21 0.1 0.11

1.007 3 1022 5.541 3 1023 2.257 3 1023

45.5 21.2 240.9

9.36 5.29 4.52

6.38 29.51 2112.17

3 3 3 3

DMTN FTH1 TNC


The amino acid sequence and mass were downloaded from the Swiss-Prot human protein database. The MATLAB bioinformatics toolbox (MathWorks, Natick, MA) was employed to calculate the isoelectric point (pI) and the charge at the physiological conditions (pH 7.4) of each protein as we previously described (11). In addition, we used SignalP 4.1 server and TMHMM 2.0 to predict signal peptides and transmembrane domains as we previously described (21).

or nonparameter tests based on the normality tests by using GraphPad Prism software version 6.02 (GraphPad Software, San Diego, CA) or MATLAB. Statistical difference was accepted when p , 0.05.


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In vitro model of IL-1b–induced synovitis As we had shown that tissue phosphoproteome was focusing on endoplasmic reticulum (ER)–/Golgi-associated secretion and proliferation, we next used our well-established IL-1b–induced synovitis in vitro model for further systems analysis (2). We could well reproduce the upregulation of IL-6 and IL-8 in the IL-1b– treated HS (Fig. 3A), justifying the consistency of the cellular model (2). We found that the PCNA expression in HS was significantly suppressed by IL-1b (Fig. 3B), implicating that the proliferation was inhibited in this cellular model. Favorably, after the HS was treated by IL-1b for 48 h, the OD of WST-1 showed significant decline (Fig. 3C). These results showed that IL-1b inhibited HS proliferation, which was consistent with the GO analysis of tissue synovium phosphoproteins.

Furthermore, we found that the FSC of the IL-1b–treated HS was significantly greater than the untreated HS (Fig. 3D), showing that the cells became bigger post–IL-1b treatment. Such a size change echoed the thickness increase of the OA synovium as shown in Fig. 2D. Additionally, we found that the phosphorylated p38 level was significantly higher in the IL-1b–treated HS (Fig. 3E), whereas p-ERK1/2 (Fig. 3F) and p-JNK2 (Fig. 3G) showed no significant difference. These results on the three MAPKs were comparable with the GO analysis shown in Fig. 2A. The above results indicate that the in vitro cellular model has certain in vivo relevance to OA synovitis. Proteomic modeling of synovitis secretome We next analyzed the secretome of the IL-1b–treated HS following the workflow shown in Fig. 4A. The harvested secretory proteins


FIGURE 2. GO analysis and biological verification of the phosphoproteome. (A) ClueGO annotation of DEPs. Functionally grouped GO/pathway term networks were computed with ClueGO referenced by the Reactome database. The circular size depicts the statistical significance based on percentage of genes per term. Edge thickness represents the degree of connectivity between terms based on the k score. (B) IHC verification of phosphorylated FLNC. Scale bar, 40 mm. (C) Statistical comparison of the pFLNC expression. The mean grayscale intensity of the DAB staining was analyzed by ImageJ software. The horizontal line indicates the median value of each group, donor n = 3, two-tailed Kolmogorov–Smirnov test. (D) Synovial tissue thickness assay. Data are shown as mean 6 SEM, donor n = 3, unpaired Student t test.

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FIGURE 3. In vitro modeling of synovitis with IL-1b–treated human synoviocytes. (A) Cytometric bead analysis. (B) Proliferation analysis. Synoviocytes were treated with IL-1b for 24 h, followed by the IB analysis of PCNA. (C) WST-1 assay. (D) Flow cytometry analysis of the cellular size as determined by the FSC. (E–G) IB assay on MAPKs, including total and phosphoproteins of p38 (E), ERK1/2 (F), and JNK 1/2 (G). All experiments shown were acquired from three independent experiments, data are shown as mean 6 SEM, unpaired Student t test. All IB blots are shown as cropped images, the whole-blot raw images can be found in Supplemental Fig. 1.

were generally separated by the SDS PAGE gel in a band manner, suggesting that no obvious degradation had occurred (Supplemental Fig. 1A). With DIA-based liquid chromatography MS/MS analysis of three biological replicates, we observed high reproducibility in protein identifications (http://www.iprox.org/); among the ∼1400 identified proteins in the HS group, 1233 were

identified across all MS analyses (Fig. 4B). Similarly, 1206 secretory proteins were consistently identified in all samples of the HS+IL-1b group (Fig. 4B). In total 86.6% proteins (1132) could be identified across all of the six samples of the two groups. These results justified that DIA MS in secretome analysis had excellent reproducibility. Furthermore, 11 secretory proteins were identified


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FIGURE 4. IL-1b induces system changes of the human synoviocyte secretome. (A) Workflow of the functionally proteomic analysis of IL-1b–induced synovitis. (B) Venn diagram comparison of the secretory proteins identified from the three independent experiments. (C) Venn diagram showing the proteins constantly identified in the secretome of IL-1b–treated HS. Numbers indicate the secretory proteins identified across all samples within each group, respectively. (D–F) Bootstrap analysis of the physical-chemical features of proteins. A total of 10,000 protein resamplings were performed from the secretomes of the HS group, the HS + IL-1b group, and the human proteome background (PE1 proteins in the neXtProt database), respectively. The isoelectric point (D), charge (E), and molecular weight (F) of these proteins were compared. Stars indicate the mean values, whereas the horizontal lines depict 95% confidence intervals. (G and H) Comparison of the proteins with signaling peptides (G) and transmembrane proteins (H). Statistical difference was examined by the x2 test.

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in all three biological replicates of the IL-1b–treated HS group, but not detected in any sample in the untreated HS group (Fig. 4C). With bootstrap analyses, we found that as compared with the background human proteome, the DIA MS tended to identify significantly more acidic proteins (Fig. 4D), with more charges (Fig. 4E); however, there was no significant difference in identifying proteins with different masses (Fig. 4F). We found that ∼38% of all secretory proteins had signaling peptides as predicted by the SignalP software, significantly more than the background human proteome (∼18%). Notably, ∼81% of the upregulated DEPs in the secretome were predicted to have signaling peptides (Fig. 4G). However, no significant difference was observed in the analysis of the transmembrane domain-containing proteins (Fig. 4H). These analyses favored the validity of DIA MS on secretome characterization in our cellular model.

Detrimental secretome of inflammatory synoviocytes We found that the secretory protein abundance determined by the DIA MS could also be well fitted by the PLGEM model, from which we discerned 68 up- and 13 downregulated DEPs (p , 0.01; http://www.iprox.org/). In addition, we counted the 11 secretory proteins shown in Fig. 4C as upregulated DEPs. With the IPA analysis, we found that the 79 upregulated DEPs significantly promoted the diseases and functions that were associated with known OA synoviocyte phenotypes, including cell movement, inflammatory response, organismal injury and abnormalities, and immune cell trafficking (z-score .1.96, Fig. 5A). However, these significantly upregulated DEPs significantly focused on the cell death and survival with z-score ,21.96 (Fig. 5A), suggesting that these DEPs have a negative impact on the function. These results


FIGURE 5. Pathway analysis of the human synoviocyte secretome. (A) IPA on the upregulated DEPs in the secretome of the IL-1b–treated HS. The significantly involved disease and functions are shown with the z-score heat map. (B) Reactome network analysis.


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reflected the inflammatory environment contributed by the secretome of inflaming synoviocytes, relevant to the HS invasion and detrimental immune responses. We found that these 79 DEPs could be enriched in four leading GO terms toward essential features of synovitis, including the endosomal/vacuolar pathway, ER/Golgi secretion, complement activation and collagen degradation, and chemokine receptors binding chemokines (Table III). With the Reactome analysis, these DEPs could be enriched and incorporated into a network with 23 nodes with a modularity of 0.7824 (Fig. 5B), suggesting that diversified protein modules were initiated by IL-1b, and the connection between these modules was loose. Most of the modules were also comparable with GO analysis, especially regarding the term-associated genes (Table III), but with tight intramodule protein-protein interactions (Fig. 5B). IL-1b can bypass TNF to induce TNF-associated protein secretion

IL-1b compromises the endocytotic ability of synoviocytes Exocytosis and endocytosis are coupled and highly interactive bioprocesses in numerous cell types (30). We then used 0.2 and 0.5 mm fluorophore-conjugated microbeads to test the clathrinand caveolae-mediated endocytosis, respectively (31, 32). Both types of endocytosis were found to be significantly decreased in the IL-1b–treated HS (Fig. 7A, 7B). However, IL-1b did not mediate changes in human normal fibroblasts in either type of endocytosis (Fig. 7C, 7D).

Discussion In this study, we report for the first time, to our knowledge, that OA synovium tends to have a completely different phosphoproteome from the fracture synovium. The differential phosphoproteins focus on regulating the ER-/Golgi-associated secretion, and inhibiting HS proliferation. As a known inducer of OA synovitis, IL-1b can significantly promote the protein secretion from HS, with diversified functional units to regulate endocytosis, secretion, complement activation, and collagen degradation. Especially, we found that IL-1b could also bypass TNF to induce TNF-associated protein secretion, whereas suppressing the endocytosis of HS. To have direct clinical relevance, we used surgery synovium samples to characterize the inflamed state maintained by the phosphoproteome. As compared with other human cells and tissues, the phosphoprotein number is quite small, ∼400 in the fracture and ∼800 in the OA groups. For example, according to our previous report, colorectal cancer cells can hold .1700 phosphoproteins, and the colorectal cancer cell–secreted exosome can pack .300 phosphoproteins (12). However, our results are comparable with a relevant report on cultured primary rheumatoid arthritis and OA synoviocytes (∼300 phosphoprotein spots in a difference gel electrophoresis analysis) (33). Even with such a small phosphoproteome size, it was interesting that we still found

Table III. GO enrichment analysis of HS secretome upon IL-1b treatment

No.

1

GO IDsa

GO:0000096

2

GO:0000233

3 4

GO:0000399 GO:0000166

5

GO:0000893

a

Leading GOTerma

Endosomal/vacuolar pathway Transport of g-carboxylated protein precursors from the ER to the Golgi apparatus Activation of C3 and C5 Collagen degradation Chemokine receptors bind chemokines

Term p Valueb 24

% Associated Genes

Associated Genes Found

9.30

[CTSS, MMP1, MMP3, TIMP1]

9.68

[IGFBP1, IGFBP4, MMP1] [C3, CD55, CFB, CFH] [AGRN, COL16A1, COL3A1, CTSD, CTSS, HAPLN1, ICAM1, LAMC2, MMP1, MMP19, MMP3] [CXCL10, CXCL3, CXCL6]

4.60 3 10

24

1.30 3 10

1.40 3 10

23

7.40 3 1025 3.90 3 1027

3.40 3 1024 4.20 3 1028

14.29 5.09

4.00 3 1023

4.20 3 1023

6.25

3.10 3 10

23

Ontology source was based on Reactome pathway (date: 09.11.2016). Benjamini–Hochberg corrected p values are shown.

b

Group p Valueb


With the upstream analysis of IPA, IL-1b was discerned as a significant upstream regulator for 15 upregulated secretory DEPs in the IL-1b–treated HS (z-score = 3.099; Fig. 6A). This meant that from these DEPs, IPA suggested IL-1b should have been added into the cell culture, which was true. Thus, such a prediction could serve as a positive control to justify the validity of our IL-1b–induced synoviocyte inflammation model. In addition, other interesting upregulators included TNF, INFG, MAPKs (p38 and JNK), IKBKB, STAT3, and IL-6. Among them, IL-6 (Fig. 3A) and phosphorylated p38 MAPK (Fig. 3E) had been experimentally verified. We next used the ConsensusPathDB tool to analyze the hierarchy of different upstream regulators by using the shortest interaction path module (26). The results showed that IL-1b could also serve as an upstream regulator of IFNG genes via STAT3 or GATA3 based on the previous source literature (27–29). No shortest interaction path between IL-1b and TNF could be built with the same ConsensusPathDB analysis. These results suggested that IFNG tended to be a downstream gene of IL1b, whereas IL-1b and TNF were independent upstream regulators. Such a prediction of TNF is comparable with our previous cellular proteome analyses, showing that IL-1b can activate intracellular TNF signaling in a TNF-independent manner (2). In this study with the secretome analysis, other than the known IL-1b downstream proteins (Fig. 6B), we detected 25 TNFassociated secretory proteins that could also be induced by IL1b (Fig. 6C), among which nine proteins were not associated with IL-1b (Fig. 6B, 6C). These nine proteins were CP, SERPINE2, COL16A1, SFRP1, HAPLN1, COL3A1, CTSC, MCAM, and SVIL (Fig. 6B, 6C). In this study, we would emphasize that IL-1b

could not induce increased TNF secretion in this cellular model according to Fig. 3A and our previous report (2). With the IPA Connect tool, we found that no connection between IL-1b and the nine TNF-associated secretory proteins could be formed per IPA knowledge base. This meant that according to current knowledge, no findings proposed the association of IL-1b with any of these nine proteins. We also validated this with the STRING analysis (https://string-db.org/) and manual literature searches. To favor our proof of concept, we showed that TNF could promote the secretion of SERPINE2 from the HS through four independent experiments (Fig. 6D). Interestingly, we found that other than SERPINE2, IL-1b could also induce the upregulated secretion of COL3A1 from the HS (Fig. 6E).

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FIGURE 6. Upstream regulator analysis and verification. (A) Upstream regulators were predicted by IPA based on the secretome DEPs. Activated (z-score .1.96) and inhibited (z-score ,21.96) regulators are highlighted in orange and blue, respectively. The IL-1b– (B) and TNF-regulated (C) HS secretory protein networks are shown. The HGNC names are provided for all node proteins. (D and E) IB verification on SERPINE2 and COL3A1 secretion from HS treated with TNF-a (D) and IL-1b (E), respectively. HS were treated with either TNF-a or IL-1b for 24 h prior to secretory protein harvests. Results were acquired from four independent experiments. All IB blots are shown as cropped images, the whole-blot raw images can be found in Supplemental Fig. 1.


82 phosphoproteins that were consistently identified in all the OA samples with shotgun MS analysis. Although HS have very similar features to fibroblasts, they are also professional secretory cells that maintain the homeostasis of synovial fluid in the joint, releasing hyaluronan (34), and chondrocyte-protective proteins such as lubricin (35) and gelsolin (36). However, with the inflaming OA microenvironment, our phosphoproteome results implicated several critical changes of OA synovium. Among them, the enhanced secretory behavior and activation of p38 MAPK were verified in our in vitro model. Interestingly, the downregulation of proliferation predicted by the downregulation of MET and EGFR phosphoproteins was also validated in the IL-1b–treated HS model. This is different from numerous other studies showing that IL-1b promotes the proliferation of human primary rheumatoid arthritis synoviocytes (37–39). Our findings suggest that the proliferation of synoviocytes in OA tends to be compromised, whereas the HS cell size can be enlarged by IL-1b. Our in vitro model indicated that IL-1b alone could switch on the massive changes in HS secretory behavior toward harming the joints. This included the significantly increased secretion of C3 and C5 complement activation regulating proteins, and numerous collagen degradation-associated proteins. Among these secretory proteins, CD55, also known as complement decay-accelerating factor, is a specific marker of HS (40). These results are comparable with another proteomics study by Melin et al. (6) showing that IL-1a–stimulated bovine cartilage releases intensively more activated C3. In numerous proteomics studies on OA synovial fluid (41–43), complement components have been deemed primary factors to differentiate OA from the health. Thus, the formation of an inflaming environment in joints involves many cell types, although the synoviocyte after exposure to IL-1b is an important contributor. Interestingly, we found that IL-1b could partially function just like TNF to regulate HS secretome. This echoes our previous comparative proteomics report on cellular analysis; in addition, such a feature of IL-1b is independent from TNF, as we have proven that IL-1b cannot induce TNF secretion in HS (2). This finding helps to understand the results from current clinical trials on biological OA therapy. Most of these trials targeted either IL-1b or TNF alone. For example, a very recent clinical trial on the TNF blocker, etanercept, has concluded that it has no significant different efficacy from the placebo in erosive inflammatory hand OA (44). As to the anti–IL-1 therapy, although most clinical trials failed to see efficacy, a fully humanized mAb to IL-1R1 AMG 108 showed improvements in the pain of patients with OA in the knee (45). Our current results and previous findings suggest that inhibiting TNF alone cannot completely block its downstream signals and resulting secretory proteins, because IL-1b could still activate TNF-associated secretions. Such a functional degeneracy of the two cytokines suggests that single-target therapy tends not to be sufficient in containing synovitis in OA. In this study, to our knowledge we not only reported the first secretome of IL-1b–induced HS, but also noted the technological advantage of DIA MS for the confident identification and quantification of secretory proteins. As the identification was referenced by the ion library, we realized 86.6% of reproducible identifications across all six samples in the three independent

acquired by the paired t test, fibroblast donor n = 3, all data are shown as mean 6 SEM. (E) Representative chart showing the gating strategy of flow cytometry.


FIGURE 7. Endocytosis analysis. (A and B) Endocytosis of 0.2 mm (A) and 0.5 mm fluorophore-beads (B) was evaluated by flow cytometry. Cells were gated in the FSC–side scatter plot to avoid analyses on debris. The untreated human synoviocytes were used as a negative control for gating. (C and D) Endocytosis assay on human fibroblasts. The experimental settings and gating strategies are the same as (A and B). All results were acquired from three independent experiments; p values were

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Disclosures The authors have no financial conflicts of interest.

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