Placenta Specific 8 Suppresses IL-18 Production through Regulation of Autophagy and Is Associated with Adult Still Disease

This information is current as Seiji Segawa, Yuya Kondo, Yuji Nakai, Akira Iizuka, of September 25, 2021. Shunta Kaneko, Masahiro Yokosawa, Kotona Furuyama, Hiroto Tsuboi, Daisuke Goto, Isao Matsumoto and Takayuki Sumida J Immunol published online 7 November 2018

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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. Published November 7, 2018, doi:10.4049/jimmunol.1800667 The Journal of Immunology

Placenta Specific 8 Suppresses IL-18 Production through Regulation of Autophagy and Is Associated with Adult Still Disease

Seiji Segawa,* Yuya Kondo,* Yuji Nakai,† Akira Iizuka,* Shunta Kaneko,* Masahiro Yokosawa,* Kotona Furuyama,* Hiroto Tsuboi,* Daisuke Goto,* Isao Matsumoto,* and Takayuki Sumida*

Adult Still disease (ASD) is a systemic disorder of unknown etiology characterized by high spiking fever, rash, and arthritis. The purpose of this study was to identify specifically associated with the active phase of the disease. In this study, we have reported that placenta specific 8 (PLAC8) was a newly specific involved in ASD. DNA microarray and validation analysis using human monocytes revealed that the expression of PLAC8 was significantly higher in active-ASD patients than in inactive-ASD patients Downloaded from and healthy controls. In ASD, PLAC8 expression level correlated with serum levels of CRP, ferritin, IL-1b, and IL-18. Stimulation of monocytes with LPS results in PLAC8 upregulation. LPS or nigericin stimulation of PLAC8-overexpressing human monocytic cell line (THP-1), but not mock THP-1 cells, was associated with a significant decrease in IL-1b and IL-18 production. PLAC8 overexpression in THP-1 cells was associated with enhanced autophagy and suppression of IL-1b and IL-18 production. There- fore, we found that PLAC8 was upregulated in activated monocytes, as was IL-1b and IL-18. The upregulated PLAC8 acts on the synthesis of inactive precursors of IL-1b and IL-18 and seemed to suppress the production of IL-1b and IL-18 by negative http://www.jimmunol.org/ feedback through enhanced autophagy, resulting in the suppression of ASD. The results highlight the role of PLAC8 in the pathogenesis of ASD and suggest its potential suitability as an activity marker and therapeutic target in ASD. The Journal of Immunology, 2018, 201: 000–000.

dult Still disease (ASD) is a rare multisystemic auto- higher levels of IL-18 correlate with ASD activity (6, 8). Thus, inflammatory disorder of unknown etiology. The clinical IL-18 is thought to be one of the most important factors in the A features correlate with systemic manifestations, such as pathogenesis of ASD. However, there is little or no information on

a high spiking fever, arthralgia or arthritis, and an evanescent the regulatory process involved in the production of IL-18 and by guest on September 25, 2021 salmon-pink maculopapular skin rash (1, 2). Although the path- other inflammatory cytokines in ASD. ogenesis of ASD is not clear, the interplay of viral infections, Activation of myeloid cells, including monocytes, macrophages, genetic factors, and immune dysregulation, including cytokine- dendritic cells, and neutrophils, is the hallmark of ASD. Especially, mediated inflammation and enhanced apoptosis, may contribute several markers and cytokines that reflect macrophage activation to the development of this disease (3). correlate with ASD activity (3). In contrast, activated monocytes Previous reports investigated the pathogenic roles of several are known to produce IL-6, TNF-a, IL-1b, and IL-18 through proinflammatory cytokines in ASD, including IL-1b, IL-6, IL-18, several stimuli including viral and bacterial components (9, 10). In TNF-a, and IFN-b (3). Among these cytokines, IL-18 is thought patients with ASD, uncontrolled activation of monocytes could to be the upstream initiator of the proinflammatory cytokine induce cytokine storms and several systemic disorders. However, cascades, and serum IL-18 levels are particularly elevated in the genes involved in the regulation of monocyte activation remain ASD, unlike in other inflammatory conditions (4–8). Furthermore, unidentified in ASD. Therefore, identification of specific genes in

*Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, University of Tsukuba, 1-1-1, Tennodai, Tsukuba, Ibaraki 305-8575, Japan. E-mail Tsukuba, Ibaraki 305-8575, Japan; and †Institute for Food Sciences, University of address: [email protected] Hirosaki, Hirosaki 038-0012, Japan The online version of this article contains supplemental material. ORCIDs: 0000-0001-8863-5534 (S.K.); 0000-0001-6646-2003 (D.G.); 0000-0002- Abbreviations used in this article: ASD, adult Still disease; BCL2A1, BCL2-related 7995-9294 (I.M.). A1; CD55, CD55 molecule; CLU, clusterin; CRP, C-reactive protein; Received for publication May 11, 2018. Accepted for publication October 3, 2018. CYP1B1, cytochrome P450 family 1 subfamily B polypeptide 1; FARMS, Factor Analysis for Robust Microarray Summarization; FCGR1B, Fc fragment of IgG high This work was supported by Research Program for Intractable Diseases, Health and affinity Ib; GO, ; HC, healthy control; IL1RN, IL-1 receptor antago- Labor Sciences research grants from the Ministry of Health, Labor and Welfare, nist; ISD, immunosuppressive drug; 3-MA, 3-methyladenine; MBL, Medical and Japan, and the Ministry of Education, Culture, Sports, Science and Technology. Biological Laboratories; mSAM, modified SAM; PCA, principal component analy- S.S. and T.S. designed the study. S.S., Y.K., A.I., S.K., M.Y., K.F., D.G., and I.M. sis; PIM1, Pim-1 proto-oncogene; PLAC8, placenta specific 8; PLAC8–THP-1, performed analysis and collected the data. Y.K. and H.T. oversaw patient recruitment. PLAC8-overexpressing THP-1; PLSCR1, phospholipid scramblase 1; PM/DM, poly- Y.N. performed DNA microarray analysis. S.S. analyzed the data and wrote the myositis/dermatomyositis; qPCR, quantitative PCR; RA, rheumatoid arthritis; manuscript. All authors have read and approved the manuscript for publication. S100A12, S100 calcium binding protein A12; SAM, Significance Analysis of Micro- arrays; SLE, systemic lupus erythematosus; SOD2, superoxide dismutase 2; SS, The microarray data presented in this article have been submitted to the Gene Sjo¨gren syndrome; STEAP4, STEAP family member 4. Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE113645. Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 Address correspondence and reprint requests to Prof. Takayuki Sumida, Division of Clinical Immunology, Department of Internal Medicine, Faculty of Medicine,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800667 2 ROLE OF PLAC8 IN ASD monocytes could help in the assessment of disease activity and (24) using the statistical language R (http://www.r-project.org/) (25) and severity and could be useful as therapeutic targets in ASD. Bioconductor (http://www.bioconductor.org/) (26). Global gene expression The present study was designed to clarify the specific genes in- profiles of all samples were evaluated with principal component analysis (PCA) (27) using the promp() function in R. To compare the genes up- volved in the progression of ASD. Using DNA microarray analysis, regulated in active ASD relative to inactive ASD, the Significance Analysis we identified placenta specific 8 (PLAC8) mRNA to be significantly of Microarrays (SAM) (28) was applied to the FARMS-normalized paired upregulated in monocytes from patients with active ASD. PLAC8 is data with modification. In brief, considering the dynamic range of changes a small protein known to be highly expressed in giant trophoblasts in gene expression, using the weight term of the weighted average dif- ference (29) statistic, the modified SAM (mSAM) statistic for the ith probe and the spongiotrophoblast layer of the mouse placenta (11, 12). In set, mSAM(i), is calculated simply as humans, PLAC8 is expressed in dendritic cells, myeloid cells, lymphoid cells, and epithelial cells in lungs and intestines (13, 14). mSAMðÞ¼i SAMi wi; Multiple functions of PLAC8 have been demonstrated in the im- mune system, such as adipocyte differentiation, infectious disease, where wi is relative average log signal intensity of the ith probe set diabetes, , and autophagy (14–18). However, little is known (i.e., weight). Then we rank the probe sets according to the mSAM statistic, and among the top-ranked 600 probe sets (approximately top about the role of PLAC8 in monocytes and how it is involved in 1% of the all probe sets on the GeneChip 2.0 Array), inflammatory conditions. The present study provides data on the probe sets with p , 0.05 were regarded as differentially expressed. To function of PLAC8 in monocytes and sheds light on the role of compare the differentially expressed probe sets between active ASD PLAC8 in the pathogenesis of ASD. and HC, the rank products method (30) was applied to the FARMS- normalized data. The probe sets with a false discovery rate ,0.05 were regarded to have different expression levels between the two groups Downloaded from Materials and Methods (i.e., differentially expressed). Then, we picked up active-ASD–spe- cific, highly expressed probe sets as commonly appeared in both dif- Patients and healthy donors ferentially expressed probe sets. Gene annotation enrichment analysis All subjects were examined at the University of Tsukuba Hospital, and each of these active-ASD–specific, highly expressed probe sets was per- provided a signed consent form for participation in the study. The study was formed according to Gene Ontology (GO) annotation using the Data- approved by the ethics committee of the University of Tsukuba. Monocytes base for Annotation, Visualization and Integrated Discovery Web tool or plasma samples were collected from patients with inactive ASD (n = 14, (https://david.ncifcrf.gov) (31). The hierarchical chart of GO terms three males and 11 females; the mean 6 SD of age was 44.7 6 13.8 y) and was constructed using the Web tool QuickGO (https://www.ebi.ac.uk/ http://www.jimmunol.org/ active ASD (n = 10, three males and seven females; the mean 6 SD of age QuickGO/) (32). was 49.5 6 17.0 y). All active-ASD patients satisfied Yamaguchi’s (19) criteria. We divided patients with ASD into active and inactive groups Validation by quantitative PCR according to clinical manifestations (Supplemental Table I). Monocytes of Quantitative PCR (qPCR) was also conducted for validation of upregulated the disease control subjects were collected from rheumatoid arthritis (RA) differentially expressed genes in active-ASD patients identified by cDNA (n = 8, one male and seven females; the mean 6 SD of age was 50.0 6 18. microarray analysis. Total RNA was extracted from monocytes isolated 3 y; the mean 6 SD of Disease Activity Score in 28 joints as measured by from active-ASD patients (n = 7, three males and four females; the mean 6 C-reactive protein [CRP] was 4.1 6 1.1; steroid use, n = 6; synthetic SD of age was 53.3 6 16.4 y), inactive-ASD patients (n = 10, two males n disease-modifying antirheumatic drug use, = 6; immunosuppressive drug and eight females; the mean 6 SD of age was 45.5 6 13.6 y), and HC (n =8, [ISD] use, n = 1; biological agent use, n =1);Sjo¨gren syndrome (SS) (n =5, three males and five females; the mean 6 SD of age was 50.1 6 8.43 y), by guest on September 25, 2021 6 6 n five females; the mean SD of age was 56.0 15.1 y; steroid use, =3); which included subjects analyzed by DNA microarray. The mRNA ex- n systemic lupus erythematosus (SLE) ( = 6, one male and five females; the pression level of the target gene was examined by qPCR using the 7500 mean 6 SD of age was 46.3 6 16.3 y; steroid use, n = 6; hydroxy- Real-Time PCR System (Applied Biosystems, Foster City, CA) with the chloroquine use, n = 3; ISD use, n = 6); and polymyositis/dermatomyositis SYBR Green PCR kit (Takara Bio). Predesigned primers specific for the n 6 (PM/DM) ( = 5, one male and four females; the mean SD of age was targeted genes (Takara Bio) were used for qPCR. The human GAPDH was 6 n n 55.8 8.73 y; steroid use, = 5; ISD use, = 4). All RA patients satisfied also examined as an internal control. The levels of gene expression were the American College of Rheumatology classification criteria (20). All the calculated from the standard curve and expressed relative to GAPDH gene patients with SS were diagnosed by rheumatologists according to the 1999 expression. The primers of 13 differentially expressed genes used for the Japanese Ministry of Health criteria for diagnosis of SS (21). All the pa- RT-PCR were as follows: clusterin (CLU)-forward, 59-CTCCCACTAGG- tients with SLE fulfilled the 1997 American College of Rheumatology GATGCAGATG-39; CLU-reverse, 59-CACAAACAGCAGCAGAGTCTT- classification criteria (22). All the patients with PM/DM fulfilled the CA-39; Fc fragment of IgG high affinity Ib (FCGR1B)–forward, 59- Bohan and Peter criteria (23). Healthy control (HC) subjects matched for GTCCTTAAGCACAGCCCTGA-39; FCGR1B-reverse, 59-AAACATTA- age were recruited as the control group. TTCCCACTGCCAGA-39; PLAC8-forward, 59-CGTTGTGACCCAACC- DNA microarray analysis of monocytes TGGAG-39; PLAC8-reverse, 59-TCCACACAGACAGCATTCATTCATA- 39; TLR1-forward, 59-CTCCCAACTTTGTCCAGAGTGAA-39; TLR1- Monocytes (CD14+ cells) were isolated from HC (n = 3, one male and two reverse, 59-TTCCAGCAAGATCAGGATTAAGCTA-39; S100 calcium females; the mean 6 SD of age was 51.0 6 7.9 y), inactive-ASD (n =3, binding protein A12 (S100A12) –forward, 59-TCTCTAAGGGTGAGCT- one male and two females; the mean 6 SD of age was 48.7 6 10.7 y), and GAAGCA-39; S100A12-reverse, 59-CAATGGCTACCAGGGATATGAA- active-ASD patients (n = 3, one male and two females; the mean 6 SD of 39; CD55 molecule (CD55)–forward, 59-TGTCTGGGTCATCCCACATT- age was 48.7 6 10.7 y) by MACS (Miltenyi Biotec, Bergisch Gladbach, TC-39; CD55-reverse, 59-ATGGTTACTAGCGTCCCAAGCA-39; Pim-1 Germany). In ASD, monocytes were isolated from the same patients in proto-oncogene (PIM1)–forward, 59-GGACAGTGCTTGATACAGGAA- active and inactive stages (the mean 6 SD interval times of active and CAAC-39; PIM1-reverse, 59-CCCGGGATATTTCAGAGTCCAG-39;BCL2- inactive was 4.33 6 1.15 mo). The purity of MACS-isolated CD14+ cells related protein A1 (BCL2A1)–forward, 59-GCCAGCTCAAGACTT- was .99%. Total RNA was extracted from each cell using Isogen (Nippon TGCTCTC-39; BCL2A1-reverse, 59-GGACCTGATCCAGGTTGTGGTA- Gene, Tokyo, Japan) and then purified using the RNeasy Micro Kit 39; superoxide dismutase 2 (SOD2)–forward, 59-CGGCCTACGTGAAC- (Qiagen, Venlo, the Netherlands) according to the instructions provided by AACCTG-39; SOD2-reverse, 59-GCTATGATTGATATGACCACCACCA- the manufacturer. Total RNA samples were prepared and processed for 39; phospholipid scramblase 1 (PLSCR1)–forward, 59-AAATAAGTGGT- microarray analysis using a GeneChip Human Genome U133 Plus 2.0 CCATGTGTTGTGTG-39; PLSCR1-reverse, 59-GTTATCAGCGTCTGT- Array (Affymetrix) according to the standard protocol supplied by the AAATGCCTCT-39; cytochrome P450 family 1 subfamily B polypeptide 1 manufacturer. All of the microarray data are Minimum Information about a (CYP1B1)–forward, 59-ACGTACCGGCCACTATCACTGAC-39;CYP1B1- Microarray Experiment–compliant and have been deposited in a Minimum reverse, 59-TGATCCAATTCTGCCTGCACTC-39; STEAP family mem- Information about Microarray Experiment–compliant database, the Na- ber 4 (STEAP4)–forward, 59-AAGCAATTCATGAAGCCTGAAGC-39; tional Center for Biotechnology Information Gene Expression Omnibus STEAP4-reverse, 59-GGCCAAAGGTCAGGTCTGGA-39; IL-1 receptor (accession no. GSE113645; http://www.ncbi.nlm.nih.gov/geo/), as detailed antagonist (IL1RN)–forward, 59-TGCTGCAGTCACAGAATGGAAA-39; on the Functional Genomics Data Society Web site (http://www.fged.org/ IL1RN-reverse, 59-AAGGTCTTCTGGTTAACATCCCAGA-39; GAPDH- projects/miame/). The obtained microarray data were quantified with the forward, 59-GCACCGTCAAGGCTGAGAAC-39; GAPDH-reverse, 59- Factor Analysis for Robust Microarray Summarization (FARMS) algorithm TGGTGAAGACGCCAGTGGA-39. The Journal of Immunology 3

In vitro cell culture studies with monocytes 10 min. Protein concentrations were measured using the BCA Protein

+ Assay Reagent Kit (Pierce, Rockford, IL) and then boiled in SDS sample Monocytes (CD14 cells) were isolated from PBMCs by MACS (Miltenyi buffer. The cell culture supernatant was concentrated with methanol/ m Biotec). The isolated cells were stimulated with LPS (1 g/ml; Sigma, chloroform, and the pellets were lysed in the SDS sample buffer. Anti- b St. Louis, MO), IL-1 (10 ng/ml; BioLegend, San Diego, CA), IL-6 PLAC8 Abs (1/1000; Cell Signaling Technology), anti–IL-1b Abs (1/500; a (10 ng/ml; BioLegend), or TNF- (10 ng/ml; BioLegend) for 3, 6, 12, Cell Signaling Technology), anti-pro–IL-18 (1/500; MBL), anti–caspase-1 24, and 48 h. Then, total RNA was extracted from each cell using Isogen (1/500; Cell Signaling Technology), anti-LC3 Abs-HRP-DirecT (1/1000; (NIPPON GENE), and PLAC8 mRNA was exacted by qPCR. At 12 and MBL), anti–b-actin Abs (1/3000; Sigma-Aldrich), anti-mouse IgG HRP 24 h after LPS stimulation, the cell lysate and culture supernatant were conjugate (Cell Signaling Technology), and goat anti-rabbit IgG HRP collected for Western blotting. conjugate (Bio-Rad, Hercules, CA) were used for immunoblotting. Cloning and establishment of the PLAC8-overexpressing Statistical analysis cell lines Allvaluesareexpressedasmean6 SD. The Mann–Whitney U test, Kruskal– The pLVSIN-AcGFP1-N1 vector (Takara Bio) was used to construct Wallis test, and Student t test were used to compare two or more independent PLAC8-overexpressing cells. The DNA sequence of human PLAC8 continuous variables. Correlation between two continuous variables was (NM_001130715.1) was obtained from human monocytes by RT-PCR assessed by the Spearman rank correlation coefficient. The p values ,0.05 and inserted into the vector by In-Fusion HD Cloning Kit (Takara Bio). were considered significant. All data were analyzed using the Statistical The primers of PLAC8 used for the RT-PCR were as follows: PLAC8- Package for Social Sciences (SPSS version 21; IBM, Chicago, IL). forward, 59-GAACTCAGATCTCGAAAAATGCAAGCTCAGGCGC-39; and PLAC8-reverse, 59-CATGACCGGTGGATCGAAAGTACGCATGG- CTCTC-39. As a control, we generated a lentiviral vector that expressed Results only AcGFP1 (mock). Lentiviruses were prepared by transfecting the

PLAC8 mRNA expression in monocytes from Downloaded from plasmid into Lenti-X293T packaging cells, which facilitate optimal len- active-ASD patients tivirus production. Lentiviral particles were obtained according to the in- structions supplied by the manufacturer and concentrated using the The purpose of this study was to identify genes specifically as- Lenti-X Concentrator kit (Takara Bio). The THP-1 cells, which are a hu- sociated with the active phase of the disease. To examine the man monocytic cell line (RBRC-RCB1189; RIKEN BioResource Research Center), were transduced with these lentiviral particles with polybrene (16 upregulated genes in active-ASD patients, monocytes were isolated mg/ml) by centrifugation for 75 min at 1200 3 g at 32˚C. Mock- and from PBMCs and analyzed by DNA microarray. Fig. 1A shows a

PLAC8-transducted THP-1 cells were selected by puromycin (0.5 mg/ml). two-dimensional plot of PCA applied to FARMS-normalized data. http://www.jimmunol.org/ For AcGFP1 detection, these cells were fixed with smear gel (Genostaff, The gene expression patterns in the three groups (HC, active ASD, Tokyo, Japan), and AcGFP1 was detected using a fluorescence microscope and inactive ASD) showed distinct clusters specific to each group. (Fluoview FV10i; Olympus, Tokyo, Japan). We next identified those genes that showed differences in ex- Stimulation with inflammasome activators for monocytes and pression among the three groups. A total of 542 and 570 probe sets THP-1 cells were identified as upregulated in active-ASD patients compared Monocytes were isolated from PBMCs by MACS (Miltenyi Biotec), and with HC and inactive-ASD patients. Furthermore, 82 probe sets these cells were plated at a density of 3 3 105 cells per well in 96-well (corresponding to 68 unique genes) were upregulated in mono- plates. These cells were primed with LPS (1 mg/ml) for 12 h in RPMI 1640 cytes from active-ASD patients compared with HC and inactive- (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Sigma- ASD patients (Supplemental Table II). We defined these 68 genes by guest on September 25, 2021 Aldrich) and 100 U/ml penicillin–streptomycin (Life Technologies). The culture medium was subsequently replaced with Opti-MEM (Invitrogen), as active-ASD–specific, highly expressed genes and subjected and the cells were stimulated with nigericin (5 mM; MilliporeSigma) for them to further analysis. 6 h. Primary THP-1, mock–, and PLAC8-overexpressing THP-1 (PLAC8– Supplemental Fig. 1 shows significantly enriched GO terms 5 THP-1) cells were plated at a density of 5 3 10 cells per well in 48-well found within 68 active-ASD–specific, highly expressed genes. With m plates. These cells were primed with LPS (1 g/ml) for 4 h in RPMI 1640 regard to the hierarchical structure of GO, the more specific GO (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and 100 U/ml penicillin–streptomycin (Life Technologies). The culture medium was term appears in the deeper hierarchy. Therefore, we focused on two subsequently replaced with Opti-MEM (Invitrogen), and the cells were GO terms, “apoptotic mitochondrial changes” and “cytokine- stimulated with nigericin (5 mM; MilliporeSigma) for 4 h. For inhibition mediated signaling pathway,” among the 14 significantly enriched of autophagy, the THP-1 cells were preincubated with 3-methyladenine GO terms within the active-ASD–specific, highly expressed genes. (3-MA; Santa Cruz Biotechnology, Dallas, TX) for 1 h before LPS or nigericin stimulation. For induction of autophagy, THP-1 cells were stimulated with Using this approach, we identified 13 genes from the two GO terms rapamycin (LC Laboratories, Woburn, MA) for 1 h after LPS stimulation. for validation by qPCR. These were CLU, FCGR1B, PLAC8, The cell lysate and culture supernatants were used for ELISA and Western TLR1, S100A12, CD55, PIM1, BCL2A1, SOD2, PLSCR1, blotting. For detection of cell death, mock– and PLAC8–THP-1 cells were CYP1B1, STEAP4, and IL1RN. stained by propidium iodide solution (BioLegend). The stained cells were The validation analysis confirmed higher mRNA expression analyzed on a BD FACSVerse flow cytometer (Becton Dickinson, Mountain View, CA), and data were processed using FlowJo software (Tree Star, levels of PLAC8 and CLU in active-ASD patients than in HC and Ashland, OR). inactive-ASD patients (Fig. 1B, p , 0.05, p , 0.05, respectively). In contrast, FCGR1B and S100A12 mRNA expression levels were Measurement of cytokines in plasma and culture supernatant significantly higher in active-ASD patients than in HC (Fig. 1B, ELISA was used to measure the concentrations of IL-1b (R&D Systems, p , 0.05, p , 0.05, respectively). The mRNA expression levels of Minneapolis, MN), IL-18 (Medical and Biological Laboratories [MBL], the other nine genes (STEAP4, PIM1, PLSCR1, BCL2A1, CD55, Nagoya, Japan), and PLAC8 (MyBioSource, San Diego, CA) in the culture supernatant and plasma. The levels of IL-1b, IL-6, and TNF-a in the SOD2, IL1RN, CYP1B1, TLR1) were not significantly different plasma samples were measured using the BD Cytometric Bead Array (BD among the three groups. Biosciences, San Jose, CA). The assays were performed in accordance To determine the disease specificity of PLAC8 and CLU mRNA with the manufacturer’s instructions. The samples were evaluated using a expression, we compared active-ASD and inactive-ASD patients BD FACSVerse flow cytometer (Becton Dickinson) and analyzed using and patients with other rheumatic diseases, including RA, SS, FCAP Array Software (BD Biosciences). SLE, and PM/DM. As shown in Fig. 1C, CLU mRNA expression Western blot analysis level was also higher in RA than in HC, inactive ASD, SS, SLE, Cells were lysed with a cell lysis buffer (50 mM Tris-HCl, 280 mM NaCl, and PM/DM. In contrast, PLAC8 mRNA expression level was 0.5% NP-40, 0.2 mM EDTA, 10% glycerol, and protease inhibitor mixture higher in active ASD than in HC, inactive ASD, RA, SS, SLE, [Cell Signaling Technology, MA]) and centrifuged at 15,000 rpm at 4˚C for and PM/DM (p , 0.05, p , 0.05, p , 0.05, p , 0.05, p , 0.05, 4 ROLE OF PLAC8 IN ASD Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. PLAC8 mRNA in monocytes. (A) Peripheral monocytes were isolated from active-ASD patients (n = 3), inactive-ASD patients (n = 3), and HC subjects (n = 3). The gene expression patterns in the three groups are indicated in PCA. In PCA, the contribution of principal component PC 1 was 33.4% and that of PC2 was 23.1%. Samples a1, a2, and a3 are from the patients with active ASD. Samples i1, i2, and i3 are from the patients with inactive ASD. Samples HC1, HC2, and HC3 are from HC. In ASD, monocytes were isolated from the same patients in active and inactive stages. To compare the genes upregulated in active ASD relative to inactive ASD, the SAM was applied to the FAMS-normalized paired data with modification as described in Materials and Methods (with correspondence). To compare the differentially expressed probe sets between active ASD and HC, the rank products method was applied to FARMS-normalized data (with no response). (B) qPCR analysis was performed using peripheral monocytes isolated from active-ASD patients (n = 7), inactive-ASD patients (n = 10), and HC (n = 8). Values are mean 6 SD. *p , 0.05 by Kruskal–Wallis test. (C) qPCR analysis was performed using peripheral monocytes isolated from active-ASD patients (n = 7), inactive-ASD patients (n = 10), HC (n = 8), RA patients (n = 8), SS patients (n = 5), SLE patients (n = 6), and PM/DM patients (n = 5). Values are mean 6 SD. *p , 0.05 by Kruskal–Wallis test. The Journal of Immunology 5 p , 0.05, respectively). These results indicated that the upregu- extracellularly from primary monocytes (Supplemental Fig. 2B). lation of PLAC8 mRNA in monocytes was specific to the patients However, the amount of IL-1b and IL-18 production induced by with active ASD. LPS and nigericin was not significantly different between HC and inactive-ASD patients (Supplemental Fig. 2B). PLAC8 mRNA expression correlates with serologic To examine the function of upregulated PLAC8 in the pathway of markers in ASD IL-1b and IL-18 production, we used the human monocytic cell Next, we examined the correlation between PLAC8 mRNA ex- line THP-1. First, we confirmed that the production of IL-1b and pression level in monocytes and serum CRP and ferritin. In patients IL-18 by THP-1 cells stimulated by LPS and nigericin was similar with ASD, the expression of PLAC8 mRNA was correlated with to primary monocytes (Supplemental Fig. 2C). Stimulation with serum CRP and ferritin levels (Fig. 2A, 2B, r = 0.855, p , 0.01, r = LPS or nigericin did not induce PLAC8 protein production in 0.674, p , 0.005, respectively). Further analysis showed signifi- THP-1 cells, as confirmed by Western blotting (Supplemental Fig. cantly higher serum concentration of IL-6, TNF-a, IL-1b, and IL- 2D). Thus, to analyze the upregulation conditions of PLAC8, 18 in active ASD than in HC (Fig. 2C, p , 0.05, p , 0.05, p , PLAC8–AcGFP1 fusion were overexpressed in THP-1 0.05, p , 0.05, respectively). In the patients with ASD, plasma cells using the lentivirus infection system (Supplemental Fig. 2E– IL-6, TNF-a, IL-1b, and IL-18 levels correlated with serum CRP G). Under steady state, PLAC8 overexpression did not affect the levels (Fig. 2D, r = 0.860, p , 0.01, r = 0.661, p , 0.01, r = proliferation of these cells (Supplemental Fig. 2H). Stimulation with 0.664, p , 0.01, r = 0.630, p , 0.01, respectively). In contrast, LPS and nigericin significantly decreased the production of IL-1b plasma IL-18 levels, but not IL-1b, IL-6, or TNF-a levels, cor- and IL-18 in PLAC8–THP-1 relative to the control (mock) THP-1 related with serum ferritin levels in ASD patients (Fig. 2E, r = cells (Fig. 4A, p , 0.05, p , 0.05, respectively). Downloaded from 0.858, p , 0.05). Moreover, serum IL-1b and IL-18 levels cor- IL-1b and IL-18 are produced from pro–IL-1b and pro–IL-18 related with PLAC8 mRNA expression levels in ASD patients by similar mechanisms through the activation of inflammasome (Fig. 2F, r = 0.635, p , 0.05, r = 0.681, p , 0.01, respectively). pathways, including pro–caspase-1 and caspase-1. We next ex- Thus, these results suggested that the expression levels of PLAC8 amined the expression of pro–IL-1b, pro–IL-18, and pro–caspase-1 mRNA seemed to be an activity or severity marker for ASD. in cell lysates and IL-1b and caspase-1 p20/p22 in culture su-

pernatants by Western blotting. As shown in Fig. 4B, the amount http://www.jimmunol.org/ PLAC8 is upregulated by TLR4 signaling cascade of IL-1b was decreased in culture supernatants from PLAC8– To confirm the upregulation of PLAC8 in monocytes, we examined THP-1 cells compared with mock–THP-1 cells after LPS and the Western blotting analysis. The amount of PLAC8 proteins was nigericin stimulation. Furthermore, stimulation with LPS de- significantly increased in active ASD than HC and inactive ASD, creased the amount of pro–IL-1b and pro–IL-18 in cell lysates of similar to the results of mRNA expression levels (Fig. 3A, p , PLAC8–THP-1 cells compared with mock–THP-1 cells (Fig. 4B, 0.05, p , 0.05, respectively). Next, to determine the mechanism p , 0.05, p , 0.05, respectively). In contrast, the amount of pro– of PLAC8 upregulation in active ASD, primary monocytes iso- caspase-1 in cell lysates and caspase-1 p20/p22 in culture super- lated from HC were stimulated by LPS, IL-1b, IL-6, and TNF-a natants were not different between mock– and PLAC8–THP-1 in vitro. PLAC8 mRNA expression was significantly increased at cells under each condition (Fig. 4B). by guest on September 25, 2021 3, 6, 12, and 24 h after LPS stimulation (Fig. 3B, p , 0.05, p , To clarify the inhibitory effects of PLAC8 on IL-1b and IL-18 0.05, p , 0.05, p , 0.05, respectively). After stimulation with production, we analyzed the induction of their mRNAs by qPCR. TNF-a, PLAC8 mRNA was slightly increased at 3 h, but this At 0, 1, 2, 3, and 4 h after LPS stimulation, the expression levels increase was transient (Fig. 3B, p , 0.05). In contrast, IL-1b and of total IL-1b and IL-18 mRNA were not different between IL-6 did not affect the expression of PLAC8 mRNA in monocytes. mock– and PLAC8–THP-1 cells (Fig. 4C). PLAC8 was localized After LPS stimulation with primary monocytes from inactive- in the cytoplasm and did not change after activation by LPS ASD patients, PLAC8 mRNA was significantly upregulated, (Fig. 4D). Furthermore, the rate of LPS-induced cell death was not similar to monocytes from HC (Fig. 3C, p , 0.05, p , 0.05, different between mock– and PLAC8–THP-1 cells (Fig. 4E). respectively). Furthermore, LPS-induced PLAC8 mRNA expres- These results suggested that the upregulated PLAC8 suppressed sion levels in monocytes were not significantly different between the production of pro–IL-1b and pro–IL-18 proteins indepen- HC and inactive-ASD patients (Fig. 3C). In addition to the change dently of these mRNA transcriptions. in mRNA expression level, we confirmed that LPS stimulation b increased the production of PLAC8 proteins at 12 and 24 h in PLAC8 induces autophagy and regulates pro–IL-1 and monocytes (Fig. 3D, p , 0.05, p , 0.05, respectively). Similar to pro–IL-18 production PLAC8 production in cell lysates, soluble PLAC8 was detected in To investigate the induction of autophagy, the expression of LC3-II, the culture supernatants of LPS-stimulated monocytes (Fig. 3D, p a marker of autophagy, was analyzed by Western blotting. LPS , 0.05, p , 0.05, respectively). However, the amount of soluble stimulation increased the rate of LC3-II expression in PLAC8– PLAC8 proteins in plasma was not significantly different between THP-1 cells compared with mock–THP-1 cells (Fig. 5A, p , HC, inactive-ASD, and active-ASD patients (Fig. 3E). These re- 0.05). Furthermore, to clarify the effects of autophagy on IL-1b sults indicated that PLAC8 was induced by TLR4 signaling and and IL-18 production, THP-1 cells were cultured with LPS, might be involved in several intracellular molecular processes. nigericin, and autophagy inhibitor 3-MA. Culture of THP-1 cells simultaneously with LPS, nigericin, and 3-MA significantly in- b PLAC8 suppresses LPS-induced IL-1 and IL-18 production creased the production of IL-1b and IL-18 in a 3-MA dose– We examined the function of PLAC8 in inflammasome-induced dependent manner (Fig. 5B). Similar to these results, IL-1b and production of cytokines, such as IL-1b and IL-18. After LPS IL-18 production by THP-1 cells was significantly increased when stimulation with primary monocytes from HC, IL-1b and IL-18 3-MA was added at the same time as LPS. However, when 3-MA mRNA levels were significantly increased at 3, 6, and 12 h was added at the same time as nigericin, the production of IL-1b (Supplemental Fig. 2A, p , 0.05, p , 0.05, p , 0.05, respec- and IL-18 from THP-1 cells did not increase. tively). Furthermore, we confirmed that the combination of LPS Finally, we examined the effects of autophagy on the production of and nigericin strongly induced the secretion of IL-1b and IL-18 IL-1b and IL-18. As shown in Fig. 5C, 3-MA inhibited autophagy 6 ROLE OF PLAC8 IN ASD Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Correlation between PLAC8 mRNA expression and serologic markers in ASD. (A and B) Peripheral monocytes and plasma were isolated from active-ASD (n = 7) and inactive-ASD patients (n = 10). The correlations between PLAC8 mRNA expression in monocytes and serum CRP and ferritin levels were examined. *p , 0.01, **p , 0.005 by Spearman correlation. (C) Plasma samples were obtained from active-ASD patients (n = 7), inactive-ASD patients (n = 10), and HC (n = 8). The plasma concentrations of IL-6, TNF-a, and IL-1b were measured by flow cytometry. The plasma concentrations of IL-18 were measured by ELISA. Values are mean 6 SD. *p , 0.05 by Kruskal–Wallis test. (D and E) Correlation between plasma cytokines and CRP and ferritin levels in active ASD (n = 7) and inactive ASD (n = 10). *p , 0.05, **p , 0.01 by Spearman correlation. (F) Peripheral monocytes and plasma were isolated from active-ASD (n = 7) and inactive-ASD patients (n = 10). Correlation between PLAC8 mRNA expression in monocytes and serum concen- trations of IL-6, TNF-a, IL-1b, and IL-18. *p , 0.05, **p , 0.01 by Spearman correlation. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. LPS stimulation upregulates PLAC8 expression. (A) Peripheral monocytes were isolated from HC (n = 4), active ASD (n = 5), and inactive ASD (n = 5). These cell lysates were collected, and the production levels of PLAC8 and b-actin were analyzed by Western blotting. *p , 0.05 by Kruskal– Wallis test. (B) Peripheral monocytes were isolated from HC (n = 3). The isolated cells were stimulated by LPS (1 mg/ml), IL-1b (10 ng/ml), IL-6 (10 ng/ml), and TNF-a (10 ng/ml). After 3, 6, 12, 24, and 48 h, the expression of PLAC8 mRNA was analyzed by qPCR. Data are representative of at least two independent experiments. Values are mean 6 SD. *p , 0.05 by Mann–Whitney U test. (C) Peripheral monocytes were isolated from HC (n = 4) and inactive ASD (n = 5). The isolated cells were stimulated by LPS (1 mg/ml) for 12 h. The expression of PLAC8 mRNA was analyzed by qPCR. *p , 0.05 by Mann–Whitney U test. n.s, not significant. (D) Peripheral monocytes were isolated from HC (n = 4) and stimulated with LPS (1 mg/ml). After 12 and 24 h, cell lysate and culture supernatant were collected and the production levels of PLAC8 and b-actin were analyzed by Western blotting. Graph indicated pooled data from four individuals. Values are mean 6 SD. *p , 0.05 by Kruskal–Wallis test. (E) Plasma samples were isolated from HC (n =8) and patients with active ASD (n = 9) and inactive ASD (n = 10). The concentration of soluble PLAC8 was measured by ELISA.

and abrogated the inhibitory effects of PLAC8 on IL-1b and IL-18 in the cell lysates was decreased by induction of autophagy (Fig. 6B). production (p , 0.05, p , 0.05, respectively). Similarly, the pro- These results indicated that upregulated PLAC8 acts on the synthesis duction of IL-1b in the culture supernatant and pro–IL-1b and pro– of inactive precursors of IL-1b and IL-18 and suppressed the pro- IL-18 in the cell lysates was increased by 3-MA dose-dependently in duction of IL-1b and IL-18 through enhanced autophagy. PLAC8–THP-1 cells (Fig. 5D, 5E). In contrast, the expression of caspase-1 in the culture supernatants and pro–caspase-1 in the cell Discussion lysates was not increased in the presence of 3-MA (Fig. 5D, 5E). This study demonstrated that PLAC8 plays a regulatory role in Furthermore, we also confirmed that the production of IL-1b and IL- IL-1b and IL-18 production through the regulation of autophagy. 18 in the culture supernatants (Fig. 6A) and pro–IL-1b and pro–IL-18 The expression of PLAC8 was upregulated in activated monocytes 8 ROLE OF PLAC8 IN ASD Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. PLAC8 overexpression inhibits IL-1b and IL-18 production. (A) Mock– and PLAC8–THP-1 cells were stimulated with LPS (1 mg/ml) for 4 h and then these cells were restimulated with nigericin (5 mM) for 6 h. The amounts of IL-1b and IL-18 in the culture supernatant were measured by ELISA. Data are representative of at least three independent experiments. Values are mean 6 SD. (B) Mock– and PLAC8–THP-1 cells were stimulated with LPS (1 mg/ml) for 4 h and then restimulated with nigericin (5 mM) for 6 h. The expression levels of pro–IL-1b, pro–IL-18, pro–caspase-1, and b-actin in cell lysate and IL-1b, caspase-1 p20/p22, and b-actin in culture supernatant were analyzed by Western blotting. Data are representative of at least two independent experiments. Right graph shows the rate of pro–IL-1b, pro–IL-18, pro–caspase-1, and b-actin in the cell lysate. Graph data were pooled from three independent experiments. Values are mean 6 SD. (C) Mock– and PLAC8–THP-1 cells were stimulated with LPS (1 mg/ml). After 1, 2, 3, and 4 h, the expression levels of total IL-1b and IL-18 mRNA were analyzed by qPCR. Data are representative of at least two independent experiments. Values are mean 6 SD. (D) Mock– and PLAC8–THP-1 cells were stimulated with LPS (1 mg/ml). After 4 h, cell death (PI+) was analyzed by FCM. Data are representative of at least three independent experiments. Values are mean 6 SD. (E) PLAC8–THP-1 cells were stimulated with LPS (1 mg/ml) for 4 h. PLAC8–AcGFP1 was detected by fluorescence microscopy. Data are representative of at least two independent experiments. *p , 0.05 by Student t test. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Induction of autophagy inhibits IL-1b and IL-18 production. (A) Mock– and PLAC8–THP-1 cells were stimulated with LPS (1 mg/ml) for 4 h. The expression levels of LC3-I/LC3-II and b-actin in cell lysate were analyzed by Western blotting. Right graph shows the rate of LC3-II and b-actin in cell lysate. Graph data were pooled from three independent experiments. Values are mean 6 SD. *p , 0.05 by Student t test. (B) THP-1 cells were stimulated with LPS (1 mg/ml) for 4 h followed by nigericin (5 mM) for 6 h simultaneously with 3-MA (0, 0.05, 0.5, 5 mM). (1) Simultaneously with LPS, nigericin, and 3-MA; (2) simultaneously with LPS and 3-MA; and (3) simultaneously with nigericin and 3-MA. The amounts of IL-1b and IL-18 in culture supernatant were measured by ELISA. Data are representative of at least two or three independent experiments. Values are mean 6 SD. *p , 0.05 by Kruskal–Wallis test. (C) Mock– and PLAC8–THP-1 cells were stimulated with LPS (1 mg/ml) for 4 h and then by nigericin (5 mM) for 6 h simultaneously with 3-MA (0, 0.05, 0.5, 5 mM). The amounts of IL-1b and IL-18 in the culture supernatant were measured by ELISA. Data are representative of at least two or three independent experiments. Values are mean 6 SD. *p , 0.05 by Kruskal–Wallis test. (D and E) Mock– and PLAC8–THP-1 cells were stimulated with LPS (1 mg/ml) for 4 h simultaneously with 3-MA (0, 0.05, 0.5, 5 mM). The expression levels of pro–IL-1b, pro–IL-18, pro–caspase-1, LC3-I/LC3-II, and b-actin in cell lysate and IL-1b and caspase-1 p20/p22 in culture supernatant were analyzed by Western blotting. Right graph shows the rates of pro–IL-1b, pro–IL-18, pro–caspase-1, LC3-II, and b-actin in cell lysate. Graph data were pooled from three independent experiments. Values are mean 6 SD. *p , 0.05 by Kruskal–Wallis test. andinmonocytesisolatedfromactive-ASD patients. In human current study, we identified high expression of PLAC8 mRNA and PLAC8-overexpressing monocytic cell lines, the production levels of PLAC8 proteins in monocytes from active-ASD patients but not in pro–IL-1b and pro–IL-18 were suppressed through the enhancement those from inactive-ASD patients. Interestingly, the expression of of autophagy pathways. These results suggest that PLAC8 seems to PLAC8 mRNA in monocytes correlated with serum levels of CRP, play a regulatory role in the production of IL-1b and IL-18 (Fig. 7). ferritin, IL-1b, and IL-18 in patients with ASD. Serum CRP and ASD is a rare multisystemic autoinflammatory disorder of ferritin are the most frequently used biomarkers for ASD diag- unknown etiology. Several mediators have been described as ac- nosis and activity (39). Furthermore, serum IL-1b and IL-18 levels tivity or severity markers for ASD, such as serum soluble intra- also correlated strongly with disease activity and are considered an cellular adhesion molecule-1 (sICAM-1) (33), macrophage important pathogenic factor in ASD (6, 8). These results support migration inhibitory factor (MIF) (34), S100A8/9 (35), S100A12 the notion that the expression of PLAC8 mRNA in monocytes can (36), calprotectin (37), and a2-glycoprotein (LRG) (38). In the be potentially a useful marker of ASD activity. 10 ROLE OF PLAC8 IN ASD Downloaded from http://www.jimmunol.org/

FIGURE 6. Reduction of IL-1b and IL-18 production by autophagy. (A) Primary THP-1 cells were stimulated with LPS (1 mg/ml) for 4 h and then with rapamycin (0, 10, 30, 100 mM) for 1 h and nigericin (5 mM) for 6 h. The amounts of IL-1b and IL-18 in culture supernatant were measured by ELISA. Data are representative of two or three independent experiments. Values are mean 6 SD. *p , 0.05 by Kruskal–Wallis test. (B) Primary THP-1 cells were stimulated with LPS (1 mg/ml) for 4 h and with rapamycin (0, 10, 30, 100 mM) for 1 h. The expression levels of pro–IL-1b, pro–IL-18, LC3-I/LC3-II, and b-actin in cell lysates were analyzed by Western blotting. Right graph shows the rates of pro–IL-1b, LC3-II and b-actin in the cell lysate. Graph data were by guest on September 25, 2021 pooled from five independent experiments. Values are mean 6 SD. *p , 0.05, **p , 0.01 by Kruskal–Wallis test.

In ASD, an uncontrolled activation of monocytes had been thought IL-1b and IL-18 production in THP-1 cells. In general, it is to induce the severe clinical features (3). After LPS stimulation, known that the production of IL-1b and IL-18 is a two-stage the expression of PLAC8 mRNA and proteins was increased in process (42). The first step is transcription and translation of monocytes from inactive-ASD patients as well as HC. Furthermore, the inactive precursors (pro–IL-1b and pro–IL-18), and this a large amount of IL-1b and IL-18 was also produced from process commonly induces the ligation of pattern-recognition monocytes after LPS and nigericin stimulation. However, the pro- receptors, particularly TLRs. The second step is the cleavage duction of PLAC8, IL-1b, and IL-18 from LPS-activated mono- of pro–IL-1b and pro–IL-18 into bioactive mature cytokines, and cytes was not significantly different between HC and inactive-ASD this second process is usually dependent on the activation of patients. Thus, these results suggested that TLR4 signaling cascade pro–caspase-1 to caspase-1 by inflammasome activation. Fur- in ASD patients was not different from that in HC. thermore, the second process also induces the cell membrane to To determine the molecular mechanisms of PLAC8 in the destroy and then release mature IL-1b, IL-18, or b-actin into the regulation of IL-1b and IL-18 production in monocytes, we used extracellular space (42). Our study showed low production of the human monocytic cell line THP-1. THP-1 cells were well used pro–IL-1b and pro–IL-18 in PLAC8–THP-1 cells compared with to examine the intracellular molecular mechanisms instead of mock–THP-1 cells after LPS stimulation. However, the amounts primary monocytes (40). After THP-1 cells were stimulated with of pro–caspase-1 and caspase-1 were not different between LPS and nigericin, we detected production of IL-1b and IL-18 PLAC8–THP-1 and mock–THP-1 cells. Furthermore, the cell similar to that of primary monocytes. In contrast, PLAC8 was not survival and mRNA expression levels of total IL-1b and IL-18 induced by LPS in THP-1 cells, unlike in primary monocytes. were not different between PLAC8–THP-1 and mock–THP-1 Previously, it was known that THP-1 cells, unlike primary cells after LPS stimulation. These findings indicate that the monocytes, express low levels of surface CD14, a part of the LPS low production of IL-1b and IL-18 in PLAC8–THP-1 cells is binding receptor (41). Thus, the low expression of CD14 on THP- independent of caspase-1 mediated by inflammasome activation, 1 cells might be insufficient for inducing PLAC8 after LPS suggesting that PLAC8 seems to suppress the pro–IL-1b and stimulation but sufficient for the production of cytokines, such as pro–IL-18 proteins independently of mRNA transcription. Pre- IL-1b and IL-18. viously, it was reported that the autophagy might be controlled Then, we generated PLAC8–THP-1 cells. We showed that the by IL-1b production, which is independent of inflammasome production of IL-1b and IL-18 was significantly decreased in activation, similar to PLAC8–THP-1 cells. Harris et al. (43) in- PLAC8–THP-1 cells compared with the control THP-1 (mock– dicated that the autophagy might control the production of IL-1b THP-1) cells, suggesting that PLAC8 overexpression inhibits through the targeting of pro–IL-1b for lysosomal degradation. The Journal of Immunology 11

FIGURE 7. Schematic diagram of the proposed function of PLAC8 in IL-1b and IL-18 production. Schematic diagram illustrating that PLAC8 suppresses IL-1b and IL-18 production via enhancement of auto- phagy. Two steps might be needed for inhibition of IL-1b and IL-18 production by PLAC8 in primary monocytes. The first step is the upregulation of PLAC8, pro–IL-1b, and pro–IL-18 in monocytes by LPS stimulation. The second step is the inhibition of pro– IL-1b and pro–IL-18 through the enhancement of autophagic machinery by upregulated PLAC8, which is independent of caspase-1.

Autophagy is a highly conserved homeostatic process for the se- Disclosures Downloaded from questration and degradation of cytosolic macromolecules, excess or The authors have no financial conflicts of interest. damaged organelles, and certain pathogens (44–46). In contrast, autophagy controls the production of the inflammatory cytokines IL- 1b and IL-18 both in vivo and in vitro (43, 47). In the current study, References b 1. Bywaters, E. G. L. 1971. Still’s disease in the adult. Ann. Rheum. Dis. 30: 121– the production levels of IL-1 and IL-18 were significantly increased 133.

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