Plasmacytoid Dendritic Cells and C1q Differentially Regulate Inflammatory Gene Induction by Lupus Immune Complexes

This information is current as Deanna M. Santer, Alice E. Wiedeman, Thomas H. Teal, of September 23, 2021. Pradipta Ghosh and Keith B. Elkon J Immunol published online 5 December 2011 http://www.jimmunol.org/content/early/2011/12/04/jimmun ol.1102797 Downloaded from

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 5, 2011, doi:10.4049/jimmunol.1102797 The Journal of Immunology

Plasmacytoid Dendritic Cells and C1q Differentially Regulate Inflammatory Gene Induction by Lupus Immune Complexes

Deanna M. Santer,* Alice E. Wiedeman,* Thomas H. Teal,† Pradipta Ghosh,† and Keith B. Elkon*,†

Immune complexes (ICs) play a pivotal role in causing inflammation in systemic lupus erythematosus (SLE). Yet, it remains unclear what the dominant blood cell type(s) and inflammation-related gene programs stimulated by lupus ICs are. To address these ques- tions, we exposed normal human PBMCs or CD14+ isolated monocytes to SLE ICs in the presence or absence of C1q and performed microarray analysis and other tests for cell activation. By microarray analysis, we identified genes and pathways regulated by SLE ICs that are both type I IFN dependent and independent. We also found that C1q-containing ICs markedly reduced expression of the majority of IFN-response genes and also influenced the expression of multiple other genes induced by

SLE ICs. Surprisingly, IC activation of isolated CD14+ monocytes did not upregulate CD40 and CD86 and only modestly Downloaded from stimulated inflammatory gene expression. However, when monocyte subsets were purified and analyzed separately, the low- abundance CD14dim (“patrolling”) subpopulation was more responsive to ICs. These observations demonstrate the importance of plasmacytoid dendritic cells, CD14dim monocytes, and C1q as key regulators of inflammatory properties of ICs and identify many pathways through which they act. The Journal of Immunology, 2012, 188: 000–000.

ystemic lupus erythematosus (SLE) is a multisystem au- plement components downstream of C3, especially release of C3a http://www.jimmunol.org/ toimmune disease characterized by the presence of high and C5a that promote chemotaxis and inflammation as well as S titer autoantibodies directed against self nucleoproteins deposition of C5b-9 together, that promote tissue injury (11–13). (reviewed in Ref. 1). Some of these Abs interact with Ags to form More recently, C1q has been shown to protect against SLE by immune complexes (ICs) that deposit in the kidneys, skin, and vas- a separate effect, via preventing the stimulation of type I IFN (14, culature (2, 3). ICs also directly engage FcgRs expressed on mac- 15). We observed that, in the absence of C1q, ICs bind to FcgRII rophages and neutrophils, resulting in the release of proinflammatory on plasmacytoid dendritic cells (pDCs) and potently stimulate cytokines, proteolytic enzymes, and reactive oxygen intermediates IFN-a production, whereas C1q-containing ICs (C1q-ICs) bound (4). Thus, ICs, especially after complement activation, are thought to predominantly to monocytes, and IFN-a production by pDCs was be the predominant inducers of tissue injury in SLE (3, 5). markedly attenuated (15). Although C1q-ICs did not stimulate by guest on September 23, 2021 Despite these findings, individuals with loss-of-function muta- monocytes to produce soluble factors such as IL-10 or TNF-a that tions of the first complement component, C1q, almost invariably could explain IFN-a suppression, we could not exclude the pos- develop SLE (reviewed in Ref. 6). This “lupus paradox” (7), in sibility that ICs induce a ligand on monocytes that engages one of which complement activation promotes tissue injury, yet comple- the many inhibitory receptors on pDCs. ment deficiency predisposes to SLE, has, in part, been reconciled To obtain comprehensive data of genes differentially regulated by studies demonstrating the protective role of classical comple- by exposure of blood cells to SLE ICs, to determine how this profile ment pathway components (C1-4) in facilitating the clearance of is altered in the presence of C1q-ICs, and to evaluate the in- lupus Ags (apoptotic debris) (8–10). In contrast, it is the com- flammatory properties of ICs on monocytes in the absence of pDCs, we performed gene expression analysis of peripheral blood as well as isolated monocytes stimulated by ICs or C1q-ICs. Our results *Department of Immunology, School of Medicine, University of Washington, Seattle, WA 98195; and †Division of Rheumatology, School of Medicine, University of enable comparison between the in vitro effects of ICs and ex vivo Washington, Seattle, WA 98195 inflammatory gene transcript profiles as well as those genes reg- Received for publication September 27, 2011. Accepted for publication November 3, ulated by C1q. They also show limited CD14+ monocyte stimu- 2011. lation by ICs in the absence of pDCs and suggest relevant genes This work was supported by National Institutes of Health Grants AR48796 and and pathways that should prove productive for future investigation NS065933. D.M.S. was supported by a Natural Sciences and Engineering Research Council of Canada postgraduate scholarship and a Kirkland scholarship. of SLE pathogenesis. The microarray data presented in this article have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE32285) under Series accession number GSE32285. Materials and Methods Reagents Address correspondence and reprint requests to Dr. Keith B. Elkon, Division of Rheumatology, University of Washington, 1959 NE Pacific Street, Box 356428, Purified C1q protein was purchased from Complement Technology. Neu- Seattle, WA 98195. E-mail address: [email protected] tralizing Ab to IFN-a was purchased from Millipore. Loxoribine was The online version of this article contains supplemental material. purchased from Invivogen. All reagents had ,0.06 EU/ml endotoxin by Abbreviations used in this article: C1q-IC, C1q-containing immune complex; GOI, Limulus amebocyte lysate clot assay (Cape Cod Associates). gene of interest; IC, immune complex; IP-10, IFN-g–inducible protein-10; ISG, IFN- stimulated gene; MFI, mean fluorescent intensity; pDC, plasmacytoid dendritic cell; Patients qRT-PCR, quantitative real-time PCR; SLC16A10, solute carrier family 16, member 10; SLE, systemic lupus erythematosus. All SLE patients fulfilled the American College of Rheumatology 1982 revised criteria for the classification of SLE (16). All serum samples were Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 collected with the respective institutions’ review board approval.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102797 2 IMMUNE COMPLEX GENE ARRAYS

Cell purification genuity software. In some cases, changes in gene expression of 2-fold or greater were analyzed separately. Functional analysis of differentially PBMCs were prepared from healthy human donors or SLE patients using expressed genes was performed using Ingenuity pathways analysis (Inge- Ficoll-Paque density gradient centrifugation. For normal donor experi- nuity Systems). Some genes considered were not eligible for Ingenuity ments, a different healthy donor was used for each independent experiment. pathway analysis, but are included in the Venn diagrams. The significance In certain experiments, pDCs were depleted from PBMCs using BDCA-4 of the association between the data set and the canonical pathway was , magnetic beads (Miltenyi Biotec) with 0.03% remaining in each ex- measured using Fisher’s exact test to calculate a p value representing the periment. As an additional control, PBMCs were mock depleted by in- probability that the association between the transcripts in the data set and cubating cells without beads, but still placed through the magnetic column. the canonical pathway was explained by chance alone. Canonical functions Total monocytes were purified from PBMCs by positive selection with were also determined, and networks were overlaid with expression data . CD14 magnetic beads (Miltenyi Biotec) with consistent purities of 95% from the data set. IFN-stimulated genes (ISGs) were identified by exam- and undetectable percentages of contaminating pDCs. In certain experi- ining eight publications (22–29) plus the interferome.org database specifi- . ments, monocyte subsets were sorted to purities of 90–95% using cally for type I IFN (30). methods described by others (17). Briefly, cells were stained with the following fluorescently labeled Abs (all from BioLegend, unless otherwise Quantitative real-time PCR noted): CD19-PE (clone HIB19), CD56-PE (clone MEM-188), NKp46-PE (clone 9E2), CD15-PE (clone H198), CD2-PE (clone RPA-2.10), HLA- First-strand cDNA was generated with the RNA-to-cDNA kit (Applied DR-PerCp/Cy5.5 (clone L243), CD16-Alexa Fluor 488 (clone 3G8), and Biosystems) using random primers. cDNA was diluted to an equivalent of CD14-allophycocyanin-Alexa Fluor 780 (clone 61D3; eBioscience). Cells 0.2 ng/ml total RNA, and 8 ml was used per reaction. Primers for the were gated for the monocyte population that lacked the PE stain (B cells, reference gene (18S) and genes of interest (GOI) were synthesized by NK cells, granulocytes, and T cells), but which was HLA-DR+; this was Integrated DNA Technologies. After melt curve and standard curve anal- further divided into three monocyte subsets that included the CD14+ ysis, primers were diluted to the most efficient concentrations for quanti- 2 + 2 dim + tative real-time PCR (qRT-PCR) using molecular grade water. BLAST CD16 , CD14 CD16 , and CD14 CD16 subsets that were sorted and Downloaded from collected live using a FACSAria flow cytometer (BD Biosciences). results of the primers show specific sequence homology only to the ref- erence gene or GOI. Reactions in duplicate or triplicate (20 ml) were run Cell stimulation on an ABI Fast 7500 system using a 1:1 mix of template/primer to Sensi- Mix SYBR low-ROX master mix (Bioline). Relative quantification was To form ICs, high dilutions of SLE serum or purified SLE IgG (5–15 mg/ml) calculated using the 22ddCT method with unstimulated cells as baseline to were used as a source of autoantibodies, and freeze-thawed U937 cells determine fold changes for each GOI. Primer sequences for genes are were used as autoantigen, as described previously (15, 18, 19). Briefly, shown in Supplemental Table I.

SLE serum (diluted 1:1000–1:2000 with RPMI 1640 media) was mixed http://www.jimmunol.org/ with U937 freeze-thawed cell extract. Cell debris was removed by cen- Cytokine detection trifugation, and the extract was added to the cell type being tested at a 1% v/v concentration. As reported previously, IFN-a production was RNA, IFN-a levels in supernatants were quantified by ELISA using commer- FcgRIIa, and TLR7 dependent (19). Although many SLE patient sera were cially available Abs, as described (15). IFN-g–inducible protein-10 (IP- used in the course of this study, the two sera used to make ICs for the 10), MCP-1, MCP-3, IL-1RA, IL-8, IL-10, IL-12p40, IL-1b, and MIP-1a microarray experiments both had the following autoantibody profile: Sm/ levels were quantified in supernatants using a custom 9-plex Bio-Plex kit RNP+,Ro2,La2, dsDNA+. ICs were added to normal PBMCs (5 3 105/ from Bio-Rad. TNF-a was measured in supernatants by ELISAs with Ab well) and left unprimed or primed with type I IFN and GM-CSF, as pre- pairs from BioLegend. viously described (15, 18, 19). In our culture system, IFN-a is only pro- Flow cytometry for activation markers duced by pDCs as Abs to BDCA-2 abrogated IFN-a production, as described previously (20), and IFN-a was not detectable in pDC-depleted Cells were left untreated (medium) or ICs were added to total PBMCs, pDC- by guest on September 23, 2021 PBMCs or purified monocyte cultures (data not shown). U937 cells were depleted PBMCs, or purified monocytes as part of the stimulation assay determined to be free of mycoplasma contamination using e-Myco My- described above. At least two different SLE patient ICs were added for each coplasma PCR detection kit (iNtRON Biotechnology). Toxicity of added independent experiment. After 2 d of stimulation, cells were washed and inhibitors was monitored by flow cytometry with LIVE/DEAD I/R (Invi- surface stained to identify monocytes (CD14 clone HCD14; BioLegend) trogen). and the level of expression of the activation markers CD86 (clone IT2.2; Biolegend) and CD40 (clone 5C3; BioLegend). Monocyte subsets were Microarray gated for activation analysis, as described above. The mean fluorescent Unprimed total PBMCs from two different healthy donors were plated in intensities (MFI) for each marker are plotted. 3 6 24-well plates at 2 10 /well in one well and left unstimulated or stim- Statistical analysis ulated with SLE ICs formed, as above, with or without C1q for ∼5 h. For 6 monocyte stimulations, a total of 1 3 10 monocytes per condition from Statistical significance between groups was determined by Mann–Whitney two different healthy donors was plated in two wells of a 96-well plate and U test, unpaired/paired t test, or one-way ANOVA, where appropriate. left unstimulated or stimulated, as above. Cells were washed three times Correlations between parameters were assessed using the Pearson corre- in PBS, and total RNA was isolated using a Qiagen RNeasy kit with lation analysis and linear regression analysis. A p value ,0.05 was con- on column DNase treatment (Qiagen). RNA (400 ng) was checked for sidered significant. Graphs and statistical analyses were performed using high quality using an Agilent 2100 Bioanalyzer (Agilent Technologies) Prism software (v 4; GraphPad Software). by the Fred Hutchinson Cancer Research Center’s Genomics Resource, and samples were labeled and hybridized to Illumina’s Human Ref-8v3 Expression BeadChips following the manufacturer’s recommendations Results (Illumina). BeadChips were scanned on an Illumina Bead Array Reader RNA-containing SLE ICs predominantly induce ISG expression System. in PBMCs Microarray data analysis ICs have been implicated in the induction of tissue inflammation in Probe-level results were generated in GenomeStudio Data Analysis Soft- SLE and many other diseases (reviewed in Ref. 31). Although it is ware’s Gene Expression Module (GSGX) Version 1.5.4 (Illumina). Bead- now well documented that SLE ICs stimulate IFN-a production Chip results were background corrected and quantile normalized using the by pDCs (15, 32–34), the full range of cytokine and other in- Bioconductor package lumi (21). A small offset was applied across all flammatory markers produced by whole blood mononuclear cells arrays to bring values above 0. Data were discarded from further analysis if the processed signal across pairwise comparisons was below 3 times the has not been studied by unbiased comprehensive approaches such mean intensity values of the negative control probes on the BeadChips. as microarray. To identify common genes stimulated by SLE ICs, Microarray data have been deposited in the Gene Expression Omnibus we incubated total PBMCs from two different donors (responder database and are accessible through the Gene Expression Omnibus Series cells), each with a different SLE IC, and then examined gene ex- accession number GSE32285 (http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE32285). Genes listed in tables had gene expression changes pression by microarray analysis after 6 h. Using Ingenuity software upregulated or downregulated .1.5-fold compared with unstimulated to analyze genes for pathway analysis, 243 genes and 218 anno- controls in both donors, which allowed detailed pathway analysis by In- tated genes were upregulated or downregulated .1.5-fold by SLE The Journal of Immunology 3

ICs in donors D1 and D2, respectively. Of the 150 genes that were regulated .1.5-fold by qRT-PCR using an additional two donors common between the two donors, 137 genes were upregulated and for a total of four independent experiments and found a significant 13 genes downregulated .1.5-fold (Fig. 1A, Table I, Supple- correlation between the fold induction obtained from qRT-PCR mental Table II). We observed a striking induction of ISGs such and the microarray data with those six genes tested (Fig. 1D, that 43 of 50 top genes regulated have previously been linked to 1E). Although 70% of all genes regulated were type I IFN ISGs, type I IFNs (bold lettering, Table I) (see Materials and Methods we also identified numerous other genes regulated by SLE ICs that for publications used to determine ISGs). Although two different include cytokines (e.g., IL-1 family member 7 [IL1F7], increased donors and ICs were used, the top genes induced were very simi- 5-fold), transcription factors (e.g., EGR1, reduced almost 3-fold), lar in the responder cells with identical top five canonical path- and cell surface receptors (e.g., CLEC12A, reduced ∼2-fold) ways and network functions (Fig. 1B,1C). We validated six genes (Table I). Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. Differential expression of genes and pathways in PBMCs after exposure to SLE ICs. A–C, SLE ICs were incubated with unprimed normal donor PBMCs for 5 h. Following RNA isolation, gene expression was quantified by microarray, as described in Materials and Methods. A, Summary of genes regulated .1.5-fold compared with unstimulated controls with the intersection representing overlapping genes in two different donors (D1 and D2). B and C, The top five canonical pathways (B) and networks (C) are shown following PBMC IC stimulation. D, Five upregulated and one downregulated gene from the microarray data were quantified by qRT-PCR with four independent normal PBMC donor stimulations. E, The fold changes from the microarray and qRT-PCR for the same samples were plotted, and the Pearson correlation coefficient was calculated (r = 0.8952, p , 0.0001). FC, fold change relative to unstimulated controls. 4 IMMUNE COMPLEX GENE ARRAYS

Table I. Top 50 genes changed upon addition of SLE ICs to total PBMC cultures

Gene Gene Name PROBE_IDa REFSEQ_ID D1 FCb D2 FCb Mean FC RSAD2c Radical S-adenosyl methionine domain ILMN_1657871 NM_080657.4 40.253 10.486 25.370 containing 2 CXCL10 Chemokine (C-X-C motif) ligand 10 ILMN_1791759 NM_001565.2 25.672 14.578 20.125 IFIT1 IFN-induced protein with tetratricopeptide repeats 1 ILMN_1707695 NM_001548.3 29.491 8.374 18.932 IFIT3 IFN-induced protein with tetratricopeptide repeats 3 ILMN_1701789 NM_001031683.1 27.491 8.199 17.845 CCL8 Chemokine (C-C motif) ligand 8 ILMN_1772964 NM_005623.2 23.576 4.798 14.187 ISG15 ISG15 ubiquitin-like modifier ILMN_2054019 NM_005101.1 16.817 6.977 11.897 IFIT2 IFN-induced protein with tetratricopeptide repeats 2 ILMN_1739428 NM_001547.4 15.324 7.076 11.200 IFI44L IFN-induced protein 44-like ILMN_1723912 NM_006820.1 13.079 4.058 8.568 IL1RN IL-1 receptor antagonist ILMN_1774874 NM_173843.1 12.476 4.305 8.390 DEFB1 Defensin, b1 ILMN_1686573 NM_005218.3 10.839 5.185 8.012 OAS1 29,59-oligoadenylate synthetase 1, 40/46 kDa ILMN_1658247 NM_002534.2 9.650 4.972 7.311 OASL 29,59-oligoadenylate synthetase-like ILMN_1681721 NM_003733.2 9.018 4.406 6.712 HES4 Hairy and enhancer of split 4 (Drosophila) ILMN_1653466 NM_021170.2 9.072 4.227 6.650 HERC5 Hect domain and RLD 5 ILMN_1729749 NM_016323.2 8.519 4.132 6.326 IFI44 IFN-induced protein 44 ILMN_1760062 NM_006417.3 8.264 3.868 6.066 CCL7 Chemokine (C-C motif) ligand 7 ILMN_1683456 NM_006273.2 9.552 2.506 6.029 TNFSF10 TNF (ligand) superfamily, member 10 (TRAIL) ILMN_1801307 NM_003810.2 8.288 3.757 6.023 IFI6 IFN, a-inducible protein 6 ILMN_2347798 NM_022872.2 7.613 3.613 5.613 Downloaded from IFITM3 IFN-induced transmembrane protein 3 (1-8U) ILMN_1805750 NM_021034.2 6.337 4.233 5.285 IL1F7 IL-1 family, member 7 (z) ILMN_1697710 NM_014439.3 7.599 2.782 5.191 EPSTI1 Epithelial stromal interaction 1 (breast) ILMN_2388547 NM_033255.2 6.860 3.061 4.960 MX2 Myxovirus (influenza virus) resistance 2 ILMN_2231928 NM_002463.1 6.736 3.114 4.925 MX1 Myxovirus (influenza virus) resistance 1 ILMN_1662358 NM_002462.2 6.253 3.077 4.665 DDX58 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 ILMN_1797001 NM_014314.3 5.045 3.937 4.491 STAT1 Signal transducer and activator of transcription ILMN_1690105 NM_007315.2 5.920 3.058 4.489 http://www.jimmunol.org/ 1, 91 kDa HERC6 Hect domain and RLD 6 ILMN_1654639 NM_017912.3 5.535 3.294 4.414 PRIC285 Peroxisomal proliferator-activated receptor A ILMN_1787509 NM_033405.2 5.503 2.995 4.249 complex 285 RTP4 Receptor (chemosensory) transporter protein 4 ILMN_2173975 NM_022147.2 4.723 3.673 4.198 EIF2AK2 Eukaryotic translation initiation factor 2-a ILMN_1706502 NM_002759.1 5.372 2.719 4.046 kinase 2 (PKR) SAMD9L Sterile a motif domain containing 9-like ILMN_1799467 NM_152703.2 4.469 3.341 3.905 TMEM158 Transmembrane protein 158 (gene/pseudogene) ILMN_1792455 NM_015444.2 2.250 5.369 3.810 CLDN23 Claudin 23 ILMN_1725338 NM_194284.2 3.611 3.956 3.783

OAS2 29-59-oligoadenylate synthetase 2, 69/71 kDa ILMN_1736729 NM_002535.2 4.777 2.711 3.744 by guest on September 23, 2021 XAF1 XIAP-associated factor 1 ILMN_2370573 NM_199139.1 4.859 2.608 3.734 LY6E Lymphocyte Ag 6 complex, locus E ILMN_1695404 NM_002346.1 4.375 3.063 3.719 DHRS9 Dehydrogenase/reductase (SDR family) member 9 ILMN_2384181 NM_005771.3 3.033 4.273 3.653 CTSL1 Cathepsin L1 ILMN_1812995 NM_001912.3 3.583 3.634 3.609 IFIT5 IFN-induced protein with tetratricopeptide ILMN_1696654 NM_012420.1 4.456 2.587 3.522 repeats 5 GBP1 Guanylate-binding protein 1, IFN inducible, ILMN_2148785 NM_002053.1 4.504 2.497 3.501 67 kDa IFIH1 IFN induced with helicase C domain 1 ILMN_1781373 NM_022168.2 4.411 2.455 3.433 IFI35 IFN-induced protein 35 ILMN_1745374 NM_005533.2 3.963 2.777 3.370 IRF7 IFN regulatory factor 7 ILMN_2349061 NM_004029.2 3.846 2.792 3.319 LAP3 Leucine aminopeptidase 3 ILMN_1683792 NM_015907.2 4.004 2.458 3.231 EMP1 Epithelial membrane protein 1 ILMN_1801616 NM_001423.1 3.087 3.215 3.151 DDX60 DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 ILMN_1795181 NM_017631.4 3.582 2.443 3.012 CXCL5 Chemokine (C-X-C motif) ligand 5 ILMN_2171384 NM_002994.3 3.042 2.894 2.968 NT5C3 59-nucleotidase, cytosolic III ILMN_2352121 NM_001002010.1 3.400 2.454 2.927 JUP Junction plakoglobin ILMN_1733811 NM_002230.1 3.149 2.676 2.912 EGR1 Early growth response 1 ILMN_1762899 NM_001964.2 22.878 22.902 22.890 MT2A Metallothionein 2A ILMN_1686664 NM_005953.2 3.755 2.018 2.886 aProbe ID from Illumina Human Ref-8v3 Expression BeadChips. bFC, fold change relative to unstimulated controls. D1 and D2, donor 1 and donor 2. Positive numbers indicate fold upregulated; negative numbers indicate fold downregulated. cBold lettering indicates type I ISGs.

The addition of C1q to ICs reduced the expression of ISGs and total, 241 genes were upregulated or downregulated .1.5-fold by modulated the expression of multiple other genes C1q-ICs compared with IC stimulation alone with 67 genes up- We recently observed that the addition of C1q to SLE ICs markedly regulated and 174 genes downregulated (Table II, Supplemental reduced IFN-a stimulation and that this effect was explained, in Table III). The top 50 genes regulated 1.5-fold or greater are shown part, by monocytes “stealing” the ICs away from pDCs (15). To in Table II. Upon analysis of the top canonical pathways regulated examine the effect of C1q on ISGs and also determine what other by C1q, the top three were directly related to type I IFNs (Fig. 2A), pathways were altered by the presence of C1q bound to ICs (C1q- and the top networks included many of these genes as well (Fig. ICs), we repeated the microarray analysis as above, except that we 2B). A strong correlation between the microarray and qRT-PCR compared SLE IC stimulation in the presence or absence of C1q. In results was observed with three genes and four different PBMC The Journal of Immunology 5

Table II. Top 50 genes changed upon addition of C1q to SLE ICs in total PBMC cultures

Mean IC Mean C1q-IC C1q-IC/IC Gene Gene Name REFSEQ_ID FCa FCa Ratiob FC C1q-IC/ICc RNASE1 RNase, RNase A family, 1 (pancreatic) NM_198235.1 1.820 0.203 0.111 28.976 CXCL10d Chemokine (C-X-C motif) ligand 10 NM_001565.2 20.125 4.560 0.227 24.414 EGR1 Early growth response 1 NM_001964.2 0.346 0.078 0.227 24.413 HES4 Hairy and enhancer of split 4 (Drosophila) NM_021170.2 6.650 1.761 0.265 23.776 RSAD2 Radical S-adenosyl methionine domain containing 2 NM_080657.4 25.370 6.750 0.266 23.758 IFIT1 IFN-induced protein with tetratricopeptide repeats 1 NM_001548.3 18.932 5.058 0.267 23.743 RNASE6 RNase, RNase A family, k6 NM_005615.4 1.010 0.275 0.272 23.674 CCL8 Chemokine (C-C motif) ligand 8 NM_005623.2 14.187 3.972 0.280 23.572 TNFSF10 TNF (ligand) superfamily, member 10 NM_003810.2 6.023 1.750 0.291 23.442 ISG15 ISG15 ubiquitin-like modifier NM_005101.1 11.897 3.686 0.310 23.227 FGL2 Fibrinogen-like 2 NM_006682.1 0.861 0.276 0.321 23.115 IFIT2 IFN-induced protein with tetratricopeptide repeats 2 NM_001547.4 11.200 3.603 0.322 23.108 PMP22 Peripheral myelin protein 22 NM_153321.1 1.320 0.449 0.340 22.940 IFITM3 IFN-induced transmembrane protein 3 (1-8U) NM_021034.2 5.285 1.861 0.352 22.839 STAB1 Stabilin 1 NM_015136.2 0.999 0.359 0.359 22.787 DHRS9 Dehydrogenase/reductase (SDR family) member 9 NM_005771.3 3.093 1.125 0.364 22.750 IL1R2 IL-1 receptor, type II NM_173343.1 0.975 0.368 0.377 22.652

RCBTB2 Regulator of chromosome condensation (RCC1) NM_001268.2 0.980 0.373 0.381 22.627 Downloaded from and BTB (POZ) CD300LF CD300 molecule-like family member f NM_139018.2 0.838 0.323 0.385 22.596 OASL 29,59-oligoadenylate synthetase-like NM_198213.1 6.569 2.618 0.399 22.509 CXCL5 Chemokine (C-X-C motif) ligand 5 NM_002994.3 2.968 7.427 2.502 2.502 SPRED1 Sprouty-related, EVH1 domain containing 1 NM_152594.1 0.883 0.362 0.410 22.440 DDAH2 Dimethylarginine dimethylaminohydrolase 2 NM_013974.1 1.381 0.587 0.425 22.352 GPR160 G protein-coupled receptor 160 NM_014373.1 1.054 0.452 0.429 22.334 IFIT3 IFN-induced protein with tetratricopeptide repeats 3 NM_001549.2 15.544 6.675 0.429 22.329 http://www.jimmunol.org/ TNFSF13B TNF (ligand) superfamily, member 13b NM_006573.3 2.388 1.030 0.431 22.318 C6ORF105 Chromosome 6 open reading frame 105 NM_032744.1 1.127 2.564 2.275 2.275 OAS1 29,59 -oligoadenylate synthetase 1, 40/46 kDa NM_001032409.1 6.404 2.846 0.444 22.250 CYP27A1 Cytochrome P450, family 27, subfamily A, NM_000784.2 0.564 1.264 2.242 2.242 polypeptide 1 HPSE Heparanase NM_006665.3 2.672 1.197 0.448 22.232 ASGR1 Asialoglycoprotein receptor 1 NM_001671.2 0.451 0.203 0.451 22.219 LMO2 LIM domain only 2 (rhombotin-like 1) NM_005574.2 1.324 0.598 0.452 22.214 DDX58 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 NM_014314.3 4.491 2.031 0.452 22.211

FPR3 Formyl peptide receptor 3 NM_002030.3 1.391 0.629 0.453 22.210 by guest on September 23, 2021 RTP4 Receptor (chemosensory) transporter protein 4 NM_022147.2 4.198 1.916 0.456 22.191 IL1A IL-1, a (IL1A) NM_000575.3 0.635 1.376 2.165 2.165 TGFBI TGF, b induced, 68 kDa NM_000358.1 1.416 0.662 0.468 22.139 MERTK c-mer proto-oncogene tyrosine kinase NM_006343.2 1.362 0.644 0.473 22.115 GBP1 Guanylate-binding protein 1, IFN inducible, 67 kDa NM_002053.1 3.501 1.664 0.475 22.104 PAICS Phosphoribosylaminoimidazole carboxylase synthetase NM_006452.3 0.901 1.894 2.101 2.101 TNFRSF21 TNF receptor superfamily, member 21 NM_014452.3 1.748 0.835 0.478 22.094 SOD2 Superoxide dismutase 2, mitochondrial NM_001024466.1 0.961 2.005 2.086 2.086 RNASE2 RNase, RNase A family, 2 (liver, eosinophil-derived NM_002934.2 1.392 0.670 0.481 22.078 neurotoxin) LYZ Lysozyme NM_000239.1 0.609 0.297 0.488 22.049 CD86 CD86 Ag (CD28 Ag ligand 2, B7-2 Ag) NM_006889.3 1.310 0.640 0.488 22.048 FAM46A Family with sequence similarity 46, member A NM_017633.2 2.654 1.303 0.491 22.037 TNS3 Tensin 3 NM_022748.10 1.282 2.590 2.021 2.021 SLC2A6 Solute carrier family 2 (facilitated glucose NM_017585.2 0.752 1.515 2.014 2.014 transporter), member 6 STS-1 Cbl-interacting protein Sts-1 NM_032873.3 0.960 1.926 2.007 2.007 CCL4L1 Chemokine (C-C motif) ligand 4-like 1 NM_001001435.2 0.685 1.362 1.988 1.988 aMean fold change (FC) from two donors relative to unstimulated controls. bRatio of fold changes from C1q-IC stimulations of PBMCs over ICs alone. cRatio converted to fold change in which positive numbers indicate fold upregulated and negative numbers indicate fold downregulated. dBold lettering indicates type I ISGs. donors (Fig. 2C,2D). Of the top 50 genes listed in Table I, the SLE ICs induce a limited inflammatory response in isolated expression of 40 of 50 was reduced by C1q (from 1.5- to 4.4-fold), and CD14+ monocytes only six of these ISGs were not changed. Interestingly, 25 of the top 50 Our results to date indicate that when whole PBMCs are exposed to genes regulated by C1q-ICs were not ISGs (Table II). Transcripts SLE ICs, the transcriptome reflects a powerful inflammatory re- encoding multiple cytokines/chemokines (e.g., CCL20, CCL23, sponse dominated by changes most closely linked to the type I IFN CXCL1), receptors (e.g., CD36, STAB1, TNFRSF4, FCGR2A), and pathway. Yet, ICs are known to engage the activating receptor enzymes (e.g., RNASE1,2,6, ITK, HPSE, SOD2, SYK) were either FcgRIIA, which is abundantly expressed on CD14+ monocytes upregulated or downregulated in the presence of C1q-ICs, reinforcing and would be expected to induce other inflammatory cytokines (4, the finding that C1q affects multiple pathways that are not direct 35, 36). To address the inflammatory potential of SLE ICs directly targetsoftypeIIFNs. on monocytes, we isolated CD14+ monocytes to .95% purity 6 IMMUNE COMPLEX GENE ARRAYS Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. Differential expression of genes and pathways in PBMCs after exposure to C1q-ICs compared with ICs alone. A and B, SLE ICs were added to the same normal donor PBMCs as in Fig. 1 in the presence or absence of purified C1q (50 mg/ml). A and B, The top five canonical pathways (A) and networks (B) are shown for the comparison of C1q-ICs to ICs alone. C, Three downregulated genes from the microarray data were quantified by qRT-PCR with a total of four independent normal PBMC donor stimulations. Results are shown as fold change compared with unstimulated controls with error bars representing the SEM. D, The fold changes from the microarray and qRT-PCR for IC-alone, C1q-IC, and C1q-alone conditions were plotted, and the Pearson correlation coefficient was calculated (r = 0.9576, p , 0.0001). *p , 0.05 with one-way ANOVA and Tukey’s multiple comparison posttest. with no contaminating pDCs detected and then incubated them from the greater degree of variation in monocytes, changes in gene with the same SLE ICs and performed microarray expression expression were more modest: only 7 and 14 genes were modu- analysis as for total PBMCs. Surprisingly, compared with whole lated 2-fold or greater in each donor’s monocytes (with three PBMCs, many fewer genes were modulated similarly in both genes commonly regulated in both donors) as compared with 121 donors’ purified monocytes (150 genes in whole PBMCs versus 21 and 85 modulated 2-fold or greater for each donor’s PBMCs (with genes in monocytes, Fig. 3A, Table III) with one top canonical 68 genes commonly regulated in both donors). The only two pathway in common (Fig. 3B). A strong correlation between the transcripts induced 2-fold or greater in both donors’ monocytes microarray and qRT-PCR results was observed with five genes were solute carrier family 16, member 10 (SLC16A10, also using four different PBMC donors (Fig. 3C,3D). Because known as monocarboxylate transporter) and CXCL5. SLC16A10 168 genes were regulated in D1 and 98 in D2 monocytes, but only is a member of a family of plasma membrane amino acid trans- 21 genes upregulated and downregulated, respectively, in both porters that mediate the transport of aromatic amino acids across donors, there was a much greater degree of individual variation in the plasma membrane. Although 9 of 21 genes regulated by SLE monocytes from different donors compared with PBMCs. Apart ICs in purified monocytes have previously been linked to type I The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. Differential expression of genes and pathways in purified CD14+ monocytes after exposure to SLE ICs. A and B, SLE ICs were added to normal donor-purified monocytes for 5 h and RNA isolated for microarray analysis, as described in Materials and Methods. A, Summary of genes dif- ferentially regulated .1.5-fold is shown, with the intersection representing overlapping genes with two different donors (D1 and D2). B, The top five canonical pathways for each donor are shown, with only one canonical pathway shared between them (in bold with genes listed in parentheses). C, Four upregulated and one downregulated gene from the microarray data were quantified by qRT-PCR with a total of four independent monocyte stimulations. Results are shown as fold upregulated or downregulated comparatively to unstimulated controls. D, The fold changes from the microarray and qRT-PCR for the same samples were plotted, and the Pearson correlation coefficient was calculated (r = 0.9753, p , 0.0001).

IFNs (bolded in Table III), these were expressed at low levels, pression in isolated monocytes exposed to C1q-ICs versus ICs and the ISG transcripts that were most strongly induced in alone. In total, 36 genes were differentially expressed (Table IV) PBMC, such as RSAD2, CXCL10, IFIT1-3, and OAS1, OASL and 11 genes were concordantly regulated compared with total were not expressed in isolated monocyte cultures exposed to ICs. PBMCs (ADAP2, C7ORF50, CCL20, CCL4L1, H1FX, IL1A, Of genes differentially expressed, seven were common between MERTK, PTMA, RCBTB2, SLC39A8, TNFRSF4). The gene PBMCs and purified monocytes with similar levels of expres- TNIP3 (also known as ABIN-3) was upregulated ∼2-fold by C1q- sion (SLC16A10, CXCL5, CCL7, CRADD, CTSL1, IL1RN, and ICs and may be related to our findings that C1q is anti-inflamma- TLR8). tory as ABIN-3 inhibits NF-kB activation induced by LPS, IL-1, C1q modulates monocyte gene expression induced by SLE ICs and TNF-a (37, 38). Another GOI that was upregulated ∼1.9-fold C1q binding to ICs increases binding to monocytes and away from by C1q-ICs compared with ICs alone was CCL20. One function of pDCs and also affects intracellular trafficking within monocytes CCL20 is to downregulate reactive oxygen species production in (15). To determine whether the composition of the IC (C1q present monocytes (39), again demonstrating that C1q affects multiple or not) affected gene induction as well, we compared gene ex- downstream anti-inflammatory pathways independent of IFN-a. 8 IMMUNE COMPLEX GENE ARRAYS

Table III. Genes regulated .1.5-fold upon addition of SLE ICs to purified monocytes

Gene Gene Name PROBE_IDa REFSEQ_ID D1 FCb D2 FCb Mean FCb SLC16A10 Solute carrier family 16, member 10 ILMN_1782938 NM_018593.3 2.667 5.842 4.255 CXCL5c Chemokine (C-X-C motif) ligand 5 ILMN_2171384 NM_002994.3 2.651 3.916 3.283 LOC150297 Hypothetical protein LOC150297 ILMN_1711067 NM_001010859.1 22.074 23.241 22.658 CCL7 Chemokine (C-C motif) ligand 7 ILMN_1683456 NM_006273.2 1.648 3.006 2.327 APOL3 Apolipoprotein L, 3 ILMN_1756862 NM_145641.1 21.569 22.361 21.965 CD93 CD93 molecule ILMN_1704730 NM_012072.3 1.991 1.672 1.832 EGR2 Early growth response 2 ILMN_1743199 NM_000399.2 22.047 21.577 21.812 RAB20 RAB20, member RAS oncogene family ILMN_1708881 NM_017817.1 1.662 1.956 1.809 CTSL1 Cathepsin L1 ILMN_1694757 NM_001912.3 1.633 1.933 1.783 CRADD CASP2 and RIPK1 domain containing ILMN_1690282 NM_003805.3 1.799 1.755 1.777 adaptor with death domain TLR8 Toll-like receptor 8 ILMN_1657892 NM_138636.2 21.621 21.889 21.755 BIRC3 Baculoviral IAP repeat-containing 3 ILMN_2405684 NM_182962.1 21.917 21.591 21.754 IL1RN IL-1R antagonist ILMN_1689734 NM_173842.1 1.637 1.775 1.706 GK5 Glycerol kinase 5 (putative) ILMN_1673892 NM_001039547.1 1.546 1.850 1.698 C19ORF59 Chromosome 19 open reading frame 59 ILMN_1762713 NM_174918.2 1.624 1.762 1.693 C21ORF7 Chromosome 21 open reading frame 7 ILMN_1699071 NM_020152.2 21.648 21.661 21.654 FFAR2 Free fatty acid receptor 2 ILMN_1797895 NM_005306.1 21.773 21.503 21.638 STEAP4 STEAP family member 4 ILMN_1772036 NM_024636.2 21.707 21.538 21.623 Downloaded from FKBP5 FK506-binding protein 5 ILMN_1778444 NM_004117.2 21.550 21.693 21.621 HIF1A Hypoxia-inducible factor 1, a subunit ILMN_1763260 NM_001530.2 1.501 1.656 1.578 LTB Lymphotoxin b (TNF superfamily, member 3) ILMN_2376205 NM_002341.1 21.505 21.550 21.528 aProbe ID from Illumina Human Ref-8v3 Expression BeadChips. bFC, fold change relative to unstimulated controls. D1 and D2, donor 1 and donor 2. Positive numbers indicate fold upregulated; negative numbers indicate fold downregulated. c

Bold lettering indicates type I ISGs. http://www.jimmunol.org/

Differential effects of SLE ICs on monocyte subsets CD16+ subset (Fig. 4D). With addition of C1q, the IC binding to all subsets increased, but binding increased 5- to 6-fold on both Microarray analysis revealed a lower magnitude of change of gene high dim expression on isolated CD14+ monocytes compared with PBMCs. of the CD14 monocyte subsets, although for the CD14 Maturation and activation of monocytes led to an upregulation of subset the increase was only 3-fold (Fig. 4D). Taken together, costimulatory molecule expression; therefore, we further inves- these observations indicate that ICs induce limited inflamma- tory gene expression or upregulation of costimulatory mole- tigated the question of the inflammatory potential of SLE ICs on + monocytes by flow cytometric analysis. In agreement with the cules on total CD14 monocytes. They do, however, upregulate by guest on September 23, 2021 CD86 expression on the CD14dim population, and C1q prefer- limited changes in gene expression observed, we found that dim exposure of total monocytes to SLE ICs did not induce signifi- entially enhances monocyte binding away from the CD14 cant upregulation of CD86 or CD40 (Fig. 4A). As a positive population. control, the TLR3 agonist poly(I:C) upregulated both CD86 and pDCs are required for strong activation of CD14+ monocytes CD40 4- to 5-fold (data not shown). If IFN-a was the sole factor by SLE ICs produced in total PBMC cultures that allowed monocytes to dim respond to SLE ICs, then priming cells with IFN-a should re- The findings above suggest that most monocytes (the CD14 subset store activation by ICs. Whereas IFN-a priming itself upregu- being the exception) are poorly activated in isolation, but are po- lated both CD86 and CD40, when we compared costimulatory tently activated in the presence of other blood cells. To determine protein expression in the presence or absence of IFN-a,weob- whether activation of pDCs is specifically required for the inflam- served no differences in CD86 or CD40 upregulation on mono- matory potential of SLE ICs on monocytes, we compared CD86 cytes when stimulated by three different SLE ICs (Fig. 4B). and CD40 expression on monocytes in total PBMCs and then again These data support the gene array findings of limited activation of following pDC depletion. As shown in Fig. 5A, monocytes in isolated CD14 monocytes by SLE ICs and that IFN-a itself plays PBMCs were strongly activated by SLE ICs after 2 d in culture, as a greater role in costimulatory molecule upregulation on mono- measured by upregulation of the expression of CD86 and CD40 by cytes. flow cytometry (Fig. 5). Very similar activation of monocytes in Monocytes are a heterogeneous population of cells that, based on PBMCs was observed if SLE ICs were formed with purified SLE CD14 and CD16 expression, can be divided into three functional IgG instead of using diluted patient serum (Supplemental Fig. 1), subsets, as follows: CD14dimCD16+, CD14+CD16+, and CD14+ and normal serum did not upregulate CD86 or CD40 expression in CD162 (17). Because Cros et al. (17) reported that it is the minor any stimulation condition (data not shown). When pDCs were de- (comprising only ∼7% of total monocytes) CD14dimCD16+ pa- pleted from PBMCs such that IFN-a was not detected in response trolling monocyte subpopulation that preferentially responds to to SLE ICs nor loxoribine (TLR7 agonist), monocytes were no TLR7 and eight agonists, we tested whether SLE ICs could up- longer potently activated by SLE ICs as determined by significantly regulate CD86 expression on this rare circulating subset. When lower levels of CD86 and CD40 (Fig. 5B). monocyte subsets were sorted to high purities and cultured with Consistent with these findings, we observed that SLE ICs in- ICs for 2 d, we found that SLE ICs did in fact upregulate CD86 duced high levels of cytokines and chemokines known to be expression on CD14dim, but not the other monocyte subsets produced by monocytes such as IL-1RA (same as IL-1RN in our (Fig. 4C). When we compared binding of ICs to each subset arrays), IP-10 (CXCL10), and MCP-1 in total PBMCs, but not within total PBMC cultures, we found that whereas all mono- in pDC-depleted cultures (Fig. 6A). MCP-3 induction varied cyte subsets bound ICs, the greatest binding was to the CD14+ depending on the donor used, and IL-8 induction was not depen- The Journal of Immunology 9

Table IV. Genes regulated .1.5-fold upon addition of C1q to SLE ICs in purified monocyte cultures

Mean IC Mean C1q-IC C1q-IC/IC C1q-IC/IC Gene Gene Name REFSEQ_ID FCa FCa Ratiob FCc C7ORF50 Chromosome 7 open reading frame 50 NM_032350.4 0.790 0.392 0.496 22.015 TNIP3d TNFAIP3-interacting protein 3 NM_024873.3 1.208 2.393 1.980 1.980 MERTK c-mer proto-oncogene tyrosine kinase NM_006343.2 1.573 0.811 0.516 21.939 CCL20 Chemokine (C-C motif) ligand 20 NM_004591.1 1.160 2.230 1.923 1.923 SLC39A8 Solute carrier family 39 (zinc transporter), member 8 NM_022154.5 1.020 1.890 1.853 1.853 IL6 IL-6 (IFN, b2) NM_000600.1 1.520 2.635 1.734 1.734 TNFRSF4 TNF receptor superfamily, member 4 NM_003327.2 0.739 1.279 1.730 1.730 EREG Epiregulin NM_001432.2 1.015 1.715 1.689 1.689 CXCL10 Chemokine (C-X-C motif) ligand 10 NM_001565.2 0.680 1.146 1.684 1.684 GCH1 GTP cyclohydrolase 1 NM_001024070.1 0.893 1.438 1.610 1.610 IL1A IL-1, a NM_000575.3 0.985 1.580 1.604 1.604 PLAC8 Placenta-specific 8 NM_016619.1 0.640 1.025 1.602 1.602 FEZ1 Fasciculation and elongation protein z 1 (zygin I) NM_005103.3 0.675 1.080 1.601 1.601 ACP5 Acid phosphatase 5, tartrate resistant NM_001611.2 1.156 0.724 0.627 21.596 CXCR4 Chemokine (C-X-C motif) receptor 4 NM_001008540.1 1.450 0.910 0.628 21.593 RGS12 Regulator of G protein signaling 12 NM_002926.3 1.067 0.674 0.632 21.583 IL17RD IL-17 receptor D NM_001080973.1 0.710 1.123 1.581 1.581

RCBTB2 Regulator of chrom condensation (RCC1) NM_001268.2 1.095 0.697 0.637 21.571 Downloaded from G0S2 G0/G1 switch 2 NM_015714.2 1.059 1.654 1.562 1.562 SERPINA1 Serpin peptidase inhibitor, clade A NM_001002236.1 0.942 1.470 1.561 1.561 IFIT3 IFN-induced protein with tetratricopeptide repeats 3 NM_001549.2 0.870 1.354 1.557 1.557 CCL4L1 Chemokine (C-C motif) ligand 4-like 1 NM_001001435.2 0.840 1.307 1.555 1.555 ODC1 Ornithine decarboxylase 1 NM_002539.1 0.879 0.567 0.645 21.551 CASP4 Caspase 4, apoptosis-related cysteine peptidase NM_033306.2 0.993 1.538 1.549 1.549 AK3L1 Adenylate kinase 3-like 1 NM_013410.2 0.852 1.315 1.543 1.543 LOC150297 Hypothetical protein LOC150297 NM_001010859.1 0.395 0.609 1.540 1.540 http://www.jimmunol.org/ CKLF Chemokine-like factor NM_001040138.1 1.490 0.969 0.650 21.539 GRAMD1A GRAM domain containing 1A NM_020895.2 0.755 1.160 1.537 1.537 RPL9 Ribosomal protein L9 (RPL9), transcript variant 2 NM_001024921.2 0.744 1.140 1.531 1.531 HSPA1A Heat shock 70-kDa protein 1A (HSPA1A) NM_005345.4 0.872 1.323 1.517 1.517 CCR7 Chemokine (C-C motif) receptor 7 NM_001838.2 1.000 1.511 1.511 1.511 TM6SF1 Transmembrane 6 superfamily member 1 NM_023003.2 1.005 0.665 0.662 21.511 ADAP2 ArfGAP with dual PH domains 2 NM_018404.2 1.004 0.665 0.663 21.509 H1FX [1H]Histone family, member X NM_006026.2 0.873 0.581 0.666 21.502 PTMA Prothymosin, a (PTMA), transcript variant 1 NM_001099285.1 0.847 1.272 1.502 1.502

EMR1 egf-like module-containing, mucin-like, hormone NM_001974.3 0.847 1.271 1.501 1.501 by guest on September 23, 2021 receptor-like 1 aMean fold change (FC) from two donors relative to unstimulated controls. bRatio of fold changes from C1q-IC stimulations of PBMCs over ICs alone. cRatio converted to fold change in which positive numbers indicate fold upregulated and negative numbers indicate fold downregulated. dBold lettering indicates type I ISGs. dent on the presence of pDCs, as similar levels were detected in Discussion both mock- and pDC-depleted cultures (Fig. 6). Using at least The goals of this study were several fold. We wished to identify three different ICs for the multiplex and .20 different ICs for common changes in gene expression by SLE ICs in total PBMCs, TNF-a stimulation in total PBMCs, the ICs did not induce IL-1b, to identify changes in genes or pathways when C1q was present IL-12p40, IL-10, MIP-1a, or TNF-a compared with unstimulated in ICs, and to determine the extent to which SLE ICs induced control cultures (data not shown). Similar to PBMC cultures, pu- common inflammatory pathways directly on monocytes. Studies rified monocytes produced low levels of MCP-1 (average of 60 pg/ were therefore performed with at least two SLE ICs and at least two ml above background) and, on average, 1259 pg/ml IL-8 above different normal donor cells. The results of these studies reveal that background when stimulated by SLE ICs (data not shown, Fig. 6B). pDCs and CD14dim monocytes are key targets of SLE ICs, that There was no induction of IP-10, IL-1RA, MCP-3, MIP-1a, IL- C1q influences IC activation of both of these cells, and we identify 12p40, IL-10, TNF-a, or IL-1b above background when ICs were multiple genes that are regulated depending upon the target cells added to purified monocytes (data not shown). Despite a small and composition of ICs. (∼1.7-fold) increase in IL-6 mRNA expression seen in the micro- Experimentally, it has been shown that ICs can induce TNF-a array following C1q addition to ICs, this increase was not confirmed (40–42) or, more recently, IFN-a (33). In our study, addition of by ELISA (data not shown, four independent experiments). SLE ICs to unprimed normal PBMCs induced a dominant type I Although pDCs were required for maximal expression of co- IFN pattern of gene transcription with little or no TNF-a pro- stimulatory molecule expression by monocytes, the addition of neu- duction, consistent with the ex vivo gene signature in SLE (43, tralizingAbtoIFN-a either had minimal effect or reduced CD86 44). When we compared genes upregulated in normal PBMCs by and CD40 expression at most 1.6- and 1.4-fold, respectively, indi- SLE ICs to published SLE patient peripheral blood ex vivo tran- cating that other factors in addition to IFN-a are important for script profiles (45), we observed that 76 of the 150 genes regulated monocyte activation by ICs. Together our data suggest pDCs are by in vitro IC exposure were represented in SLE patients’ PBMC required for SLE ICs to efficiently stimulate the major monocyte transcriptome. These findings are therefore consistent with the 2 populations (CD14+CD16 or CD14+CD16+), whereas the rare sub- idea that circulating ICs account to a significant degree for the IFN set of CD14dim monocytes is able to respond directly to SLE ICs. signature observed in SLE patients’ blood cells. The lack of full 10 IMMUNE COMPLEX GENE ARRAYS Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. SLE ICs upregulate CD86 only on CD14dim monocytes. A and B, SLE ICs were incubated with total CD14+ purified monocytes (.96% pure) in medium alone (A) or with or without 4-h priming with 500 U/ml type I IFN (B). After 2 d in culture, CD86 and CD40 expression was quantified by flow cytometry. Results in A are expressed as MFI from three independent experiments with two to three different SLE patient ICs per experiment. Results in B are representative of three independent experiments with three different SLE patient ICs added per experiment. C, The three different monocyte subsets, CD14dimCD16+, CD14+CD16+, and CD14+CD162, were isolated by flow sorting and exposed to SLE ICs, as in A. CD86 expression was quantified and expressed as the ratio of MFI following IC stimulation/medium (no IC) control. D and E, SLE ICs were formed using fluorescently labeled SmRNP Ag in the absence (D) or presence of C1q (20 mg/ml) (E) and allowed to bind to PBMCs for 30 min on ice. D, Left panel, Representative histogram with the MFI of ICs bound to each monocyte subset shown in the legend below. Right panel, Compiled MFI data of SLE IC binding to each monocyte subset with one to six SLE ICs per experiment for a total of three independent experiments. E, Left panel, Representative experiment with four SLE ICs and the binding to monocyte subsets within PBMCs with or without C1q. Right panel, Compiled data for SLE IC binding to monocyte subsets expressed as a MFI ratio of C1q-IC to IC binding in the absence of C1q (IC alone) from three experiments with a total of 11 SLE patient ICs. Horizontal lines are mean 6 SEM. n.s., not significant; *p , 0.05, **p , 0.01 with unpaired t test (A) and one-way ANOVA with Tukey’s multiple comparison posttest (C–E). concordance could be explained by the acute nature of in vitro ICs induced the expression of multiple genes and pathways that stimulation versus chronic stimulation in vivo. In addition, certain are implicated in the pathogenesis of SLE. For example, upregu- autoantigens released during apoptosis may not be present in our lation of nucleic acid receptors and associated adaptor molecules freeze-thawed extract to make ICs. Another possible explanation that are known ISGs (DDX58 [RIG-I], TRIM25, UNC93B1, IRF7, is that DNA released from neutrophil nets can form ICs and di- IFIHI [MDA5], MyD88) would serve as a positive feedback loop rectly stimulate TLR9 in pDCs, but neutrophils were not added in to enhance responses to ICs or other nucleic acid stimuli in SLE. our PBMC cultures (46, 47). Whether other potent IFN inducers SLE ICs also upregulated the expression of two recently identified such as endogenous (48) or exogenous (49) virus infection con- anti-inflammatory molecules that are not known to be regulated by tribute to type I IFN stimulation as well remains to be determined. IFN-a: IL1F7 (also known as IL-37) 5-fold and IL-4–induced The Journal of Immunology 11

FIGURE 5. SLE ICs upregulate monocyte CD86 and CD40 expression in PBMC cultures, but increased ex- pression requires the presence of pDCs. SLE ICs were incubated with total PBMC cultures, as in Fig. 1. A, After 2 d, CD14+ monocytes were analyzed for CD86 and CD40 expression by flow cytometry. Results from four independent experiments with three to five dif- ferent SLE patient ICs per experiment are expressed as MFI in which each line is a different SLE IC stim- ulation. B, Monocyte expression of CD86 and CD40 following SLE IC or loxoribine (200 mM) stimulation was analyzed by flow cytometry, as in A, except that PBMCs were either mock depleted or depleted of pDCs

(pDC dep) with BDCA-4 magnetic beads (representa- Downloaded from tive depletion shown in upper panel). MFI results from three independent experiments with at least two dif- ferent SLE patient ICs per experiment are shown in the lower panel. Loxo, loxoribine. MED, medium. *p , 0.05, **p , 0.01, ***p , 0.001 with unpaired t test (A) and paired t test (B). http://www.jimmunol.org/

gene 1 (IL4I1) 2-fold. IL-37 exerts an anti-inflammatory effect in monocyte-derived dendritic cells in vitro (61). Because we ob- response to LPS stimulation, and small interfering RNA knock- served an increase in RNASE1 gene expression with ICs alone, by guest on September 23, 2021 down led to enhanced production of multiple proinflammatory PBMCs may respond to exposure to RNA containing ICs by in- cytokines (50). IL4Il inhibits T cell proliferation and an optimal creasing RNase expression. Increased expression of RNASE2 has Th1 response by inhibiting IFN-g and other inflammatory cyto- been observed previously in expression arrays in SLE patient kines (51, 52). These genes may therefore serve in a negative PBMCs (43, 44) and also noted to be upregulated in inflammatory feedback loop, and their function would be well worth exploring bowel disease and rheumatoid arthritis patient PBMCs (62, 63). in SLE patients. Other IC-induced genes (PPARG, ∼2-fold and RNASE6 has 47% amino acid sequence similarity to RNASE2 RASGRP3, 1.8-fold) that are not known to be IFN inducible have (64) and is most highly expressed in pDCs, monocytes, neu- been implicated in lupus susceptibility or pathogenesis. IC stim- trophils, and B cells (64–66). Because RNASE6 was modestly ulation could potentially explain peroxisome proliferator-activated upregulated by SLE ICs and strongly downregulated by C1q, receptor g elevation in patients with active SLE (53). RASGRP future studies will be required to determine the role of this was recently discovered to be a SLE susceptibility gene associated protein in the immune response. Because none of the RNASE with the development of malar rash, discoid rash, and antinuclear genes were regulated in purified monocyte cultures, induction Ab positivity (54, 55). of transcription is most likely controlled by inflammatory Consistent with previously published results on IFN-a sup- cytokines released by other immune cell types. Genes that were pression by C1q-ICs (14, 15), addition of C1q to ICs resulted in a upregulated by C1q-ICs, such as the antioxidant, SOD2, could marked suppression of ISGs and multiple other genes. In total, 81 also be important in the anti-inflammatory response. Whereas previously identified type I IFN response genes were downregu- our study has focused on C1q, C4 and C2 deficiency is also lated by C1q-ICs compared with ICs alone. Two prominent gene associated with increased risk for the development of SLE, families whose expression was reduced were TNF family mem- although the risk is not as high (reviewed in Ref. 67). Prelim- bers and RNASEs. TNFSF13B (BAFF) and TNFSF10 (TRAIL) inary data suggested that C2 was not required for inhibition of have been implicated in SLE (56, 57), and the expression of IFN-a induced by SLE ICs (15), and purified C4 protein did not TNFRSF21 (DR6) impacts B and T cell responses (58). Reduction inhibit IC stimulation (data not shown). In vivo it is likely that in expression of these genes by C1q would therefore be predicted the early classical complement proteins, C1q, C2, and C4, may to be beneficial in SLE patients. In contrast, RNases are less well act in a common pathway leading to the assembly of C3bi on studied. RNASE1 (the homolog of bovine RNase A) is expressed apoptotic cells, which we, and others, previously showed is im- in the pancreas, endothelial cells (59), as well as immune cells portant for the clearance of dying cells (8, 9). Because C1q such as activated monocytes (60). Of interest, RNASE1 secretion deficiency is associated with a much higher penetrance of SLE, was shown to increase in response to extracellular RNA (but additional immune regulation effects, as we have shown in this not DNA), and addition of RNase 1 or RNase 2 (also known study, plus the importance of C1q in IC and apoptotic cell as eosinophil-derived neurotoxin) induced activation of human clearance, are most likely involved in disease pathogenesis. 12 IMMUNE COMPLEX GENE ARRAYS

FIGURE 6. pDCs are required for maximal in- duction IL-1RA, IP-10, MCP-1, and MCP-3, but not IL-8, in response to SLE ICs. A and B, Cytokines/ Chemokines were measured by multiplex analy- sis, as described in Materials and Methods, after mock-depleted (Mock) or pDC-depleted (pDC dep) PBMCs (A) or purified monocytes (B) were exposed to SLE ICs for 2 d of stimulation. Raw data in pg/ml are plotted from two independent experiments with two different donors and three (A) or two (B) dif- Downloaded from ferent SLE ICs per experiment. No IC indicates the presence of freeze-thawed Ag alone. http://www.jimmunol.org/

Surprisingly, IC stimulation of purified total CD14+ monocytes colleagues (17) recently identified a rare monocyte subset that resulted in lower changes in gene expression compared with in vivo is thought to patrol endothelial surfaces, where they re- by guest on September 23, 2021 PBMCs and little to no upregulation of CD40 or CD86. Only the spond rapidly to microbial stimuli. When experiments were per- monocarboxylate transporter, SLC16A10, and the neutrophil- formed to examine differences in the responses of the three attracting chemokine, CXCL5, were induced 2-fold or greater monocyte subsets, we observed that the CD14dim population did in both donors’ monocytes. Although expression of other mono- appear to be more responsive to SLE ICs as determined by up- carboxylate transporters has been studied in monocytes (68), regulation of CD86 expression, although the maximum TNF-a little is known of the function of SLC16A10 in these cells. production was very low (0–60 pg/ml in three experiments, data CXCL5, the ligand for the chemokine receptor CXCR2, stimulates not shown). Perhaps of further relevance to protection from in- the chemotaxis of neutrophils, and therefore can be implicated in flammation (15), the presence of C1q in ICs favored the binding of the inflammatory properties of SLE ICs on monocytes. The low ICs away from the CD14dim population to CD14high monocytes. gene induction by SLE ICs on isolated CD14+ monocytes is con- Of considerable interest, differences were noted in the response sistent with the finding that pDC depletion of PBMCs resulted in to SLE ICs between two different normal monocyte donors. It will very limited stimulation of monocytes, as determined by costimu- be interesting to examine monocyte gene stimulation in a larger latory molecule expression. This may be explained by the much number of normal individuals as well as SLE patients in remission lower expression of TLR7 in monocytes as compared with pDCs to see whether stable patterns are observed and whether patients can (69). Experiments utilizing either addition of type I IFN to mono- be segregated based on the response. Although we confirmed some cyte cultures as well as IFN-a neutralization in PBMCs suggested chemokine mRNA expression changes by multiplex analysis, fur- that type I IFN as well as other factors were required for full ther work is needed to confirm changes in gene expression at the monocyte activation. A similar requirement for pDCs for stimula- protein level. In addition, our studies have focused on IC responses tion of monocytes was shown by Hornung et al. (69) with the TLR9 in PBMCs. Future experiments to test the physiological conse- agonist CpG. In addition, human B cells require the presence of quences of C3b-coated IC handling in whole blood where RBCs pDCs and IFN-a for maximal stimulation by TLR7 agonists (70). and neutrophils could play important roles will be important to ICs are considered to be potent activators of inflammatory determine (47, 71). In summary, these studies identify pDCs and responses in FcgRII-bearing cells of the myeloid lineage associ- CD14dim monocytes as the most important cell targets for SLE ated with the production of TNF-a (35, 36, 40, 41). Several ICs and define genes and pathways stimulated in total PBMCs. explanations for the differences between our studies compared This information will help to identify additional biomarkers and to with some prior studies of the activating effect of ICs on whole design targeted therapies for SLE. monocytes are possible. Most used heat-aggregated IgG or tetanus toxoid as ICs and/or did not exclude endotoxin contamination as Acknowledgments a possible cause of TNF-a stimulation. ICs may also have dif- We thank Edward Clark, Grant Hughes for helpful discussion, Thomas ferent effects in tissues in vivo. In this context, Geissmann and Mo¨ller for assistance with cytokine multiplex analysis, and Ryan Basom, The Journal of Immunology 13

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Supplementary Figure 1. SLE ICs formed with either diluted serum or purified IgG activate monocytes within PBMCs. SLE ICs were formed with 1:2000 diluted serum or 5-15 μg/ml purified IgG and 1% freeze thawed antigen and added to total PBMCs for 2 d. The expression of CD86 and CD40 was measured by flow cytometry and plotted as mean fluorescent intensities (MFI). A total of 4 (CD86) and 3

(CD40) independent experiments with at least 2 different SLE ICs per experiment are plotted. **, P<0.01;

***, P<0.001. n.s., not significant with one-way ANOVA with Tukey’s multiple comparison post-test.

Supplementary Table I: Quantitative real-time PCR primers

Gene Primer Sequence

18S F 5' GAG GGA GCC TGA GAA ACG G 3'

R 5' GTC GGG AGT GGG TAA TTT GC 3' CCL7 F 5' ATG AAA GCC TCT GCA GCA CT 3' R 5' TCT GTA GCA GCA GGT AGT TGA AGT 3'

CTSL1 F 5' ACA TCC CTA AGC AGG AGA AGG 3' R 5' ACA GTC TGG CTC AAA ATA AAT GC 3'

IL1RN F 5' GAA GAT GTG CCT GTC CTG TGT 3' R 5' CGC TCA GGT CAG TGA TGT TA 3'

CXCL5 F 5' TCT GCA AGT GTT CGC CAT AG 3' R 5' AAA CTT TTC CAT GCG TGC TC 3' TRIM21 F 5' CTC AGG AGT GTG TGC CAT GT 3'

R 5' AGG TAT GCT CTG CTG GGT GT 3' CX3CR1 F 5' CAG ATC CAG AGG TTC CCT TG 3' R 5' AGA CCA CGA TGT CCC CAA TA 3' MERTK F 5’GGG TCC AGA ACC ATG AGA TG 3’ R 5’GCA GCC TCA ATA CTG AAA AGG 3’ RNASE 1 F 5’TGT CCT GAT ACT GCT GGT GCT 3’ R 5’TAC AGT AGG TGG AGC TGC TGC T 3’ RNASE 6 F 5’ATA CAC ACA GGG CTC GAA GG 3’ R 5’CCC ATA GAA CCA GCA GCA GT 3’ APOL3 F 5' AAG AGT TTC CCC AAG TCA AGA GG 3' R 5' GCA AGG GAC ATG ATG CCA GA 3' Supplementary Table II: Remaining 100 genes changed upon addition of SLE ICs to total PBMC cultures

Mean Gene Name PROBE_IDa REFSEQ_ID D1 FCb D2 FCb Gene FC

SAMD9c sterile alpha motif domain containing 9 ILMN_1814305 NM_017654.2 3.439 2.282 2.861 poly (ADP-ribose) polymerase family, PARP9 ILMN_2053527 NM_031458.1 3.198 2.264 2.731 member 9 HPSE heparanase ILMN_2092850 NM_006665.3 2.941 2.403 2.672

FAM46A family with sequence similarity 46, member A ILMN_1740466 NM_017633.2 2.849 2.459 2.654

SP110 SP110 nuclear body protein ILMN_2415144 NM_004510.2 3.339 1.948 2.644 poly (ADP-ribose) polymerase family, PARP12 ILMN_1718558 NM_022750.2 3.262 1.929 2.595 member 12 ENDOGL1 endonuclease G-like 1 ILMN_1709747 NM_005107.2 3.179 2.011 2.595 ATF3 activating transcription factor 3 ILMN_2374865 NM_001040619.1 3.150 1.975 2.562 LHFPL2 lipoma HMGIC fusion partner-like 2 ILMN_1747744 NM_005779.1 2.214 2.877 2.545 LGALS9 lectin, galactoside-binding, soluble, 9 ILMN_2412214 NM_009587.2 2.659 2.348 2.504 TDRD7 tudor domain containing 7 ILMN_1705241 NM_014290.1 2.721 2.282 2.501 DHX58 DEXH (Asp-Glu-X-His) box polypeptide 58 ILMN_1678422 NM_024119.2 3.101 1.878 2.489 pro-platelet basic protein (chemokine (C-X-C PPBP ILMN_1767281 NM_002704.2 1.535 3.388 2.462 motif) ligand 7) FBXO6 F-box protein 6 ILMN_1701455 NM_018438.4 2.815 2.091 2.453 TRIM25 tripartite motif-containing 25 ILMN_1813625 NM_005082.4 2.793 2.089 2.441 HMOX1 heme oxygenase (decycling) 1 ILMN_1800512 NM_002133.1 2.066 2.806 2.436 PLSCR1 phospholipid scramblase 1 ILMN_1745242 NM_021105.1 3.062 1.774 2.418 MT1A metallothionein 1A ILMN_1691156 NM_005946.2 2.979 1.825 2.402 TOR1B torsin family 1, member B (torsin B) ILMN_1724333 NM_014506.1 3.026 1.772 2.399 TRIM21 tripartite motif-containing 21 ILMN_1678054 NM_003141.3 2.464 2.332 2.398 KIAA1618 ring finger protein 213 (aka RNF213) ILMN_2289093 NM_020954.2 2.835 1.942 2.388 tumor necrosis factor (ligand) superfamily, TNFSF13B ILMN_2066858 NM_006573.3 2.349 2.426 2.388 member 13b (BAFF) ZC3HAV1 zinc finger CCCH-type, antiviral 1 ILMN_1729973 NM_024625.3 2.733 2.003 2.368

TRIM5 tripartite motif-containing 5 ILMN_1704972 NM_033034.1 2.928 1.767 2.348 CASP2 and RIPK1 domain containing adaptor CRADD ILMN_1690282 NM_003805.3 1.917 2.774 2.346 with death domain TRIM22 tripartite motif-containing 22 ILMN_1779252 NM_006074.3 2.825 1.807 2.316 poly (ADP-ribose) polymerase family, member PARP10 ILMN_1721411 XM_001127571.1 2.575 2.046 2.311 10 (PARP10) poly (ADP-ribose) polymerase family, PARP14 ILMN_1691731 NM_017554.1 2.668 1.940 2.304 member 14 HSPA1B heat shock 70kDa protein 1B ILMN_1660436 NM_005346.3 2.932 1.650 2.291 DUSP5 dual specificity phosphatase 5 ILMN_1656501 NM_004419.3 2.667 1.889 2.278 C5ORF29 chromosome 5 open reading frame 29 ILMN_1675191 NM_152687.2 -1.891 -2.604 -2.248 interferon stimulated exonuclease gene ISG20 ILMN_1659913 NM_002201.4 2.622 1.868 2.245 20kDa ASGR1 asialoglycoprotein receptor 1 ILMN_1769013 NM_001671.2 -2.131 -2.313 -2.222 DUSP19 dual specificity phosphatase 19 ILMN_1722492 NM_080876.2 2.915 1.521 2.218 BST2 bone marrow stromal cell antigen 2 ILMN_1723480 NM_004335.2 2.438 1.996 2.217 CCL2 chemokine (C-C motif) ligand 2 ILMN_1720048 NM_002982.3 2.899 1.525 2.212 IFI16 interferon, gamma-inducible protein 16 ILMN_1710937 NM_005531.1 2.469 1.898 2.183 IL4I1 interleukin 4 induced 1 ILMN_1659960 NM_172374.1 2.525 1.722 2.124 SP100 SP100 nuclear antigen ILMN_2284998 NM_001080391.1 2.125 2.099 2.112 LAMP3 lysosomal-associated membrane protein 3 ILMN_2170814 NM_014398.2 2.553 1.607 2.080 GBP5 guanylate binding protein 5 ILMN_2114568 NM_052942.2 2.412 1.737 2.074 solute carrier family 7, (cationic amino acid SLC7A11 ILMN_1655229 NM_014331.3 2.140 1.942 2.041 transporter, y+ system) member 11 UBE2L6 ubiquitin-conjugating enzyme E2L 6 ILMN_1769520 NM_004223.3 2.303 1.776 2.040 SCARB2 scavenger receptor class B, member 2 ILMN_1814726 NM_005506.2 1.893 2.162 2.028 NCOA7 nuclear receptor coactivator 7 ILMN_1687768 NM_181782.2 2.507 1.533 2.020 HSH2D hematopoietic SH2 domain containing ILMN_1788017 NM_032855.2 2.236 1.804 2.020 SOCS1 suppressor of cytokine signaling 1 ILMN_1774733 NM_003745.1 2.512 1.525 2.018 ZBP1 Z-DNA binding protein 1 ILMN_1765994 NM_030776.1 2.255 1.780 2.018 CLEC12A C-type lectin domain family 12, member A ILMN_1663142 NM_201623.2 -2.034 -1.878 -1.956 interferon induced transmembrane protein 2 IFITM2 ILMN_1673352 NM_006435.2 2.269 1.630 1.950 (1-8D) NMI N-myc (and STAT) interactor ILMN_1739541 NM_004688.1 2.084 1.816 1.950 HAVCR2 hepatitis A virus cellular receptor 2 ILMN_1693826 NM_032782.3 1.807 2.092 1.949 SP140 SP140 nuclear body protein ILMN_2246882 NM_001005176.1 2.068 1.785 1.927 UNC93B1 unc-93 homolog B1 (C. elegans) ILMN_2193591 NM_030930.2 1.917 1.933 1.925 peroxisome proliferator-activated receptor PPARG ILMN_1800225 NM_015869.4 1.971 1.874 1.922 gamma solute carrier family 16, member 10 (aromatic SLC16A10 ILMN_1782938 NM_018593.3 1.854 1.972 1.913 amino acid transporter) C19ORF66 chromosome 19 open reading frame 66 ILMN_1750400 NM_018381.2 1.912 1.907 1.910 ACOT9 acyl-CoA thioesterase 9 ILMN_1658995 NM_001033583.2 1.839 1.931 1.885 CD69 CD69 molecule ILMN_2188333 NM_001781.1 2.210 1.553 1.881 LAG3 lymphocyte-activation gene 3 ILMN_1813338 NM_002286.4 2.200 1.554 1.877 CCR1 chemokine (C-C motif) receptor 1 ILMN_1678833 NM_001295.2 2.099 1.655 1.877 TMEM140 transmembrane protein 140 ILMN_1736863 NM_018295.2 2.017 1.721 1.869 ZNFX1 zinc finger, NFX1-type containing 1 ILMN_1745148 NM_021035.2 2.189 1.519 1.854 VNN2 vanin 2 ILMN_1678939 NM_004665.2 -1.572 -2.123 -1.847 CX3CR1 chemokine (C-X3-C motif) receptor 1 ILMN_2088437 NM_001337.3 -1.733 -1.923 -1.828 CCL23 chemokine (C-C motif) ligand 23 ILMN_1686109 NM_145898.1 -1.616 -2.029 -1.823 CKAP4 cytoskeleton-associated protein 4 ILMN_1790891 NM_006825.2 2.047 1.593 1.820 RNASE1 ribonuclease, RNase A family, 1 (pancreatic) ILMN_2333670 NM_198235.1 1.533 2.106 1.820 TRAFD1 TRAF-type zinc finger domain containing 1 ILMN_1758250 NM_006700.1 2.002 1.636 1.819 SPHK1 sphingosine kinase 1 ILMN_2357134 NM_021972.2 1.521 2.112 1.817 protein kinase, AMP-activated, gamma 2 PRKAG2 ILMN_1797531 NM_024429.1 1.826 1.780 1.803 non-catalytic subunit RGS1 regulator of G-protein signaling 1 ILMN_1656011 NM_002922.3 1.685 1.919 1.802 TPST1 tyrosylprotein sulfotransferase 1 ILMN_1651950 NM_003596.2 1.533 2.063 1.798 signal transducer and activator of STAT2 ILMN_1690921 NM_005419.2 1.909 1.680 1.794 transcription 2, 113kDa SAT1 spermidine/spermine N1-acetyltransferase 1 ILMN_1753342 NM_002970.1 1.893 1.689 1.791 DR1-associated protein 1 (negative cofactor 2 DRAP1 ILMN_2112301 NM_006442.2 1.792 1.774 1.783 alpha) RAS guanyl releasing protein 3 (calcium and RASGRP3 ILMN_1727045 NM_170672.1 1.832 1.702 1.767 DAG-regulated) myeloid differentiation primary response MYD88 ILMN_1738523 NM_002468.3 1.900 1.616 1.758 gene (88) ZNF467 zinc finger protein 467 ILMN_1779015 NM_207336.1 -1.819 -1.679 -1.749 TLR8 toll-like receptor 8 ILMN_1657892 NM_138636.2 -1.615 -1.870 -1.743 NAPSB napsin B aspartic peptidase pseudogene ILMN_2109416 NR_002798.1 1.829 1.651 1.740 N4BP1 NEDD4 binding protein 1 ILMN_2201966 NM_153029.3 1.926 1.554 1.740 TREM1 triggering receptor expressed on myeloid cells 1 ILMN_1688231 NM_018643.2 1.883 1.583 1.733 EHD4 EH-domain containing 4 ILMN_1720083 NM_139265.2 1.853 1.605 1.729 DFNA5 deafness, autosomal dominant 5 ILMN_1670145 NM_004403.2 1.563 1.892 1.728 MGST1 microsomal glutathione S-transferase 1 ILMN_2355168 NM_145792.1 1.624 1.803 1.714 SQRDL sulfide quinone reductase-like (yeast) ILMN_1667199 NM_021199.2 1.792 1.615 1.703 LOC201175 hypothetical protein LOC201175 ILMN_1656361 NM_174919.2 1.722 1.654 1.688 GPR162 G protein-coupled receptor 162 ILMN_1730816 NM_019858.1 -1.508 -1.828 -1.668 GCH1 GTP cyclohydrolase 1 ILMN_1812759 NM_001024071.1 1.804 1.531 1.667 CHRNB1 cholinergic receptor, nicotinic, beta 1 ILMN_1741131 NM_000747.2 1.600 1.732 1.666 GBP4 guanylate binding protein 4 ILMN_1771385 NM_052941.3 1.711 1.578 1.645 CRTAP cartilage associated protein ILMN_1665235 NM_006371.3 -1.684 -1.556 -1.620 ribosome binding protein 1 homolog 180kDa RRBP1 ILMN_2360784 NM_001042576.1 1.712 1.503 1.607 (dog) DYNLT1 dynein, light chain, Tctex-type 1 ILMN_1678766 NM_006519.1 1.645 1.552 1.599 TMEM62 transmembrane protein 62 ILMN_1745807 NM_024956.3 1.598 1.583 1.591 OGFR opioid growth factor receptor ILMN_1728224 NM_007346.2 1.575 1.604 1.589 IL23A interleukin 23, alpha subunit p19 ILMN_1715603 NM_016584.2 -1.564 -1.577 -1.571 solute carrier family 36 (proton/amino acid SLC36A1 ILMN_2124471 NM_078483.2 1.531 1.535 1.533 symporter), member 1 CCL20 chemokine (C-C motif) ligand 20 (CCL20) ILMN_1657234 NM_004591.1 -2.436 1.657 -0.390 a Probe ID from Illumina Human Ref-8v3 Expression BeadChips b FC, fold change relative to unstimulated controls. D1 and D2, donor 1 and donor 2. Positive numbers indicate fold upregulated, negative numbers indicate fold downregulated c Bold lettering indicates type I IFN stimulated genes (ISGs)

Supplementary Table III: Remaining 191 genes changed upon addition of C1q to SLE ICs in total PBMC cultures

Mean Mean C1q- C1q-IC/IC FC Gene REFSEQ_ID Gene Name IC FCa IC FCa Ratiob C1q-IC/ICc

CCL23 chemokine (C-C motif) ligand 23 NM_145898.1 0.556 1.096 1.972 1.972 protein kinase (cAMP-dependent, catalytic) PKIB NM_032471.4 0.940 0.479 0.509 -1.964 inhibitor beta C4ORF18 chromosome 4 open reading frame 18 NM_016613.4 0.768 0.392 0.510 -1.962 myxovirus (influenza virus) resistance 2 MX2d NM_002463.1 4.925 2.514 0.510 -1.959 (mouse) CCR1 chemokine (C-C motif) receptor 1 NM_001295.2 1.877 0.960 0.512 -1.954 IFI6 interferon, alpha-inducible protein 6 NM_022872.2 5.613 2.898 0.516 -1.937 GBP5 guanylate binding protein 5 NM_052942.2 2.074 1.071 0.516 -1.937 HERC5 hect domain and RLD 5 NM_016323.2 6.326 3.271 0.517 -1.934 TOR1B torsin family 1, member B (torsin B) NM_014506.1 2.399 1.243 0.518 -1.930 CKLF-like MARVEL transmembrane domain CMTM8 NM_178868.3 0.987 1.901 1.925 1.925 containing 8 CLEC7A C-type lectin domain family 7, member A NM_197950.2 1.072 0.562 0.524 -1.908 FPR1 formyl peptide receptor 1 NM_002029.3 1.200 0.637 0.531 -1.885 CCL20 chemokine (C-C motif) ligand 20 NM_004591.1 1.034 1.945 1.882 1.882 IFI44 interferon-induced protein 44 NM_006417.3 6.066 3.236 0.533 -1.874 TRIM21 tripartite motif-containing 21 NM_003141.3 2.398 1.286 0.536 -1.865 NT5C3 5'-nucleotidase, cytosolic III NM_001002010.1 2.927 1.577 0.539 -1.856 ZNF467 zinc finger protein 467 NM_207336.1 0.573 0.309 0.539 -1.854 FVT1 follicular lymphoma variant translocation 1 NM_002035.1 0.889 1.647 1.853 1.853 SAMD9 sterile alpha motif domain containing 9 NM_017654.2 2.861 1.551 0.542 -1.845 AIF1 allograft inflammatory factor 1 NM_032955.1 0.967 0.524 0.542 -1.845 IFI44L interferon-induced protein 44-like NM_006820.1 8.568 4.654 0.543 -1.841 C1ORF162 chromosome 1 open reading frame 162 NM_174896.2 0.834 0.456 0.547 -1.829 FUCA1 fucosidase, alpha-L- 1, tissue NM_000147.3 0.750 0.411 0.548 -1.824

NCOA7 nuclear receptor coactivator 7 NM_181782.2 2.020 1.109 0.549 -1.822 thromboxane A synthase 1 (platelet, cytochrome TBXAS1 NM_001061.2 0.906 0.498 0.550 -1.819 P450, family 5, subfamily A) BOP1 block of proliferation 1 NM_015201.3 0.858 1.558 1.817 1.817 H1F0 H1 histone family, member 0 NM_005318.2 0.942 0.519 0.551 -1.815 LGMN legumain NM_001008530.1 1.337 0.738 0.552 -1.811 Fc fragment of IgG, low affinity IIa, receptor FCGR2A NM_021642.2 0.973 0.543 0.558 -1.792 (CD32) nucleotide-binding oligomerization domain NOD2 NM_022162.1 1.493 0.834 0.558 -1.791 containing 2 H1FX H1 histone family, member X NM_006026.2 0.920 0.515 0.560 -1.787 SP140 SP140 nuclear body protein NM_001005176.1 1.879 1.054 0.561 -1.782 peroxisomal proliferator-activated receptor A PRIC285 NM_033405.2 4.249 2.386 0.561 -1.781 interacting complex 285 C1ORF122 chromosome 1 open reading frame 122 NM_198446.1 1.000 1.774 1.774 1.774 CHN2 chimerin (chimaerin) 2 NM_004067.2 1.095 0.618 0.564 -1.774 chemokine (C-X-C motif) ligand 1 (melanoma CXCL1 NM_001511.1 1.283 2.271 1.771 1.771 growth stimulating activity, alpha) tumor necrosis factor receptor superfamily, TNFRSF4 NM_003327.2 1.005 1.777 1.768 1.768 member 4 LAP3 leucine aminopeptidase 3 NM_015907.2 3.231 1.828 0.566 -1.768 NAPSB napsin B aspartic peptidase pseudogene NR_002798.1 1.740 0.988 0.568 -1.761 EMP1 epithelial membrane protein 1 NM_001423.1 3.151 1.803 0.572 -1.748 MT2A metallothionein 2A NM_005953.2 2.886 1.651 0.572 -1.748 LRRN3 leucine rich repeat neuronal 3 NM_018334.3 0.774 0.443 0.573 -1.746 C22ORF28 chromosome 22 open reading frame 28 NM_014306.3 1.743 1.007 0.578 -1.730 PTMA prothymosin, alpha NM_001099285.1 0.997 1.724 1.729 1.729 HSPA1B heat shock 70kDa protein 1B NM_005346.3 2.291 1.330 0.580 -1.723 ectonucleoside triphosphate diphosphohydrolase ENTPD1 NM_001098175.1 0.884 0.515 0.582 -1.717 1 HERC6 hect domain and RLD 6 NM_017912.3 4.414 2.572 0.583 -1.716 ADORA2A adenosine A2a receptor NM_000675.3 0.884 1.518 1.716 1.716 CEBPD CCAAT/enhancer binding protein NM_005195.3 0.759 0.443 0.584 -1.713 NAMPT nicotinamide phosphoribosyltransferase NM_005746.2 0.985 1.685 1.711 1.711 ATF3 activating transcription factor 3 NM_001040619.1 2.562 1.502 0.586 -1.706 IL1RN interleukin 1 receptor antagonist NM_173842.1 7.043 4.132 0.587 -1.704 TRAFD1 TRAF-type zinc finger domain containing 1 NM_006700.1 1.819 1.068 0.587 -1.702 FBXO6 F-box protein 6 NM_018438.4 2.453 1.442 0.588 -1.701 IKZF1 IKAROS family zinc finger 1 NM_006060.3 0.953 1.617 1.697 1.697 NCF4 neutrophil cytosolic factor 4, 40kDa NM_000631.3 0.818 0.482 0.590 -1.696 UNC93B1 unc-93 homolog B1 NM_030930.2 1.925 1.137 0.590 -1.694 CD36 CD36 molecule (thrombospondin receptor) NM_000072.2 0.849 0.503 0.592 -1.689 EPSTI1 epithelial stromal interaction 1 (breast) NM_033255.2 4.960 2.942 0.593 -1.686 CCL3 chemokine (C-C motif) ligand 3 NM_002983.1 0.694 1.169 1.686 1.686 guanine nucleotide binding protein-like 3 GNL3 NM_206826.1 0.894 1.503 1.682 1.682 (nucleolar) OSCAR osteoclast-associated receptor NM_133169.2 0.950 0.566 0.595 -1.680 RPP40 ribonuclease P/MRP 40kDa subunit NM_006638.2 0.824 1.382 1.678 1.678 IFI35 interferon-induced protein 35 NM_005533.2 3.370 2.010 0.596 -1.677 APOB48R apolipoprotein B48 receptor NM_018690.2 1.016 0.606 0.597 -1.675 REXO2 REX2, RNA exonuclease 2 homolog NM_015523.2 0.838 1.401 1.672 1.672 HK3 hexokinase 3 (white cell) NM_002115.1 1.235 0.739 0.598 -1.671 ITK IL2-inducible T-cell kinase NM_005546.3 0.884 1.476 1.671 1.671 DUSP5 dual specificity phosphatase 5 NM_004419.3 2.278 1.364 0.599 -1.670 FAM89A family with sequence similarity 89, member A NM_198552.1 1.053 1.755 1.667 1.667 RNF19B ring finger protein 19B NM_153341.1 1.523 0.917 0.602 -1.662 C10ORF54 chromosome 10 open reading frame 54 NM_022153.1 0.630 0.380 0.602 -1.660 CXCR4 chemokine (C-X-C motif) receptor 4 NM_003467.2 0.763 1.265 1.658 1.658 ADAP2 ArfGAP with dual PH domains 2 NM_018404.2 1.220 0.736 0.603 -1.657 CLC Charcot-Leyden crystal protein NM_001828.4 0.877 0.530 0.604 -1.655 ATPase, Ca++ transporting, plasma membrane ATP2B4 NM_001684.3 0.831 1.375 1.655 1.655 4 TMEM62 transmembrane protein 62 NM_024956.3 1.591 0.962 0.605 -1.654 RRAS related RAS viral (r-ras) oncogene homolog NM_006270.3 0.876 0.530 0.605 -1.654 CST3 cystatin C NM_000099.2 1.188 0.719 0.605 -1.653 GLIPR1 GLI pathogenesis-related 1 NM_006851.2 0.997 0.605 0.606 -1.649 IRAK2 interleukin-1 receptor-associated kinase 2 NM_001570.3 1.066 1.756 1.648 1.648 NCF1C neutrophil cytosolic factor 1C pseudogene NR_003187.1 1.667 1.012 0.607 -1.648 GPR68 G protein-coupled receptor 68 NM_003485.3 0.869 1.432 1.647 1.647 CUL4A cullin 4A NM_003589.2 0.839 1.379 1.644 1.644 acyl-CoA synthetase long-chain family member ACSL1 NM_001995.2 1.121 1.842 1.643 1.643 1 CORO1B coronin, actin binding protein, 1B NM_001018070.1 0.902 0.549 0.609 -1.643 TXNRD1 thioredoxin reductase 1 NM_001093771.1 1.333 2.190 1.643 1.643 KIAA1618 KIAA1618 NM_020954.2 2.388 1.454 0.609 -1.642 CXCR7 chemokine (C-X-C motif) receptor 7 NM_020311.2 1.155 0.704 0.610 -1.639 LY6E lymphocyte antigen 6 complex, locus E NM_002346.1 3.719 2.270 0.610 -1.639 interferon-induced protein with IFIT5 NM_012420.1 3.522 2.150 0.610 -1.638 tetratricopeptide repeats 5 lectin, galactoside-binding, soluble, 3 (galectin LGALS3 NM_002306.1 0.965 1.579 1.636 1.636 3) proteolipid protein 2 (colonic epithelium- PLP2 NM_002668.1 1.161 0.712 0.613 -1.630 enriched) non-metastatic cells 1, protein (NM23A) NME1 NM_000269.2 0.950 1.547 1.629 1.629 expressed in (NME1) poliovirus receptor-related 2 (herpesvirus entry PVRL2 NM_002856.2 1.432 0.879 0.614 -1.629 mediator B) CD163 CD163 molecule NM_004244.4 1.590 0.980 0.616 -1.623 FAM113B family with sequence similarity 113, member B NM_138371.1 1.073 1.738 1.619 1.619 leukocyte immunoglobulin-like receptor, LILRB3 subfamily B (with TM and ITIM domains), NM_006864.2 1.358 0.839 0.618 -1.618 member 3 Gardner-Rasheed feline sarcoma viral (v-fgr) FGR NM_001042729.1 0.900 1.455 1.617 1.617 oncogene homolog SULF2 sulfatase 2 NM_018837.2 0.782 0.484 0.619 -1.615 ZC3HAV1 zinc finger CCCH-type, antiviral 1 NM_024625.3 2.297 1.423 0.619 -1.614 ASCL2 achaete-scute complex homolog 2 (Drosophila) NM_005170.2 1.366 0.848 0.621 -1.611 BRSK1 BR serine/threonine kinase 1 NM_032430.1 1.557 0.968 0.622 -1.608 C3ORF59 chromosome 3 open reading frame 59 NM_178496.2 1.193 0.742 0.622 -1.608 DHX58 DEXH (Asp-Glu-X-His) box polypeptide 58 NM_024119.2 2.489 1.549 0.622 -1.607 SAMD9L sterile alpha motif domain containing 9-like NM_152703.2 3.905 2.431 0.623 -1.606 MYO1F myosin IF NM_012335.2 0.900 0.560 0.623 -1.606 H2AFJ H2A histone family, member J NM_177925.2 1.002 0.624 0.623 -1.605 CCL7 chemokine (C-C motif) ligand 7 NM_006273.2 6.029 3.757 0.623 -1.605 leukocyte immunoglobulin-like receptor, LILRA5 NM_021250.2 1.217 0.760 0.624 -1.602 subfamily A (with TM domain), member 5 DYNLT1 dynein, light chain, Tctex-type 1 NM_006519.1 1.599 0.999 0.625 -1.600 general transcription factor IIF, polypeptide GTF2F1 NM_002096.1 1.355 0.848 0.625 -1.599 1, 74kDa signal transducer and activator of STAT1 NM_007315.2 4.489 2.808 0.626 -1.599 transcription 1, 91kDa MTMR11 myotubularin related protein 11 NM_181873.2 0.791 0.496 0.627 -1.595 DPH2 DPH2 homolog (S cerevisiae) NM_001384.4 0.897 1.430 1.594 1.594 RAB31 RAB31, member RAS oncogene family NM_006868.2 1.106 0.694 0.627 -1.594 nucleolar protein 5A (56kDa with KKE/D NOL5A NM_006392.2 0.786 1.253 1.593 1.593 repeat) MXD1 MAX dimerization protein 1 NM_002357.2 1.145 0.719 0.628 -1.592 myxovirus (influenza virus) resistance 1, MX1 NM_002462.2 4.665 2.936 0.629 -1.589 interferon-inducible protein p78 ST8 alpha-N-acetyl-neuraminide alpha-2,8- ST8SIA4 NM_005668.3 1.118 0.704 0.630 -1.587 sialyltransferase 4 O-linked N-acetylglucosamine (GlcNAc) OGT NM_181672.1 0.759 1.205 1.586 1.586 transferase membrane-spanning 4-domains, subfamily A, MS4A6A NM_022349.2 0.674 0.425 0.631 -1.585 member 6A GPR35 G protein-coupled receptor 35 NM_005301.2 1.089 1.725 1.585 1.585 MT1A metallothionein 1A NM_005946.2 2.402 1.517 0.632 -1.583 v-maf musculoaponeurotic fibrosarcoma MAF NM_005360.3 0.984 0.621 0.632 -1.583 oncogene homolog (avian) WD repeat domain, phosphoinositide WIPI1 NM_017983.4 1.283 0.811 0.632 -1.581 interacting 1 leukocyte immunoglobulin-like receptor, LILRA3 subfamily A (without TM domain), member NM_006865.2 1.572 0.994 0.632 -1.581 3 killer cell immunoglobulin-like receptor, two KIR2DL3 NM_014511.3 1.099 0.695 0.633 -1.580 domains, long cytoplasmic tail, 3 solute carrier family 39 (zinc transporter), SLC39A8 NM_022154.5 1.068 1.686 1.579 1.579 member 8 LST1 leukocyte specific transcript 1 NM_205840.1 0.790 0.501 0.634 -1.577 SCO cytochrome oxidase deficient homolog 2 SCO2 NM_005138.1 1.554 0.987 0.635 -1.576 (yeast) cysteine-rich secretory protein LCCL domain CRISPLD2 NM_031476.2 1.240 0.787 0.635 -1.575 containing 2 TSPAN3 tetraspanin 3 NM_005724.4 0.907 1.426 1.573 1.573 NOL1 nucleolar protein 1, 120kDa NM_001033714.1 0.868 1.365 1.572 1.572 RNF144B ring finger 144B NM_182757.2 0.790 1.241 1.571 1.571 Notch homolog 1, translocation-associated NOTCH1 NM_017617.3 0.891 1.399 1.571 1.571 (Drosophila) HIST2H2AC histone cluster 2, H2ac NM_003517.2 1.042 0.664 0.637 -1.569 EHD4 EH-domain containing 4 NM_139265.2 1.729 1.103 0.638 -1.567 major histocompatibility complex, class II, DO HLA-DOA NM_002119.3 0.996 0.637 0.639 -1.564 alpha TMEM140 transmembrane protein 140 NM_018295.2 1.869 1.195 0.640 -1.564 DHX9 DEAH (Asp-Glu-Ala-His) box polypeptide 9 NM_001357.3 0.822 1.284 1.563 1.563 RNF135 ring finger protein 135 NM_197939.1 1.167 0.747 0.640 -1.562 5-aminoimidazole-4-carboxamide ATIC ribonucleotide formyltransferase/IMP NM_004044.4 0.875 1.365 1.559 1.559 cyclohydrolase JAK1 Janus kinase 1 (a protein tyrosine kinase) NM_002227.2 0.878 1.369 1.559 1.559 C5AR1 complement component 5a receptor 1 NM_001736.3 1.296 0.831 0.641 -1.559 RIN2 Ras and Rab interactor 2 NM_018993.2 2.442 1.567 0.642 -1.559 TRIM5 tripartite motif-containing 5 NM_033034.1 2.348 1.506 0.642 -1.559 IRF7 interferon regulatory factor 7 NM_004029.2 3.319 2.133 0.643 -1.556 FCGRT Fc fragment of IgG, receptor, transporter, alpha NM_004107.3 1.097 0.705 0.643 -1.555 polymerase (RNA) III (DNA directed) POLR3H NM_001018052.1 0.849 1.319 1.555 1.555 polypeptide H (22 9kD) tumor necrosis factor (ligand) superfamily, TNFSF14 NM_172014.1 0.886 1.374 1.551 1.551 member 14 DENR density-regulated protein NM_003677.3 0.868 1.346 1.550 1.550 peroxisome proliferator-activated receptor PPRC1 NM_015062.3 0.871 1.350 1.550 1.550 gamma, coactivator-related 1 TRIB2 tribbles homolog 2 (Drosophila) NM_021643.1 0.960 1.487 1.549 1.549 ALOX5 PREDICTED: arachidonate 5-lipoxygenase XM_001127464.1 0.650 0.420 0.647 -1.546 P2RY11 purinergic receptor P2Y, G-protein coupled, 11 NM_002566.4 0.887 1.369 1.544 1.544 DEAD (Asp-Glu-Ala-Asp) box polypeptide DDX21 NM_004728.2 1.003 1.547 1.543 1.543 21 solute carrier family 11 (proton-coupled SLC11A2 NM_000617.1 0.903 1.392 1.542 1.542 divalent metal ion transporters), member 2 TMEM167A transmembrane protein 167A NM_174909.3 1.433 0.930 0.649 -1.541 solute carrier family 3 (activators of dibasic SLC3A2 NM_001013251.1 1.099 1.691 1.538 1.538 and neutral amino acid transport), member 2 PLXDC2 plexin domain containing 2 NM_032812.7 0.998 0.649 0.650 -1.538 OAS2 2'-5'-oligoadenylate synthetase 2, 69/71kDa NM_016817.2 3.237 2.109 0.651 -1.535 FKBP14 FK506 binding protein 14, 22 kDa NM_017946.2 0.836 1.283 1.534 1.534 6-phosphofructo-2-kinase/fructose-2,6- PFKFB4 NM_004567.2 1.103 0.719 0.652 -1.534 biphosphatase 4 GRN granulin NM_002087.2 1.045 0.681 0.652 -1.533 SYK spleen tyrosine kinase NM_003177.3 1.036 0.676 0.652 -1.533 WARS tryptophanyl-tRNA synthetase NM_173701.1 1.561 1.019 0.653 -1.532 CASP2 and RIPK1 domain containing adaptor CRADD NM_003805.3 2.346 1.532 0.653 -1.531 with death domain FLJ22662 hypothetical protein FLJ22662 NM_024829.4 0.671 0.438 0.653 -1.531 ZFP36L2 zinc finger protein 36, C3H type-like 2 NM_006887.3 0.716 1.095 1.528 1.528 NRGN neurogranin (protein kinase C substrate, RC3) NM_006176.1 1.100 0.721 0.655 -1.526 CSTB cystatin B (stefin B) NM_000100.2 1.355 2.060 1.521 1.521 solute carrier family 40 (iron-regulated SLC40A1 NM_014585.3 0.956 0.629 0.658 -1.520 transporter), member 1 ANTXR2 anthrax toxin receptor 2 NM_058172.3 1.312 0.865 0.659 -1.518 interferon stimulated exonuclease gene ISG20 NM_002201.4 2.245 1.481 0.660 -1.516 20kDa ZBP1 Z-DNA binding protein 1 NM_030776.1 2.018 1.337 0.662 -1.510 HSH2D hematopoietic SH2 domain containing NM_032855.2 2.020 1.339 0.663 -1.509 IRAK3 interleukin-1 receptor-associated kinase 3 NM_007199.1 0.903 1.361 1.507 1.507 ADCY3 adenylate cyclase 3 NM_004036.3 0.898 1.353 1.507 1.507 KRT73 keratin 73 NM_175068.2 1.089 0.723 0.664 -1.506 NCF1 neutrophil cytosolic factor 1 NM_000265.4 1.439 0.956 0.664 -1.505 C7ORF50 chromosome 7 open reading frame 50 NM_032350.4 0.937 0.623 0.665 -1.505 aldehyde dehydrogenase 2 family ALDH2 NM_000690.2 0.864 0.574 0.665 -1.504 (mitochondrial) B-cell CLL/lymphoma 6 (zinc finger protein BCL6 NM_001706.2 1.034 1.554 1.504 1.504 51) EIF5A eukaryotic translation initiation factor 5A NM_001970.3 0.943 1.416 1.502 1.502 PUS1 pseudouridylate synthase 1 NM_001002019.1 0.856 1.286 1.502 1.502 KIAA0513 KIAA0513 NM_014732.2 1.039 0.692 0.666 -1.502 CD44 CD44 molecule (Indian blood group) NM_001001392.1 0.839 1.259 1.502 1.502 IFI30 interferon, gamma-inducible protein 30 NM_006332.3 1.215 0.809 0.666 -1.502 WTAP Wilms tumor 1 associated protein NM_004906.3 0.837 1.256 1.501 1.501 RAB37 RAB37, member RAS oncogene family NM_175738.3 0.867 0.578 0.666 -1.501

a Mean fold change (FC) from 2 donors relative to unstimulated controls b Ratio of fold changes from C1q-IC stimulations of PBMCs over ICs alone c Ratio converted to fold change where positive numbers indicate fold upregulated and negative numbers indicate fold downregulated d Bold lettering indicates type I IFN stimulated genes (ISGs)