MAFB Determines Human Macrophage Anti-Inflammatory

Home , APOL3, CRHBP, EMP2, FAM129A, ITM2C, LIN7A

MAFB Determines Human Macrophage Anti-Inflammatory Polarization: Relevance for the Pathogenic Mechanisms Operating in Multicentric Carpotarsal Osteolysis This information is current as of September 25, 2021. Víctor D. Cuevas, Laura Anta, Rafael Samaniego, Emmanuel Orta-Zavalza, Juan Vladimir de la Rosa, Geneviève Baujat, Ángeles Domínguez-Soto, Paloma Sánchez-Mateos, María M. Escribese, Antonio Castrillo, Valérie Cormier-Daire, Miguel A. Vega and Ángel L. Corbí Downloaded from J Immunol published online 16 January 2017 http://www.jimmunol.org/content/early/2017/01/15/jimmun ol.1601667 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2017/01/15/jimmunol.160166 Material 7.DCSupplemental

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 16, 2017, doi:10.4049/jimmunol.1601667 The Journal of Immunology

MAFB Determines Human Macrophage Anti-Inﬂammatory Polarization: Relevance for the Pathogenic Mechanisms Operating in Multicentric Carpotarsal Osteolysis

Vıćtor D. Cuevas,* Laura Anta,† Rafael Samaniego,‡ Emmanuel Orta-Zavalza,* Juan Vladimir de la Rosa,x Genevie`ve Baujat,{,‖ A´ ngeles Domıńguez-Soto,* Paloma Sańchez-Mateos,‡ Marıá M. Escribese,# Antonio Castrillo,x Vale´rie Cormier-Daire,{,‖ Miguel A. Vega,* and A´ ngel L. Corbı´*

Macrophage phenotypic and functional heterogeneity derives from tissue-specific transcriptional signatures shaped by the local microenvironment. Most studies addressing the molecular basis for macrophage heterogeneity have focused on murine cells, whereas the factors Downloaded from controlling the functional specialization of human macrophages are less known. M-CSF drives the generation of human monocyte-derived macrophages with a potent anti-inflammatory activity upon stimulation. We now report that knockdown of MAFB impairs the acquisition of the anti-inflammatory profile of human macrophages, identify the MAFB-dependent gene signature in human macrophages and il- lustrate the coexpression of MAFB and MAFB-target genes in CD163+ tissue-resident and tumor-associated macrophages. The contribution of MAFB to the homeostatic/anti-inflammatory macrophage profile is further supported by the skewed polarization of monocyte-derived macrophages from multicentric carpotarsal osteolysis (Online Mendelian Inheritance in Man #166300), a pathology http://www.jimmunol.org/ caused by mutations in the MAFB gene. Our results demonstrate that MAFB critically determines the acquisition of the anti- inflammatory transcriptional and functional profiles of human macrophages. The Journal of Immunology, 2017, 198: 000–000.

acrophage heterogeneity derives from the existence of bition of tumor cell growth and production of LPS-induced cy- tissue-specific factors that control macrophage differ- tokines (7, 14). Specifically, GM-CSF primes macrophages M entiation and functional maturation. M-CSF and IL-34 (GM-MØ) to gain immunogenic activity and to produce inflam- control macrophage differentiation in most tissues (1, 2), whereas matory cytokines upon TLR stimulation, whereas M-CSF primes GM-CSF drives the generation of alveolar macrophages (3). Cir- macrophages (M-MØ) with tissue repair and proangiogenic

culating monocytes are recruited to damaged tissues under in- functions, and with potent TLR-induced IL-10–producing ability by guest on September 25, 2021 flammatory conditions (4) and acquire specialized functions (7, 15). Accordingly, human GM-MØ and M-MØ are considered (macrophage polarization) under the influence of extracellular as proinflammatory and anti-inflammatory macrophages (6, 16). cues (5). The macrophage sensitivity to the surrounding milieu is MAFB is a transcription factor of the large MAF subfamily exemplified by the ability of GM-CSF and M-CSF to induce the (MAFA, cMAF, MAFB, NRL) that binds to a specific DNA acquisition of distinct effector functions (6, 7). Previous studies element (MARE); heterodimerizes with cMAF, JUN, and FOS have demonstrated that GM-CSF–primed macrophages (GM-MØ) (17–19); and associates and functionally inhibits MYB (19), and M-CSF–primed macrophages (M-MØ) exhibit distinct cellu- MITF, and NFATc1 (20). MAFB controls lens development (21), lar phenotypes (8–11), a distinct metabolic state (12, 13), and lymphangiogenesis (22), pancreatic a and b cell differentiation (23, display opposite effector functions like activin A–mediated inhi- 24), skin cell differentiation (25), chondrocyte matrix formation and

*Laboratorio de Ce´lulas Mieloides, Centro de Investigaciones Biolo´gicas, Consejo Grant S2010/BMD-2350 (to A´ .L.C. and A.C.), and a FormaciońdePersonalInves- Superior de Investigaciones Cientı´ficas, 28040 Madrid, Spain; †Servicio de Cirugıá tigador predoctoral fellowship from MINECO through Grant BES-2012-053864 Ortope´dica y Traumatologıá, Complejo Hospitalario de Santiago de Compostela, (to V.D.C.). 15706 Santiago de Compostela, Spain; ‡Laboratorio de Inmuno-Oncologıá, Unidad V.D.C., L.A., M.M.E., A.C., M.A.V., and A´ .L.C. designed the research; V.D.C., L.A., de Microscopıá Confocal, Instituto de Investigacioń Sanitaria Gregorio Maranõń, x R.S., E.O.-Z., J.V.d.l.R., G.B., A´ .D.-S., P.S.-M., and V.C.-D. performed experiments, 28007 Madrid, Spain; Instituto de Investigaciones Biomedicas Alberto Sols, Consejo { recruited patients, and analyzed data; V.D.C. and A´ .L.C. wrote the manuscript. Superior de Investigaciones Cientı´ficas, 28029 Madrid, Spain; Unidad de Biomedi- cina, Instituto de Investigaciones Biome´dicas–Universidad de Las Palmas de Gran The sequences presented in this article have been submitted to Gene Expression Canaria (ULPGC), Instituto Universitario de Investigaciones Biomedicas y Sanitarias Omnibus under accession number GSE84622. de la ULPGC, 35001 Las Palmas, Spain; ‖De´partement de Geńe´tique, INSERM Address correspondence and reprint requests to Dr. A´ ngel L. Corbı´ and Dr. Miguel A. U781, Universite´ Paris Descartes-Sorbonne Paris Cite´, Institut Imagine, Hoˆpital Vega, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Necker Enfants Malades, 75015 Paris, France; and #Institute for Applied Molecular Cientı´ficas, Calle Ramiro de Maeztu 9, 28040 Madrid, Spain. E-mail addresses: Medicine, School of Medicine, University CEU San Pablo, Madrid, Spain [email protected] (A´ .L.C.) and [email protected] (M.A.V.) ORCIDs: 0000-0002-2816-8070 (V.D.C.); 0000-0002-4196-5218 (L.A.); 0000-0002- The online version of this article contains supplemental material. 3081-7332 (R.S.); 0000-0003-1443-7548 (J.V.d.l.R.); 0000-0003-1980-5733 (A´ .L.C.). Abbreviations used in this article: GM-MØ, GM-CSF–primed macrophages; GSEA, Received for publication September 26, 2016. Accepted for publication December gene set enrichment analysis; MARE, MAF recognition element; MCTO, multicen- 16, 2016. tric carpotarsal osteolysis; M-MØ, M-CSF–primed macrophages; RANKL, receptor This work was supported by Ministerio de Economıá y Competitividad (MINECO) activator for NF-kB ligand; siControl, small interfering RNA control; siMAFB, Grant SAF2014-52423-R and Instituto de Salud Carlos III Red de Investigacioń MAFB-specific small interfering RNA; TRAP, tartrate-resistant acid phosphatase. en Enfermedades Reuma´ticas Grant RIER RD12/009 (to A´ .L.C. and M.A.V.), MINECO Grant SAF2014-56819-R (to A.C.), Comunidad Autońoma de Madrid/ Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 Fonds Europeén de De´veloppement E´ conomique et Re´gional RAPHYME Program

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601667 2 MAFB-DIRECTED MACROPHAGE ANTI-INFLAMMATORY POLARIZATION development (26), and podocyte generation (27–30), and also reg- that were provided by M. Palomero (Oncology Department, Hospital Gen- ulates type I IFN production through recruitment of coactivators to eral Universitario Gregorio Maranõń). Macrophage supernatants were IFN regulatory factor 3 (31). Within the murine myeloid lineage, assayed for the presence of cytokines using commercial ELISA kits for CCL2 (BD Biosciences) and IL-10 (BioLegend), according to the proto- MafB is preferentially expressed in most tissue-resident macro- cols supplied by the manufacturers. Mouse bone marrow–derived macrophages, whose specific enhancers contain an overrepresentation of phages were generated using human M-CSF (10 ng/ml; ImmunoTools). All MARE sequences (32). MAFB restricts the ability of M-CSF to animal procedures were approved by the Comite´ E´ tico de Experimentacioń instruct myeloid cell proliferation, promotes macrophage differen- Animal (Ethical Committee for Animal Experimentation) of the Consejo Superior de Investigaciones Cientı´ficas and conducted in accordance with tiation (33) through repression of self-renewal enhancers in mac- the approved guidelines. rophages in vivo (34), and negatively regulates osteoclast generation via inhibition of FOS, MITF, and NFATc1 (20). Quantitative real-time RT-PCR The deregulated expression/function of MAFB gives rise to various Total RNAwas extracted using the NucleoSpin RNA/Protein kit (Macherey- human pathologies. Specifically, heterozygous missense MAFB mu- Nagel, Duren,€ Germany), retrotranscribed, and amplified using the Uni- tations result in multicentric carpotarsal osteolysis (MCTO; Online versal Human Probe Library (Roche Diagnostics, Mannheim, Germany). Mendelian Inheritance in Man #166300), a rare osteolytic syndrome Oligonucleotides for selected genes were designed according to the Roche software for quantitative real-time PCR (Roche Diagnostics, Mannheim, with kidney affectation (35–38), whereas MAFB overexpression is a Germany). Assays were made in triplicate, and results were normalized common feature in multiple myeloma (39) and is linked to Dupuytren’s according to the expression levels of TBP or 18S rRNA for macrophage or disease (40), a fibroblastic proliferation of the palmar fascia. Loss of monocyte samples, respectively. Custom-made microfluidic gene cards MAFB function has recently been shown to cause Duane retraction (Roche Diagnostics) were designed to analyze the expression of a set of genes whose expression is differentially modulated by LPS in GM-MØ and syndrome (41). Besides, animal models of disease revealed that MafB Downloaded from M-MØ (V.D. Cuevas and A.L. Corbı´, unpublished observations). Specifically, deficiency ameliorates atherosclerotic lesions (42) and accelerates the gene cards included 10 genes upregulated by LPS in both GM-MØ and obesity (43), whereas MafB mutations cause kidney-associated dis- M-MØ, 16 genes upregulated by LPS exclusively in GM-MØ, 37 genes eases that result from altered podocyte differentiation (44). upregulatedbyLPSexclusivelyinM-MØ,3genesdownregulatedbyLPS Despite its pathological relevance, the association of MAFB to exclusively in GM-MØ, and 20 genes downregulated by LPS exclusively in M-MØ. Assays were made in duplicate on three independent samples of each human macrophage polarization states and the identity of MAFB- type, and the results were normalized according to the mean of the expression regulated genes in human macrophages are mostly unknown. We level of endogenous reference genes HPRT1, TBP,andRPLP0. In all cases http://www.jimmunol.org/ report that MAFB determines the anti-inflammatory gene signature (quantitative real-time PCR or gene cards), the results were expressed using of M-CSF–primed human macrophages, identify the range of the DD cycle threshold method for quantitation. MAFB-dependent genes in macrophages both in vitro and in vivo, Western blot and demonstrate that MCTO monocyte-derived macrophages exhibit an exacerbated anti-inflammatory profile that might con- Cell lysates were obtained in radioimmunoprecipitation assay buffer (10 mM Tris-HCl [pH 8], 150 mM NaCl, 1% Nonidet P-40, 2 mM Pfabloc, tribute to the pathological consequences of deregulated MAFB 2 mg/ml aprotinin/antipain/leupeptin/pepstatin, 10 mM NaF, and 1 mM expression or function. Na3VO4). A total of 10 mg cell lysate was subjected to SDS-PAGE under reducing (MAFB) or nonreducing (CLEC5A) conditions and transferred

Materials and Methods onto an Immobilon polyvinylidene difluoride membrane (Millipore). by guest on September 25, 2021 Generation of monocyte-derived macrophages Protein detection was carried out using Abs against MAFB (sc-10022; Santa Cruz) and CLEC5A (MAB2384; R&D Systems). Protein loading The methods were carried out in accordance with the approved guidelines. was normalized using a mAb against GAPDH (sc-32233; Santa Cruz). Human PBMC were isolated from buffy coats from normal donors over a Lymphoprep (Nycomed Pharma, Oslo, Norway) gradient according to standard MAFB expression vectors, site-directed mutagenesis, and procedures approved by Ethical Board of the Consejo Superior de Inves- reporter assays tigaciones Cientı´ficas. Blood leukocyte preparations were purchased from Comunidad de Madrid Blood Center. Monocytes were purified from PBMC by The coding sequence of MAFB was amplified by PCR from reverse-transcribed magnetic cell sorting using anti-CD14 microbeads (Miltenyi Biotec, Bergisch cDNA from a healthy donor and was sequenced to confirm the absence of Gladbach, Germany) (.95% CD14+ cells). Monocytes (0.5 3 106 cells/ml, mutations. The oligonucleotides used for amplification of the MAFB coding .95% CD14+ cells) were cultured in RPMI 1640 supplemented with 10% region were 59-CGGAATTCCGATGGCCGCGGAGCTGAGC-39 and 59- FBS for 7 d in the presence of 1000 U/ml GM-CSF, 10 ng/ml M-CSF GCTCTAGAGCTCACAGAAAGAACTCGGGAGAG-39, which include (ImmunoTools, Friesoythe, Germany), or 10 ng/ml IL-34 (BioLegend) to EcoRI and XbaI restriction sites for subsequent cloning into EcoRI- and XbaI-cut generate GM-CSF–polarized macrophages (GM-MØ), M-CSF–polarized pCDNA3.1(+) expression vector (pMAFBwt). Site-directed mutagenesis was macrophages (M-MØ), or IL-34–polarized macrophages, respectively. Cyto- carried out using the QuikChange site-directed mutagenesis kit (Stratagene) to 54 kines were added every 2 d. Cells were cultured in 21% O2 and 5% CO2. generate MAFB expression constructs with the Ser Leu mutation (161C . T, Monocyte-derived osteoclasts were generated by culturing monocytes for 12 d found in MCTO#1 patient) (pMAFB161T), the Pro63Argmutation(188C. G) on glass coverslips in the presence of M-CSF (25 ng/ml) and receptor activator (pMAFB188G) (36), or the Pro71Ser mutation (211C . T) (pMAFB211T) (38). for NF-kB ligand (RANKL; 30 ng/ml). After fixing, osteoclast generation was The oligonucleotides used for mutagenesis were S54L forward (59-CACGC- verified by staining for tartrate-resistant acid phosphatase (TRAP) using the CTGCAGCCAGCCGGCTTGGTGTCCTCCAC-39), S54L reverse (59-GTGG- Leukocyte Acid Phophatase kit (Sigma-Aldrich). Determination of osteoclast- AGGACACCAAGCCGGCTGGCTGCAGGCGTG-39), P63R forward (59-CC- mediated degradation of human bone collagen (type I) was done using the GCTCAGCACTCGGTGTAGCTCCGTGCCCTCGTC-39), P63R reverse (59- OsteoLyse Assay kit (human collagen; Lonza, Walkersville, MD). Generation GACGAGGGCACGGAGCTACACCGAGTGCTGAGCGG-39), P71S forward of monocyte-derived macrophages from MCTO patients was done using a (59-GTAGCTCCGTGCCCTCGTCGTCCAGCTTCAGCCCGACCGAAC-39), similar procedure and following the Medical Ethics Committee procedures of and P71S reverse (59-GTTCGGTCGGGCTGAAGCTGGACGACGAGGGCA- Hospital Clıńico Universitario, Santiago de Compostela (patient MCTO#1), CGGAGCTAC-39). The resulting plasmids were verified by sequencing. and of Hoˆpital Necker Enfants Malades, Paris (two patients referred to as To check the transcriptional activity of MAFB MCTO-causing muta- MCTO#2). Informed consents were obtained from all subjects. MCTO#1 tions, HEK293T cells were seeded in 24-well plates at 4 3 104 cells per patient was found to contain a Ser54Leu (161C . T) mutation, whereas pa- well and transfected with SuperFect (Qiagen) with 200 ng each expression tients MCTO#2 contain a Pro63Leu (188C . T) mutation (36). For MAFB vector and 1 mg33MARE-Luc reporter vector. Each transfection also knockdown, M-MØ (106 cell/ml) were transfected with a MAFB-specific small included 25 ng construct expressing the Renilla luciferase for normaliza- interfering RNA (siMAFB, 50 nM; Life Technologies) using Hiperfect (Qia- tion of transfection efficiency. After 24 h, firefly and Renilla luciferase gen). As a control, cells were transfected with a nonspecific small interfering activities were determined by using the Dual-Luciferase Reporter Assay RNA control (siControl; Life Technologies). After transfection, cells were System (Promega). The 33MARE-Luc reporter construct was generated cultured for the indicated times in RPMI 1640 supplemented with 10% FBS. by inserting three multimerized MAF-recognition elements (MARE) (18) Where indicated, cells were treated with 10 ng/ml Escherichia coli 055:B5 into HindIII- and XhoI-cut pXP2-TATA plasmid. The sequences of LPS (Sigma-Aldrich) or exposed to tumor ascitic fluids of different origins (11) the oligonucleotides used to generate the 33MARE-Luc construct were The Journal of Immunology 3

59-agctttcgacccgaaaggTGCTGAcgTCAGCAgctagccctcgacccgaaaggTGCTGAcgT- levels dropped at early time points and peaked 24–48 h after CAGCAgctagccctcgacccgaaaggTGCTGAcgTCAGCAgctagcccc-39 and 59- M-CSF treatment (Fig. 1C, upper panel). The MAFB protein levels tcgaggggcta gcTGCTGAcgTCAGCAcctttcgggtcgagggctagcTGCTGAcgTC- were greatly increased along M-MØ differentiation, with maximal AGCAcctttcgggtcgagggctagcTGCTGAcgTCAGCAcctttcgggtcgaa-39,in which capital letters indicate the MAFB binding sites. levels observed 24–48 h after the initial M-CSF treatment (Fig. 1C, To assess the protein stability of MAFB MCTO-causing mutants, lower panel). Interestingly, maximal MAFB protein levels preceded HEK293T cells were transfected with 1 mg pMAFBwt, pMAFB161T, the maximal level of expression of paradigmatic M-MØ–specific pMAFB188G, or pMAFB211T plasmids using SuperFect (Qiagen). After 24 h, genes like HTR2B, F13A1, OLFML2B, IL10,andCD163L1,which cells were treated with 10 mg/ml cycloheximide, and cell lysates were generated at the indicated times using radioimmunoprecipitation assay buffer. were expressed at high levels 72 h after addition of M-CSF (Supplemental Fig. 1A). Therefore, the presence of high levels of Microarray analysis MAFB precedes the maximal expression of M-MØ–specific genes. Global gene expression analysis was performed on RNA obtained from High levels of MAFB mRNA were also detected in IL-34–polarized siControl-transfected M-MØ, siMAFB-transfected M-MØ, and M-MØ macrophages (Fig. 1D), and MAFB levels were upregulated in generated from either MCTO#1 or two healthy individuals. In the case of monocytes exposed to M-CSF–containing tumor cell-conditioned M-MØ from MCTO#1, two RNA aliquots were analyzed in parallel. RNA media (Fig. 1E). Therefore, M-CSF–, IL-34–, and M-CSF–con- was isolated using the RNeasy Mini kit (Qiagen, Germantown, MD) and analyzed with a whole human genome microarray from Agilent Technol- taining ascitic fluids increase MAFB expression in human mono- ogies (Palo Alto, CA). Only probes with signal values .60% quantile in at cytes. Immunohistochemistry analysis revealed that MAFB ex- least one condition were considered for the differential expression and pression is absent in CD163+ placenta macrophages, but readily statistical analysis. Statistical analysis for differential gene expression was detected in vivo in tissue-resident CD163+ macrophages from colon carried out using empirical Bayes moderated t test implemented in the limma package (http://www.bioconductor.org). For the gene expression (submucosa and muscle) and skin, as well as in tumor-associated Downloaded from + analysis of the siMAFB-transfected M-MØ, a paired t test was used. The CD163 macrophages in melanoma samples (Fig. 1F), thus con- p values were further adjusted for multiple hypotheses tested using the firming the expression of MAFB in certain tissue-resident macro- Benjamini-Hochberg method to control the false discovery rate (45). All of phages and in macrophages with anti-inflammatory potential under the above procedures were coded in R (http://www.r-project.org). Micro- pathological conditions. These results indicate that MAFB expres- array data were deposited in the Gene Expression Omnibus (http://www. ncbi.nlm.nih.gov/geo/) under accession number GSE84622. The differen- sion characterizes human macrophages with homeostatic/anti- tially expressed genes in both microarray experiments were analyzed for inflammatory functions both in vitro and in vivo. http://www.jimmunol.org/ annotated gene set enrichment using the online tool ENRICHR (http:// amp.pharm.mssm.edu/Enrichr/) (46, 47). Enrichment terms were consid- MAFB controls the acquisition of the anti-inflammatory ered significant when they had a Benjamini-Hochberg–adjusted p value transcriptional profile of M-MØ ,0.05. For gene set enrichment analysis (GSEA) (48), the gene sets contained in the Molecular Signature databases available at the GSEA Web To assess the role of MAFB in human macrophage anti-inflammatory site and the previously defined proinflammatory gene set and anti- polarization, MAFB expression was silenced using MAFB-specific inflammatory gene set (49), which contain the top and bottom 150 probes small interfering RNA (siMAFB, Fig. 2A), and the effects of from the GM-MØ versus M-MØ limma analysis of the microarray data in MAFB silencing on the M-MØ–specific transcriptome were deter- GSE68061 (ranked on the basis of the value of the t statistic), were used. mined. MAFB knockdown resulted in diminished expression of Fluorescence confocal microscopy most M-MØ–specific genes (Fig. 2B), including HTR2B, IGF1, by guest on September 25, 2021 Human biopsied samples were obtained from patients undergoing surgical STAB1,andCD163L1, whose expression is associated with mac- treatment and following the Medical Ethics Committee procedures of rophage anti-inflammatory functions (9, 10, 49–51). A similar result Hospital General Universitario Gregorio Maranõń, Madrid, and Hospital was observed after Mafb knockdown in M-CSF–dependent bone Clinic, Barcelona. Frozen samples were cryosectioned (5 mm), fixed with marrow–derived mouse macrophages (Supplemental Fig. 1B, 1C). acetone, blocked with human Igs, and simultaneously stained with different primary Abs at 1–5 mg/ml. Imaging was performed with the glycerol Therefore, MAFB controls the expression of genes associated with ACS APO 320 NA 0.60 immersion objective of a confocal fluorescence anti-inflammatory macrophage polarization. To identify the whole microscope (SPE; Leica Mycrosystems). For quantification of in vivo range of MAFB-dependent genes in human macrophages, we next protein expression, mean fluorescence intensities of the proteins of inter- determined the transcriptome of MAFB-deficient M-MØ (Fig. 3A). est (MAFB, CD163L1, HTR2B, and CXCL12) were quantified at seg- + Defective MAFB expression significantly (adjusted p value ,0.05) mented CD163 macrophages using the FIJI software (National Institutes of Health) and background subtracted data from at least three different fields altered the expression of 284 probes (247 annotated genes) in displayed as scatter plots (GraphPad). The Abs used were the following: M-MØ (Supplemental Table I). Specifically, MAFB knockdown led goat polyclonal anti-MAFB (sc-10022; Santa Cruz), mouse monoclonal anti- to downregulation of 147 genes and upregulation of 100 genes in CD163 (K0147-4; MBL), rabbit polyclonal anti-CD163L1 (HPA015663; M-MØ (Fig. 3B, Supplemental Table I). These results were later Sigma-Aldrich), rabbit polyclonal anti-CXCL12 (P87B; PeproTech), and rabbit polyclonal anti-HTR2B (sc-25647; Santa Cruz). confirmed by quantitative PCR, which showed that MAFB silencing reduces the expression of M-MØ–specific genes (e.g., IL10, CCL2, Statistical analysis EMR1, SLC40A1) and also increases the expression of genes asso- For comparison of means, and unless otherwise indicated, statistical ciated with GM-CSF–driven proinflammatory polarization (e.g., significance of the generated data was evaluated using the Student t test. In CLEC5A) (50) (Fig. 3C, 3D). Gene Ontology analysis of the genes all cases, a p value , 0.05 was considered statistically significant. downregulated upon MAFB knockdown revealed a significant enrichment of genes upregulated by IL-10 (adjusted p =7.43 10223) Results and IFN-b (adjusted p =9.83 10232), but downregulated upon MAFB expression in human macrophages under homeostatic MYB overexpression (adjusted p =5.83 10214), thus supporting and anti-inflammatory conditions the link between MAFB and the expression of anti-inflammatory Transcriptional analysis of M-MØ and GM-MØ (GSE27792) (14) genes, and in line with previously reported MAFB functions and revealed the preferential expression of the MAFB gene in mac- interactions (Supplemental Fig. 2A, 2B). Regarding the genes whose rophages with anti-inflammatory potential, as M-MØ express 19 expression increases after MAFB knockdown, ENRICHR revealed times higher levels of MAFB RNA than GM-MØ (p , 0.0005) enrichment of genes with functional MYB binding sites (adjusted (Fig. 1A). In agreement with the transcriptional data, M-MØ p =5.13 10216) as well as vitamin D3– or retinoic acid–responsive exhibited much higher levels of MAFB protein than GM-MØ genes (adjusted p =3.43 1023 and 5.4 3 1023, respectively) (Fig. 1B). During M-CSF–driven differentiation, MAFB mRNA (Supplemental Fig. 2A, 2B), two compounds that control osteoclast 4 MAFB-DIRECTED MACROPHAGE ANTI-INFLAMMATORY POLARIZATION Downloaded from http://www.jimmunol.org/

FIGURE 1. MAFB expression in homeostatic and anti-inflammatory macrophages in vitro and in vivo. (A) MAFB mRNA expression as determined by quantitative RT-PCR. Results are indicated as the expression in M-MØ relative to GM-MØ (n =6,p = 0.0055). (B) MAFB protein expression as determined by Western blot. A representative experiment of three independent experiments is shown. (C) MAFB mRNA (upper panel) and protein (lower panel) expression in monocytes differentiated in the presence of M-CSF. The upper panel shows mean 6 SD of the mRNA levels at the indicated time points and relative to the levels detected at the 24-h time point (n =4,*p , 0.05, ***p , 0.005). The lower panel illustrates the result of one of two independent by guest on September 25, 2021 experiments. (D) MAFB mRNA expression in IL-34 MØ. Shown is a representative experiment run in triplicates by quantitative RT-PCR. Results are indicated as the levels of MAFB mRNA relative to the levels of housekeeping HPRT1, TBP, and RPLP0 genes. (E) MAFB protein in untreated monocytes (lane -) and monocytes exposed for 48 h to ascitic fluids from patients with tumors of distinct origin: bladder (lane 1), colon (lanes 2, 3, and 6), stomach (lane 4), ovary (lane 5), bile duct (lane 7), and thyroid gland (lane 8). (F) MAFB (green) expression in CD163+ (red) macrophages in tissues. DAPI staining is shown in blue. Melanoma #274 refers to a biopsy from a lymph node melanoma metastasis sample. proliferation and differentiation (52). Moreover, MAFB knockdown The M-MØ–specific macrophage transcriptome is altered in downregulated the expression of 69 genes whose mouse orthologous macrophages derived from MCTO monocytes have been identified as MAFB targets by ChIP-sequencing (34) Mutations within the MAFB transcriptional activation domain (Supplemental Fig. 2C, 2D). cause MCTO (Online Mendelian Inheritance in Man #166300), a GSEA using the gene sets that best define the proinflammatory GM- very rare autosomal dominant disorder (35–38). Because MCTO- MØ– and anti-inflammatory M-MØ–specific signatures (49) revealed causing MAFB mutations map to the GSK3 phosphorylation sites that MAFB knockdown significantly downregulates the expression of (35–38) that determine large MAF protein stability (53, 54), we the genes within the M-MØ–specific gene set while increasing the undertook the characterization of MCTO monocyte-derived expression of the GM-MØ–specific gene set (Fig. 3E). In fact, 19.4% macrophages as a means to address the consequences of an en- of the genes within the M-MØ–specific gene set (33 of 170) were hanced expression of MAFB within a pathological context. Spe- downregulated upon MAFB knockdown, whereas no gene within the cifically, we derived macrophages from a case of MCTO GM-MØ–specific gene set was downregulated after MAFB silencing previously characterized from the clinical point of view (harboring (Fig. 3F). These results were validated at the protein level, as MAFB a heterozygous 161C . T mutation [Ser54Leu], and hereafter knockdown reduced the basal production of both CCL2 (Fig. 3G) termed MCTO#1) (Fig. 4A) (55) and two previously described and IL-10 (Fig. 3H) from M-MØ. Altogether, the above results patients (harboring a heterozygous 188C . Tmutation[Pro63Leu], demonstrate that MAFB is a critical factor for the acquisition/ both referred to as MCTO#2) (36, 38) (Fig. 4B). MAFB protein maintenance of the anti-inflammatory transcriptome of human mac- levels were higher in both MCTO#1 monocytes (Fig. 4C) and rophages, as it positively controls the expression of M-MØ–specific MCTO#1 monocyte-derived macrophages (Fig. 4D), suggesting genes and impairs the expression of genes associated with the GM- that MCTO MAFB mutations enhance protein stability. In line CSF–directed proinflammatory polarization. Such a conclusion is with this hypothesis, MAFB mutants containing the mutations further supported by the significant enrichment of the hallmark- found in MCTO#1 and two additional patients (188C . G, [Pro63 inflammatory response, hallmark IFN-g response, and hallmark Arg] and 211C . T, [Pro71Ser], respectively) (36, 38) exhibited a IFN-a response GSEA gene sets within the genes upregulated after considerably extended t1/2 after transfection in HEK293T cells siMAFB transfection (false discovery rate q value = 0). (Fig. 4E). Besides, and in agreement with previous results on The Journal of Immunology 5

inhibitory actions of MAFB (20). Further supporting the presence of elevated MAFB levels in MCTO M-MØ, the set of genes with lower expression in MCTO#1 M-MØ was enriched in genes positively regulated by RANKL (adjusted p = 1.9 3 10213), a major driver of osteoclast generation (20) (Fig. 5B). Interestingly, the set of genes with higher expression in MCTO M-MØ was enriched in genes downregulated in systemic juvenile idiopathic arthritis (adjusted p = 2.5 3 1022), a pathology that mimics MCTO (56). Further validation of microarray data through quantitative RT-PCR on independent samples of MCTO#1 and MCTO#2 M-MØ allowed the identification of 14 genes whose expression is significantly different between control and MCTO- derived M-MØ (Fig. 5C). The global transcriptional effect of MCTO-causing MAFB mutations was also assessed by GSEA using the gene sets that best define GM-MØ– and M-MØ–specific signatures. MCTO#1 M-MØ exhibited a significant reduction in the expression of proinflammatory (GM-MØ–specific) genes as well as a significant in-

crease in the expression of anti-inflammatory (M-MØ–specific) Downloaded from genes (Fig. 5D), with genes such as IL-10, HTR2B, and CD209 critically contributing to these significant changes. In fact, the expression of these and other M-MØ–specific genes was also significantly enhanced in M-MØ from MCTO#1 and MCTO#2 monocytes (Fig. 5E). Conversely, and in line with the GSEA re-

sults, the expression of genes that best define proinflammatory http://www.jimmunol.org/ GM-MØ polarization (TNF, CCR2, INHBA, EGLN3) (9, 12, 14) appeared reduced, whereas IL10 was enhanced, in MCTO#1 GM- MØ (Fig. 5F). Moreover, in agreement with the inhibitory effect of the MCTO#1 MAFB mutation on the expression of RANKL- regulated genes (Fig. 5B), MCTO#1 monocytes exposed to M-CSF and RANKL exhibited an impaired ability to differentiate into multinucleated osteoclasts, as shown by TRAP5 staining (Fig. 5H), expression of osteoclast differentiation gene markers (CTSK, OCSTAMP, DCSTAMP, and the TRAP-encoding gene by guest on September 25, 2021 ACP5) (Fig. 5I), and collagen-degradation ability (Fig. 5J). Al- together, these results indicate that macrophages from MCTO patients exhibit an enhanced anti-inflammatory profile and that heterozygous MCTO-causing MAFB mutations drive macrophages toward the upregulation of genes associated with anti-inflammatory macrophage polarization, thus reinforcing the contribution of MAFB to the acquisition and maintenance of the anti-inflammatory FIGURE 2. The expression of M-MØ–specific genes is MAFB depen- gene signature in human macrophages (Fig. 5G). A dent. ( ) MAFB Western blot in siControl or siMAFB M-MØ for the in- MAFB also influences the LPS responsiveness of human dicated times. (B) Quantitative RT-PCR of the indicated genes after macrophages transfection for 24 h. Results are indicated as the mRNA levels of each gene in siMAFB M-MØ relative to siControl cells (n =4,*p , 0.05, **p , Next, the involvement of MAFB in the LPS-induced activation of 0.005, ***p , 0.0005). M-MØ was assessed through the analysis of the M-MØ–specific transcriptional response to LPS, which differs from that of MAF and MAFA (17, 54), MCTO MAFB mutants exhibited re- GM-MØ and includes CCL19, ARNT2, MAOA,andPDGFA up- duced transactivation activity (Fig. 4F). Therefore, MCTO regulation (V.D. Cuevas and A.L. Corbı´, unpublished observations). monocytes constitute a useful, and pathologically relevant, system MAFB knockdown altered the LPS responsiveness of M-MØ, as it to test the transcriptional consequences of an enhanced MAFB reduced the LPS-dependent upregulation of CCL19, ARNT2, expression in human macrophages. Accordingly, M-MØ and PDGFA,andMAOA (Fig. 6A) and significantly diminished the GM-MØ were generated from MCTO#1 monocytes, and the LPS-induced expression of IL10 (Fig. 6B). By contrast, the LPS- transcriptomic profile of MCTO#1 M-MØ was determined. mediated increase of these genes was higher in MCTO#1 M-MØ Comparison of MCTO#1 and control M-MØ gene signatures than in control M-MØ (Fig. 6C), again illustrating the opposite revealed the differential expression (adjusted p , 0.19, corre- consequences of MAFB silencing and MCTO-causing MAFB sponding to unadjusted p , 0.005) of 321 annotated genes, with mutations. Therefore, MAFB also contributes to the acquisition of 231 genes downregulated and 90 genes upregulated in MCTO#1 the gene expression profile of LPS-activated M-MØ. M-MØ (Fig. 5A, Supplemental Table II). Analysis of the genes with lower expression in MCTO#1 M-MØ revealed a significant Coexpression of MAFB and MAFB-regulated genes in human enrichment of genes containing functional MYB (adjusted p =4.53 macrophages in vivo 1029), MITF (adjusted p = 7.5 3 10213), and JUN binding sites To gain additional evidence for the physiological significance of the (adjusted p = 6.5 3 1028) (Fig. 5B), what fits with the known MAFB-dependent transcriptome in human macrophages, we next 6 MAFB-DIRECTED MACROPHAGE ANTI-INFLAMMATORY POLARIZATION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. MAFB controls the global anti-inflammatory transcriptional signature of M-MØ. (A) Experimental design. (B) Number of genes whose expression is higher (UP) or lower (DOWN) in siMAFB M-MØ compared with siControl M-MØ (adjusted p , 0.05). (C) Validation of microarray results by quantitative RT-PCR in independent samples of siMAFB and siControl M-MØ (n =4,*p , 0.05, **p , 0.005, ***p , 0.0005). Data are indicated as mean 6 SD of mRNA levels in siMAFB M-MØ relative to siControl M-MØ. (D) CLEC5A Western analysis in M-MØ treated as indicated. Shown is one experiment of three performed on independent samples. (E) GSEA analysis on the t statistic–ranked list of genes obtained from the siMAFB–M-MØ versus siControl–M-MØ limma analysis, using the proinflammatory (top) and anti-inflammatory (bottom) gene sets previously defined (49). Red and blue arrows indicate the location in the ranked list of those genes whose expression is higher or lower, respectively, in siMAFB M-MØ. (F) Venn diagram analysis of the genes with significantly lower expression in siMAFB M-MØ compared with the GM-MØ– and M-MØ–specific gene signatures (genes that are differentially expressed between GM-MØ and M-MØ by .8-fold, GSE68061). (G and H) ELISA of CCL2 and IL-10 protein levels in culture media from M-MØ treated as indicated (n = 4 for CCL2, n = 7 for IL-10, *p , 0.05). determined whether a correlation exists between the expression of CD163L1, and CXCL12 (Fig. 7B). Therefore, MAFB is a primary MAFB and proteins encoded by MAFB-dependent genes in human driver of the anti-inflammatory gene profile of human M-CSF– macrophages in vivo. To that end, we performed immunofluo- dependent macrophages, and the presence of MAFB-regulated rescence on melanoma, in which accumulation of tumor-promoting genes provides useful markers for the in vivo identification of and immunosuppressive macrophages associates with a poor anti-inflammatory macrophages. clinical outcome (57), as well as in other tissues in which macrophages display anti-inflammatory/homeostatic functions (58). Discussion Coexpression of MAFB and CD163L1 was observed in CD163+ Whereas most studies on the functional heterogeneity of macro- macrophages from colon and dermis, where MAFB expression phages have focused on mouse tissue-resident macrophages (32, had been detected (Fig. 7A). Moreover, CD163+ tumor-associated 59, 60), determining the factors that underlie human macrophage macrophages from melanoma samples coexpressed MAFB and heterogeneity under homeostatic and pathological conditions is the proteins encoded by MAFB-dependent genes such as HTR2B, still required for translational approaches to be undertaken (61). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Mutations responsible for MCTO affect the stability and transcriptional activity of MAFB. (A) Nucleotide sequence of the mutation hotspot in the MAFB gene in MCTO. Sequencing of a healthy donor (top) and the MCTO#1 patient (down) is shown. Red arrow marks the heterozygous mutation (161C . T) in MCTO#1. (B) Representation of the MAFB protein. Red dots indicate amino acids mutated in MCTO. Black arrows indicate the amino acids mutated in the MCTO MAFB expression vectors and the amino acids affected in the MCTO#1 and MCTO#2 patients. (C and D) MAFB mRNA and protein expression in monocytes (C) and M-MØ (D) from MCTO#1 and three healthy controls, as determined by quantitative RT-PCR and Western blot. Relative mRNA expression indicates the MAFB mRNA levels relative to the TBP mRNA levels in each macrophage sample. (E) MAFB protein levels were determined by Western blot in HEK293T transfected with the indicated expression vectors after 1–5 h of cycloheximide (CHX) treatment. (F) MAFB- dependent MARE-speciﬁc transcriptional activity in HEK293T cells transfected with the indicated MAFB expression vectors. The MARE-speciﬁc luciferase activity was determined by expressing the luciferase activity produced by each MAFB construct on the 33MARE-Luc reporter relative to the luciferase activity produced by the same expression vector in the presence of the promoter-less TATA-pXP2 plasmid. In all cases, the MARE sp. act. of each MCTO- causing MAFB mutant is referred to the activity produced by pMAFBwt (arbitrarily set to 1) (n =3,*p , 0.05; for pMAFB161T, p = 0.12).

We now report the identification of the MAFB-dependent tran- group, it is worth mentioning IL-10, the paradigmatic anti- scriptome in IL-10–producing anti-inflammatory macrophages. inflammatory cytokine (62); CCL2, which impairs the produc- The analysis of the MAFB-dependent gene set in control and tion of inflammatory cytokines (9, 63, 64) and is controlled by MCTO macrophages has led us to demonstrate that MAFB con- MAFB in mouse myeloid cells (65); and HTR2B, whose ligation trols the acquisition of the transcriptional signature and effector promotes an anti-inflammatory macrophage differentiation (10). functions that characterize anti-inflammatory human macrophages The MAFB dependency of CD163L1 expression is relevant be- in vivo and in vitro. In line with the preferential expression of cause CD163L1 marks anti-inflammatory IL-10–producing tissue- MAFB in IL-10–producing macrophages, the MAFB tran- resident macrophages in healthy secondary lymphoid organs, liver, scriptome of human M-MØ includes genes with anti-inflammatory and gut, and also characterizes melanoma-associated macrophages activity and genes whose expression associates with the acquisi- (50). MAFB also positively regulates the expression of the EMR1 tion of macrophage anti-inflammatory activity. Among the first gene, whose mouse ortologue (the F4/80 receptor-encoding Emr1 8 MAFB-DIRECTED MACROPHAGE ANTI-INFLAMMATORY POLARIZATION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. MCTO MAFB mutations significantly affect the M-MØ transcriptome. (A) Number of genes whose expression is higher (MCTO1 . control) or lower (MCTO1 , control) in MCTO#1 M-MØ than in control M-MØ (p , 0.005; adjusted p , 0.192; log2 fold MCTO#1/control . 0.7), as determined by microarray analysis. (B) Gene ontology analysis of genes downregulated in MCTO#1, using the ENRICHR tool. The results extracted from the indicated databases are shown (combined score = log(p value) 3 z-score). (C) Validation of microarray results by quantitative RT-PCR in M-MØ from MCTO#1 and MCTO#2 patients. Results are indicated as mean 6 SD of mRNA levels in MCTO M-MØ relative to control M-MØ (n =3,*p , 0.05, **p , 0.005, ***p , 0.0005). (D) GSEA analysis of the t statistic–ranked list of genes obtained from the MCTO#1 M-MØ versus control M-MØ limma analysis, using the proinflammatory (top) and anti-inflammatory (bottom) gene sets previously defined. (E) Expression of the indicated M-MØ–specific genes in M-MØ derived from MCTO#1 and MCTO#2 monocytes relative to control M-MØ, as determined by quantitative RT-PCR. Shown are the mean 6 SD of three independent experiments (n =3,*p , 0.05). (F) Expression of the indicated GM-MØ–specific genes in MCTO#1 GM-MØ relative to control GM-MØ, as determined by quantitative (Figure legend continues) The Journal of Immunology 9

FIGURE 6. MAFB impacts the LPS response of M-MØ. (A) mRNA expression levels in LPS-treated (10 ng/ml, 4 h) siMAFB M-MØ, as determined by quantitative RT-PCR using custom-made microﬂuidic gene cards. Results are indicated as the mRNA levels of each gene in LPS-treated siMAFB M-MØ relative to LPS-treated siControl M-MØ (n =3,*p , 0.05, **p , 0.005, ***p , 0.0005). (B) IL10 mRNA expression in LPS-treated siMAFB M-MØ as determined by quantitative RT-PCR. Results are shown relative to the IL10 mRNA level detected in siControl-transfected LPS- treated M-MØ (arbitrarily set to 1) (n =5,*p , 0.05). (C) mRNA expression levels in LPS-treated MCTO#1 Downloaded from M-MØ, as determined by quantitative RT-PCR. Results are indicated as the change in the expression of each gene in LPS-treated MCTO#1 M-MØ relative to the change of the same gene in LPS-treated control M-MØ. http://www.jimmunol.org/

gene) is required for the induction of T regulatory cells in peripheral agrees with the ability of murine MafB to antagonize the phenotypic tolerance (66) and whose expression depends on the Mafb- alteration of microglia induced by GM-CSF (68). Whether the by guest on September 25, 2021 heterodimerizing factor c-Maf (27, 67). Conversely, MAFB nega- MAFB/GM-CSF antagonism operates in all types of macrophages is tively regulates genes associated with proinflammatory polarization still unknown. However, we have observed that MCTO mutations like CLEC5A, which codes for a lectin preferentially expressed in limit the acquisition of GM-CSF–inducible genes in GM-MØ, a TNF-a–producing macrophages in physiological and pathological finding that might be relevant in the case of macrophages whose settings (50). Gene ontology analysis further supports the link be- development is critically dependent on GM-CSF (lung alveolar tween MAFB expression and macrophage anti-inflammatory po- macrophages) (69). Although no lung-associated pathology has been larization, as MAFB knockdown leads to a global downregulation to date reported in MCTO patients, it is worth noting that a significant of genes preferentially expressed in anti-inflammatory macrophages number of genes aberrantly expressed in pulmonary diseases (pul- (anti-inflammatory gene set) and to a significant upregulation of the monary sarcoidosis, asthma, and tuberculosis) exhibit an altered ex- proinflammatory gene set. Conversely, and in agreement with the pression in MCTO M-MØ, a finding that might be related to the fact higher t1/2 of MCTO-causing MAFB mutants, the MCTO M-MØ that mature alveolar macrophages, identified as intermediate positive gene signature revealed a global upregulation of the anti-inflammatory for the Emr1-encoded F4/80, are reduced in the bronchoalveolar la- gene set. All of these findings indicate that MAFB is a critical vage of mice expressing a dominant-negative MafB in macrophages determinant for the acquisition of the anti-inflammatory profile of (70). Given these antecedents, the analysis of further MCTO patients, human macrophages. it is certainly worthy as a means to get a deeper knowledge of the Our results on in vitro monocyte-derived macrophages clearly molecular mechanisms operating in this and other related osteolytic point to a link between M-CSF–driven differentiation and MAFB syndromes as well as to more clearly delineate the role of MAFB in expression: M-CSF–driven macrophage differentiation results in the acquisition of the human macrophage anti-inflammatory profile. high levels of MAFB, whereas the presence of GM-CSF lowers Although we have used MCTO-derived macrophages as a tool to the basal levels of MAFB found in human monocytes. This result unravel the role of MAFB in human macrophage polarization, the

RT-PCR. Results from a single experiment are shown. (G) Schematic representation of the correlation between MAFB protein levels and the expression of GM-MØ– and M-MØ–specific genes in control and MCTO macrophages. (H) MCTO#1 and control osteoclasts, as determined by phase-contrast microscopy, on cells stained for TRAP. Two independent experiments were done on monocytes from the MCTO#1 patient, and one of them is shown. Original magnification 340. (I) Relative mRNA expression of the indicated osteoclast-associated genes along the M-CSF + RANKL–induced osteoclast differentiation of monocytes from either two independent healthy controls (control) or the MCTO#1 patient. Shown are the mean (6SD) of the mRNA level of each gene at the indicated time points and relative to the mRNA level of the corresponding gene in M-CSF + RANKL–treated MCTO#1 monocytes at day 9. Results from a single experiment are shown. (J) Collagen-degradation activity of monocytes (from two healthy controls and the MCTO#1 patient) subjected to the M-CSF + RANKL–induced osteoclast differentiation procedure. Two experiments were performed with similar results, and one of them is shown. Empty and filled bars indicate monocytes differentiated in the presence of M-CSF alone or M-CSF + RANKL, respectively. 10 MAFB-DIRECTED MACROPHAGE ANTI-INFLAMMATORY POLARIZATION

FIGURE 7. Human tissue-resident macrophages coexpress MAFB and MAFB-dependent genes. (A)Immu- noﬂuorescence of MAFB (green) and CD163L1 (L1, red) in tissues, as determined by confocal microscopy. Right panel: correlation between MAFB and CD163L1 expression in CD163+ macrophages. Red and blue + + mark tissues with CD163 MAFB Downloaded from or CD163+MAFB2 macrophages, respectively. (B) Expression of the indicated proteins in samples of primary melanoma (PMel #276). Yellow arrows mark CD163+ MAFB+ CD163L1+ HTR2B+ CXCL12+ macrophages, + whereas white arrows mark CD163 http://www.jimmunol.org/ MAFB2 CD163L12 HTR2B2 CXCL122 macrophages. Bottom panels show the positive correlation between MAFB expression and the expression of CD163L1, HTR2B, and CXCL12 in CD163+ tumor-associated macrophages from primary melanoma (Pmel #276), lymph node metastatic melanoma (MMel #274), and skin metastatic melanoma (MMel #255) by guest on September 25, 2021 samples.

information we have gathered from MCTO monocytes/macrophages Disclosures might shed light on the pathogenic mechanisms operating in MCTO The authors have no financial conflicts of interest. and other diseases in which osteolysis is a defining pathological feature. In the case of MCTO, we have identified a number of genes whose expression is altered not only in MCTO-derived macrophages, but also References 1. Greter, M., I. Lelios, P. Pelczar, G. Hoeffel, J. Price, M. Leboeuf, T. M. Kundig,€ in peripheral blood monocytes from the three analyzed MCTO patients K. Frei, F. Ginhoux, M. Merad, and B. Becher. 2012. Stroma-derived interleukin- (data not shown). Although analysis of additional patients is still re- 34 controls the development and maintenance of Langerhans cells and the quired to propose their use as potential MCTO diagnostic markers, a maintenance of microglia. Immunity 37: 1050–1060. 2. Wang, Y., K. J. Szretter, W. Vermi, S. Gilfillan, C. Rossini, M. Cella, disease commonly misdiagnosed (56), our results suggest that MCTO- A. D. Barrow, M. S. Diamond, and M. Colonna. 2012. IL-34 is a tissue-restricted causing mutations affect monocyte differentiation into tissue-resident ligand of CSF1R required for the development of Langerhans cells and micro- macrophages, as exemplified by their inability to differentiate into glia. Nat. Immunol. 13: 753–760. 3. Hashimoto, D., A. Chow, C. Noizat, P. Teo, M. B. Beasley, M. Leboeuf, functional osteoclasts in vitro. Whether the profound anti-inflammatory C. D. Becker, P. See, J. Price, D. Lucas, et al. 2013. Tissue-resident macrophages skewing of MCTO-derived M-MØ is responsible for their inability to self-maintain locally throughout adult life with minimal contribution from cir- generate osteoclasts in vitro is an issue that we are currently pursuing. culating monocytes. Immunity 38: 792–804. 4. Epelman, S., K. J. Lavine, and G. J. Randolph. 2014. Origin and functions of tissue macrophages. Immunity 41: 21–35. Acknowledgments 5. Mosser, D. M., and J. P. Edwards. 2008. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8: 958–969. We gratefully acknowledge Dr. Lizbeth Estrada-Capetillo and Dr. Amaya 6. Hamilton, J. A. 2008. Colony-stimulating factors in inflammation and autoim- Puig-Kro¨ger for kindly providing useful reagents and samples. munity. Nat. Rev. Immunol. 8: 533–544. The Journal of Immunology 11

7. Verreck, F. A., T. de Boer, D. M. Langenberg, M. A. Hoeve, M. Kramer, 30. Dong, L., S. Pietsch, Z. Tan, B. Perner, R. Sierig, D. Kruspe, M. Groth, E. Vaisberg, R. Kastelein, A. Kolk, R. de Waal-Malefyt, and T. H. Ottenhoff. R. Witzgall, H. J. Gro¨ne, M. Platzer, and C. Englert. 2015. Integration of cis- 2004. Human IL-23-producing type 1 macrophages promote but IL-10- tromic and transcriptomic analyses identifies Nphs2, Mafb, and Magi2 as Wilms’ producing type 2 macrophages subvert immunity to (myco)bacteria. Proc. tumor 1 target genes in podocyte differentiation and maintenance. J. Am. Soc. Natl. Acad. Sci. USA 101: 4560–4565. Nephrol. 26: 2118–2128. 8. Puig-Kro¨ger, A., E. Sierra-Filardi, A. Domıńguez-Soto, R. Samaniego, 31. Kim, H., and B. Seed. 2010. The transcription factor MafB antagonizes antiviral M. T. Corcuera, F. Go´mez-Aguado, M. Ratnam, P. Sańchez-Mateos, and responses by blocking recruitment of coactivators to the transcription factor A. L. Corbı´. 2009. Folate receptor beta is expressed by tumor-associated mac- IRF3. Nat. Immunol. 11: 743–750. rophages and constitutes a marker for M2 anti-inflammatory/regulatory macro- 32. Lavin, Y., D. Winter, R. Blecher-Gonen, E. David, H. Keren-Shaul, M. Merad, phages. Cancer Res. 69: 9395–9403. S. Jung, and I. Amit. 2014. Tissue-resident macrophage enhancer landscapes are 9. Sierra-Filardi, E., C. Nieto, A. Domıńguez-Soto, R. Barroso, P. Sańchez-Mateos, shaped by the local microenvironment. Cell 159: 1312–1326. A. Puig-Kroger, M. Lo´pez-Bravo, J. Joven, C. Ardavıń, J. L. Rodrı´guez-Fernańdez, 33. Sarrazin, S., N. Mossadegh-Keller, T. Fukao, A. Aziz, F. Mourcin, L. Vanhille, et al. 2014. CCL2 shapes macrophage polarization by GM-CSF and M-CSF: L. Kelly Modis, P. Kastner, S. Chan, E. Duprez, et al. 2009. MafB restricts identification of CCL2/CCR2-dependent gene expression profile. J. Immunol. M-CSF-dependent myeloid commitment divisions of hematopoietic stem cells. 192: 3858–3867. Cell 138: 300–313. 10. de las Casas-Engel, M., A. Domıńguez-Soto, E. Sierra-Filardi, R. Bragado, 34. Soucie, E. L., Z. Weng, L. Geirsdottir, K. Molawi, J. Maurizio, R. Fenouil, C. Nieto, A. Puig-Kroger, R. Samaniego, M. Loza, M. T. Corcuera, F. Go´mez- N. Mossadegh-Keller, G. Gimenez, L. VanHille, M. Beniazza, et al. 2016. Aguado, et al. 2013. Serotonin skews human macrophage polarization through Lineage-specific enhancers activate self-renewal genes in macrophages and HTR2B and HTR7. J. Immunol. 190: 2301–2310. embryonic stem cells. Science 351: aad5510. 11. Domıńguez-Soto, A., E. Sierra-Filardi, A. Puig-Kro¨ger, B. Pe´rez-Maceda, 35. Dworschak, G. C., M. Draaken, A. Hilger, M. Born, H. Reutter, and M. Ludwig. F. Go´mez-Aguado, M. T. Corcuera, P. Sańchez-Mateos, and A. L. Corbı´. 2011. 2013. An incompletely penetrant novel MAFB (p.Ser56Phe) variant in autoso- Dendritic cell-specific ICAM-3-grabbing nonintegrin expression on M2-polarized mal dominant multicentric carpotarsal osteolysis syndrome. Int. J. Mol. Med. 32: and tumor-associated macrophages is macrophage-CSF dependent and enhanced 174–178. by tumor-derived IL-6 and IL-10. J. Immunol. 186: 2192–2200. 36. Mehawej, C., J. B. Courcet, G. Baujat, R. Mouy, M. Ge´rard, I. Landru, 12. Escribese, M. M., E. Sierra-Filardi, C. Nieto, R. Samaniego, C. Sańchez-Torres, M. Gosselin, P. Koehrer, C. Mousson, S. Breton, et al. 2013. The identification of T. Matsuyama, E. Calderon-Go´mez, M. A. Vega, A. Salas, P. Sańchez-Mateos, MAFB mutations in eight patients with multicentric carpo-tarsal osteolysis Downloaded from and A. L. Corbı´. 2012. The prolyl hydroxylase PHD3 identifies proinflammatory supports genetic homogeneity but clinical variability. Am. J. Med. Genet. A macrophages and its expression is regulated by activin A. J. Immunol. 189: 161A: 3023–3029. 1946–1954. 37. Mumm, S., M. Huskey, S. Duan, D. Wenkert, K. L. Madson, G. S. Gottesman, 13. Izquierdo, E., V. D. Cuevas, S. Fernańdez-Arroyo, M. Riera-Borrull, E. Orta- A. R. Nenninger, R. M. Laxer, W. H. McAlister, and M. P. Whyte. 2014. Mul- Zavalza, J. Joven, E. Rial, A. L. Corbi, and M. M. Escribese. 2015. Reshaping of ticentric carpotarsal osteolysis syndrome is caused by only a few domain-specific human macrophage polarization through modulation of glucose catabolic path- mutations in MAFB, a negative regulator of RANKL-induced osteoclasto- ways. J. Immunol. 195: 2442–2451. genesis. Am. J. Med. Genet. A 164A: 2287–2293.

14. Sierra-Filardi, E., A. Puig-Kro¨ger, F. J. Blanco, C. Nieto, R. Bragado, 38. Zankl, A., E. L. Duncan, P. J. Leo, G. R. Clark, E. A. Glazov, M. C. Addor, http://www.jimmunol.org/ M. I. Palomero, C. Bernabeú, M. A. Vega, and A. L. Corbı´. 2011. Activin A skews T. Herlin, C. A. Kim, B. P. Leheup, J. McGill, et al. 2012. Multicentric carpo- macrophage polarization by promoting a proinflammatory phenotype and inhibiting tarsal osteolysis is caused by mutations clustering in the amino-terminal tran- the acquisition of anti-inflammatory macrophage markers. Blood 117: 5092–5101. scriptional activation domain of MAFB. [Published erratum appears in 2014 Am. 15. Lacey, D. C., A. Achuthan, A. J. Fleetwood, H. Dinh, J. Roiniotis, G. M. Scholz, J. Hum. Genet. 94: 643.] Am. J. Hum. Genet. 90: 494–501. M. W. Chang, S. K. Beckman, A. D. Cook, and J. A. Hamilton. 2012. Defining 39. Stralen, E., R. J. Leguit, H. Begthel, L. Michaux, A. Buijs, H. Lemmens, GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro J. M. Scheiff, C. Doyen, P. Pierre, F. Forget, et al. 2009. MafB oncoprotein models. J. Immunol. 188: 5752–5765. detected by immunohistochemistry as a highly sensitive and specific marker for 16. Gordon, S., and F. O. Martinez. 2010. Alternative activation of macrophages: the prognostic unfavorable t(14;20) (q32;q12) in multiple myeloma patients. mechanism and functions. Immunity 32: 593–604. Leukemia 23: 801–803. 17. Eyche`ne, A., N. Rocques, and C. Pouponnot. 2008. A new MAFia in cancer. Nat. 40. Lee, L. C., A. Y. Zhang, A. K. Chong, H. Pham, M. T. Longaker, and J. Chang. Rev. Cancer 8: 683–693. 2006. Expression of a novel gene, MafB, in Dupuytren’s disease. J. Hand Surg. 18. Kataoka, K., K. T. Fujiwara, M. Noda, and M. Nishizawa. 1994. MafB, a new Am. 31: 211–218. by guest on September 25, 2021 Maf family transcription activator that can associate with Maf and Fos but not 41. Park, J. G., M. A. Tischfield, A. A. Nugent, L. Cheng, S. A. Di Gioia, with Jun. Mol. Cell. Biol. 14: 7581–7591. W. M. Chan, G. Maconachie, T. M. Bosley, C. G. Summers, D. G. Hunter, et al. 19. Tillmanns, S., C. Otto, E. Jaffray, C. Du Roure, Y. Bakri, L. Vanhille, S. Sarrazin, 2016. Loss of MAFB function in humans and mice causes Duane syndrome, R. T. Hay, and M. H. Sieweke. 2007. SUMO modification regulates MafB-driven aberrant extraocular muscle innervation, and inner-ear defects. Am. J. Hum. macrophage differentiation by enabling Myb-dependent transcriptional repres- Genet. 98: 1220–1227. sion. Mol. Cell. Biol. 27: 5554–5564. 42. Hamada, M., M. Nakamura, M. T. Tran, T. Moriguchi, C. Hong, T. Ohsumi, 20. Kim, K., J. H. Kim, J. Lee, H. M. Jin, H. Kook, K. K. Kim, S. Y. Lee, and T. T. Dinh, M. Kusakabe, M. Hattori, T. Katsumata, et al. 2014. MafB promotes N. Kim. 2007. MafB negatively regulates RANKL-mediated osteoclast differ- atherosclerosis by inhibiting foam-cell apoptosis. Nat. Commun. 5: 3147. entiation. Blood 109: 3253–3259. 43. Tran, M. T., M. Hamada, M. Nakamura, H. Jeon, R. Kamei, Y. Tsunakawa, 21. Reza, H. M., A. Urano, N. Shimada, and K. Yasuda. 2007. Sequential and K. Kulathunga, Y. Y. Lin, K. Fujisawa, T. Kudo, and S. Takahashi. 2016. MafB combinatorial roles of maf family genes define proper lens development. Mol. deficiency accelerates the development of obesity in mice. FEBS Open Bio 6: Vis. 13: 18–30. 540–547. 22. Dieterich, L. C., S. Klein, A. Mathelier, A. Sliwa-Primorac, Q. Ma, Y. K. Hong, 44. Sadl, V., F. Jin, J. Yu, S. Cui, D. Holmyard, S. Quaggin, G. Barsh, and S. Cordes. J. W. Shin, M. Hamada, M. Lizio, M. Itoh, et al. 2015. DeepCAGE tran- 2002. The mouse Kreisler (Krml1/MafB) segmentation gene is required for scriptomics reveal an important role of the transcription factor MAFB in the differentiation of glomerular visceral epithelial cells. Dev. Biol. 249: 16–29. lymphatic endothelium. Cell Rep. 13: 1493–1504. 45. Hochberg, Y., and Y. Benjamini. 1990. More powerful procedures for multiple 23. Conrad, E., C. Dai, J. Spaeth, M. Guo, H. A. Cyphert, D. Scoville, J. Carroll, significance testing. Stat. Med. 9: 811–818. W. M. Yu, L. V. Goodrich, D. M. Harlan, et al. 2016. The MAFB transcription 46. Chen, E. Y., C. M. Tan, Y. Kou, Q. Duan, Z. Wang, G. V. Meirelles, N. R. Clark, factor impacts islet a-cell function in rodents and represents a unique signature and A. Ma’ayan. 2013. Enrichr: interactive and collaborative HTML5 gene list of primate islet b-cells. Am. J. Physiol. Endocrinol. Metab. 310: E91–E102. enrichment analysis tool. BMC Bioinformatics 14: 128. 24. Hang, Y., and R. Stein. 2011. MafA and MafB activity in pancreatic b cells. 47. Kuleshov, M. V., M. R. Jones, A. D. Rouillard, N. F. Fernandez, Q. Duan, Trends Endocrinol. Metab. 22: 364–373. Z. Wang, S. Koplev, S. L. Jenkins, K. M. Jagodnik, A. Lachmann, et al. 2016. 25. Lopez-Pajares, V., K. Qu, J. Zhang, D. E. Webster, B. C. Barajas, Z. Siprashvili, Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. B. J. Zarnegar, L. D. Boxer, E. J. Rios, S. Tao, et al. 2015. A LncRNA-MAF: Nucleic Acids Res. 44: W90–W97. MAFB transcription factor network regulates epidermal differentiation. Dev. 48. Subramanian, A., P. Tamayo, V. K. Mootha, S. Mukherjee, B. L. Ebert, Cell 32: 693–706. M. A. Gillette, A. Paulovich, S. L. Pomeroy, T. R. Golub, E. S. Lander, and 26. Zhang, Y., and A. C. Ross. 2013. Retinoic acid and the transcription factor MafB J. P. Mesirov. 2005. Gene set enrichment analysis: a knowledge-based approach act together and differentially to regulate aggrecan and matrix metalloproteinase for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA gene expression in neonatal chondrocytes. J. Cell. Biochem. 114: 471–479. 102: 15545–15550. 27. Moriguchi, T., M. Hamada, N. Morito, T. Terunuma, K. Hasegawa, C. Zhang, 49. Gonza´lez-Domıńguez, E´ ., A´ . Domıńguez-Soto, C. Nieto, J. L. Flores-Sevilla, T. Yokomizo, R. Esaki, E. Kuroda, K. Yoh, et al. 2006. MafB is essential for M. Pacheco-Blanco, V. Campos-Penã, M. A. Meraz-Rıós, M. A. Vega, renal development and F4/80 expression in macrophages. Mol. Cell. Biol. 26: A. L. Corbı´, and C. Sańchez-Torres. 2016. Atypical activin A and IL-10 pro- 5715–5727. duction impairs human CD16+ monocyte differentiation into anti-inflammatory 28. Morito, N., K. Yoh, M. Ojima, M. Okamura, M. Nakamura, M. Hamada, macrophages. J. Immunol. 196: 1327–1337. H. Shimohata, T. Moriguchi, K. Yamagata, and S. Takahashi. 2014. Over- 50. Gonza´lez-Domıńguez, E´ ., R. Samaniego, J. L. Flores-Sevilla, S. F. Campos- expression of Mafb in podocytes protects against diabetic nephropathy. J. Am. Campos, G. Go´mez-Campos, A. Salas, V. Campos-Penã, A. L. Corbı´,P.Sańchez- Soc. Nephrol. 25: 2546–2557. Mateos, and C. Sańchez-Torres. 2015. CD163L1 and CLEC5A discriminate 29. Kann, M., S. Ettou, Y. L. Jung, M. O. Lenz, M. E. Taglienti, P. J. Park, subsets of human resident and inflammatory macrophages in vivo. J. Leukoc. Biol. B. Schermer, T. Benzing, and J. A. Kreidberg. 2015. Genome-wide analysis of 98: 453–466. Wilms’ tumor 1-controlled gene expression in podocytes reveals key regulatory 51. Kzhyshkowska, J., G. Workman, M. Cardo´-Vila, W. Arap, R. Pasqualini, mechanisms. J. Am. Soc. Nephrol. 26: 2097–2104. A. Gratchev, L. Krusell, S. Goerdt, and E. H. Sage. 2006. Novel function of 12 MAFB-DIRECTED MACROPHAGE ANTI-INFLAMMATORY POLARIZATION

alternatively activated macrophages: stabilin-1-mediated clearance of SPARC. J. 61. Schultze, J. L. 2016. Reprogramming of macrophages: new opportunities for Immunol. 176: 5825–5832. therapeutic targeting. Curr. Opin. Pharmacol. 26: 10–15. 52. Henning, P., H. H. Conaway, and U. H. Lerner. 2015. Retinoid receptors in bone 62. Gabrysova´, L., A. Howes, M. Saraiva, and A. O’Garra. 2014. The regulation of and their role in bone remodeling. Front. Endocrinol. (Lausanne) 6: 31. IL-10 expression. Curr. Top. Microbiol. Immunol. 380: 157–190. 53. Niceta, M., E. Stellacci, K. W. Gripp, G. Zampino, M. Kousi, M. Anselmi, 63. Suzuki, H. I., M. Arase, H. Matsuyama, Y. L. Choi, T. Ueno, H. Mano, A. Traversa, A. Ciolfi, D. Stabley, A. Bruselles, et al. 2015. Mutations impairing K. Sugimoto, and K. Miyazono. 2011. MCPIP1 ribonuclease antagonizes dicer GSK3-mediated MAF phosphorylation cause cataract, deafness, intellectual and terminates microRNA biogenesis through precursor microRNA degradation. disability, seizures, and a down syndrome-like facies. Am. J. Hum. Genet. 96: Mol. Cell 44: 424–436. 816–825. 64. Matsushita, K., O. Takeuchi, D. M. Standley, Y. Kumagai, T. Kawagoe, 54. Rocques, N., N. Abou Zeid, K. Sii-Felice, L. Lecoin, M. P. Felder-Schmittbuhl, T. Miyake, T. Satoh, H. Kato, T. Tsujimura, H. Nakamura, and S. Akira. 2009. A. Eyche`ne, and C. Pouponnot. 2007. GSK-3-mediated phosphorylation en- Zc3h12a is an RNase essential for controlling immune responses by regulating hances Maf-transforming activity. Mol. Cell 28: 584–597. mRNA decay. Nature 458: 1185–1190. 55. Anta Martıńez, L., A. Vieitez Rey, S. Pombo Expo´sito, and F. Sines Castro. 65. Zhang, Y., Q. Chen, and A. C. Ross. 2012. Retinoic acid and tumor necrosis 2011. Osteolisis Carpo-Tarsal Multiceńtrica. In XX Congreso Hispano Luso de factor-a induced monocytic cell gene expression is regulated in part by induction Cirugıá de la mano. GEYSECO Ed., Valencia, Spain, p. 152. of transcription factor MafB. Exp. Cell Res. 318: 2407–2416. 56. Faber, M. R., R. Verlaak, T. J. Fiselier, B. C. Hamel, M. J. Franssen, and 66. Lin, H. H., D. E. Faunce, M. Stacey, A. Terajewicz, T. Nakamura, J. Zhang- G. P. Gerrits. 2004. Inherited multicentric osteolysis with carpal-tarsal local- Hoover, M. Kerley, M. L. Mucenski, S. Gordon, and J. Stein-Streilein. 2005. The isation mimicking juvenile idiopathic arthritis. Eur. J. Pediatr. 163: 612–618. macrophage F4/80 receptor is required for the induction of antigen-specific ef- 57. Ruffell, B., and L. M. Coussens. 2015. Macrophages and therapeutic resistance ferent regulatory T cells in peripheral tolerance. J. Exp. Med. 201: 1615–1625. in cancer. Cancer Cell 27: 462–472. 67. Nakamura, M., M. Hamada, K. Hasegawa, M. Kusakabe, H. Suzuki, 58. Rivollier, A., J. He, A. Kole, V. Valatas, and B. L. Kelsall. 2012. Inflammation D. R. Greaves, T. Moriguchi, T. Kudo, and S. Takahashi. 2009. c-Maf is essential switches the differentiation program of Ly6Chi monocytes from antiin- for the F4/80 expression in macrophages in vivo. Gene 445: 66–72. flammatory macrophages to inflammatory dendritic cells in the colon. J. Exp. 68. Koshida, R., H. Oishi, M. Hamada, and S. Takahashi. 2015. MafB antagonizes Med. 209: 139–155. phenotypic alteration induced by GM-CSF in microglia. Biochem. Biophys. Res. 59. Okabe, Y., and R. Medzhitov. 2016. Tissue biology perspective on macrophages. Commun. 463: 109–115. Nat. Immunol. 17: 9–17. 69. Shibata, Y., P. Y. Berclaz, Z. C. Chroneos, M. Yoshida, J. A. Whitsett, and Downloaded from 60. Gosselin, D., V. M. Link, C. E. Romanoski, G. J. Fonseca, D. Z. Eichenfield, B. C. Trapnell. 2001. GM-CSF regulates alveolar macrophage differentiation and N. J. Spann, J. D. Stender, H. B. Chun, H. Garner, F. Geissmann, and C. K. Glass. innate immunity in the lung through PU.1. Immunity 15: 557–567. 2014. Environment drives selection and function of enhancers controlling tissue- 70. Aida, Y., Y. Shibata, S. Abe, S. Inoue, T.Kimura,A.Igarashi,K.Yamauchi, specific macrophage identities. [Published erratum appears in 2015 Cell 160: 351.] K.Nunomiya,H.Kishi,T.Nemoto,etal.2014.Inhibitionofelastase-pulmonary Cell 159: 1327–1340. emphysema in dominant-negative MafB transgenic mice. Int. J. Biol. Sci. 10: 882–894. http://www.jimmunol.org/ by guest on September 25, 2021

Table S1. Probes and annotated genes with altered expression in siMAFB M-MØ

probe log2 siMAFB-M-MØ/siControl-M-MØ P value adj. P value Gene ID GeneSymbol A_24_P125335 -2.8967 0 0.0088 6357 CCL13 A_23_P7144 -2.76 4.00E-04 0.0362 2919 CXCL1 A_23_P167328 -2.7133 1.00E-04 0.0158 952 CD38 A_23_P25994 -2.61 0 0.0063 5641 LGMN A_23_P152605 -2.57 0 0.0149 284110 GSDMA A_23_P89431 -2.5 3.00E-04 0.034 6347 CCL2 A_23_P18452 -2.4567 6.00E-04 0.0436 4283 CXCL9 A_23_P202448 -2.4133 0 0.0051 6387 CXCL12 A_23_P17345 -2.2267 0 0.0051 9935 MAFB A_33_P3375859 -2.1533 1.00E-04 0.019 3580 CXCR2P1 A_33_P3220837 -1.9933 0 0.0051 9935 MAFB A_33_P3354607 -1.99 9.00E-04 0.048 6351 CCL4 A_23_P119478 -1.92 9.00E-04 0.0469 10148 EBI3 A_33_P3317580 -1.9167 0 0.0141 2675 GFRA2 A_23_P376557 -1.9 0 0.0051 64386 MMP25 A_33_P3215803 -1.89 0 0.0051 2015 EMR1 A_33_P3236881 -1.87 0 0.0053 100532736 MINOS1-NBL1 A_33_P3395605 -1.8633 0 0.0115 338773 TMEM119 A_23_P61466 -1.8567 3.00E-04 0.0329 283316 CD163L1 A_23_P164047 -1.81 0 0.0063 23531 MMD A_23_P78037 -1.8067 0 0.0088 6354 CCL7 A_33_P3416097 -1.7267 7.00E-04 0.0456 2162 F13A1 A_23_P15146 -1.72 1.00E-04 0.0208 9235 IL32 A_23_P41217 -1.7067 0 0.0081 131450 CD200R1 A_23_P152838 -1.67 0 0.0117 6352 CCL5 A_24_P183150 -1.6567 0 0.0102 2921 CXCL3 A_33_P3401243 -1.56 2.00E-04 0.0278 25903 OLFML2B A_33_P3330264 -1.5533 0 0.0088 2919 CXCL1 A_23_P126735 -1.5333 2.00E-04 0.0311 3586 IL10 A_23_P200073 -1.53 0 0.0088 57095 PITHD1 A_33_P3232955 -1.5233 8.00E-04 0.0456 9002 F2RL3 A_23_P210482 -1.5033 0 0.0088 100 ADA A_32_P140139 -1.4967 3.00E-04 0.034 2162 F13A1 A_23_P7827 -1.4967 8.00E-04 0.0456 441168 FAM26F A_33_P3400273 -1.49 0 0.0088 6402 SELL A_33_P3331511 -1.48 2.00E-04 0.0246 100287569 LINC00173 A_23_P137689 -1.45 7.00E-04 0.0456 25903 OLFML2B A_23_P30567 -1.43 0 0.0149 1393 CRHBP A_23_P102391 -1.43 4.00E-04 0.0358 30061 SLC40A1 A_33_P3222451 -1.43 5.00E-04 0.0396 84335 AKT1S1 A_33_P3380383 -1.4067 6.00E-04 0.0435 497189 TIFAB A_33_P3712341 -1.3833 0 0.0129 6387 CXCL12 A_23_P45871 -1.38 2.00E-04 0.0247 10964 IFI44L A_24_P379750 -1.3733 0 0.0088 4084 MXD1 A_23_P2431 -1.37 0 0.0088 719 C3AR1 A_33_P3423941 -1.3633 1.00E-04 0.0173 8519 IFITM1 A_23_P143621 -1.35 2.00E-04 0.0247 1414 CRYBB1 A_23_P374322 -1.34 0 0.0129 144811 LACC1 A_23_P33723 -1.3367 0 0.0088 9332 CD163 A_23_P163567 -1.3167 4.00E-04 0.0358 55512 SMPD3 A_24_P257416 -1.3067 1.00E-04 0.0223 2920 CXCL2 A_23_P17481 -1.3067 8.00E-04 0.0456 6614 SIGLEC1 A_33_P3332112 -1.2967 5.00E-04 0.0396 355 FAS A_33_P3346826 -1.2967 6.00E-04 0.043 9235 IL32 A_33_P3299898 -1.28 2.00E-04 0.0314 1517 CTSLP2 A_23_P151805 -1.2467 1.00E-04 0.0204 10516 FBLN5 A_33_P3235053 -1.2433 0 0.0115 91107 TRIM47 A_33_P3348288 -1.24 3.00E-04 0.032 9886 RHOBTB1 A_23_P340848 -1.2333 1.00E-04 0.0165 5739 PTGIR A_23_P81441 -1.2033 1.00E-04 0.0236 140947 C5orf20 A_23_P63896 -1.1933 9.00E-04 0.0484 355 FAS A_24_P131522 -1.19 1.00E-04 0.0208 84168 ANTXR1 A_23_P376449 -1.18 1.00E-04 0.0204 XLOC_l2_002049 A_23_P315364 -1.18 2.00E-04 0.0314 2920 CXCL2 A_33_P3282489 -1.1767 1.00E-04 0.0208 2650 GCNT1 A_23_P435521 -1.1667 0 0.0112 113277 TMEM106A A_23_P68031 -1.15 2.00E-04 0.0247 6775 STAT4 A_32_P4262 -1.15 8.00E-04 0.0459 26074 C20orf26 A_24_P228130 -1.1467 0.001 0.0488 414062 CCL3L3 A_24_P335305 -1.1367 1.00E-04 0.0158 4940 OAS3 A_33_P3262124 -1.1333 3.00E-04 0.034 64386 MMP25 A_32_P54553 -1.12 4.00E-04 0.0358 373856 USP41 A_23_P252962 -1.1167 0 0.0149 6453 ITSN1 A_24_P120934 -1.1067 0 0.0149 10912 GADD45G A_23_P500381 -1.0933 1.00E-04 0.021 3363 HTR7 A_23_P321388 -1.0533 1.00E-04 0.0208 127544 RNF19B A_32_P201979 -1.0533 1.00E-04 0.0236 143279 HECTD2 A_33_P3489737 -1.05 3.00E-04 0.034 57486 NLN A_23_P12554 -1.05 5.00E-04 0.0396 9033 PKD2L1 A_23_P205489 -1.0467 3.00E-04 0.032 23428 SLC7A8 A_23_P105794 -1.0467 4.00E-04 0.0362 94240 EPSTI1 A_23_P404821 -1.0433 1.00E-04 0.0208 57189 KIAA1147 A_23_P105465 -1.04 5.00E-04 0.0418 1240 CMKLR1 A_33_P3371089 -1.0233 3.00E-04 0.0329 54557 SGTB A_33_P3225512 -1.0167 1.00E-04 0.0194 4939 OAS2 A_33_P3421626 -1.0167 1.00E-04 0.0203 57189 KIAA1147 A_33_P3397840 -1.0133 3.00E-04 0.0328 644150 WIPF3 A_23_P129064 -1.01 2.00E-04 0.0311 2628 GATM A_23_P46351 -1.0033 1.00E-04 0.0166 11022 TDRKH A_24_P766716 -0.9967 7.00E-04 0.0456 1240 CMKLR1 A_23_P100001 -0.9933 9.00E-04 0.0484 400451 FAM174B A_33_P3370305 -0.9733 1.00E-04 0.021 113277 TMEM106A A_32_P44394 -0.9733 0.001 0.0488 9447 AIM2 A_23_P75800 -0.9467 4.00E-04 0.0346 5866 RAB3IL1 A_24_P66027 -0.94 7.00E-04 0.0439 9582 APOBEC3B A_33_P3214586 -0.9333 2.00E-04 0.0314 9048 ARTN A_24_P366509 -0.9333 3.00E-04 0.0329 146850 PIK3R6 A_23_P94533 -0.93 1.00E-04 0.0222 1514 CTSL A_23_P347632 -0.93 4.00E-04 0.0362 9788 MTSS1 A_33_P3311863 -0.9267 3.00E-04 0.0329 161 AP2A2 A_32_P163125 -0.92 6.00E-04 0.0435 259230 SGMS1 A_23_P204087 -0.9133 1.00E-04 0.0208 4939 OAS2 A_33_P3252394 -0.9033 2.00E-04 0.0247 10912 GADD45G A_23_P66241 -0.8967 7.00E-04 0.0439 4499 MT1M A_23_P116942 -0.8933 2.00E-04 0.0314 3902 LAG3 A_23_P145631 -0.89 2.00E-04 0.0312 474344 GIMAP6 A_23_P214222 -0.89 8.00E-04 0.0468 4082 MARCKS A_24_P131222 -0.8867 4.00E-04 0.0352 23400 ATP13A2 A_33_P3234697 -0.8833 3.00E-04 0.034 56925 LXN A_32_P101860 -0.8833 6.00E-04 0.0435 113277 TMEM106A A_24_P304423 -0.8833 9.00E-04 0.0469 3479 IGF1 A_24_P932418 -0.87 4.00E-04 0.0352 161 AP2A2 A_24_P140788 -0.8667 2.00E-04 0.0314 221472 FGD2 A_23_P125680 -0.8633 3.00E-04 0.0329 55026 TMEM255A A_24_P173754 -0.86 2.00E-04 0.0245 81563 C1orf21 A_23_P64828 -0.8567 4.00E-04 0.0358 4938 OAS1 A_33_P3258346 -0.8533 2.00E-04 0.0245 54739 XAF1 A_24_P142118 -0.85 9.00E-04 0.0484 7057 THBS1 A_33_P3411075 -0.8467 3.00E-04 0.034 6624 FSCN1 A_23_P204850 -0.8433 4.00E-04 0.0358 5925 RB1 A_23_P13907 -0.84 0.001 0.0492 3479 IGF1 A_23_P29257 -0.8367 9.00E-04 0.0483 3005 H1F0 A_23_P167005 -0.8333 3.00E-04 0.0329 26996 GPR160 A_33_P3306973 -0.8333 8.00E-04 0.0456 A_33_P3393821 -0.8267 6.00E-04 0.0438 715 C1R A_23_P67367 -0.8267 0.001 0.0488 27294 DHDH A_23_P150768 -0.8167 7.00E-04 0.0452 11309 SLCO2B1 A_32_P46214 -0.8133 8.00E-04 0.0459 285195 SLC9A9 A_23_P27332 -0.8033 7.00E-04 0.0439 6925 TCF4 A_23_P35995 -0.8 3.00E-04 0.034 79827 CLMP A_23_P16953 -0.7967 4.00E-04 0.0362 3357 HTR2B A_33_P3292679 -0.7933 3.00E-04 0.034 58533 SNX6 A_33_P3253394 -0.7933 8.00E-04 0.0459 3903 LAIR1 A_33_P3317058 -0.7867 3.00E-04 0.0322 10734 STAG3 A_23_P87545 -0.7833 2.00E-04 0.0314 10410 IFITM3 A_23_P54000 -0.7833 4.00E-04 0.0358 58533 SNX6 A_33_P3228322 -0.7833 5.00E-04 0.0402 10068 IL18BP A_24_P33982 -0.7733 3.00E-04 0.034 284021 MILR1 A_23_P145024 -0.77 7.00E-04 0.0439 154 ADRB2 A_24_P11315 -0.7667 7.00E-04 0.0445 56944 OLFML3 A_23_P145657 -0.7633 5.00E-04 0.0402 10734 STAG3 A_33_P3264926 -0.7633 5.00E-04 0.0402 23034 SAMD4A A_23_P47777 -0.7633 6.00E-04 0.0429 92979 09-Mar A_24_P59667 -0.76 9.00E-04 0.0483 3718 JAK3 A_33_P3355230 -0.7567 4.00E-04 0.0362 3903 LAIR1 A_23_P55270 -0.7533 8.00E-04 0.0459 6362 CCL18 A_33_P3298062 -0.7467 4.00E-04 0.0362 10057 ABCC5 A_33_P3327847 -0.7333 4.00E-04 0.0349 8879 SGPL1 A_23_P133868 -0.7233 8.00E-04 0.0456 387032 ZKSCAN4 A_33_P3400389 -0.72 5.00E-04 0.0372 26010 SPATS2L A_32_P100109 -0.71 6.00E-04 0.0439 9185 REPS2 A_24_P15502 -0.7067 5.00E-04 0.0377 A_33_P3375368 -0.7067 9.00E-04 0.0488 2859 GPR35 A_24_P287043 -0.7033 4.00E-04 0.037 10581 IFITM2 A_24_P354724 -0.6933 6.00E-04 0.0423 117289 TAGAP A_23_P75071 -0.6833 6.00E-04 0.043 9585 KIF20B A_24_P212539 -0.6833 8.00E-04 0.0456 130589 GALM A_23_P111583 -0.68 6.00E-04 0.043 948 CD36 A_32_P174572 -0.68 8.00E-04 0.0456 93164 HTR7P1 A_23_P97064 -0.6733 6.00E-04 0.0439 26270 FBXO6 A_24_P16124 -0.6733 7.00E-04 0.0439 340198 IFITM4P A_23_P168951 -0.67 6.00E-04 0.0439 22882 ZHX2 A_33_P3248967 -0.66 8.00E-04 0.0468 64398 MPP5 A_33_P3281795 -0.6567 8.00E-04 0.0456 11343 MGLL A_33_P3316323 -0.6533 9.00E-04 0.0484 5937 RBMS1 A_23_P56734 -0.65 8.00E-04 0.0456 3176 HNMT A_24_P393838 -0.6333 8.00E-04 0.0459 9804 TOMM20 A_24_P56194 -0.6233 0.001 0.0493 1389 CREBL2 A_23_P252541 -0.6167 0.001 0.0493 338382 RAB7B A_33_P3417452 0.6067 0.001 0.0498 100125288 ZGLP1 A_32_P180265 0.62 9.00E-04 0.0484 A_23_P99397 0.6233 0.001 0.0493 253832 ZDHHC20 A_23_P26905 0.63 8.00E-04 0.0467 11232 POLG2 A_23_P154234 0.6333 8.00E-04 0.0468 56655 POLE4 A_32_P88905 0.6433 8.00E-04 0.0456 A_33_P3360942 0.6433 9.00E-04 0.0488 9522 SCAMP1 A_23_P365817 0.65 7.00E-04 0.0456 26472 PPP1R14B A_23_P350574 0.66 7.00E-04 0.0439 127943 FCRLB A_33_P3248265 0.66 8.00E-04 0.0456 4050 LTB A_24_P365327 0.6633 6.00E-04 0.0435 26065 LSM14A A_23_P211212 0.6633 8.00E-04 0.0456 80781 COL18A1 A_24_P389491 0.67 9.00E-04 0.0482 51117 COQ4 A_33_P3419691 0.6867 6.00E-04 0.0438 352954 GATS A_23_P406438 0.7033 6.00E-04 0.0439 6733 SRPK2 A_24_P124624 0.7033 0.001 0.0488 4973 OLR1 A_23_P206310 0.7067 9.00E-04 0.0484 9764 KIAA0513 A_23_P325690 0.71 9.00E-04 0.048 148741 ANKRD35 A_23_P105862 0.7133 7.00E-04 0.0452 10129 FRY A_23_P329152 0.72 7.00E-04 0.0439 3609 ILF3 A_23_P501722 0.72 8.00E-04 0.0468 10077 TSPAN32 A_33_P3540143 0.72 0.001 0.0488 23765 IL17RA A_23_P143147 0.7233 4.00E-04 0.0366 10732 TCFL5 A_33_P3225843 0.7233 5.00E-04 0.0418 91050 CCDC149 A_33_P3320212 0.7267 5.00E-04 0.0388 A_24_P71700 0.7267 6.00E-04 0.043 92999 ZBTB47 A_23_P97700 0.73 5.00E-04 0.0418 10628 TXNIP A_33_P3334220 0.7367 4.00E-04 0.0358 32 ACACB A_33_P3272390 0.7367 9.00E-04 0.0484 5903 RANBP2 A_23_P152791 0.74 0.001 0.0491 9120 SLC16A6 A_23_P28697 0.7433 4.00E-04 0.0346 23498 HAAO A_33_P3209960 0.7433 7.00E-04 0.0451 10235 RASGRP2 A_33_P3244122 0.75 3.00E-04 0.034 23498 HAAO A_23_P77048 0.7567 3.00E-04 0.034 123096 SLC25A29 A_32_P195401 0.7633 4.00E-04 0.0362 150864 FAM117B A_33_P3702055 0.7767 8.00E-04 0.0459 6556 SLC11A1 A_23_P140668 0.7867 2.00E-04 0.0314 3419 IDH3A A_33_P3321657 0.79 0.001 0.0488 3339 HSPG2 A_24_P239811 0.7933 8.00E-04 0.0459 91544 UBXN11 A_32_P172864 0.7967 3.00E-04 0.0336 284611 FAM102B A_23_P97990 0.7967 7.00E-04 0.0439 5654 HTRA1 A_33_P3394404 0.8067 8.00E-04 0.0468 10077 TSPAN32 A_23_P218456 0.81 3.00E-04 0.034 3609 ILF3 A_24_P163237 0.81 8.00E-04 0.0459 56977 STOX2 A_24_P322741 0.82 8.00E-04 0.0459 3588 IL10RB A_33_P3330608 0.8233 2.00E-04 0.0258 84106 PRAM1 A_24_P645765 0.8233 4.00E-04 0.0359 57542 KLHL42 A_23_P94095 0.8233 4.00E-04 0.0362 157567 ANKRD46 A_33_P3272209 0.8267 4.00E-04 0.0358 54842 MFSD6 A_24_P551028 0.8267 6.00E-04 0.0439 339745 SPOPL A_23_P30275 0.83 6.00E-04 0.0418 78991 PCYOX1L A_23_P104692 0.8433 2.00E-04 0.0247 246330 PELI3 A_23_P43326 0.8467 6.00E-04 0.0423 10558 SPTLC1 A_33_P3328426 0.8467 6.00E-04 0.0439 55129 ANO10 A_33_P3423270 0.85 5.00E-04 0.0388 55287 TMEM40 A_23_P6413 0.85 9.00E-04 0.0469 140606 SELM A_24_P252996 0.86 3.00E-04 0.034 2352 FOLR3 A_33_P3331451 0.89 3.00E-04 0.0329 7046 TGFBR1 A_23_P61487 0.89 7.00E-04 0.0452 55222 LRRC20 A_33_P3222203 0.8933 2.00E-04 0.0278 165140 OXER1 A_24_P192914 0.8933 9.00E-04 0.0482 120425 AMICA1 A_23_P23171 0.9133 2.00E-04 0.0247 192670 AGO4 A_23_P76823 0.92 9.00E-04 0.0484 122622 ADSSL1 A_23_P166408 0.9233 5.00E-04 0.0402 5008 OSM A_23_P82929 0.9233 0.001 0.0488 4856 NOV A_24_P916614 0.93 1.00E-04 0.0222 9991 PTBP3 A_24_P941643 0.95 6.00E-04 0.043 23236 PLCB1 A_23_P62967 0.9633 1.00E-04 0.021 27185 DISC1 A_23_P64058 0.9633 5.00E-04 0.0402 10235 RASGRP2 A_23_P152181 0.9667 1.00E-04 0.0236 55718 POLR3E A_23_P6099 0.9667 2.00E-04 0.0311 23236 PLCB1 A_32_P56001 0.9667 4.00E-04 0.0358 22918 CD93 A_24_P32085 0.97 1.00E-04 0.0204 79817 MOB3B A_23_P56505 1.0133 1.00E-04 0.0187 3676 ITGA4 A_33_P3316878 1.0133 7.00E-04 0.0456 79586 CHPF A_23_P25503 1.02 1.00E-04 0.0236 22862 FNDC3A A_33_P3286041 1.03 3.00E-04 0.0324 55718 POLR3E A_23_P167276 1.03 3.00E-04 0.0329 152559 PAQR3 A_23_P133474 1.0367 3.00E-04 0.034 2878 GPX3 A_33_P3353816 1.04 1.00E-04 0.0165 3676 ITGA4 A_33_P3394075 1.05 2.00E-04 0.0247 6016 RIT1 A_23_P34597 1.0533 3.00E-04 0.0329 978 CDA A_33_P3215676 1.06 7.00E-04 0.0452 100287284 MANSC4 A_32_P161762 1.0767 1.00E-04 0.0203 860 RUNX2 A_33_P3343210 1.08 1.00E-04 0.0208 51312 SLC25A37 A_23_P218549 1.08 2.00E-04 0.0254 84658 EMR3 A_32_P178966 1.0833 1.00E-04 0.0208 100113407 TMEM170B A_24_P200000 1.0833 3.00E-04 0.0329 55240 STEAP3 A_23_P253791 1.09 1.00E-04 0.0222 820 CAMP A_23_P92842 1.1 0 0.0149 51128 SAR1B A_24_P64100 1.1033 0 0.0135 51312 SLC25A37 A_23_P38795 1.11 7.00E-04 0.0456 2357 FPR1 A_23_P429977 1.1367 1.00E-04 0.0208 3784 KCNQ1 A_33_P3713357 1.14 1.00E-04 0.0204 214 ALCAM A_24_P196851 1.2067 0 0.0112 7094 TLN1 A_23_P52676 1.2233 1.00E-04 0.0204 117144 CATSPER1 A_23_P121956 1.2267 0 0.0102 54974 THG1L A_24_P248053 1.2667 1.00E-04 0.0222 116447 TOP1MT A_32_P19294 1.2967 1.00E-04 0.0222 144423 GLT1D1 A_23_P144959 1.35 0 0.0115 1462 VCAN A_23_P91390 1.36 0 0.0112 7056 THBD A_23_P122852 1.36 0 0.0141 6604 SMARCD3 A_23_P304682 1.37 0 0.0088 2013 EMP2 A_33_P3210561 1.4367 0 0.0088 A_33_P3350863 1.4567 0 0.0076 56729 RETN A_24_P347378 1.4767 0 0.0076 241 ALOX5AP A_23_P206733 1.49 4.00E-04 0.0362 1066 CES1 A_23_P90601 1.5067 4.00E-04 0.037 55240 STEAP3 A_23_P106682 1.51 0 0.0086 2013 EMP2 A_23_P304356 1.56 1.00E-04 0.0222 23601 CLEC5A A_23_P250245 1.5933 0 0.0115 971 CD72 A_23_P140384 1.65 3.00E-04 0.0329 1511 CTSG A_23_P101434 1.6533 0 0.0112 91662 NLRP12 A_23_P21485 1.7233 0 0.0063 55022 PID1 A_33_P3241269 1.7533 1.00E-04 0.0208 1066 CES1 Table S2. Probes and annotated genes with altered expression in MCTO#1 M-MØ (p<0.005)

probe log2 MCTO#1 M-MØ/Control M-MØ P value adj. P value Gene ID GeneSymbol A_23_P152605 -5.43 0 0.0111 284110 GSDMA A_33_P3401902 -3.835 0 0.0199 441430 ANKRD20A2 A_24_P281395 -3.62 0 0.0111 A_24_P852756 -3.5 0 0.0111 3118 HLA-DQA2 A_23_P42718 -3.455 3.00E-04 0.0646 9603 NFE2L3 A_33_P3242623 -3.005 1.00E-04 0.0438 23657 SLC7A11 A_23_P157007 -2.68 0 0.0161 28959 TMEM176B A_23_P140290 -2.65 0 0.0199 6252 RTN1 A_23_P23639 -2.635 0 0.0161 255231 MCOLN2 A_33_P3351180 -2.63 0 0.0161 A_33_P3346327 -2.525 0 0.0161 387628 KGFLP1 A_33_P3342111 -2.415 5.00E-04 0.0882 169841 ZNF169 A_33_P3333982 -2.345 0.0012 0.1186 9659 PDE4DIP A_23_P252082 -2.315 0 0.0199 55365 TMEM176A A_23_P55270 -2.295 7.00E-04 0.1009 6362 CCL18 A_24_P49597 -2.29 0 0.0199 A_32_P187617 -2.28 1.00E-04 0.048 81550 TDRD3 A_33_P3354604 -2.245 9.00E-04 0.111 9560 CCL4L2 A_33_P3367850 -2.235 1.00E-04 0.0486 1132 CHRM4 A_33_P3286157 -2.22 0 0.0236 7293 TNFRSF4 A_24_P289404 -2.195 0 0.0199 6231 RPS26 A_32_P105083 -2.135 0 0.0304 348751 FTCDNL1 A_33_P3415191 -2.135 8.00E-04 0.1079 5205 ATP8B1 A_23_P122724 -2.13 1.00E-04 0.0433 8875 VNN2 A_24_P353638 -2.125 0 0.0236 57823 SLAMF7 A_23_P138541 -2.115 1.00E-04 0.0486 8644 AKR1C3 A_23_P17914 -2.09 7.00E-04 0.1009 80339 PNPLA3 A_33_P3280157 -2.065 1.00E-04 0.0322 727708 SNORD116-19 A_23_P208182 -2.05 3.00E-04 0.0615 89790 SIGLEC10 A_24_P299685 -2.045 2.00E-04 0.0551 10630 PDPN A_32_P206839 -2.035 1.00E-04 0.0433 100288911 LOC100288911 A_24_P233786 -1.995 1.00E-04 0.0339 116496 FAM129A A_33_P3405424 -1.965 3.00E-04 0.062 259307 IL4I1 A_33_P3368313 -1.94 1.00E-04 0.0486 4496 MT1H A_23_P116694 -1.88 1.00E-04 0.0444 6231 RPS26 A_23_P42144 -1.86 2.00E-04 0.0509 5190 PEX6 A_23_P165598 -1.85 5.00E-04 0.0882 92196 DAPL1 A_33_P3316273 -1.84 1.00E-04 0.048 6348 CCL3 A_24_P355145 -1.84 1.00E-04 0.048 85479 DNAJC5B A_33_P3323298 -1.84 4.00E-04 0.0816 3725 JUN A_33_P3514859 -1.8 0.0019 0.1369 100506342 LOC100506342 A_23_P44421 -1.77 1.00E-04 0.048 203100 HTRA4 A_33_P3233645 -1.755 2.00E-04 0.0587 4495 MT1G A_33_P3337896 -1.74 5.00E-04 0.0882 A_33_P3280502 -1.735 4.00E-04 0.0779 A_23_P15146 -1.73 0.0034 0.1713 9235 IL32 A_23_P150693 -1.725 4.00E-04 0.0753 24147 FJX1 A_23_P52207 -1.71 0.0019 0.1376 25805 BAMBI A_23_P82929 -1.705 9.00E-04 0.1092 4856 NOV A_33_P3390107 -1.695 0.0039 0.1784 100008588 RNA18S5 A_33_P3398912 -1.68 1.00E-04 0.048 11182 SLC2A6 A_33_P3296181 -1.66 3.00E-04 0.0692 414062 CCL3L3 A_24_P166663 -1.65 4.00E-04 0.0753 1021 CDK6 A_23_P48056 -1.65 6.00E-04 0.0883 10970 CKAP4 A_33_P3376821 -1.64 2.00E-04 0.0587 3001 GZMA A_23_P134176 -1.635 2.00E-04 0.0557 6648 SOD2 A_24_P228130 -1.635 8.00E-04 0.1092 414062 CCL3L3 A_33_P3399571 -1.635 0.0022 0.1427 8876 VNN1 A_23_P165624 -1.63 2.00E-04 0.0554 7130 TNFAIP6 A_33_P3229918 -1.625 1.00E-04 0.0493 171558 PTCRA A_23_P209954 -1.6 3.00E-04 0.0692 10578 GNLY A_33_P3371305 -1.585 0.0011 0.1186 644090 LOC644090 A_33_P3342628 -1.57 2.00E-04 0.0551 57801 HES4 A_23_P146325 -1.565 0.0012 0.1186 29065 ASAP1-IT1 A_23_P201808 -1.54 0.0017 0.1339 8613 PPAP2B A_33_P3265016 -1.53 2.00E-04 0.0554 5190 PEX6 A_33_P3336527 -1.51 0.0021 0.1404 23042 PDXDC1 A_23_P63209 -1.495 0.0025 0.1513 3290 HSD11B1 A_23_P376557 -1.49 6.00E-04 0.0899 64386 MMP25 A_33_P3393766 -1.485 0.0029 0.1593 100170841 C17orf96 A_32_P61684 -1.475 4.00E-04 0.0753 55824 PAG1 A_23_P40956 -1.475 0.0043 0.1836 51738 GHRL A_23_P46936 -1.45 0.0011 0.1186 1959 EGR2 A_23_P211561 -1.435 2.00E-04 0.0568 150365 MEI1 A_33_P3242543 -1.435 3.00E-04 0.0646 4128 MAOA A_23_P203475 -1.43 0.0025 0.1513 112464 PRKCDBP A_23_P57667 -1.425 2.00E-04 0.0612 5361 PLXNA1 A_23_P398566 -1.41 6.00E-04 0.0925 8013 NR4A3 A_33_P3474859 -1.405 5.00E-04 0.0858 203274 LINC00537 A_33_P3381943 -1.4 0.0029 0.1583 9988 DMTF1 A_23_P26024 -1.395 6.00E-04 0.0911 84419 C15orf48 A_33_P3454679 -1.395 0.0011 0.1186 A_33_P3234697 -1.39 3.00E-04 0.0643 56925 LXN A_33_P3412900 -1.38 4.00E-04 0.0761 643866 CBLN3 A_33_P3269803 -1.36 8.00E-04 0.1017 9746 CLSTN3 A_33_P3368750 -1.36 0.0021 0.1409 54852 PAQR5 A_33_P3364869 -1.355 6.00E-04 0.0883 10135 NAMPT A_33_P3255290 -1.34 0.0034 0.1713 9832 JAKMIP2 A_23_P74609 -1.31 6.00E-04 0.0882 50486 G0S2 A_23_P99253 -1.31 0.0018 0.1369 8825 LIN7A A_23_P398491 -1.29 0.0025 0.1503 90416 C15orf57 A_23_P104464 -1.285 5.00E-04 0.0858 240 ALOX5 A_32_P108655 -1.28 0.0039 0.1784 205 AK4 A_23_P146233 -1.275 6.00E-04 0.0882 4023 LPL A_23_P321703 -1.275 0.0032 0.1644 597 BCL2A1 A_23_P8640 -1.265 5.00E-04 0.0858 2852 GPER1 A_23_P59877 -1.26 0.0015 0.128 2171 FABP5 A_23_P126486 -1.26 0.0039 0.1784 84809 CROCCP2 A_33_P3394140 -1.255 0.001 0.1186 283050 ZMIZ1-AS1 A_23_P34915 -1.255 0.0018 0.1369 467 ATF3 A_33_P3267799 -1.25 4.00E-04 0.0816 11006 LILRB4 A_33_P3404097 -1.25 0.0017 0.1339 595135 PGM5P2 A_33_P3286774 -1.245 5.00E-04 0.0858 A_23_P123853 -1.245 7.00E-04 0.0993 6363 CCL19 A_23_P50678 -1.235 0.001 0.1186 4145 MATK A_33_P3231414 -1.23 9.00E-04 0.1094 10859 LILRB1 A_23_P103932 -1.23 0.0011 0.1186 2268 FGR A_24_P370472 -1.225 8.00E-04 0.1042 3126 HLA-DRB4 A_33_P3567967 -1.22 0.0032 0.165 55338 FLJ11292 A_33_P3432961 -1.215 9.00E-04 0.111 401320 LOC401320 A_33_P3287388 -1.215 0.0019 0.1376 A_33_P3413701 -1.21 0.0025 0.1513 51752 ERAP1 A_24_P673063 -1.21 0.0045 0.1864 2171 FABP5 A_23_P79518 -1.205 6.00E-04 0.0883 3553 IL1B A_33_P3342443 -1.205 0.0011 0.1186 100271927 RASA4B A_23_P50946 -1.205 0.0012 0.1186 10267 RAMP1 A_24_P286114 -1.2 7.00E-04 0.0994 6507 SLC1A3 A_23_P108948 -1.2 0.002 0.1402 55686 MREG A_23_P22352 -1.2 0.0022 0.1427 55691 FRMD4A A_23_P138137 -1.195 0.0029 0.1593 115209 OMA1 A_33_P3363091 -1.19 7.00E-04 0.1009 55697 VAC14 A_23_P420610 -1.19 7.00E-04 0.1017 115548 FCHO2 A_24_P59667 -1.185 8.00E-04 0.1078 3718 JAK3 A_23_P129903 -1.185 0.0017 0.1346 147166 TRIM16L A_23_P326142 -1.18 9.00E-04 0.1092 27099 SND1-IT1 A_33_P3366120 -1.18 0.0011 0.1186 2316 FLNA A_23_P98350 -1.18 0.0034 0.1705 330 BIRC3 A_23_P354805 -1.17 6.00E-04 0.0885 11278 KLF12 A_23_P90626 -1.165 0.0018 0.1356 9595 CYTIP A_33_P3216890 -1.165 0.0041 0.1806 55824 PAG1 A_23_P133293 -1.16 0.0044 0.1853 79772 MCTP1 A_32_P356316 -1.155 0.0012 0.1186 3111 HLA-DOA A_32_P128258 -1.14 0.0022 0.143 284367 SIGLEC17P A_23_P132956 -1.14 0.0024 0.1484 7345 UCHL1 A_23_P40847 -1.13 7.00E-04 0.098 9435 CHST2 A_23_P62764 -1.12 0.0011 0.1186 79140 CCDC28B A_23_P44724 -1.12 0.0018 0.1353 1466 CSRP2 A_33_P3269069 -1.12 0.0038 0.1771 9653 HS2ST1 A_33_P3267263 -1.105 0.0012 0.1186 26863 RNVU1-18 A_24_P807031 -1.09 9.00E-04 0.1092 92270 ATP6AP1L A_33_P3382105 -1.085 0.0015 0.128 A_23_P342131 -1.075 9.00E-04 0.111 220002 CYB561A3 A_33_P3289865 -1.075 0.0011 0.1186 5334 PLCL1 A_23_P125078 -1.075 0.0012 0.1186 284129 SLC26A11 A_24_P248240 -1.075 0.0014 0.128 23208 SYT11 A_24_P68908 -1.075 0.0025 0.1521 344887 LOC344887 A_33_P3718269 -1.07 0.004 0.1797 406938 MIR146A A_33_P3290443 -1.065 0.0015 0.128 619383 SCARNA9 A_23_P384044 -1.06 0.0016 0.1339 149111 CNIH3 A_23_P97141 -1.06 0.0023 0.1447 5996 RGS1 A_33_P3713357 -1.06 0.003 0.1618 214 ALCAM A_33_P3338121 -1.055 0.0019 0.1369 3914 LAMB3 A_33_P3233906 -1.055 0.0022 0.1427 10267 RAMP1 A_23_P315364 -1.045 0.0011 0.1186 2920 CXCL2 A_32_P168464 -1.04 0.0015 0.131 8573 CASK A_23_P103110 -1.04 0.0016 0.1339 23764 MAFF A_33_P3268564 -1.04 0.0042 0.1807 8440 NCK2 A_33_P3244998 -1.035 0.0013 0.1244 57489 ODF2L A_33_P3361422 -1.03 0.0019 0.1374 1593 CYP27A1 A_23_P206501 -1.025 0.0011 0.1186 497190 CLEC18B A_23_P426021 -1.025 0.0014 0.128 23231 SEL1L3 A_23_P125748 -1.025 0.0034 0.1705 84460 ZMAT1 A_33_P3321781 -1.025 0.0046 0.1902 645460 FLJ44342 A_23_P111804 -1.02 0.0015 0.128 64761 PARP12 A_33_P3265956 -1.02 0.0021 0.1404 9139 CBFA2T2 A_23_P408094 -1.015 0.0014 0.128 4084 MXD1 A_33_P3226542 -1.015 0.0035 0.1718 26851 SNORD3B-1 A_33_P3246833 -1.01 0.0014 0.128 3557 IL1RN A_23_P78037 -1.005 0.0013 0.1216 6354 CCL7 A_23_P210690 -1.005 0.0015 0.128 57761 TRIB3 A_23_P212497 -1.005 0.0042 0.1813 84129 ACAD11 A_33_P3308481 -1 0.0037 0.1762 57061 HYMAI A_23_P76488 -0.995 0.0015 0.128 2012 EMP1 A_23_P50508 -0.995 0.0018 0.1369 8605 PLA2G4C A_23_P85693 -0.995 0.0022 0.1422 2634 GBP2 A_23_P427760 -0.995 0.0033 0.1681 143689 PIWIL4 A_33_P3272231 -0.99 0.0014 0.128 84879 MFSD2A A_24_P173754 -0.985 0.0013 0.1244 81563 C1orf21 A_24_P161293 -0.98 0.0017 0.1339 A_23_P161076 -0.975 0.0047 0.1902 914 CD2 A_33_P3228102 -0.97 0.0028 0.1581 27031 NPHP3 A_33_P3273584 -0.965 0.0017 0.1339 677766 SCARNA2 A_23_P500271 -0.965 0.0028 0.1571 3663 IRF5 A_23_P210131 -0.965 0.0029 0.1583 A_23_P345942 -0.955 0.0027 0.1566 91942 NDUFAF2 A_24_P931443 -0.95 0.0017 0.1339 8111 GPR68 A_33_P3275255 -0.95 0.002 0.1402 A_23_P103672 -0.95 0.0023 0.1447 10763 NES A_33_P3338440 -0.95 0.0035 0.1718 A_24_P67898 -0.95 0.0035 0.1719 10724 MGEA5 A_33_P3254380 -0.95 0.0037 0.1762 84679 SLC9A7 A_23_P133133 -0.95 0.0044 0.1863 80216 ALPK1 A_23_P414958 -0.945 0.002 0.1402 10154 PLXNC1 A_33_P3413962 -0.945 0.0028 0.1583 5214 PFKP A_23_P394064 -0.945 0.0038 0.1777 284119 PTRF A_23_P75430 -0.94 0.0019 0.1376 56935 SMCO4 A_23_P146456 -0.94 0.0021 0.1404 1515 CTSV A_33_P3264612 -0.94 0.0031 0.1621 219931 TPCN2 A_33_P3325558 -0.935 0.0026 0.1523 9910 RABGAP1L A_33_P3296707 -0.935 0.004 0.1797 441518 FAM127C A_23_P35066 -0.93 0.0024 0.1503 51375 SNX7 A_24_P351283 -0.925 0.002 0.1402 55686 MREG A_24_P366509 -0.925 0.002 0.1402 146850 PIK3R6 A_33_P3345036 -0.92 0.0018 0.1369 5443 POMC A_33_P3244931 -0.92 0.0019 0.1369 1622 DBI A_23_P392942 -0.92 0.0027 0.1566 4481 MSR1 A_32_P86763 -0.92 0.0035 0.1718 7052 TGM2 A_23_P213102 -0.92 0.0047 0.1902 23022 PALLD A_24_P416997 -0.915 0.0019 0.1375 80833 APOL3 A_24_P238143 -0.915 0.002 0.1402 474170 LRRC37A2 A_23_P414913 -0.915 0.0021 0.1409 152007 GLIPR2 A_33_P3531828 -0.915 0.0037 0.1765 51520 LARS A_33_P3214105 -0.915 0.0048 0.1907 467 ATF3 A_24_P136551 -0.91 0.0022 0.1422 55666 NPLOC4 A_33_P3327956 -0.9 0.0025 0.1503 100289635 ZNF605 A_24_P283341 -0.895 0.0026 0.154 64780 MICAL1 A_23_P211680 -0.89 0.0025 0.1518 23209 MLC1 A_24_P152649 -0.89 0.0026 0.154 644189 LOC644189 A_33_P3219572 -0.885 0.0031 0.1621 A_23_P157875 -0.885 0.0041 0.1806 2219 FCN1 A_23_P47682 -0.88 0.0029 0.1593 56675 NRIP3 A_23_P106362 -0.88 0.0047 0.1902 366 AQP9 A_23_P200728 -0.875 0.004 0.1797 2214 FCGR3A A_23_P206806 -0.87 0.0027 0.1566 3683 ITGAL A_23_P254271 -0.87 0.0048 0.1909 84617 TUBB6 A_33_P3225983 -0.865 0.0036 0.176 A_33_P3223660 -0.865 0.0047 0.1907 100128782 LINC00476 A_33_P3397795 -0.86 0.0029 0.1583 64430 PCNXL4 A_23_P14515 -0.86 0.0037 0.1762 122970 ACOT4 A_33_P3357580 -0.855 0.0027 0.1566 51154 MRTO4 A_23_P56590 -0.855 0.0028 0.1581 10438 C1D A_33_P3245539 -0.855 0.0043 0.1822 9240 PNMA1 A_33_P3324884 -0.845 0.0038 0.1781 64780 MICAL1 A_33_P3645805 -0.845 0.0039 0.1784 A_23_P54918 -0.84 0.0033 0.1677 197257 LDHD A_33_P3336760 -0.84 0.0034 0.1705 5244 ABCB4 A_32_P83465 -0.84 0.0036 0.176 100132406 NBPF10 A_33_P3372526 -0.835 0.0034 0.1706 130752 MDH1B A_23_P46928 -0.835 0.0049 0.1912 5214 PFKP A_23_P102000 -0.825 0.0037 0.1771 7852 CXCR4 A_33_P3235330 -0.815 0.0039 0.1784 8897 MTMR3 A_24_P39639 -0.81 0.0048 0.1907 118460 EXOSC6 A_24_P401870 -0.805 0.0042 0.1809 401563 C9orf139 A_23_P143374 -0.8 0.0041 0.1806 22981 NINL A_23_P137665 -0.8 0.0042 0.1809 1116 CHI3L1 A_23_P12554 -0.795 0.0037 0.1762 9033 PKD2L1 A_33_P3298024 -0.795 0.0039 0.1784 8714 ABCC3 A_33_P3279708 -0.795 0.0041 0.1806 6066 RNU2-1 A_24_P145316 -0.795 0.0043 0.1822 84062 DTNBP1 A_33_P3362891 -0.795 0.0043 0.1836 3181 HNRNPA2B1 A_33_P3405068 -0.79 0.0049 0.1912 89796 NAV1 A_23_P25674 -0.785 0.0047 0.1902 1152 CKB A_24_P241183 -0.78 0.0044 0.1853 29121 CLEC2D A_23_P30805 -0.78 0.0044 0.186 8363 HIST1H4J A_23_P393401 -0.78 0.0049 0.1912 283970 PDXDC2P A_33_P3283828 -0.755 0.0049 0.1912 645811 CCDC154 A_24_P96961 -0.745 0.0049 0.1912 80176 SPSB1 A_33_P3423570 0.77 0.0046 0.1902 79006 METRN A_23_P256561 0.77 0.0047 0.1902 10333 TLR6 A_23_P12733 0.78 0.004 0.1796 55506 H2AFY2 A_23_P217269 0.785 0.0041 0.1806 11326 VSIG4 A_23_P55356 0.8 0.004 0.1796 284013 VMO1 A_33_P3358957 0.81 0.0048 0.1909 390928 PAPL A_23_P119196 0.83 0.0033 0.1705 10365 KLF2 A_23_P94754 0.83 0.0048 0.1909 9966 TNFSF15 A_23_P318284 0.845 0.0041 0.1806 23171 GPD1L A_33_P3287611 0.855 0.0048 0.191 3850 KRT3 A_24_P287043 0.86 0.0032 0.1638 10581 IFITM2 A_33_P3395081 0.86 0.004 0.1797 54988 ACSM5 A_23_P165989 0.865 0.0027 0.1566 140825 NEURL2 A_23_P347632 0.865 0.0038 0.1784 9788 MTSS1 A_23_P160849 0.875 0.0023 0.1464 2207 FCER1G A_24_P402690 0.88 0.0027 0.1558 81618 ITM2C A_23_P44684 0.9 0.0024 0.1486 1894 ECT2 A_23_P45871 0.905 0.0021 0.1422 10964 IFI44L A_24_P379820 0.905 0.0022 0.1424 81618 ITM2C A_24_P243749 0.905 0.0031 0.1621 5166 PDK4 A_23_P29303 0.915 0.0027 0.1566 27341 RRP7A A_24_P354724 0.915 0.0039 0.1784 117289 TAGAP A_33_P3214943 0.92 0.0031 0.1621 9806 SPOCK2 A_23_P127663 0.935 0.0024 0.1501 79056 PRRG4 A_24_P397294 0.935 0.003 0.1607 4056 LTC4S A_24_P129588 0.95 0.0046 0.1902 338557 FFAR4 A_24_P373768 0.95 0.0047 0.1902 2696 GIPR A_32_P36694 0.965 0.0017 0.1339 221895 JAZF1 A_33_P3255677 0.97 0.0028 0.1566 A_23_P217917 0.98 0.0031 0.1621 2948 GSTM4 A_23_P371765 0.98 0.0047 0.1902 84221 SPATC1L A_33_P3392892 0.985 0.0017 0.1339 54986 ULK4 A_23_P126735 0.985 0.0019 0.1369 3586 IL10 A_23_P326893 0.985 0.0029 0.1593 115948 CCDC151 A_24_P382187 0.995 0.0043 0.1836 3487 IGFBP4 A_33_P3276207 1 0.0039 0.1784 91775 NXPE3 A_23_P101093 1.005 0.0041 0.1806 51226 COPZ2 A_23_P252193 1.02 0.0034 0.1705 3680 ITGA9 A_33_P3221748 1.03 0.0017 0.1339 864 RUNX3 A_33_P3281572 1.04 0.0019 0.1374 8418 CMAHP A_23_P4160 1.045 0.0012 0.1186 10230 NBR2 A_33_P3268472 1.05 0.0012 0.1186 1075 CTSC A_23_P254842 1.055 0.0037 0.1765 8226 HDHD1 A_33_P3316786 1.06 0.0011 0.1186 1602 DACH1 A_23_P76538 1.065 0.0012 0.1186 54997 TESC A_23_P205489 1.065 0.0024 0.1503 23428 SLC7A8 A_23_P30666 1.085 0.0011 0.1186 27242 TNFRSF21 A_33_P3412087 1.085 0.0014 0.128 80129 CCDC170 A_23_P74252 1.1 0.001 0.1186 29092 LINC00339 A_23_P43276 1.11 0.0012 0.1186 25960 GPR124 A_33_P3283601 1.155 0.0016 0.1337 389033 LOC389033 A_23_P35444 1.165 0.0049 0.1912 9118 INA A_23_P142631 1.19 5.00E-04 0.0882 2281 FKBP1B A_33_P3232557 1.2 6.00E-04 0.0882 58512 DLGAP3 A_32_P224522 1.2 0.0016 0.1339 79085 SLC25A23 A_23_P10647 1.205 0.0011 0.1186 54360 CYTL1 A_23_P218579 1.205 0.0017 0.1339 79411 GLB1L A_33_P3387696 1.21 0.003 0.1607 51643 TMBIM4 A_23_P400378 1.215 9.00E-04 0.1094 151306 GPBAR1 A_23_P351467 1.215 0.0012 0.1186 8418 CMAHP A_33_P3252695 1.22 5.00E-04 0.0858 54360 CYTL1 A_23_P3963 1.23 0.0013 0.122 30850 CDR2L A_23_P140563 1.235 0.0028 0.1566 64927 TTC23 A_33_P3318696 1.275 0.0016 0.1339 56267 CCBL2 A_24_P10657 1.28 6.00E-04 0.0882 57153 SLC44A2 A_23_P360209 1.28 6.00E-04 0.0882 4537 ND3 A_33_P3330549 1.28 0.0015 0.128 57153 SLC44A2 A_33_P3260053 1.28 0.0028 0.1581 83543 AIF1L A_24_P31235 1.295 4.00E-04 0.0816 1984 EIF5A A_23_P357760 1.3 0.0045 0.1881 414 ARSD A_23_P150549 1.315 0.0022 0.1422 643834 PGA3 A_23_P330461 1.35 0.0012 0.1186 147798 TMC4 A_23_P423309 1.355 0.0037 0.1762 51294 PCDH12 A_33_P3281567 1.365 9.00E-04 0.1092 8418 CMAHP A_33_P3753757 1.445 4.00E-04 0.0763 158402 LOC158402 A_23_P109427 1.495 5.00E-04 0.0858 2953 GSTT2 A_33_P3350863 1.55 2.00E-04 0.0583 56729 RETN A_24_P245379 1.605 1.00E-04 0.048 5055 SERPINB2 A_32_P4262 1.605 2.00E-04 0.0587 26074 C20orf26 A_24_P331560 1.62 0.0018 0.1356 412 STS A_23_P41217 1.65 0.0048 0.1909 131450 CD200R1 A_33_P3236881 1.685 2.00E-04 0.0554 100532736 MINOS1-NBL1 A_24_P165864 1.69 1.00E-04 0.0438 9934 P2RY14 A_23_P74088 1.715 0.003 0.1607 8510 MMP23B A_23_P119222 1.72 2.00E-04 0.0573 56729 RETN A_23_P94819 1.735 1.00E-04 0.0438 9501 RPH3AL A_24_P945113 1.87 1.00E-04 0.048 94 ACVRL1 A_33_P3289045 1.9 0.0026 0.154 653689 GSTT2B A_23_P3911 2.03 1.00E-04 0.0437 57125 PLXDC1 A_33_P3424222 2.235 2.00E-04 0.0554 3119 HLA-DQB1 A_33_P3250612 2.3 0 0.0283 4337 MOCS1 A_23_P19510 2.395 0.0017 0.1346 3120 HLA-DQB2 A_23_P140384 2.72 0 0.0161 1511 CTSG A_23_P164773 2.82 0 0.026 2208 FCER2 A_24_P339858 3.2 0.0017 0.1339 114043 C21orf90 A_24_P213161 3.26 0 0.0161 55655 NLRP2 A_23_P26325 3.59 0 0.0161 6361 CCL17 A_23_P136683 5.77 0 0.0161 3119 HLA-DQB1