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Effects of Etanercept Are Distinct from Infliximab in Modulating Proinflammatory in Activated Human Leukocytes Asifa S. Haider1, Irma R. Cardinale1, Julia A. Whynot1 and James G. Krueger1

Proinflammatory diseases like , Crohn’s disease, and psoriasis have been treated by the (TNF) antagonists infliximab and etanercept with different degrees of success. Although these agents are widely used in humans, little is known about their mechanisms of action or why etanercept and infliximab have differences in clinical activity. In this study, we define leukocyte genes that are suppressed by etanercept within 24 hours of exposure. Compared to previous work with infliximab, fewer immune-related genes are suppressed by etanercept. Importantly, the range of genes suppressed by these alternative TNF inhibitors is only partially overlapping, suggesting each has unique immune modulating effects. In sharp contrast to etanercept, infliximab strongly suppresses genes associated with ‘‘Type 1’’ immune responses (IFN-g and the IL-12-receptor beta 2 subunit), providing a clear mechanism for clinically relevant immune suppression. Journal of Investigative Dermatology Symposium Proceedings (2007) 12, 9–15. doi:10.1038/sj.jidsymp.5650032

INTRODUCTION range of -regulated products is induced. By Two biologic tumor necrosis factor (TNF) inhibitors, etaner- blocking ‘‘inductive’’ effects of secreted in these cept and infliximab, are effective in treating psoriasis and cultures with monoclonal antibodies, we have been able to psoriatic arthritis (Gottlieb, 2005), but differences exist in the define genes that are selectively regulated by IFN-g or TNF in rapidity of therapeutic improvement, the overall fraction of leukocytes. To extend this work, we have now studied patients that respond to the agents, and the range of other the range of genes that are regulated by etanercept in mixed inflammatory diseases, for example, Crohns disease, that can leukocyte cultures. A relatively small set of genes (n ¼ 29) be effectively treated. Although etanercept and infliximab are was commonly suppressed by etanercept and a TNF antibody structurally different, that is, etanercept is a TNF-receptor- (infliximab), whereas 55 genes were uniquely suppressed immunoglobulin fusion (Mohler et al., 1993), and by etanercept versus 513 genes uniquely suppressed infliximab is a chimeric antibody specific for TNF (Knight by infliximab. Overall, these results establish distinct biologic et al., 1993), each of these molecules can effectively inhibit activities of etanercept and infliximab and they suggest a TNF-mediated responses in vitro (Scallon et al., 2002). potential explanation for differences in pharmacologic However, in vivo pharmacologic mechanisms of these activity across the range of diseases treated with these alternative TNF inhibitors are poorly understood, as is the agents. range of inflammatory genes that may be regulated by these biologic agents. RESULTS In a recent study, we used gene arrays to define a broad set Human peripheral blood mononuclear leukocytes were of genes, which are induced in peripheral blood mono- cultured for 24 hours with bead-bound antibodies to CD3 nuclear leukocytes through activating T cells with antibodies and CD28 in order to activate T cells. We confirmed to CD3 and CD28 (Haider et al., submitted). In this mixed activation of T cells by measuring CD3/CD69 þ cells by cell culture, cytokines produced by activated T-cells (IFN-g, flow cytometry. There were 98.6% cells that were activated TNF, and many others) have secondary effects on other cell in anti-CD3/CD28 costimulated peripheral blood mono- types, for example, monocytes, such that an extremely wide nuclear cells (PBMCs) as compared to 4.62% of non-

1Laboratory of Investigative Dermatology, Rockefeller University, New York, New York, USA Correspondence: Dr James G. Krueger, Laboratory for Investigative Dermatology, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA. E-mail: [email protected] Abbreviations: LTA, lymphotoxin alpha; mRNA, messenger RNA; PBMC, peripheral blood mononuclear cell; PTS, 6-pyruvoyltetrahydropterin synthase; TARC, thymus and activation-regulated chemokine; TNF, tumor necrosis factor; XLC1 and XCL2, chemokines (SCYC1, SCM1a) and (SCYC2, SCM1b) Received 20 September 2006; accepted 28 September 2006

& 2007 The Society for Investigative Dermatology www.jidonline.org 9 AS Haider et al. Etanercept and Infliximab in Human Leukocytes

stimulated PBMCs. In some cultures, etanercept was added +Anti-CD3/CD28

during this activation period. T-cell activation was not Non- Activated+ generally suppressed by etanercept, as 491% of T cells stimulated Activated etanercept became CD69 þ in etanercept-treated cultures. In addition, a we observed no significant increase in of T cells (assessed by Annexin V and propidium-iodide staining) in etanercept-treated cultures (data not shown). Abundance of cellular mRNA was quantified by hybridi- zation of complementary DNA to Affymetrix U95Av2 gene arrays that contain B12,000 genes. There were 41,700 genes that were significantly upregulated by activation (Po0.05, after correction for multiple measures). Figure 1 shows heatmaps of genes that are induced in activated mononuclear leukocytes and also 86 genes whose expression is significantly suppressed by etanercept treatment (Figure 1, upper panel; Table 1 for a list of the top 36 genes and associated P-values). We noted that out of six donors there were some differences in response to etanercept – two donors responded more strongly than the other four. Etanercept downregulated the in these two donors down to normal levels as seen by the heatmap tiles in >1.2 < Activated genes downregulated by etanercept (86 genes) etanercept-treated versus non-stimulated PBMCs (Figure 1). Also as shown in Figure 1 (bottom panel), many other b activation-associated genes were not significantly suppressed

by etanercept. For example, CD69 mRNA was upregulated changes Expression in fold 4.1-fold in CD3/CD28 stimulated cultures and 4.2-fold in etanercept exposed cultures – similar levels of CD69 were confirmed by measuring protein expression by flow cytometry. We detected several proinflammatory genes that were Genes activated by anti-CD3/CD28 co-stimulation and regulated by etanercept. Etanercept binds equally to TNF and not regulated by etanercept (>1,700) lymphotoxin alpha (LTA) (Schall et al., 1990). Interestingly, Figure 1. Genes downregulated by etanercept in activated PBMCs. mRNA we detected reduced expression of LTA mRNA in etanercept- from cultured PBMCs was hybridized to individual oligonucleotide arrays treated cultures (Figure 2). Three chemokines of interest, containing B12,000 human genes (Affymetrix HG-U95Av2 chips). Heatmaps XCL1 (SCYC1, SCM1a), XCL2 (SCYC2, SCM1b), and TARC show unsupervised hierarchical clusters of genes regulated by etanercept in (CC chemokine 17, thymus and activation-regulated chemo- PBMCs using similarity measure: Pearson’s correlation. Gene lists were kine) and several genes of type 2 pathway like IL-4, -5, and -9 created after significance analysis as defined by a paired Welch test, with a were regulated by etanercept. TARC has been shown to be subsequent correction for multiple gene comparisons by the Benjamini and Hochberg method (Po0.05). The genes regulated by 41.2-fold mean regulated by TNF (Sumyoshi et al., 2003) and produced by expression levels are represented with red lines (upregulated) as compared to monocytes (Imai et al., 1999) that are treated with green lines (downregulated). Genes in PBMCs activated with anti-CD3/CD28 granulocyte–macrophage colony-stimulating factor. In our (ACT) are compared to non-stimulated and activated in the presence of TNF analysis granulocyte–macrophage colony-stimulating factor inhibitor etanercept, n ¼ 6. (a) Heatmap of 86 genes significantly was significantly downregulated by treatment of activated downregulated by treatment with etanercept. (b) Heatmap representing PBMCs (ACT) with etanercept (ET) (ACT: 34.17710.3, ET: 41,700 genes upregulated by activation but not affected after treatment with etanercept. The top 36 genes downregulated by etanercept are listed in Table 20.976.0; n ¼ 6. Po0.05). We confirmed the expression of S1 available online with a complete list of etanercept-regulated genes, with several type 2 genes IL-4, -5, and -9 and TARC to be fold changes and P-values. exclusively regulated by etanercept (Table 1). This suggests that etanercept is affecting the cytokines produced mainly by Th2 T cells. Infliximab, on the other hand, downregulated type 1 genes like IFN-g and IL-12-receptor beta 2. LTA-TNF regions (Criswell et al., 2004). LTA is involved in the development of follicular dendritic cells and induces DISCUSSION signals that lead to lymphoid neo-organogenesis, phenomena This study illustrates a pharmacogenomic approach to observed in like rheumatoid arthritis elucidate the effects of etanercept and infliximab in a model (Takemura et al., 2001) and tumors (Criswell et al., 2004). of ‘‘inflammation’’. One finding is the suppressive effect of This suggests that etanercept could suppress lymphoid neo- etanercept on LTA gene expression. There is not much known organogenesis, which is consistent with reduced numbers of about the pharmacology of LTA; however, there are reports of mature dendritic cells in psoriasis lesions treated for several association of response to etanercept with genetic variation in months with etanercept (Gottlieb et al., 2005).

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Table 1. Activation genes in PBMCs downregulated by etanercept Activation (ACT) genes downregulated by P-value, corr., etanercept (ET): fold activation versus change ACT versus ET etanercept treated Symbol Gene product

4.6 0.0144 IL9 interleukin 9 2.6 0.00521 GMCSF human granulocyte-macrophage colony-stimulating factor (CSF1) 2.5 0.00852 LTA lymphotoxin alpha (TNF superfamily, member 1) 2.5 6.37E-05 FEZ1 fasciculation and elongation protein zeta 1 (zygin I) 2.2 0.0069 XCL2 Homo sapiens DNA for SCM-1beta precursor, complete cds 2.1 0.0328 IL4 interleukin 4 1.9 0.00125 HOP homeodomain-only protein 1.8 0.00304 MAOA monoamine oxidase A 1.7 0.0255 IGSF3 immunoglobulin superfamily, member 3 1.7 0.0053 XCL1 chemokine (C motif) ligand 1 1.7 0.0344 LMNA lamin A/C 1.5 0.00736 PTS putative; Homo sapiens PTS gene, complete cds 1.5 0.043 MCM2 MCM2 minichromosome maintenance deficient 2, mitotin (S. cerevisiae) 1.5 0.00033 ACYP1 acylphosphatase 1, erythrocyte (common) type 1.5 0.0274 AF1Q ALL1-fused gene from 1q 1.5 0.049 IL5 interleukin 5 (colony-stimulating factor, eosinophil) 1.5 0.0391 BATF Homo sapiens B-ATF gene, complete cds 1.5 0.0104 RUVBL2 RuvB-like 2 (E. coli) 1.5 0.0206 PA2G4 proliferation-associated 2G4, 38 kDa 1.4 0.0428 CKS1B CDC28 protein kinase regulatory subunit 1B 1.4 0.0168 UROD uroporphyrinogen decarboxylase 1.4 0.00799 RFC5 replication factor C (activator 1) 5, 36.5 kDa 1.4 0.00401 HIRIP3 HIRA interacting protein 3 1.4 0.0227 ATP6V0A2 ATPase, H+ transporting, lysosomal V0 subunit a isoform 2 1.4 0.0342 RPP30 ribonuclease P (30kD) 1.4 0.0115 ANP32E acidic (leucine-rich) nuclear phosphoprotein 32 family, member E 1.4 0.0378 PCCB propionyl coenzyme A carboxylase, beta polypeptide 1.4 0.0331 NPM3 nucleophosmin/nucleoplasmin, 3 1.4 0.00883 PRG2 proteoglycan 2, bone marrow 1.4 0.00961 PSMD1 (prosome, macropain) 26S subunit, non-ATPase, 1 1.4 0.0388 CDKN3 cyclin-dependent kinase inhibitor 3 1.4 0.0244 RRM1 ribonucleotide reductase M1 polypeptide 1.4 0.0113 NUP155 nucleoporin 155 kDa 1.4 0.00787 SPAG5 sperm associated antigen 5 1.4 0.0455 SNRPA1 small nuclear ribonucleoprotein polypeptide A’ 1.4 0.0217 TIMM44 translocase of inner mitochondrial membrane 44 homolog (yeast) Selected list of genes (out of total 86 genes) downregulated in activated PBMCs as compared to treatment with ET for 24 h. Fold changes are ratios of mean gene expressions in PBMCs that are activated with anti-CD3/28 as compared to activation in the presence of ET. P-value of ACT versus ET after correction for multiplicity is listed. Complete list with P-values and fold changes is in Table S1. Gene symbols and products are described in annotations.

The two chemokines downregulated by etanercept, XCL1 lymphocytes and natural killer cells (Maghazachi et al., and XCL2, have been identified as two isoforms of 1997). The expression of lymphotactin is upregulated in CD3/ lymphotactin (Yoshida et al., 1998). They are produced by CD28 costimulated T cells in a calcineurin-dependent CD8 þ T cells and natural killer cells and are chemotactic for manner (Cristillo et al., 2003). Classical anti-psoriasis drugs

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120 LTA 10 IL-4 100 8 80 6 60 4 40 * * 20 2 (normalized to HARP) 0 (normalized to HARP) 0 Relative mRNA expression Relative NS ACT Infliximab Etanercept mRNA expression Relative NS ACT Infliximab Etanercept

IL-9 IL-5 35 35 30 30 25 25 20 20 15 15 10 10 5 * 5 * (normalized to HARP) 0 (normalized to HARP) 0

Relative mRNA expression Relative NS ACT Infliximab Etanercept Relative mRNA expression Relative NS ACT Infliximab Etanercept

TARC 35 30 25 20 15 10 * 5

(normalized to HARP) 0

Relative mRNA expression Relative NS ACT Infliximab Etanercept

IFN- IL-12R2 1,000 60 50 750 40 500 30 20 250 * 10 * (normalized to HARP) 0 (normalized to HARP) 0 Relative mRNA expression Relative NS ACT Infliximab Etanercept mRNA expression Relative NS ACT Infliximab Etanercept

Figure 2. Real-time RT-PCR confirmation of genes regulated by etanercept as compared to infliximab: IL-4, -5, -9 (IL-4, -5, and -9), IL-12-receptor b 2, IFN-c, LTA, and TARC. mRNA from PBMCs activated with anti-CD3/CD28 antibody (ACT) was compared to non-stimulated PBMCs (NS), activation in the presence of anti-TNF antibodies etanercept or infliximab. Mean of relative mRNA expression normalized to human acidic ribosomal protein is plotted on column bars. Significance of ACT versus treatment is indicated with asterix (*): n ¼ 6, Po0.01.

like cyclosporine that inhibit T cell cytokine production as comparison with etanercept revealed that most of the genes well as T cell proliferation target the calcineurin pathway. regulated by infliximab are unique to that agent (Venn This may suggest that etanercept is regulating some of the diagram shown in Figure 3 compares etanercept versus cytokines produced by T cells. infliximab-regulated genes). We have listed the top B10 TARC has been shown to be regulated by TNF (Sumyoshi genes in each category of genes unique to effects of each drug et al., 2003) and produced by monocytes (Imai et al., 1999) as well as genes that are commonly regulated by the that are treated with GM-CSF. This may suggest that two inhibitors. A complete list of each category is available etanercept, by reducing the GM-CSF, is inhibiting mono- online in supplement Tables S2–S4. The surprising finding in cytes that produce TARC in a mixed cell population. Together this comparison is that there are very few commonly with IL-4, -5, and -9, TARC has also been shown to regulated genes by the two inhibitors. Although there be upregulated in type 2 diseases like atopic dermatitis are several regulating genes in the list, of particular (Jenmalm et al., 2001; Uchida et al., 2002; Klunker et al., interest in the immune response are nuclear basic leucine 2003). This suggests that etanercept is affecting cytokines zipper (BATF) and a negative regulator produced by Th2 T cells. of AP-1 activity and 6-pyruvoyltetrahydropterin synthase (PTS). Basic leucine zipper protein and a negative regulator Comparison of genes regulated by etanercept versus infliximab of AP-1 activity forms heteromers with Jun We have identified genes suppressed by infliximab using the and blocks AP-1 trans-activation in vivo and in vitro. same methods as in this study (Haider et al., submitted). Our Overexpression of this gene in mice is known to disrupt the

12 Journal of Investigative Dermatology Symposium Proceedings (2007), Volume 12 AS Haider et al. Etanercept and Infliximab in Human Leukocytes

Genes regulated by etanercept as protective immunity would be less affected in patients treated compared to infliximab with etanercept. Genes downregulated Genes downregulated In conclusion, the effects of infliximab are much more by etanercept by infliximab prominent on gene expression of proinflammatory type 1 Fold changes Fold changes genes as compared to etanercept. Effects of etanercept may Activation/ Activation/ etanercept infliximab possibly involve suppression of lymphoid neo-organogenesis. IL-9 4.6 IFN- 5.9 Potentially, disease improvement in psoriasis or psoriatic GMCSF 2.6 55 29 513 MMP12 5.8 LTA 2.5 arthritis is mediated by inhibition of ‘‘common’’ inflamma- IL-12R2 3.2 FEZ1 2.5 IL-1 tory pathways. The common effects of both inhibitors are on XCL2 2.2 2.7 IL-4 2.1 CCL18 2.6 T cell development and the nitric oxide pathway. This study HOP 1.9 CXCL11 2.6 describes the pharmacogenomics of the two inhibitors and MAOA 1.8 IL-2R 2.6 IGSF3 1.7 GZMB 1.9 elucidates the possible differences observed in the clinical XCL1 1.7 LAMP3 1.9 IL-5 1.5 and safety profiles of these drugs. Genes commonly downregulated by infliximab and etanercept MATERIALS AND METHODS Fold changes Fold changes The drawing of blood for these studies was approved by the Activation/ Activation/ infliximab etanercept Rockefeller University Review Board and volunteers or patients FEZ1 2.2 2.5 signed consent for their donation according to the Declaration of BATF 1.6 1.5 Helsinki Principles. CKS1B 1.5 1.4 RRM1 1.5 1.4 HRMT1L2 1.5 1.3 MCM2 1.5 1.5 Samples used in the study MAOA 1.4 1.8 PBMCs were isolated from freshly drawn human blood (in heparin NACA 1.4 1.3 ANP32E 1.4 1.4 tube) using Ficoll-Paque Plus according to standard protocol. PBMCs RPP30 1.3 1.4 were resuspended at 1 106/ml in Rosewell Park Memorial Institute, PTS 1.3 1.5 medium (Invitrogen, Cat No. 21870) þ 10% human serum (Hyclone, Figure 3. Comparison of effects of etanercept and infliximab on gene Logan, UT, Cat No. SH30071.03) and PenStrep (Penicillin 10 KU/ml activation. Venn diagram shows comparison of genes regulated by etanercept and Streptomycin 10 mg/ml) – (Invitrogen, Carlsbad, CA, USA, Cat (left circle) with genes regulated by infliximab (right circle). Fifty-five genes are regulated exclusively by etanercept, 513 genes are regulated by infliximab No. 15140–148). Cultures were set up in 20 ml/dish (100 20 mm, and both inhibitors commonly regulate 29 genes. Selected list of genes with Corning, Cat No. 25020) in six different experiments for the four fold changes are listed in the figure. conditions. Non-stimulated PBMCs (NS) served as control to activation (ACT) with anti-CD3/CD28 using Dynabeadss CD3/ CD28 T Cell Expander (Dynal Biotech, 111.31), activation in the development of thymus-derived natural killer T cells (Wil- presence of TNF alpha inhibitors etanercept (Et) (100 mg/ml, Amgen) liams et al., 2003); thus, both inhibitors may be regulating the and infliximab (I) (100 mg/ml, Centacor,). All reagents were added at development of the T cells. Enhanced 6-pyruvoyltetrahy- time 0 and cells were collected at 24-hour-post-stimulation. dropterin synthase expression is necessary for optimized Floating cells were collected and Dynabeads were removed with tetrahydrobiopterin (BH4) synthesis in IL-1b, and to some a magnet. An aliquot of at least 500,000 cells was taken to study extent TNF and IFN-g-dependent manner. BH4 is an essential phenotype and apoptosis. Adherent cells were lysed with 2 ml/dish cofactor for nitric oxide synthesis (Franscini et al., 2003). In of Qiagen’s RLT buffer (plus 10 ml/ml 2-mercapto-ethanol), and the human monocytes/macrophages, 6-pyruvoyltetrahydropterin lysates were transferred to the corresponding suspended cell pellets, synthase may be a rate-limiting enzyme in the BH4 synthesis. followed by vortexing and storage at 801C for later RNA We have previously shown that nitric oxide synthase is an preparation. enzyme that is downregulated in skin biopsies of patients treated with TNF-inhibitor etanercept (Gottlieb et al., 2005). Measurement of gene expression in RNA The suppression of 6-pyruvoyltetrahydropterin synthase Target preparation. The microarrays used for this study were mRNA thus provides a mechanism for reducing expression U95Av2-set GeneChip probe arrays (Affymetrix Inc, Santa Clara) that of iNOS that could be independent of IFN-g or other contain probe sets representing approximately 12,000 genes. inducers. Fragmentation, array hybridization, scanning, and quality con- It is striking to note that there is a strong signature of type 1 trol, GeneChip expression value analysis, hierarchical clustering, genes regulated by infliximab. We confirmed the mRNA and heatmaps are described in our recent publication (Haider et al., expressions of IFN-g or IL-12-receptor beta 2 by real-time RT- 2006). PCR (Figure 2). Cytokines such as IFN-g made by Th1 T cells Briefly, for individual transcripts, fold change in expression was are important for immune responses to intracellular organ- the ratio of the means of expression levels in each condition as isms such as Mycobacterium tuberculosis. Hence, blockade compared to appropriate control (n ¼ 6). The heatmap of the of TNF by infliximab, which also may result in less synthesis computed tree represent unsupervised hierarchical clustering and of IFN-g, could underlie re-activation of tuberculosis which is presented as red and green lines. Each line represents genes with has been observed in some infliximab-treated patients (Keane relative upregulated (red) or downregulated (green) fold changes in et al., 2001). Conversely, one might postulate that this form of expression values.

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Statistical comparisons of microarray analysis ribosomal protein, a , was used to normalize each To obtain a list with the biologically most interesting genes used in sample and each gene. The data were analyzed and samples quantified this study, multiple statistical analyses were performed. Based on the by the software provided with the Applied Biosystems PRISM 7700 full data set, one-way analysis of variance was performed to assess (Sequence Detection Systems, ver.1.7). significantly different genes between each condition. Genes with Po0.05 that passed the Benjamini and Hochberg criterion and fold Statistical analysis of gene expression measured by real-time change differences of 41.2 between each condition, were further RT-PCR subjected to paired Welch tests (t-test for unequal variance) to Statistical comparisons of expression level, pair wise activation distinct activation versus non-stimulation and activation versus versus non-stimulation or activation in the presence of etanercept or etanercept treatment. Owing to the multiple testing problem infliximab between each condition were performed using a two- encountered in the analysis of microarray data, we chose to declare tailed, Student’s t-test. those genes with a false discovery rate of at most 5% as having a significant effect. In this study we considered genes that passed the CONFLICT OF INTEREST Benjamini and Hochberg criterion as biologically most interesting. Drs Haider and Krueger received honoraria from Amgen Inc. for contributing Complete lists of genes with P-values and description of relevant to this article. Dr Krueger has consultancy agreements with Amgen Inc. and functions of genes are provided in Tables S1–S4. Centocor. GeneOntology annotations of differentially expressed genes were collected from LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink). SUPPLEMENTARY MATERIAL Table S1. Genes downregulated by Etanercept. Comparison of etanercept-regulated genes with Table S2. Genes downregulated by Etanercept only. infliximab-regulated genes Table S3. Genes commonly regulated by Etanercept and Infliximab. The significantly downregulated genes after treatment with etaner- Table S4. Genes downregulated by Infliximab only when compared to cept were further compared to genes that are significantly down- Etanercept. regulated by infliximab, described elsewhere (Haider et al. manuscript submitted). 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