Chronic Exposure to Glucocorticoids Shapes Gene Expression and Modulates Innate and Adaptive Activation Pathways in Macrophages with Distinct Changes in This information is current as Leukocyte Attraction of September 29, 2021. Martijn D. B. van de Garde, Fernando O. Martinez, Barbro N. Melgert, Machteld N. Hylkema, René E. Jonkers and Jörg Hamann J Immunol published online 6 January 2014 Downloaded from http://www.jimmunol.org/content/early/2014/01/05/jimmun ol.1302138 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2014/01/05/jimmunol.130213 Material 8.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 6, 2014, doi:10.4049/jimmunol.1302138 The Journal of Immunology

Chronic Exposure to Glucocorticoids Shapes Gene Expression and Modulates Innate and Adaptive Activation Pathways in Macrophages with Distinct Changes in Leukocyte Attraction

Martijn D. B. van de Garde,*,1 Fernando O. Martinez,†,1 Barbro N. Melgert,‡,x || Machteld N. Hylkema,x,{ Rene´ E. Jonkers, and Jo¨rg Hamann*

Glucocorticoids (GCs) have been used for more than 50 y as immunosuppressive drugs, yet their efficacy in macrophage-dominated disorders, such as chronic obstructive pulmonary disease, is debated. Little is known how long-term GC treatment affects macrophage responses in inflammatory conditions. In this study, we compared the transcriptome of human macrophages, matured in the presence or absence of fluticasone propionate (FP), and their ability to initiate or sustain classical activation, mimicked using acute LPS and

chronic IFN-g stimulation, respectively. We identified macrophage gene expression networks, modulated by FP long-term exposure, Downloaded from and specific patterns of IFN-g– and LPS-induced genes that were resistant, inhibited, or exacerbated by FP. Results suggest that long-term treatment with GCs weakens adaptive immune signature components of IFN-g and LPS gene profiles by downmodulating MHC class II and costimulatory molecules, but strengthens innate signature components by maintaining and increasing expression of chemokines involved in phagocyte attraction. In a mouse model of chronic obstructive pulmonary disease, GC treatment induced higher chemokine levels, and this correlated with enhanced recruitment of leukocytes. Thus, GCs do not generally suppress

macrophage effector functions, but they cause a shift in the innate–adaptive balance of the immune response, with distinct changes http://www.jimmunol.org/ in the chemokine–chemokine receptor network. The Journal of Immunology, 2014, 192: 000–000.

acrophages are innate immune cells with well-established of a developing concept: macrophage plasticity (3). Plasticity is roles in tissue homeostasis, primary response to patho- naturally the basis of macrophage heterogeneity in basal and M gens, coordination of adaptive immunity, and even wound inflammatory conditions; it is also the basis of worldwide efforts repair (1, 2). Macrophages accomplish these varied roles by adapting to treat diseases by subverting aberrant macrophage activation. their gene and protein expression programs in response to endoge- Macrophage-mediated inflammation is increasingly recognized nous and exogenous environmental cues, such as cytokines and as contributing to chronic inflammatory disorders, such as chronic pathogen-associated molecular patterns. The ability of macrophages obstructive pulmonary disease (COPD), severe asthma, rheumatoid by guest on September 29, 2021 to change their gene and protein signatures falls under the umbrella arthritis, and multiple sclerosis (4). The best-characterized macrophage activation pathway has been called classical macrophage activation, and it is triggered by *Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; †Sir William Dunn School of the recognition of pathogen-associated molecular patterns by TLRs, Pathology and Kennedy Institute of Rheumatology, University of Oxford, Oxford supported by further elicitation of IFN pathways (2). Classically ‡ OX1 3RE, United Kingdom; Department of Pharmacokinetics, Toxicology and activated macrophages produce microbicidal enzymes, such as Targeting, Groningen Research Institute for Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands; xGroningen Research Institute for Asthma inducible NO synthase, inflammatory cytokines, such as TNF, IL-6, and COPD, University Medical Center Groningen, University of Groningen, IL-1b, IL-12, and IFN-b, various chemoattractants, and matrix 9713 GZ Groningen, The Netherlands; {Department of Pathology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The metallopeptidases (MMPs). These mediators support IFN-g–mediated Netherlands; and ||Department of Pulmonology, Academic Medical Center, University Th1 responses, which are important for sustained antimicrobial of Amsterdam, 1105 AZ Amsterdam, The Netherlands activity but are deleterious in chronic diseases, because they lead to 1 M.D.B.v.d.G. and F.O.M. contributed equally to this work. tissue damage and chronic inflammation. Received for publication August 14, 2013. Accepted for publication November 25, To limit macrophage activation and restore homeostasis, endog- 2013. enous glucocorticoids (GCs) induce anti-inflammatory programs via This work was supported by a grant from the J.K. de Cock Stichting and Prof. Dirkje Postma through a Spinoza grant from the Dutch Government. the GC receptor (GCR) (5, 6). GCR agonists are prescribed for a wide range of inflammatory disorders, and their use is crucial to Address correspondence and reprint requests to Dr. Fernando O. Martinez or Dr. Jo¨rg Hamann, Kennedy Rheumatology Institute, Nuffield Department of Orthopaedics, treat diseases of the respiratory tract, especially asthma (6). Drugs Rheumatology and Musculoskeletal Sciences, Botnar Research Centre, University such as prednisolone, dexamethasone, budesonide, and fluticasone of Oxford, Windmill Road, Headington, Oxford, OX37 LD, U.K. (F.O.M.) or De- partment of Experimental Immunology, Academic Medical Center, University of propionate (FP) share great GCR specificity albeit different phar- Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (J.H.). E-mail macodynamics (7). The remarkable efficacy of GCs in suppressing addresses: [email protected] (F.O.M.) or j.hamann@amc. uva.nl (J.H.) inflammation and decreasing lymphocyte activation, proliferation, The online version of this article contains supplemental material. and survival is well appreciated. However, our knowledge of the interactions of GC and inflammatory pathways in human mac- Abbreviations used in this article: COPD, chronic obstructive pulmonary disease; FP, fluticasone propionate; GC, glucocorticoid; GCR, GC receptor; IPA, Ingenuity Path- rophages remains limited (8), and recent studies have shown that GCs ways Analysis; MDM, monocyte-derived macrophage; MEV, Multiple Experiment may have little effect in controlling inflammation in macrophage- Viewer; MMP, matrix metalloproteinase. dominated diseases, and their long-term use is associated with Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 persisting complications (9–16).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302138 2 GLUCOCORTICOID ACTION ON MACROPHAGES

In this study, we investigated from a whole genome point of view study we focused our analysis on data generated on human cells backed the effects of chronic GCR ligation on the maturation of human up by experimental evidence. Data compliant for Minimum Information macrophages and on their ability to initiate and sustain classical About a Microarray Experiment for the datasets used in this study are deposited in Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) pro- activation that is important for Th1 inflammation, dissected by files dataset GSE49852. This dataset is part of a superseries—GSE35495— acute and chronic stimulation with the TLR4 ligand LPS and IFN-g. that covers microarrays over the spectrum of macrophage activation, in- We show that long-term treatment with FP modulates classical cluding IFN-g, IL-4, IL-10, IL-13, and dexamethasone (18). activation in human inflammatory macrophage models in a com- For comparison, CEL files generated by Ehrchen et al. (19) in a microarray analysis of monocytes stimulated with FP for 16 h were summarized as plex and not solely suppressive manner by maintaining or even Robust Multiarray Averaging, and quantiles were normalized using Affy increasing the expression of chemokines involved in leukocyte package, R. recruitment, while clearly affecting their ability to initiate adaptive Protein expression analysis by flow cytometry immune response programs by decreasing expression of Ag- presenting MHC class II, costimulatory, and cell adhesion mol- For flow cytometric determination of cell surface marker expression, cells ecules. To confirm these results in vivo, we evaluated the effects of were fixed in 1% paraformaldehyde for at least 10 min at room temperature GCR ligation in a mouse model of COPD-like airways disease, and resuspended in cold FACS buffer consisting of PBS, 0.5% BSA, 10% 0.13 M (pH 7) trisodium citrate dehydrate, and 4 mM EDTA. Immuno- induced by chronic exposure to tobacco smoke. In this model, staining was performed using mouse mAbs of the IgG1 isotype, unless GCR ligation led to higher levels of pulmonary chemokines, which stated otherwise, specific for: CD32 (3D3-PE), CD45 (2D1-PerCP), CD80 correlated with more cellular infiltration, including macrophages. (L307.4-PE), CD86 (FUN-1-FITC), CD163 (GHI/61-PE), CD206 (19.2- With this global approach, we have identified pathways that remain APC; all from BD Biosciences, San Jose, CA), CD16 (LNK16-AF647), CD200R (OX108-AF647; both AbD Serotec, Oxford, U.K.), CD64 (10.1- active or worsen upon GC treatment and have confirmed these AF488), HLA-DR (IgG2a, L243-AF488; both Biolegend San Diego, CA), Downloaded from pathways to play a role in vivo as well. These results could explain CD14 (61D3-PerCP-Cy5.5; eBioscience, San Diego, CA). Mouse isotype in part the lack of efficacy of GC treatment in macrophage- control Abs were IgG1 (clone MOPC-21-PerCP-Cy5.5, PE, or APC; BD dominated disorders. Biosciences) and IgG2a (MOPC-173-AF488; BioLegend). Flow cytometric analysis was performed using a FACSCanto II (BD Biosciences) and FlowJo (Tree Star, Ashland, OR). Statistics were calculated using one-way or two- Materials and Methods way ANOVA with Bonferroni correction for multiple comparisons.

Generation of monocyte-derived macrophages http://www.jimmunol.org/ Mouse model of lung inflammation Monocytes were isolated from buffy coats of healthy blood donors (San- quin, Amsterdam, The Netherlands) by Lymphoprep (Axis-Shield, Oslo, Male C57BL/6j mice (aged 8–10 wk, n = 6 per group) were obtained from Norway) density gradient centrifugation followed by plastic adherence in Harlan (Zeist, The Netherlands) and were held under specific pathogen- IMDM (Lonza, Basel, Switzerland) containing 10% human pool serum, free conditions. The mice were fed ad libitum with standard food and 1% penicillin/streptomycin (Invitrogen, San Diego, CA), and 0.25% cipro- water and were held under specific pathogen-free conditions in groups of 6 floxacin (Bayer, Leverkusen, Germany). PBMCs were incubated, and mice per cage. The animal procedures, approved by the Institutional An- nonadherent cells were removed after 2 h incubation by extensive washing imal Care and Use Committee of the University of Groningen (application with PBS. Subsequently, adherent monocytes were detached using PBS number 2625), were performed under strict governmental and international containing 10 mM EDTA at room temperature and plated for macrophage guidelines. 5 2 maturation at a density of 1 3 10 cells/cm in IMDM containing 10% For the induction of a COPD-like phenotype, we used a model of by guest on September 29, 2021 human pool serum, 1% penicillin/streptomycin, and 0.25% ciprofloxacin. cigarette smoke–induced lung inflammation as published previously (20, 21). In short, mice were exposed nasally to mainstream smoke for 5 d/wk, Macrophage-activation assays two sessions per day for 4 mo. As a break-in period, three puffs of smoke two times from 2R1 Reference Cigarettes (University of Kentucky, Lex- For protein measurement, monocyte-derived macrophages (MDMs) were ington, KY) were administered on the first 3 d. Each day thereafter, the generated over a period of 3–7 d (at least n = 3). Where relevant, the number of puffs was increased with one puff up to 24 puffs two times. For media was supplemented with FP (100 nM unless indicated otherwise; the remainder of the 4 mo, 24 puffs two times were administered daily, GlaxoSmithKline, Brentford, U.K.), dexamethasone (100 nM unless in- which equals four cigarettes. The smoking machine was checked for the dicated otherwise; Sigma-Aldrich, St. Louis, MO), IFN-g (12.5 ng/ml; delivery of total particulate matter as described by Griffith et al. (22, 23) Sigma-Aldrich), IL-4 (50 ng/ml; Peprotech, Rocky Hill, NY), or IL-10 and calibrated before every smoking session to ensure accurate and stan- (50 ng/ml; Peprotech). For microarray analysis of the acute endotoxin dardized smoke delivery. On average, each mouse was exposed to 6.4 6 challenge model, MDMs were cultured for 7 d with FP (100 nM) or no 0.1 mg of total particulate matter per smoking session, which is compa- addition. At day 7, LPS (10 ng/ml; Salmonella minnesota R595; Alexis rable to what has been described in other studies using this setup (22, 23). Biochemicals, Lausen, Switzerland) was added 6 h prior to harvesting the Actual smoke exposure was assessed by measuring carboxyhemoglobin cells (n = 3). In the chronic inflammation model, MDMs were cultured levels. Smoking mice on average had carboxyhemoglobin levels of 23.0 6 for 5 d with FP (100 nM), IFN-g (12.5 ng/ml), both, or no addition, fol- 1.7% directly after smoking, which is common in mice directly after lowed by an additional 2 d with the same culture conditions or FP plus smoking (24). Control nonexposed mice had levels of 2.0 6 0.5%. IFN-g (n = 2). The FP profiles were extracted from n =5. Smoking mice were treated nasally with nebulized budesonide (Pul- Microarray transcriptional profiling micort Respules; a gift from AstraZeneca, Zoetermeer, The Netherlands) once per day, five times per week for 4 mo. Pulmicort Respules were diluted RNA was isolated using a Macherey-Nagel (Du¨ren, Germany) total RNA to 0.125 mg/ml, and an aerosol was delivered to a Perspex exposure isolation kit NucleoSpin II, following the manufacturer’s instructions. chamber (9 l) with a Aeroneb nebulizer (type Lab; Aerogen, Galway, Quality control, RNA labeling, hybridization onto the Illumina HumanHT- Ireland) for 85 s. Six mice were then allowed to breath in the aerosol for 12 v4 Expression BeadChips covering 31,000 annotated genes with .47,000 1 min by nasal exposure. This procedure was repeated for each batch of probes (Illumina, San Diego, CA), and data extraction was performed at six mice. The estimated dose given to the animals was 0.2 mg/mouse, which ServiceXS (Leiden, The Netherlands). is equivalent to a 500-mg dose for an adult person. Untreated smoking Expression results were normalized by quantiles, using R (Bioconductor, mice were exposed to saline in a similar manner. Seattle, WA). Genes differentially expressed were selected using ANOVA, Lung cell isolation and flow cytometric analysis with a p value, based on permutations, ,0.01 and a false discovery rate ,0.05, using Multiple Experiment Viewer (MEV; Dana-Farber Cancer Single-cell suspensions were obtained from lungs for flow cytometric Institute, Boston, MA). Principal component analysis was performed on analysis as described previously (25). Cells were quantified using a Coulter the samples using MEV. Genes were filtered for a fold $2 in at least one Counter Z1 (Coulter, Hialeah, FL). Expression of CD3, CD4, B220, GR1, condition. The transcripts were further organized with K-means clustering F4/80, and CD11c was examined with flow cytometry to determine the after median centering using MEV. Pathways and gene ontology over- frequencies of T and B lymphocytes, neutrophils, and macrophages. All representation were assessed using Ingenuity Pathways Analysis (IPA; Abs were obtained from BD Biosciences and were conjugated with fluo- Ingenuity Systems, Redwood City, CA) (17). IPA database collects data rochromes. T cells were defined by positive staining for CD3 and B cells from literature; the database covers all cell types and species, and for this by positive staining for B220. Neutrophils were defined by bright, positive The Journal of Immunology 3 staining for Gr1 and intermediate staining for CD11c. Macrophages were common carbohydrate residues found in microbes (26), a role for 4 defined by positive staining for F4/80 and CD11c. Cell populations (4 3 10 gliomedin in macrophages is not described. Secreted RNase 1 is events) were analyzed using an Epics Elite flow cytometer (Coulter), and able to regulate the metabolism of granulation tissue fibroblasts data analysis was performed using FlowJo. by increasing RNA degradation, affecting metabolic state (27), Cytokine measurement in lung homogenates which can also have consequences for the plasticity of the mac- Lung tissue was homogenized (20% w/v) in 50 mm Tris-HCl buffer rophage response. ADORA3 also is one of the most strongly up- (pH 7.5), containing 150 mm NaCl, 0.002% Tween-20, and protease in- regulated genes in smoking-dependent reprogramming of alveolar hibitor (Sigma-Aldrich), and was subsequently centrifuged at 12,000 3 g for macrophages in COPD (28). Adenosine A3 receptor activation 10 min to remove any insoluble material. The concentrations of cytokines results in anti-inflammatory effects with a reduction in IL-1b, were measured by multiplex ELISA (Lincoplex Systems, St. Charles, MO) on a Luminex 100 system using Starstation software (Applied Cytometry TNF, and IL-6, and decreased superoxide production in other Systems, Sheffield, U.K.). phagocytes (29, 30). MMP7 (matrilysin) sheds proteoglycan– chemokine complexes from the mucosal surface and confines Statistical analysis neutrophil influx to sites of acute lung injury (31). These highly Statistical analyses were done with Mann–Whitney U test unless stated regulated biomarkers may be of relevance to monitor drug ef- otherwise. ficacyinpatients. The 165 genes modulated by chronic FP exposure could be Results organized into 13 functional and regulation-related networks Chronic GCR ligation in vitro remodels human macrophage (Supplemental Table I). Upstream regulator analysis detected as transcriptome and phenotype in a time- and dose-dependent theme, as expected, GCR activation with 48 genes previously Downloaded from fashion associated with dexamethasone; also highlighting the novelty of GC analogs are especially important for the treatment of chronic these gene signatures, revealing 117 new GC-target genes in hu- diseases (6); however, the effects of chronic GCR stimulation at man macrophages (Fig. 1A). Interestingly, many of these genes the whole genome level in human macrophages are hitherto not can also be linked to IL10, TGFBR2, IL1RN, and HIF1A, pointing described. We matured human peripheral monocytes of five do- to putative autocrine subpathways, all previously associated to

nors into macrophages for 7 d in the presence of FP, which has the resolution of inflammation (Supplemental Table I). Many of the FP- http://www.jimmunol.org/ longest GC half-life in vitro (7). At day 7, we conducted whole regulated genes are also linked to IFN receptor (38 genes) and genome analysis using microarrays covering 31,000 annotated TLR4 pathways (56 genes), and the analysis suggests inhibition genes and found 165 unique genes significantly regulated by FP of the basal levels of STAT3 (21 genes), IRF1 (8 genes), and IRF7 (Table I, Supplemental Table I) (26–31). Among the genes most (8 genes), among other mediators, which can affect macrophage upregulated by FP were GLDN (gliomedin), RNASE1 (RNase 1), ability to initiate classical activation (Fig. 1A). and ADORA3 (adenosine A3 receptor), the latter was also upreg- In terms of functional pathways (Fig. 1B), FP treatment down- ulated in monocytes (Table II) (19), whereas CCL22, FCN1 (fico- regulated eight members of the MHC II complex and affected co- lin), and MMP7 were the most downregulated. Both gliomedin and agulation pathways with inhibition of prothrombotic CCL22 and ficolin are collagen domain proteins. Although ficolin can recognize increased levels of TFP1 (tissue factor pathway inhibitor) and by guest on September 29, 2021

Table I. Selection of genes modulated by chronic FP treatment in human macrophages

Symbol Gene Name Type Fold Change GLDN Gliomedin Other 15.467 RNASE1 RNase, RNase A family, 1 (pancreatic) Enzyme 9.149 ADORA3 Adenosine A3 receptor GPCR 7.422 TFPI Tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) Other 7.049 FKBP5 FK506 binding protein 5 Enzyme 5.429 IL1R2 IL-1 receptor, type II TM receptor 4.781 CD163 CD163 molecule TM receptor 3.716 HTRA1 HtrA serine peptidase 1 Peptidase 3.671 ADAMDEC1 ADAM-like, decysin 1 Peptidase 3.018 CCL8 CCL8 Cytokine 2.231 CCL7 CCL 7 Cytokine 2.222 PTGER2 PGE receptor 2 (subtype EP2) GPCR 2.152 MMP19 MMP19 Peptidase 2.057 HLA-F MHC, class I, F TM receptor 22.071 HLA-DRA MHC, class II, DR a TM receptor 22.107 CD4 CD4 molecule TM receptor 22.212 PLAU Plasminogen activator, urokinase Peptidase 22.392 CLEC4A C-type lectin domain family 4, member A TM receptor 22.417 HLA-DPB1 MHC, class II, DP b 1 TM receptor 22.417 HLA-DOA MHC, class II, DO a TM receptor 22.764 HLA-DPA1 MHC, class II, DP a 1 TM receptor 23.145 CD74 CD74 molecule, MHC, class II invariant chain TM receptor 23.185 TNFSF13B TNF (ligand) superfamily, member 13b Cytokine 24.029 HLA-DRB4 MHC, class II, DR b 4 TM receptor 24.079 MMP9 MMP9 (gelatinase B) Peptidase 24.871 HLA-DQA1 MHC, class II, DQ a 1 TM receptor 25.158 MMP7 MMP7 (matrilysin) Peptidase 25.686 FCN1 Ficolin (collagen/fibrinogen domain containing) 1 Other 26.933 CCL22 CCL22 Cytokine 213.365 GPCR, G protein-coupled receptor; MMP, matrix metallopeptidase; TM, transmembrane. 4 GLUCOCORTICOID ACTION ON MACROPHAGES

Table II. Thirty-five genes modulated by FP treatment in human monocytes (16 h) and macrophages (5 d)

Symbol Gene Name Type Fold Change Monocytes Fold Change Macrophages ADM Adrenomedullin Other 2.1 3.7 ADORA3 Adenosine A3 receptor GPCR 4.7 7.4 ALOX15B Arachidonate 15-lipoxygenase, type B Enzyme 4.8 2.7 C1QB Complement component 1, q subcomponent, B chain Other 3.1 2.2 CD163 CD163 molecule TM receptor 2.4 3.7 DDIT4 DNA-damage-inducible transcript 4 Other 5.1 4.0 FBLN5 Fibulin 5 Other 2.2 4.5 FKBP5 FK506 binding protein 5 Enzyme 6.8 5.4 HPGD hydroxypg dehydrogenase 15-(NAD) Enzyme 2.2 3.1 HTRA1 HtrA serine peptidase 1 Peptidase 3.2 3.7 IL1R2 IL-1 receptor, type II TM receptor 12.5 4.8 IRS2 Insulin receptor substrate 2 Enzyme 3.8 2.4 MERTK c-mer proto-oncogene tyrosine kinase Kinase 4.3 3.0 METTL7A Methyltransferase like 7A Other 2.3 2.4 MFGE8 Milk fat globule-EGF factor 8 protein Other 2.4 6.4 PDK4 Pyruvate dehydrogenase kinase, isozyme 4 Kinase 4.8 3.1 RNASE1 RNase, RNase A family, 1 (pancreatic) Enzyme 2.2 9.1 SERPINE1 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator Other 2.7 2.6 inhibitor type 1), member 1 Downloaded from SESN1 Sestrin 1 Other 4.6 2.6 SLA Src-like-adaptor Other 2.1 2.2 SLC16A6 Solute carrier family 16, member 6 (monocarboxylic acid Transporter 2.9 3.4 transporter 7) SLC1A3 Solute carrier family 1 (glial high affinity glutamate transporter), Transporter 5.2 2.2 member 3 SRPX Sushi-repeat containing protein, X-linked Other 5.3 5.9 http://www.jimmunol.org/ TBC1D16 TBC1 domain family, member 16 Other 7.7 2.6 TCN2 Transcobalamin II Transporter 2.9 2.1 TFPI Tissue factor pathway inhibitor (lipoprotein-associated Other 4.1 7.0 coagulation inhibitor) TPST1 Tyrosylprotein sulfotransferase 1 Enzyme 4.5 3.0 TSC22D3 TSC22 domain family, member 3 Other 2.4 4.1 VSIG4 V-set and Ig domain containing 4 Other 11.2 6.0 ZCCHC6 Zinc finger, CCHC domain containing 6 Enzyme 2.6 2.3 CHI3L1 Chitinase 3-like 1 (cartilage glycoprotein-39) Enzyme 24.0 22.4 FCN1 Ficolin (collagen/fibrinogen domain containing) 1 Other 22.3 26.9

IFIT1 IFN-induced protein with tetratricopeptide repeats 1 Other 22.1 22.3 by guest on September 29, 2021 MMP9 MMP 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) Peptidase 22.7 24.9 RARRES1 Retinoic acid receptor responder (tazarotene induced) 1 Other 22.1 24.8 Results of this study were compared with a previous study by Ehrchen et al. (18). Genes modulated with a fold $ 2 and t test p value , 0.05 in both datasets were considered for the comparison. GPCR, G protein-coupled receptor; TM, transmembrane.

SERPINF1 (serine peptidase inhibitor F1). The synthesis, transport, response at 100 nM—levels achieved in the tissue of patients in- or recognition of lipids also appears decreased with downregula- haling FP (33). tion of ABCG1, ACAT2, CYP27B1, HSD11B1, PPARGC1A, and The effect on these four genes, CD163, HLA-DR, CD64, and PTGER2. The detailed analysis alsoshowedstrongchangesin CD86, was further corroborated in a time course. We cultured molecules for the recruitment of phagocytes (e.g., upregulation of monocytes with FP or in medium alone for 3, 5, or 7 d, followed the chemokines CCL7 and CCL8, downregulation of CCL22) and by flow cytometric analysis, and we found that the pattern of changes in the phagocytosis capacity of macrophages, exemplified regulation for every gene was slightly different, suggesting dif- by increased expression of ADORA3, BIN1, CAMP, MERTK, ferential regulation from gene expression to protein modification MFGE8, SERPINE1, and THBS1, and corresponding with previous and expression. FP increased the expression of CD163 until day 3, reports (32). when it reached a plateau, whereas HLA-DR followed a bell- Gene expression remodeling by FP in human macrophages is of shaped curve. The regulation was subtle for CD64 and CD86 functional relevance, and the effects observed at the transcriptome (Fig. 1D). GCR involvement was confirmed in all experiments level translated into phenotype changes as demonstrated by flow because dexamethasone had comparable effects to those of FP, cytometric analysis of CD163 and HLA-DR protein expression, with the only difference being a slight superiority of FP in the which were upregulated and slightly downregulated respectively dose and time response (Fig. 1C–D). (Fig. 1C). Given the suggestion that IRF3, IRF7, and STAT3 basal activity may be diminished, we also investigated the expression Acute endotoxin challenge in chronically GC-treated of two important inflammation-mediated molecules regulated by macrophages reveals complex interplay with innate and TLR4 and IFN pathways, namely CD64 and CD86. The first is adaptive immune activation responsible for immune complex responses, and the second is an Classical macrophage activation involves the exposure to patho- important costimulatory signal for T cell activation. For both gens, which induces IFN-b and IL-12 expression that primes Th1 molecules, we could confirm protein level changes despite a lack cells to undergo expansion, while activating strong microbicidal of regulation at the mRNA level. The kinetics of FP effects on programs in macrophages (2). This pathogen stimulation has also CD64 and CD86 protein expression were comparable to CD163 been termed “innate activation” and can be recapitulated by TLR4 and HLA-DR, with clearly visible effects at 10 nM and a saturated triggering (34). To investigate the effects of GCR ligation during The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Remodeling of the transcriptome of human macrophages by chronic FP treatment. Human peripheral monocytes from five donors were matured into macrophages in the presence or absence of FP. At day 7, RNA was extracted for transcriptome analyses using Illumina microarray. (A) Interactome analysis of genes modulated by FP performed using IPA. Genes appear pseudocolored according to the regulation by FP: red = upregulated, green = downregulated. Genes marked with an asterisk indicate that multiple identifiers in the dataset file map to a single gene in the IPA Global Molecular Network. The selection of the interactome shown here highlights that many FP-regulated genes have not been associated with dexamethasone regulation previously in human macrophages or other cells, which are visible because of the lack of connecting lines, and shows that a great proportion of the genes have been associated previously with LPS and IFN signaling. (B) Selection of IPA canonical pathways, which are modulated by FP in human macrophages, corroborates FP effects on important immune pathways, such as Ag presentation, LPS response, and IFN response (Supplemental Table I). (C) Dose response (upper panels) and time kinetics (lower panels) of the regulation of selected surface markers in peripheral blood monocytes, stimulated for 5 d with FP (dark blue lines) or dexamethasone (light blue lines) at a concentration ranging from 0.1–1000 nM and for 3, 5, and 7 d with 100 nM FP (dark blue lines), 100 nM dexamethasone, or left untreated (gray lines). Protein expression, measured using flow cytometry, is provided relative to isotype control. **p , 0.01, ***p , 0.001. innate activation, we defined the transcriptome of MDMs from consist of triplicates of each condition. The largest difference is three donors, matured for 7 d with or without FP, after challenging seen in LPS-treated and untreated macrophages; however, a clear them for 6 h—concurring with an inflammatory peak (35)—with distinction remains between untreated and FP-treated cells. A total 10 ng/ml LPS (Fig. 2A). Principal component analysis (Fig. 2B) of 1685 transcripts representing 1500 genes were significantly re- showed that FP-treated macrophages are able to respond to LPS gulated in the data set (Fig. 2C). Of 630 genes upregulated or but only reach an intermediate state, with a significant shift toward downregulated by LPS, 193 were significantly influenced by FP, an FP activation-like state, represented by negative movements in which corresponds to observations in mouse peritoneal macro- the y-axis. The graph clustered 12 samples in four groups, which phages exposed for 6 h to LPS and dexamethasone (36). More- 6 GLUCOCORTICOID ACTION ON MACROPHAGES Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Complex changes in gene expression induced by LPS in FP-polarized macrophages. (A) Experimental design: Human peripheral monocytes from three donors were matured into macrophages in the presence (red bar) or absence (gray bar) of FP. Prior to RNA extraction for transcriptome analyses using the Illumina microarray at day 7, cells were stimulated for 6 h with LPS (green bars) as indicated. (B) Principal component analysis showed remote locations for medium-, FP-, LPS-, and FP+LPS-treated MDMs, representing distinct phenotypes. Components 1 and 2, comprising 72.8% of the total variance, were used to create the graph. (C) Venn diagrams providing numbers of transcripts upregulated or downregulated greater than twofold over untreated by FP, LPS, or FP→LPS treatment. (D) Median-centered K-means clustering of genes significantly regulated in the dataset revealed six distinctive expression patterns. The y-axis indicates divergence from median values with intensity of gene expression illustrated in colors: low (black), intermediate (blue to green to yellow), high (red). Cluster 1 consists of 396 genes that are upregulated by LPS and fairly resistant to FP. Cluster 2 includes 323 genes that are downregulated by LPS and fairly resistant to FP. Cluster 3 shows 185 genes that are upregulated by LPS and inhibited by FP. In cluster 4, 190 genes were upregulated by LPS in FP-primed MDMs. Alternatively, cluster 5 contains 238 genes that are downregulated by LPS stimulation in FP-primed cells. Finally, cluster 6 shows 168 genes that are induced by FP and mildly counteracted by LPS. Gene clusters were assessed for pathways and upstream regulators using IPA. For details see Supplemental Table II. (E) Hierarchical clustering heat map of Ag presentation–related molecules and chemokines. over, 409 genes, not significantly changed by LPS or FP alone, MyD88 pathway. This hypothesis is strengthened by the fact that were regulated by FP plus LPS. A summary of genes regulated by the IL-1B increase was accompanied by increases in MyD88- the combination of FP and LPS is provided in Table III. regulated CCL20, CCL8, IL-8, CXCL1, CCL2, and CCL17, whereas The genes regulated in the dataset could be divided into six the CXCL10 decrease was accompanied by downregulation of IFN- distinct major gene patterns using K-median clustering (Fig. 2D–E; dependent IFIT2, IFIT1, IFIT3, CXCL9, CD80, and IFIH1. Cluster for comprehensive details about molecular pathways, gene net- 2 contained 323 genes downregulated in response to LPS, regard- works, and upstream regulators, see Supplemental Table II). Cluster 1 less of the presence of FP. This cluster comprised macrophage identified 396 genes modulated by LPS and not FP, and the com- markers, and upstream regulator analysis revealed possible inhi- bination marginally decreased levels for 25% of the genes. This bition of signaling by receptors for the lineage-determining factors cluster included inflammatory cytokines such as IL-1B, TNF, IL-6, GM-CSF and M-CSF, downregulating CD68, DOK2, GDF15, and IL-15. Upstream regulators analysis showed clear links with IFNGR1, PCNA, and PLAU. MyD88 (modulates 45 genes), IFN (modulates . 70 genes), and Cluster 3 constituted the only LPS-dependent pathway com- IL-6 pathways (modulates 51 genes). In this study, FP pretreatment pletely abrogated by FP treatment. The cluster contained 185 genes decreased CXCL10 and increased IL-1B, suggesting that FP neg- enriched in lymphocyte activation molecules such as CD80, CD83, atively affected the IFN pathway while positively affecting the CD86, IL10, IL15, IL18, CD274, and TNFSF13B. Interestingly, The Journal of Immunology 7

Table III. Selection of genes regulated by the combination of FP and LPS

Fold Change

Symbol Gene Name Type FP LPS+FP LPS CCL20 CCL20 Cytokine 1.346 85.178 20.286 IL1B IL-1b Cytokine 21.112 83.968 36.773 CCL4L2 CCL4-like 1 Other 2.014 67.507 47.831 CCL8 CCL8 Cytokine 2.189 66.669 31.013 SERPINB2 Serpin peptidase inhibitor, clade B (OVA), member 2 Other 1.288 43.689 5.85 SLAMF1 Signaling lymphocytic activation molecule family member 1 TM receptor 1.195 39.851 27.412 EBI3 EBV induced 3 Cytokine 2.5 32.823 19.231 IL8 IL-8 Cytokine 1.195 28.994 5.7 ANGPTL4 Angiopoietin-like 4 Other 1.328 26.979 6.613 CFB Complement factor B Peptidase 21.107 25.849 17.81 NAMPT Nicotinamide phosphoribosyltransferase Cytokine 1.046 21.729 12.559 MT1A Metallothionein 1A Other 4.725 19.824 5.709 MT2A Metallothionein 2A Other 5.367 15.328 6.632 IL1A IL-1a Cytokine 21.01 11.533 1.32 MT13 Metallothionein 13 Other 1.769 10.04 1.397 CXCL1 CXCL1 Cytokine 21.038 8.068 2.074

TIMP1 TIMP metallopeptidase inhibitor 1 Other 2.92 6.715 1.477 Downloaded from CCL2 CCL2 Cytokine 2.103 6.574 1.645 CCL7 CCL7 Cytokine 1.787 6.001 1.826 CXCL2 CXCL2 Cytokine 21.202 4.35 1.887 CXCL5 CXCL5 Cytokine 1.008 3.489 1.145 MDFIC MyoD family inhibitor domain containing Other 21.026 22.075 1.453 IRF8 IFN regulatory factor 8 Transcription regulator 21.079 22.104 1.438 SGK3 Serum/glucocorticoid regulated kinase family, member 3 Kinase 1.285 22.155 1.027 ARHGAP25 Rho GTPase activating protein 25 Other 21.361 22.175 1.017 http://www.jimmunol.org/ ADAM9 ADAM metallopeptidase domain 9 Peptidase 1.021 22.403 1.023 HLA-DPA1 MHC, class II, DP a 1 TM receptor 22.466 22.491 1.012 TPM3 Tropomyosin 3 Other 21.228 22.905 1.25 CCL22 CCL22 Cytokine 24.953 23.135 1.022 TM, Transmembrane. dexamethasone now appeared as upstream regulator of at least were kept stable or switched from FP or IFN-g, respectively, to FP 43 genes, which are also linked to IFN-dependent pathways, more plus IFN-g (Fig. 3A). than to MyD88. Cluster 4 comprised 190 genes upregulated by Principal component analysis (Fig. 3B) showed remote locations by guest on September 29, 2021 LPS stimulation in FP-treated MDM exclusively. Upstream anal- for medium-, FP-, IFN-g–, and FP plus IFN-g–treated macro- ysis showed that at least 50 of these genes have been associated phages, representing distinct phenotypes. Cells cultured with FP with dexamethasone treatment. This cluster is enriched in metal- or IFN-g for 5 d, followed by the combination of both stimuli for lothioneins (e.g., MT1M, MT1A, MT1E, MT1G, MT13, MT1F), 2 d, developed intermediate yet distinct gene expression profiles which participate in stress responses and apoptosis modulation, thatcloselyresembledtheprofileofcellsexposedfor7dtothe and the chemokines CXCL1, CXCL2, CXCL5, CCL2, CCL7, IL- combination of FP plus IFN-g. The graph shows six distinct clus- 1A, and IL-8, which participate in cell recruitment. Cluster 5 com- ters, correlating with the experimental conditions and comprising prised 238 genes expressed in resting MDM, downregulated by the donor duplicates. FP and IFN-g clusters are highly diverse from combination of FP and LPS. This group strongly associated with each other. The FP-plus-IFN–g cluster is equally distant to FP MHC class II–mediated Ag presentation (e.g., HLA-DMA, HLA- and IFN-g alone. Samples in which stimuli were changed after DMB, HLA-PA1, HLA-PB1, HLA-DQA1, HLA-DRA, HLA-DRB3, 5 d clustered at intermediate positions; 1327 transcripts, rep- HLA-DRB4, CD74) and appeared to be cluster 2 dependent on resenting 1119 genes, were significantly regulated in the dataset M-CSF and GM-CSF. Finally, cluster 6 contained 168 genes in- (Fig. 3C). Of the 362 genes regulated by IFN-g, 145 were sig- duced by LPS and further enhanced by FP, with overrepresen- nificantly affected by FP; 400 genes not significantly changed by tation of complement system pathways (e.g., C1QA, C1QB, C1QC, IFN-g or FP alone were regulated by FP plus IFN-g. A summary C3AR1, C5AR1) and cytokine recognition (e.g., IL1R1, IL1R2, of genes regulated by the combination of FP and IFN-g is pro- IL13RA1, IL27RA). Dexamethasone, retinoic acid, and PPARD vided in Table IV. appeared as major regulators. The regulated genes could be divided into six distinct clusters by K-means clustering (Fig. 3D–E; for comprehensive details about GC/IFN receptor–mediated interplay in gene expression the genes, molecular pathways, gene networks, and upstream highlights partial plasticity affecting chronic events in regulators in each cluster, see Supplemental Table III). Cluster 1 macrophage classical activation and 2 comprised 87 and 97 genes, respectively, regulated pri- LPS stimulation provided clues about how FP-treated macrophages marily by FP and overlapping largely with the genes regulated by initiate altered classical activation programs, with the IFN path- FP alone that we described in the first section of Results and in ways being the most affected. Next, we investigated how FP Supplemental Table I. IFN-g oppositely regulated these genes, treatment affected long-term IFN-g responses, by treating before, in combination and alone, which adds to the theme of opposed during, or after IFN-g challenge, to mimic treatment in chronic crosstalk between GCR and TLR4 and IFN receptor pathways. inflammatory settings. We analyzed mRNA expression profiles of Cluster 3 represented 285 genes induced by IFN-g, refractory to MDMs of two donors cultured for 5 d with medium alone, FP, FP treatment in all variations and slightly exacerbated in the IFN-g, or both, followed by 2 d during which culture conditions cotreatment. These genes support Th1 attraction and activation 8 GLUCOCORTICOID ACTION ON MACROPHAGES Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Distinct yet reversible effects of FP and IFN-g on gene expression in macrophages. (A) Experimental design: Human peripheral monocytes from two donors were treated with FP (red bar), IFN-g (blue bar), both (red and blue bar), or no stimulus (gray bar). After 5 d, MDM were exposed to the same or changed stimuli for an additional 2 d as indicated, after which RNA was extracted for transcriptome analyses using Illumina microarray. (B) Principal component analysis showed remote locations for medium-, FP-, IFN-g–, and FP+IFN-g–treated cells, representing distinct MDM phenotypes. Components 1 and 2, comprising 79.8% of the total variance, were used to create the graph. (C) Venn diagrams providing numbers of transcripts up- regulated or downregulated greater than twofold over untreated by FP, IFN-g, or FP+IFN-g treatment. (D) Median-centered K-means clustering of the significantly regulated genes in the dataset revealed six distinctive expression patterns. The y-axis indicates divergence from median values with intensity of gene expression illustrated in colors: low (black), intermediate (blue to green to yellow), high (red). Cluster 1 consists of 87 genes upregulated by FP. Cluster 2 includes 97 genes downregulated by FP. Most genes in cluster 1 and 2 are oppositely regulated by IFN-g. The 285 genes in cluster 3 are up- regulated by IFN-g and are resistant to FP-mediated downregulation. Clusters 4 and 5 comprise 305 and 128 genes, respectively, which showed an augmented effect with long-term exposure to the combination of FP+IFN-g. Cluster 6 comprises 217 genes downregulated only in FP-treated MDMs. Gene clusters were assessed for pathways and upstream regulators using IPA. For details see Supplemental Table III. (E) Hierarchical clustering heat map of Ag presentation–related molecules and chemokines. with inflammatory mediators, such as TNF, the chemokines CXCL9 proteins (APOC1, APOC2, CD36, FABP3, FABP4, FABP5, and CXCL10, and the MHC class I–mediated Ag-presentation FABP5L2, FABP5L3) were affected. The scavenger receptor cluster HLA-A, HLA-C, HLA-E, HLA-F, and HLA-G. The down- CD36 (decreased by FP) is also involved in bacterial phagocytosis regulation of MHC class II in TLR stimulation and preservation of during pneumococcal pneumonia (39). Other functional pathways, class I activation upon IFN receptor stimulation suggests that FP which appear decreased, are cell adhesion (ITGAE, ITGAX, spares antiviral and not antibacterial responses, which may con- ITGA3, ITGB, ITGB1, ITGB5, ITGB1BP1) and chemotaxis with tribute to the enhanced incidence of pneumonia in patients with downregulation of CCL2, CCL7, and CCL3. Cluster 5 is the op- COPD treated long term with FP (9, 37, 38). Long-term exposure posite of cluster 4, with 128 genes upregulated by long-term ex- to the combination of FP plus IFN-g exclusively downregulated posure to FP plus IFN-g. This cluster is related to the inflammatory 305 genes, as shown in cluster 4. PPRG was downregulated 2.5- response; its predicted upstream regulators include LPS, dexa- fold in this cluster, and genes that depend on it such as lipid-binding methasone, and IFN over MyD88. Among the genes included are The Journal of Immunology 9

Table IV. Selection of genes regulated by the combination of FP and IFN-g

Fold Change

Symbol Gene Name Type FP IFN-g+FP IFN-g S100A8 S100 calcium binding protein A8 Other 1.127 18.525 2.874 RNASE2 RNase, RNase A family, 2 (liver, eosinophil-derived neurotoxin) Enzyme 1.019 6.906 1.057 SERPINA1 Serpin peptidase inhibitor, clade A (a-1 antiproteinase, antitrypsin), Other 21.274 6.770 4.578 member 1 PLAC8 Placenta-specific 8 Other 21.086 6.457 1.315 ALOX5 Arachidonate 5-lipoxygenase Enzyme 1.047 5.029 1.771 SIGLEC10 Sialic acid binding Ig-like lectin 10 Other 1.135 4.986 1.260 ITGB7 Integrin, b 7 TM receptor 21.369 4.954 2.597 OLR1 Oxidized low density lipoprotein (lectin-like) receptor 1 TM receptor 1.983 4.356 1.093 MUC1 Mucin 1, cell surface associated Transcription regulator 21.183 4.325 1.321 OLFML3 Olfactomedin-like 3 Other 21.261 4.123 1.127 CLEC12A C-type lectin domain family 12, member A Other 21.328 4.013 2.541 GAPT GRB2-binding adaptor protein, transmembrane Other 21.156 3.195 1.146 CSF2RA CSF 2 receptor, a, low-affinity (granulocyte-macrophage) TM receptor 1.089 2.686 1.249 FCN1 Ficolin (collagen/fibrinogen domain containing) 1 Other 27.093 21.264 1.795 CCL7 CCL7 Cytokine 2.213 22.645 21.330

CCL2 CCL2 Cytokine 1.498 23.881 2.031 Downloaded from SEPP1 Selenoprotein P, plasma, 1 Other 1.671 25.247 21.860 CD36 CD36 molecule (thrombospondin receptor) TM receptor 1.894 26.550 21.607 CD83 CD83 molecule Other 21.034 27.126 21.861 CHIT1 Chitinase 1 (chitotriosidase) Enzyme 21.622 211.295 24.330 IL1RN IL-1 receptor antagonist Cytokine 21.256 212.333 23.028 EMP1 Epithelial membrane protein 1 Other 1.319 216.277 23.414 LPL Lipoprotein lipase Enzyme 1.804 221.094 23.944 AQP9 Aquaporin 9 Transporter 1.115 221.650 23.952 http://www.jimmunol.org/ SPP1 Secreted phosphoprotein 1 Cytokine 1.606 230.607 22.708 FABP4 Fatty acid binding protein 4, adipocyte Transporter 1.457 246.129 22.236 TM4SF19 TM4 L six family member 19 Other 21.164 260.890 22.320 MMP9 MMP9 (gelatinase B) Peptidase 24.934 2132.508 23.313 MMP, Matrix metallopeptidase; TM, transmembrane

complement members C1QC, C1QB, and C5, the multifunctional GCs. Importantly, GCs seem to alter the response to otherwise pair S100A8 and S100A9, and the chemokine receptors for CCL2 polarizing cytokines. by guest on September 29, 2021 and CXCL12, CCR2 and CXCR4, respectively. Cluster 6 was similar to cluster 4 and contained 217 genes, downregulated GC treatment enhances immune cell recruitment in a mouse only in FP-treated macrophages. Forty-three of these genes have model of COPD-like lung inflammation previously been associated with TLR4 stimulation and are generally To investigate the effects of GC treatment in vivo, we used a mouse IFN independent; MMP7, MMP9, CCL2, and PLAU are downreg- model of cigarette smoke-induced lung inflammation. It consisted ulated and add to a vast list of inflammatory genes in the cluster. of 4 mo of tobacco smoke exposure to generate COPD-like fea- To investigate macrophage plasticity further at a protein level, tures, such as airway remodeling, mild emphysema, and chronic we compared FP effects on IFN-g and two other important cytokines inflammation (20, 21). This model differs from the human disease, for macrophages—alternatively activating IL-4 and anti-inflammatory but is useful to investigate cellular and molecular mechanisms IL-10—by following the behavior of model genes. Of the 10 underlying the development and progression of COPD. The GC molecules with a reported differential expression in human mac- analog used for this experiment was budesonide, which has an rophages (40–42) that we validated in detail (Fig. 4A), three were intermediate pharmacodynamics activity compared with flutica- studied further, namely CD64, CD163, and CD206 (Fig. 4B). sone and dexamethasone (7); it is retained to a greater extent than CD163 is an interesting marker because it was induced by FP and FP in the mucosa of the airways (43) and, unlike FP, shows no IL-10, but not modulated by IFN-g or IL-4. IL-10 and FP coop- differences in plasma level in healthy or diseased groups (44). erated to increase CD163 expression even further. However, IL-10 As described previously, smoking induced a Th17 type of airway is not that similar to FP because it increased CD64 expression, inflammation, characterized by accumulation of neutrophils, mac- which was decreased by FP. Regulation of CD206 (mannose re- rophages, and B cells, and increased levels of IL-17, IL-6, GM-CSF, ceptor), which is important for complications such as fibrosis (4), G-CSF, and CCL2 (21). After GC treatment, total macrophages and depended on the cytokine context. FP appeared to leave IL-4– B cells were significantly higher in lung tissue of the smoking mice driven CD206 expression unaffected, but opened possibilities for as compared with saline-treated smoking animals (Fig. 5A). The cytokine cooperation. Combined treatment with FP and IFN-g transcriptome study in human macrophages predicted that GCR further increased IL-4–mediated CD206 expression, whereas IFN-g ligation would affect chemokine expression. Despite interspecies does not induce CD206. Moreover, FP with IFN-g and IL-10 en- differences, we found that GC treatment affected the expression of abled CD206 expression, whereas the separate treatments had no key chemokines in this model, which correlated with higher overall effect. In conclusion, FP effects on macrophage-polarizing cytokines leukocyte numbers in the lungs (Fig. 5B). In lung tissue, these are not simply inhibitory. Through model genes, it is possible chemokines can be produced by many cell types, and they have to appreciate the many phenotypes elicited by treatment with a pleiotropic effect. Given the correlation with macrophages, 10 GLUCOCORTICOID ACTION ON MACROPHAGES Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. FP affects the interaction between classical and alternative macrophage activation. Peripheral blood monocytes were cultured for 5 d in the presence of 12.5 ng/ml IFN-g, 50 ng/ml IL-4, 50 ng/ml IL-10, 100 nM FP, or combinations thereof, followed by flow cytometric assessment of the ex- pression of surface proteins. (A) Macrophages that are classically activated with IFN-g upregulate CD64; macrophages that are alternatively activated with IL-4 upregulate HLA-DR, CD86, CD200R, and CD206; and regulatory macrophages that are generated in the presence of IL-10 upregulate CD14, CD16, CD64, and CD163. Macrophages that are generated with FP upregulate CD163. Expression of CD32 and CD80 was not affected by the investigated stimuli (data not shown). (B) Combinations of IFN-g, IL-4, IL-10, and FP regulate expression of CD64, CD163, and CD206 in a complex manner. FP neutralizes induction of CD64 by IFN-g and IL-10, but enhances expression of CD163 by IL-10. Notably, FP enabled expression of CD206, even when none of the individual stimuli (IFN-g and IL-10) had an effect by themselves. Protein expression is provided relative to isotype control. Underscores in (B) indicate a significant difference in expression caused by a combination compared with the respective underlined stimulus. *p , 0.05, **p , 0.01, ***p , 0.001. we consider this a significant trend, which will require further the fact that FP modulates genes in an acute and a chronic fashion. investigation. These differences add to the emergent concept of intermodel and interspecies differences found in the response of macrophages to Discussion other immune relevant stimuli, such as GM-CSF, M-CSF, LPS, The role of macrophages in the etiology and pathology of chronic and IL-4 (18, 35, 45). The genes already associated with FP in inflammatory disorders is being recognized increasingly (4). Many monocytes at 16 h, also modulated after 7 d of FP, included CD163, of these diseases are treated with GCs, but recent studies have MERTK, and MFGE8. Early transient genes modulated after 16 h, shown that GCs can have little effect in controlling inflammation but not after 7 d, included the GC-associated formyl peptide in macrophage-dominated diseases (9, 16). This lack of efficacy in receptor 1 and IL-10 (19, 46). Induction of IL-10 by GCs in diseases, like COPD, might be explained by our finding that GCs monocytes but not MDMs is in line with earlier findings (47). The do not generally suppress macrophage effector functions, but cause a shift in the innate–adaptive balance of immune responses. chronic FP transcriptome comprises functionally relevant genes, FP significantly altered the expression of 165 genes in macro- such as TFPI, CAMP (cathelicidin antimicrobial peptide), THBS1 phages matured for 7 d. When comparing the effects of long-term (thrombospondin 1), and the transcription factor KLF9, which FP exposure on gene expression with the profile obtained by binds GC-box elements and has not been linked to FP treatment in Ehrchen et al. (19) in monocytes stimulated for 16 h with FP, we macrophages. We conclude that FP regulates genes in a time- found a modest correlation (r2 = 0.3850; p , 0.0001), pointing to dependent fashion; therefore, chronic stimulation of macrophages The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/

FIGURE 5. GC enhances leukocyte recruitment and chemokine expression in a mouse model of COPD-like lung inflammation. Mice (n = 6 per group) were exposed to cigarette smoke 5 d per week for 4 mo and treated five times per week with either budesonide (closed symbols) or saline (open symbols). by guest on September 29, 2021 Lung leukocyte infiltration and expression of cytokines and chemokines were measured with flow cytometry and ELISA, respectively. (A) GC treatment led to significantly higher numbers of macrophages and B lymphocytes, being present in lung tissue of smoking mice. A similar trend was observed for neutrophils and T lymphocytes. (B) GC treatment led to significantly higher levels of key chemokines in lung tissue of smoking mice. These differences were not found for IFN-g, IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, and GM-CSF. *p , 0.05, **p , 0.01, as tested with Mann–Whitney U test. might not be directly translatable to studies in circulating tiation of adaptive immune responses, whereas innate immune monocytes. responses can develop and might be enhanced by FP (51). The transcriptome analysis revealed the expected overlap of the Although GCs differentially regulate various immune gene FP profiles with dexamethasone, methylprednisolone, prednisolone, signatures (52, 53), the regulation of chemokines by FP emerged and suggested a strong negative relationship between GCR gene as a strong theme; this recapitulates previously identified trends in signatures and TLR4 and IFN receptor pathways. Anticipating that monocytes (19). FP treatment significantly augmented LPS- the true effects of GCR ligation on macrophages are better observed induced IL-8 (CXCL8), CCL2, CCL8, and CCL20 expression, in inflammatory conditions, we explored the consequences of FP and moderately enhanced CXCL1, CXCL2, CXCL5, CCL3L1, treatment on the macrophage transcriptome upon LPS-mediated or and CCL7 expression, whereas it inhibited LPS-induced upregu- IFN-g–mediated activation. As expected, FP massively affected lation of CXCL9, CXCL10, CCL19, CCL22, and (moderately) gene expression induced by LPS or IFN-g (36, 48). Transcriptome CXCL11. These chemokines activate distinct sets of receptors. analysis revealed distinct behavioral patterns, of which the most The inhibited chemokines bind CXCR3A, CXCR3B, CCR7, and interesting ones comprised induced genes resistant to FP, inhibited CCR4, which are mainly expressed on T cells; the overexpressed by FP, or exacerbated upon FP treatment. Notably, IFN-g–induced chemokines bind CXCR1, CXCR2, CCR1, CCR2, CCR3, CCR5, genes related to the development of an innate immune response were and CCR6, which are primarily found on monocytes and gran- fairly resistant to FP. In contrast, FP counteracted IFN-g–induced ulocytes, and only some T cells (54). The dysregulation of the genes associated with lymphocyte activation. Illustrative is the ability chemokine system by GCR ligation in macrophages is gaining of GCs to repress Ag-presentation pathways, which is well estab- interest. Recently, Kent et al. (55) studied the transcriptome of COPD lished (49, 50). We found that FP suppresses both MHC class I and macrophages exposed to LPS and dexamethasone, and found 23 class II genes. Whereas MHC class II genes remained suppressed by genes that were insensitive to GCR ligation, including IL-1b, IL- FP upon IFN-g stimulation, genes encoding MHC class I molecules 18, and CCL5. Importantly, the chromosome 4 chemokine cluster were upregulated by IFN-g, regardless of the presence of FP. The members CXCL1, CXCL2, CXCL3, and CXCL8 were all GC- decreased expression of MHC class II is likely to hamper the ini- resistant in our and their study (55). 12 GLUCOCORTICOID ACTION ON MACROPHAGES

The contribution of resident versus inflammatory macrophages 11. Hakim, A., I. M. Adcock, and O. S. Usmani. 2012. Corticosteroid resistance and novel anti-inflammatory therapies in chronic obstructive pulmonary disease: is not clear in COPD and other lung diseases, and mimicking the current evidence and future direction. Drugs 72: 1299–1312. milieu of such a complex organ is not straightforward. Studies done 12. Jen, R., S. I. Rennard, and D. D. Sin. 2012. Effects of inhaled corticosteroids on on MDMs are important, although it cannot be excluded that these airway inflammation in chronic obstructive pulmonary disease: a systematic review and meta-analysis. Int. J. Chron. Obstruct. Pulmon. Dis. 7: 587–595. profiles may be more representative of inflammatory, newly recruited 13. Baschant, U., N. E. Lane, and J. Tuckermann. 2012. The multiple facets of monocytes, rather than resident tissue macrophages, nor the fact that glucocorticoid action in rheumatoid arthritis. Nat. Rev. Rheumatol. 8: 645–655. in the lung environment GCs may act differently. Furthermore, dis- 14. Gomes, J. A., R. D. Stevens, J. J. Lewin, III, M. A. Mirski, and A. Bhardwaj. 2005. Glucocorticoid therapy in neurologic critical care. Crit. Care Med. 33: 1214–1224. eased human tissue has limited possibilities when studying immune 15. Ciccone, A., S. Beretta, F. Brusaferri, I. Galea, A. Protti, and C. Spreafico. 2008. responses. To investigate the relevance of our in vitro results in Corticosteroids for the long-term treatment in multiple sclerosis. Cochrane a more physiologic manner, we used a mouse model of COPD, in Database Syst. Rev. (1):CD006264. 16. Sorrells, S. F., J. R. Caso, C. D. Munhoz, and R. M. Sapolsky. 2009. The stressed which resident and inflammatory macrophages are known to play CNS: when glucocorticoids aggravate inflammation. Neuron 64: 33–39. a role (21). In this model, we found sustained increases of mac- 17. Martinez, F. O. 2012. Analysis of gene expression and gene silencing in human macrophages. Curr. Protoc. Immunol. Chapter 14: Unit 23. rophages and a set of chemokines and cytokines upon GC treat- 18. Martinez, F. O., L. Helming, R. Milde, A. Varin, B. N. Melgert, C. Draijer, ment with budesonide. The increased chemokine levels correlated B. Thomas, M. Fabbri, A. Crawshaw, L. P. Ho, et al. 2013. Genetic programs with enhanced leukocyte numbers, supporting the predictions of the expressed in resting and IL-4 alternatively activated mouse and human macro- phages: similarities and differences. Blood 121: e57–e69. chemokine–chemokine receptor network. Previously, in two mouse 19. Ehrchen, J., L. Steinmu¨ller, K. Barczyk, K. Tenbrock, W. Nacken, M. Eisenacher, studies using either LPS or Mycoplasma pneumonia in combination U. Nordhues, C. Sorg, C. Sunderko¨tter, and J. Roth. 2007. Glucocorticoids induce with corticosteroid treatment, CCL5, CXCL1, CXCL2, and neu- differentiation of a specifically activated, anti-inflammatory subtype of human monocytes. Blood 109: 1265–1274. trophils were found to be upregulated by dexamethasone treatment 20. van der Strate, B. W., D. S. Postma, C. A. Brandsma, B. N. Melgert, M. A. Luinge, Downloaded from (56, 57). Interestingly, in the bronchoalveolar lavage of patients with M. Geerlings, M. N. Hylkema, A. van den Berg, W. Timens, and H. A. Kerstjens. COPD treated with budesonide, the number of macrophages was also 2006. Cigarette smoke-induced emphysema: A role for the B cell? Am.J.Respir. Crit. Care Med. 173: 751–758. increased (12). These unexpected effects of corticosteroids are there- 21. Melgert, B. N., W. Timens, H. A. Kerstjens, M. Geerlings, M. A. Luinge, fore reported more often, and they deserve further attention (58). J. P. Schouten, D. S. Postma, and M. N. Hylkema. 2007. Effects of 4 months of This study puts forward a set of GCR/TLR/IFN receptor- smoking in mice with ovalbumin-induced airway inflammation. Clin. Exp. Allergy 37: 1798–1808. regulated macrophage markers that could be used as biomarkers 22. Griffith, R. B. 1984. A simple machine for smoke analytical studies and total http://www.jimmunol.org/ to monitor GC efficacy and further investigate the interaction of particulate matter collection for biological studies. Toxicology 33: 33–41. 23. Griffith, R. B., and R. Hancock. 1985. Simultaneous mainstream-sidestream GCs and macrophages in human disease. Furthermore, our results smoke exposure systems I. Equipment and procedures. Toxicology 34: 123–138. add to the emerging concept of GC-insensitive and GC-exacerbated 24. Watson, E. S., A. B. Jones, M. K. Ashfaq, and J. T. Barrett. 1987. Spectro- pathways in macrophages that could explain their lack of efficacy in photometric evaluation of carboxyhemoglobin in blood of mice after exposure to marijuana or tobacco smoke in a modified Walton horizontal smoke exposure macrophage-dominated disorders, such as COPD. Therapeutically, machine. J. Anal. Toxicol. 11: 19–23. the innate–adaptive immune response shift induced by GCs in mac- 25. Melgert, B. N., D. S. Postma, M. Geerlings, M. A. Luinge, P. A. Klok, B. W. van rophages and its subdivision in pathways could offer novel views der Strate, H. A. Kerstjens, W. Timens, and M. N. Hylkema. 2004. Short-term smoke exposure attenuates ovalbumin-induced airway inflammation in allergic on other anti-inflammatory therapeutic strategies. mice. Am. J. Respir. Cell Mol. Biol. 30: 880–885. 26. Liu, Y., Y. Endo, D. Iwaki, M. Nakata, M. Matsushita, I. Wada, K. Inoue, by guest on September 29, 2021 M. Munakata, and T. Fujita. 2005. Human M-ficolin is a secretory protein that Acknowledgments activates the lectin complement pathway. J. Immunol. 175: 3150–3156. We thank Marie Geerlings, Nicole Stowell, Anuk Das, and Don Griswold 27. Aho, S., P. Lehtinen, and E. Kulonen. 1980. Effects of purified macrophage for experimental help; the Laboratory of Viral Immune Pathogenesis (Ac- RNase on granuloma fibroblasts with reference to silicosis. Acta Physiol. Scand. 109: 275–281. ademic Medical Center, Amsterdam, The Netherlands), Dr. Rene´ Lutter, 28. Shaykhiev, R., A. Krause, J. Salit, Y. Strulovici-Barel, B. G. Harvey, and Prof. Peter Sterk for supporting this project; and Prof. Siamon Gordon T. P. O’Connor, and R. G. Crystal. 2009. Smoking-dependent reprogramming of for reading the manuscript and helpful discussions. alveolar macrophage polarization: implication for pathogenesis of chronic ob- structive pulmonary disease. J. Immunol. 183: 2867–2883. 29. Sajjadi, F. G., K. Takabayashi, A. C. Foster, R. C. Domingo, and G. S. Firestein. Disclosures 1996. Inhibition of TNF-alpha expression by adenosine: role of A3 adenosine GlaxoSmithKline financially supported this study. receptors. J. Immunol. 156: 3435–3442. 30. Hasko´, G., C. Szabo´,Z.H.Ne´meth, V. Kvetan, S. M. Pastores, and E. S. Vizi. 1996. Adenosine receptor agonists differentially regulate IL-10, TNF-alpha, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. References J. Immunol. 157: 4634–4640. 1. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. 31. Li, Q., P. W. Park, C. L. Wilson, and W. C. Parks. 2002. Matrilysin shedding of Nat. Rev. Immunol. 5: 953–964. syndecan-1 regulates chemokine mobilization and transepithelial efflux of neu- 2. Mosser, D. M., and J. P. Edwards. 2008. Exploring the full spectrum of mac- trophils in acute lung injury. Cell 111: 635–646. rophage activation. Nat. Rev. Immunol. 8: 958–969. 32. Zahuczky, G., E. Kristo´f, G. Majai, and L. Fe´su¨s. 2011. Differentiation and 3. Sica, A., and A. Mantovani. 2012. Macrophage plasticity and polarization: glucocorticoid regulated apopto-phagocytic gene expression patterns in human in vivo veritas. J. Clin. Invest. 122: 787–795. macrophages. Role of Mertk in enhanced phagocytosis. PLoS ONE 6: e21349. 4. Murray, P. J., and T. A. Wynn. 2011. Protective and pathogenic functions of 33. Esmailpour, N., P. Ho¨gger, K. F. Rabe, U. Heitmann, M. Nakashima, and macrophage subsets. Nat. Rev. Immunol. 11: 723–737. P. Rohdewald. 1997. Distribution of inhaled fluticasone propionate between 5. Glass, C. K., and K. Saijo. 2010. Nuclear receptor transrepression pathways human lung tissue and serum in vivo. Eur. Respir. J. 10: 1496–1499. that regulate inflammation in macrophages and T cells. Nat. Rev. Immunol. 10: 34. Mukhopadhyay, S., L. Peiser, and S. Gordon. 2004. Activation of murine mac- rophages by Neisseria meningitidis and IFN-gamma in vitro: distinct roles of 365–376. class A scavenger and Toll-like pattern recognition receptors in selective mod- 6. Barnes, P. J. 2011. Glucocorticosteroids: current and future directions. Br. J. ulation of surface phenotype. J. Leukoc. Biol. 76: 577–584. Pharmacol. 163: 29–43. 35. Schroder, K., K. M. Irvine, M. S. Taylor, N. J. Bokil, K. A. Le Cao, 7. Esmailpour, N., P. Hogger, and P. Rohdewald. 1998. Binding kinetics of ¨ K. A. Masterman, L. I. Labzin, C. A. Semple, R. Kapetanovic, L. Fairbairn, et al. budesonide to the human glucocorticoid receptor. Eur. J. Pharm. Sci. 6: 219– 2012. Conservation and divergence in Toll-like receptor 4-regulated gene ex- 223. pression in primary human versus mouse macrophages. Proc. Natl. Acad. Sci. 8. Yona, S., and S. Gordon. 2007. Inflammation: Glucocorticoids turn the monocyte USA 109: E944–E953. switch. Immunol. Cell Biol. 85: 81–82. 36. Ogawa, S., J. Lozach, C. Benner, G. Pascual, R. K. Tangirala, S. Westin, 9. Calverley, P. M., J. A. Anderson, B. Celli, G. T. Ferguson, C. Jenkins, A. Hoffmann, S. Subramaniam, M. David, M. G. Rosenfeld, and C. K. Glass. P. W. Jones, J. C. Yates, and J. Vestbo; TORCH investigators. 2007. Salmeterol 2005. Molecular determinants of crosstalk between nuclear receptors and toll- and fluticasone propionate and survival in chronic obstructive pulmonary dis- like receptors. Cell 122: 707–721. ease. N. Engl. J. Med. 356: 775–789. 37. Lapperre, T. S., J. B. Snoeck-Stroband, M. M. Gosman, D. F. Jansen, A. van 10. Rabe, K. F., and J. A. Wedzicha. 2011. Controversies in treatment of chronic Schadewijk, H. A. Thiadens, J. M. Vonk, H. M. Boezen, N. H. Ten Hacken, obstructive pulmonary disease. Lancet 378: 1038–1047. J. K. Sont, et al; Groningen Leiden Universities Corticosteroids in Obstructive The Journal of Immunology 13

Lung Disease Study Group. 2009. Effect of fluticasone with and without sal- 48. Flammer, J. R., J. Dobrovolna, M. A. Kennedy, Y. Chinenov, C. K. Glass, meterol on pulmonary outcomes in chronic obstructive pulmonary disease: L. B. Ivashkiv, and I. Rogatsky. 2010. The type I interferon signaling pathway is a randomized trial. Ann. Intern. Med. 151: 517–527. a target for glucocorticoid inhibition. Mol. Cell. Biol. 30: 4564–4574. 38. Thornton Snider, J., Y. Luna, K. S. Wong, J. Zhang, S. S. Chen, P. J. Gless, and 49. Snyder, D. S., and E. R. Unanue. 1982. Corticosteroids inhibit murine macro- D. P. Goldman. 2012. Inhaled corticosteroids and the risk of pneumonia in phage Ia expression and interleukin 1 production. J. Immunol. 129: 1803–1805. Medicare patients with COPD. Curr. Med. Res. Opin. 28: 1959–1967. 50. Schwiebert, L. M., R. P. Schleimer, S. F. Radka, and S. J. Ono. 1995. Modulation 39. Sharif, O., U. Matt, S. Saluzzo, K. Lakovits, I. Haslinger, T. Furtner, of MHC class II expression in human cells by dexamethasone. Cell. Immunol. B. Doninger, and S. Knapp. 2013. The scavenger receptor CD36 downmodulates 165: 12–19. 51. Franchimont, D. 2004. Overview of the actions of glucocorticoids on the im- the early inflammatory response while enhancing bacterial phagocytosis during mune response: a good model to characterize new pathways of immunosup- pneumococcal pneumonia. J. Immunol. 190: 5640–5648. pression for new treatment strategies. Ann. N. Y. Acad. Sci. 1024: 124–137. 40. Martinez, F. O., A. Sica, A. Mantovani, and M. Locati. 2008. Macrophage ac- 52. Warren, M. K., and S. N. Vogel. 1985. Opposing effects of glucocorticoids on tivation and polarization. Front. Biosci. 13: 453–461. interferon-gamma-induced murine macrophage Fc receptor and Ia antigen ex- 41. Koning, N., M. van Eijk, W. Pouwels, M. S. Brouwer, D. Voehringer, I. Huitinga, pression. J. Immunol. 134: 2462–2469. R. M. Hoek, G. Raes, and J. Hamann. 2010. Expression of the inhibitory CD200 53. Galon, J., D. Franchimont, N. Hiroi, G. Frey, A. Boettner, M. Ehrhart-Bornstein, receptor is associated with alternative macrophage activation. J. Innate Immun. 2: J. J. O’Shea, G. P. Chrousos, and S. R. Bornstein. 2002. Gene profiling reveals 195–200. unknown enhancing and suppressive actions of glucocorticoids on immune cells. 42. Ambarus, C. A., S. Krausz, M. van Eijk, J. Hamann, T. R. Radstake, FASEB J. 16: 61–71. K. A. Reedquist, P. P. Tak, and D. L. Baeten. 2012. Systematic validation of specific 54. Viola, A., and A. D. Luster. 2008. Chemokines and their receptors: drug targets phenotypic markers for in vitro polarized human macrophages. J. Immunol. in immunity and inflammation. Annu. Rev. Pharmacol. Toxicol. 48: 171–197. Methods 375: 196–206. 55. Kent, L. M., L. J. Smyth, J. Plumb, C. L. Clayton, S. M. Fox, D. W. Ray, 43. Petersen, H., A. Kullberg, S. Edsba¨cker, and L. Greiff. 2001. Nasal retention of S. N. Farrow, and D. Singh. 2009. Inhibition of lipopolysaccharide-stimulated budesonide and fluticasone in man: formation of airway mucosal budesonide- chronic obstructive pulmonary disease macrophage inflammatory gene expres- esters in vivo. Br. J. Clin. Pharmacol. 51: 159–163. sion by dexamethasone and the p38 mitogen-activated protein kinase inhibitor 44. Harrison, T. W., and A. E. Tattersfield. 2003. Plasma concentrations of flutica- N-cyano-N’-(2-[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8- Downloaded from sone propionate and budesonide following inhalation from dry powder inhalers dihydropyrido[2,3-d] pyrimidin-2-yl]aminoethyl)guanidine (SB706504). J. Pharmacol. Exp. Ther. 328: 458–468. by healthy and asthmatic subjects. Thorax 58: 258–260. 56. Aoki, K., Y. Ishida, N. Kikuta, H. Kawai, M. Kuroiwa, and H. Sato. 2002. Role 45. Lacey, D. C., A. Achuthan, A. J. Fleetwood, H. Dinh, J. Roiniotis, G. M. Scholz, of CXC chemokines in the enhancement of LPS-induced neutrophil accumula- M. W. Chang, S. K. Beckman, A. D. Cook, and J. A. Hamilton. 2012. Defining tion in the lung of mice by dexamethasone. Biochem. Biophys. Res. Commun. GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro 294: 1101–1108. models. J. Immunol. 188: 5752–5765. 57. Tagliabue, C., C. M. Salvatore, C. Techasaensiri, A. Mejias, J. P. Torres, K. Katz, 46. Sawmynaden, P., and M. Perretti. 2006. Glucocorticoid upregulation of the annexin- A. M. Gomez, S. Esposito, N. Principi, and R. D. Hardy. 2008. The impact of A1 receptor in leukocytes. Biochem. Biophys. Res. Commun. 349: 1351–1355. steroids given with macrolide therapy on experimental Mycoplasma pneumoniae http://www.jimmunol.org/ 47. Mozo, L., A. Sua´rez, and C. Gutie´rrez. 2004. Glucocorticoids up-regulate con- respiratory infection. J. Infect. Dis. 198: 1180–1188. stitutive interleukin-10 production by human monocytes. Clin. Exp. Allergy 34: 58. Wilckens, T., and R. De Rijk. 1997. Glucocorticoids and immune function: 406–412. unknown dimensions and new frontiers. Immunol. Today 18: 418–424. by guest on September 29, 2021